Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT METHODS, COMPOSITIONS, AND KITS INCLUDING CELL-PENETRATING AGENTS CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63/538,596, filed on September 15, 2023, the content of which is hereby incorporated by reference in its entirety. SEQUENCE LISTING This application contains a Sequence Listing that has been submitted electronically as an XML file named 50887-0007WO1_ST26_SL.XML.” The XML file, created on August 30, 2024, is 238,166 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety. BACKGROUND Biomolecules and macromolecules are valuable assets for diagnostic and therapeutic applications. Antibodies alone have become a major therapeutic modality, achieving exceptional success in treating a variety of diseases, including cancers, immune disorders, and infectious diseases. In the past four decades, more than one hundred antibody-based drugs have been approved by the FDA for the treatment of these diseases. However, many biomolecules and macromolecules (including antibodies) are limited in their ability to enter cells. As a consequence, the therapeutic and diagnostic applications for these molecules have been largely limited to binding to and/or modulating extracellular targets, such as cell-surface receptors. Despite the successes achieved to date by modulating extracellular targets, intracellular targets also play crucial roles in disease development and progression. Treatments based on modulating these intracellular targets have traditionally utilized small molecules. However, many intracellular targets are extremely difficult to modulate with small molecules, and in some cases these targets are referred to in the literature as “undruggable.” By facilitating the intracellular delivery of payloads that bind to and/or modulate these intracellular targets, the therapeutic and diagnostic potential of biomolecules and/or macromolecules can be greatly expanded for many
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT biomedical applications, including diagnostics, therapeutics, and fundamental research. Current intracellular delivery systems have not achieved meaningful success in delivering payloads directed at intracellular targets. Thus, new systems and methods are needed to facilitate the intracellular delivery of payload molecules. SUMMARY Provided herein is a cell membrane internalizing peptide (CMIP) comprising an amino acid sequence selected from Formulas (IA) to (IH): X
1-X
2-X
3-X
4-X
5-X
6-X
7-F-S-G-K-A-A-A-K-X
16-E-A-K-X
20-X
21-X
22-X
23-X
24-X
25 (IA); I-W-L-T-A-L-X
7-F-X
9-X
10-X
11-X
12-X
13-X
14-X
15-A-X
17-X
18-X
19-Q-F-X
22-X
23-X
24-X
25 (IB); I-W-X
3-X
4-X
5-X
6-K-X
8-S-X
10-X
11-H-X
13-X
14-X
15-A-E-X
18-X
19-X
20-X
21-L-S-K-L (IC); X
1-W-X
3-T-A-X
6-X
7-X
8-X
9-G-K-A-X
13-A-K-A-X
17-A-K-Q-X
21-X
22-S-K-X
25 (ID); I-X
2-X
3-X
4-X
5-L-K-F-X
9-X
10-X
11-X
12-A-A-K-X
16-E-X
18-K-Q-F-X
22-X
23-X
24-L (IE); X
1-W-L-T-A-X
6-X
7-X
8-S-G-X
11-X
12-A-A-X
15-A-X
17-X
18-K-X
20-F-X
22-X
23-X
24-X
25 (IF); S-X
2-L-T-A-X
6-X
7-X
8-X
9-X
10-K-H-X
13-I-T-Q-X
17-X
18-K-R-R-X
22-X
23-X
24-X
25 (IG); R-R-L-T-X
5-L-F-K-X
9-G-X
11-X
12-X
13-X
14-E- X
16-I-X
18-X
19-X
20-F (IH); wherein X
1 X
2, X
3, X
4, X
5, X
6, X
7, X
8, X
9, X
10, X
11, X
12, X
13, X
14, X
15, X
16, X
17, X
18, X
19, X
20, and X
21 are each independently a natural or unnatural amino acid residue; and X
22, X
23, X
24, and X
25 are each independently absent or are each independently a natural or unnatural amino acid residue; or a pharmaceutically acceptable salt thereof. In some embodiments, at least one of (a) to (c) is present in the CMIP sequence: (a) X
21 is selected from alanine, arginine, lysine, phenylalanine, and tryptophan; (b) X
9 is serine and X
12 is selected from alanine, arginine, glutamic acid, glutamine, phenylalanine, and tryptophan; or (c) X
5 is histidine and X
3 is selected from alanine, glutamic acid, leucine, lysine, phenylalanine, and tryptophan. In some aspects, the present disclosure provides a cell membrane internalizing peptide (CMIP) comprising an amino acid sequence selected from Formulas (IA) to (IH): X
1-X
2-X
3-X
4-X
5-X
6-X
7-F-S-G-K-A-A-A-K-X
16-E-A-K-X
20-X
21-X
22-X
23-X
24-X
25 (IA);
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT I-W-L-T-A-L-X
7-F-X
9-X
10-X
11-X
12-X
13-X
14-X
15-A-X
17-X
18-X
19-Q-F-X
22-X
23-X
24-X
25 (IB); I-W-X
3-X
4-X
5-X
6-K-X
8-S-X
10-X
11-H-X
13-X
14-X
15-A-E-X
18-X
19-X
20-X
21-L-S-K-L (IC); X
1-W-X
3-T-A-X
6-X
7-X
8-X
9-G-K-A-X
13-A-K-A-X
17-A-K-Q-X
21-X
22-S-K-X
25 (ID); I-X
2-X
3-X
4-X
5-L-K-F-X
9-X
10-X
11-X
12-A-A-K-X
16-E-X
18-K-Q-F-X
22-X
23-X
24-L (IE); X
1-W-L-T-A-X
6-X
7-X
8-S-G-X
11-X
12-A-A-X
15-A-X
17-X
18-K-X
20-F-X
22-X
23-X
24-X
25 (IF); S-X
2-L-T-A-X
6-X
7-X
8-X
9-X
10-K-H-X
13-I-T-Q-X
17-X
18-K-R-R-X
22-X
23-X
24-X
25 (IG); R-R-L-T-X
5-L-F-K-X
9-G-X
11-X
12-X
13-X
14-E- X
16-I-X
18-X
19-X
20-F (IH); wherein each of X
1 through X
25 is independently an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; or a pharmaceutically acceptable salt thereof. In some aspects, the present disclosure provides a cell membrane internalizing peptide (CMIP) comprising an amino acid sequence selected from Formulas (IIA) to (IID): I-X
2-X
3-T-A-L-X
7-F-X
9-G-X
11-A-A-X
14-K-X
16-E-A-X
19-Q-F-L-X
23-X
24-L (IIA); ; ; wherein X
21 and
X
25 are either absent or each independently a natural or unnatural amino acid residue; or a pharmaceutically acceptable salt thereof. In another aspect, the present disclosure provides a CMIP comprising an amino acid sequence having a formula selected from Formula (IIIA) to Formula (IIIC): I-X
2-X
3-T-A-L-X
7-F-X
9-G-X
11-A-A-X
14-K-X
16-E-A-X
19-Q-F-L-X
23-X
24-L (IIIA); I-W-X
3-X
4-A-L-X
7-F-X
9-G-X
11-X
12-X
13-A-X
15-A-E-A-X
19-Q-F-X
22-S-X
24-L (IIIB); X
1-X
2-L-T-A-L-K-F-S-X
10-K-A-A-A-X
15-A-E-A-K-Q-X
21-L-S-X
24-L (IIIC); wherein X
1 is Isoleucine or lysine; X
2 is arginine or tryptophan; X
3 is arginine, glutamic acid, leucine, or tryptophan; X
4 is threonine or tryptophan; X
7 is arginine, isoleucine, or lysine; X
9 is arginine, histidine, or serine; X
10 is glycine or tyrosine; X
11 is arginine, isoleucine, lysine, or
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT threonine; X
12 is alanine or glutamine; X
13 is alanine or serine; X
14 is alanine, histidine, leucine, or valine; X
15 is arginine, glutamic acid, or lysine; X
16 is alanine or lysine; X
18 is alanine, methionine, or tryptophan; X
19 is arginine or lysine; X
21 is arginine or phenylalanine; X
22 is arginine, leucine, or tryptophan; X
23 is arginine or serine; and X
24 is arginine, lysine, or tryptophan; or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP consists essentially of an amino acid sequence selected from Formulas (IA) to (IH), Formulas (IIA) to (IID), and Formulas (IIIA) to (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from Formulas (IA) to (IH), Formulas (IIA) to (IID), and Formulas (IIIA) to (IIIC). Also provided herein are nucleic acids encoding any of the CMIPs described herein. Also provided herein are vectors including any of the nucleic acids described herein. Also provided herein are cells including any of the CMIPs described herein, any of the nucleic acids described herein, and/or any of the vectors described herein. In some embodiments, the cell is a mammalian cell. Also provided herein are methods of producing any of the CMIPs described herein, the methods including culturing a cell including a nucleic acid encoding the CMIP in a liquid culture medium under conditions that allow the cell to produce the CMIP; and harvesting the CMIP from the cell or the liquid culture medium. Also provided herein are pharmaceutical compositions including any of the CMIPs described herein and a pharmaceutically acceptable excipient. Also provided herein are cell-penetrating agents (CPA) including (i) a CMIP and (ii) a payload molecule. In some embodiments, the CMIP is an M-lycotoxin derivative. In some embodiments, the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In some embodiments, the CMIP is covalently linked to the payload molecule. In some embodiments, the CMIP is non-covalently linked to the payload molecule. In some embodiments, the cell-penetrating agent includes a linker connecting the CMIP to the payload molecule. In some embodiments, the linker is covalently linked to both the CMIP and the payload molecule. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker includes a polypeptide. In some embodiments, the linker includes an amino acid sequence selected from Table 4. In some embodiments, the payload molecule is a biomolecule. In some embodiments, the biomolecule including a carbohydrate, a lipid, a nucleic acid, a peptide nucleic acid, a peptidomimetic, a protein, or a peptide. In some embodiments, the payload molecule is a macromolecule. In some embodiments, the payload molecule has a molecular weight of about 5 kD to about 1000 kD. In some embodiments, the payload includes a proteinaceous molecule. In some embodiments, the proteinaceous molecule is about 100 to about 1500 amino acids. In some embodiments, the proteinaceous molecule is an is an antibody, an enzyme, a protein, or a peptide. In some embodiments, the proteinaceous molecule is linked to the CMIP via a polypeptide linker. In some embodiments, the cell-penetrating agent is a fusion protein. In some embodiments, the cell-penetrating agent is encoded by a nucleic acid encoding a CMIP and a payload molecule or a portion thereof. In some embodiments, the payload molecule or the portion thereof is linked to the C-terminus of the CMIP. In some embodiments, the payload molecule or the portion thereof is linked to the N-terminus of the CMIP. In some embodiments, the payload molecule is an antibody or a portion thereof. In some embodiments, the antibody or a portion thereof includes a heavy chain. In some embodiments, the CMIP is covalently linked to a C-terminus of the heavy chain or a functional portion thereof. In some embodiments, the CMIP is covalently linked to a N-terminus of the heavy chain or a functional portion thereof. In some embodiments, the antibody or portion thereof includes a light chain. In some embodiments, the CMIP is covalently linked to a C-terminus of the light chain or a functional portion thereof. In some embodiments, the CMIP is covalently linked to a N- terminus of the light chain or a functional portion thereof. In some embodiments, the heavy chain of the antibody or a portion thereof and the CMIP are expressed as a single polypeptide. In
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT some embodiments, the light chain of the antibody or a portion thereof and the CMIP are expressed as a single polypeptide. In some embodiments, the antibody or fragment thereof specifically binds to a target protein. In some embodiments, the target protein is a cytosolic protein. In some embodiments, the target protein is a membrane-associated protein. In some embodiments, the target protein is an enzyme. In some embodiments, the target protein is a cytoskeletal protein. In some embodiments, the target protein is a beta-actin protein. In some embodiments, the target protein is an alpha tubulin protein. In some embodiments, the target protein is an oxidoreductase. In some embodiments, the target protein is an endoplasmic reticulum oxidoreductin-1-like (EROL1L) protein. In some embodiments, the target protein is a mitochondrial protein. In some embodiments, the target protein is a mitochondrial transport protein. In some embodiments, the target protein is a translocase of outer mitochondrial membrane 20 (TOMM20) protein. In some embodiments, the target protein is a nuclear membrane protein. In some embodiments, the target protein is an outer nuclear membrane protein. In some embodiments, the target protein is a nuclear envelope spectrin repeat protein-1 (Nesprin-1) protein. In some embodiments, the payload molecule is an enzyme or a functional portion thereof. In some embodiments, the enzyme is a kinase. In some embodiments, the enzyme is a serine/threonine kinase. In some embodiments, the serine/threonine kinase is GSK3β. In some embodiments, the enzyme is a protease. In some embodiments, the protease is a cysteine protease. In some embodiments, the cysteine protease is a non-lysosomal cysteine protease. In some embodiments, the non-lysosomal cysteine protease is calpain. In some embodiments, the enzyme is a bioluminescent protein. In some embodiments, the bioluminescent protein is a luciferase. In some embodiments, the CMIP is linked to the C- terminus of the enzyme or a functional portion thereof. In some embodiments, the CMIP is linked to the N-terminus of the enzyme or a functional portion thereof. In some embodiments, the cell-penetrating agent is a fusion protein. In some embodiments, the CMIP and the enzyme are expressed as a single polypeptide. Also provided herein are pharmaceutical compositions including any of the cell- penetrating agents described herein and a pharmaceutically acceptable excipient. Also provided herein are kits including any of the pharmaceutical compositions described herein.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Also provided herein are nucleic acids encoding at least a portion of any of the cell- penetrating agents described herein. In some embodiments, the nucleic acid encodes the CMIP. In some embodiments, the nucleic acid encodes the CMIP and at least a portion of the payload molecule. In some embodiments, the nucleic acid encodes the CMIP, a polypeptide linker, and at least a portion of the payload molecule. In some embodiments, the nucleic acid encodes the CMIP, the polypeptide linker, and the payload molecule. Also provided herein are vectors including any of the nucleic acids described herein. Also provided herein are cells including any of the cell-penetrating agents described herein, any of the nucleic acids described herein, or any of the vectors described herein. In some embodiments, the cell is a mammalian cell. Also provided herein are methods of producing any of the cell-penetrating agents described herein, the method including: culturing a cell including a nucleic acid encoding the CMIP and at least a portion of the payload molecule in a liquid culture medium under conditions that allow the cell to produce a polypeptide including the CMIP and at least a portion of the payload molecule; and harvesting the polypeptide from the cell or the liquid culture medium. Also provided herein are methods of delivering a payload molecule into a cell, including contacting any of the cell-penetrating agents described herein with the cell, thereby resulting in the internalization of the payload molecule into the cell and transfer of the payload molecule to the cytosol. Also provided herein are methods of delivering an antibody into a cell, including contacting any of the cell-penetrating agents described herein with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol. Also provided herein are methods of binding an intracellular target protein in a cell, the method including: (a) contacting any of the cell-penetrating agents described herein with the cell thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; and (b) binding of the cell-penetrating agent to the intracellular target protein. Also provided herein are methods of modulating at least one activity of an intracellular target (a) contacting any of the cell-penetrating agents described herein with the cell thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT penetrating agent to the cytosol; and (b) binding of the cell-penetrating agent to the intracellular target protein, thereby directly or indirectly modulating at least one activity of an intracellular target protein in the cell. In some embodiments, modulating includes reducing an activity of the intracellular target protein. In some embodiments, modulating includes increasing an activity of the intracellular target. In some embodiments, modulating includes altering the site of activity of the intracellular target. In some embodiments, modulating includes altering the turnover of the intracellular target. Also provided herein are methods of inhibiting at least one activity of an intracellular target protein in a cell, the method including: (a) contacting any of the cell-penetrating agents described herein with the cell thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; and (b) binding of the cell- penetrating agent to the intracellular target protein, thereby inhibiting at least one activity of an intracellular target protein in the cell. Also provided herein are methods of delivering an enzyme into a cell, including contacting any of the cell-penetrating agents described herein with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol. Also provided herein are methods of increasing intracellular enzyme activity in a cell, the method including contacting any of the cell-penetrating agents described herein with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the enzyme portion of the cell-penetrating agent is enzymatically active in the cytosol. Also provided herein are methods of restoring intracellular enzyme activity in a cell having reduced activity of an intracellular enzyme, the method including contacting any of the cell-penetrating agents described herein with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the enzyme portion of the cell-penetrating agent performs the same or a similar enzymatic reaction as the intracellular enzyme and the enzyme portion of the cell-penetrating agent performs is active in the cytosol. Also provided herein are methods of delivering a payload protein sequence to the cytosol of a cell, the method including contacting the cell with a polypeptide including a CMIP and the
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT payload protein sequence, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. Also provided herein are methods of delivering a payload protein sequence to the nucleus of a cell, the method including contacting the cell with a polypeptide including a CMIP and the payload protein sequence. In some embodiments, the CMIP is an M-lycotoxin derivative. In some embodiments, the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. In some embodiments, the payload protein sequence has a molecular weight of about 5 kD to about 1000 kD. In some embodiments, the payload protein sequence is about 100 to about 1500 amino acids. Also provided herein are methods of delivering an antigen-binding domain to the cytosol of a cell, the method including contacting the cell with a polypeptide including a CMIP and the antigen-binding domain, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG),
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. Also provided herein are methods of delivering an antibody to the cytosol of a cell, the method including contacting the cell with a polypeptide including a CMIP and the antibody, wherein the CMIP comprises an amino acid sequence selected the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. Also provided herein are methods of inhibiting an intracellular target protein in a cell, the method including: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the cell-penetrating agent includes: (i) an antibody that specifically binds the intracellular target protein; and (ii) a CMIP covalently linked to the antibody; and (b) binding of the cell-penetrating agent to the intracellular target protein. In some embodiments, the target protein is selected from a cytosolic protein, a mitochondrial protein, an endoplasmic reticulum lumen protein, and a nuclear membrane protein. In some embodiments, the target protein is selected from a protease, a cytoskeletal protein, an oxidoreductase, a mitochondrial transport protein, and an outer nuclear membrane protein. In some embodiments, the target protein is selected from beta-actin, alpha tubulin, ERO1L, TOMM20, and Nesprin-1. In some embodiments, the target protein is selected from beta-actin, alpha tubulin, ERO1L, and TOMM20. In some embodiments, the target protein is a beta-actin protein. In some embodiments, the target protein is an alpha tubulin protein. In some embodiments, the target protein is an EROL1L protein. In some embodiments, the target protein is a TOMM20 protein. In some embodiments, the target protein is a Nesprin-1 protein. Also provided herein are method for inhibiting an intracellular TOMM20 protein in a cell including: (a) contacting a cell-penetrating agent with the cell thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer to the cytosol, the cell-
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT penetrating agent including: (i) an antibody that specifically binds to the TOMM20 protein; and (ii) a CMIP derived from M-lycotoxin; and (b) binding the antibody of the cell-penetrating agent to the intracellular TOMM20 protein, thereby inhibiting the intracellular TOMM20 protein. Also provided herein are methods for inhibiting an intracellular beta-actin protein in a cell including: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer to the cytosol, the cell- penetrating agent including: (i) an antibody that specifically binds to the beta-actin protein; and (ii) a CMIP derived from M-lycotoxin; and (b) binding the antibody of the cell-penetrating agent to the intracellular beta-actin protein. Also provided herein are methods for inhibiting polymerization of an intracellular alpha- tubulin protein in a cell including: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer to the cytosol, the cell-penetrating agent including: (i) an antibody that specifically binds to the alpha- tubulin protein; and (ii) a CMIP derived from M-lycotoxin; and (b) binding the antibody of the cell-penetrating agent to the intracellular alpha-tubulin protein. Also provided herein are methods for inhibiting an intracellular endoplasmic reticulum oxidoreductin-1-like protein (ERO1L) protein in a cell including: (a) contacting a cell- penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer to the cytosol, the cell-penetrating agent including: (i) an antibody that specifically binds to the ERO1L protein; and (ii) a CMIP derived from M-lycotoxin; and (b) binding the antibody of the cell-penetrating agent to the intracellular ERO1L protein. Also provided herein are methods for inhibiting an intracellular translocase of outer mitochondrial membrane 20 (TOMM20) protein in a cell including: (a) contacting a cell- penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer to the cytosol, the cell-penetrating agent including: (i) an antibody that specifically binds to the TOMM20 protein; and (ii) a CMIP derived from M-lycotoxin; and (b) binding of the cell-penetrating agent to the intracellular TOMM20 protein. Also provided herein are methods for inhibiting an intracellular Nesprin-1 protein in a cell including: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer to the cytosol, the cell- penetrating agent including: (i) an antibody that specifically binds to the Nesprin-1 protein; and
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT (ii) a CMIP derived from M-lycotoxin; and (b) binding of the cell-penetrating agent to the intracellular Nesprin-1 protein. In some embodiments, the CMIP is covalently linked to the antibody. In some embodiments, the CMIP is covalently linked to the antibody via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. Also provided herein are methods of delivering an enzyme to the cytosol of a cell, the method including contacting the cell with a polypeptide including a CMIP and the enzyme. In some embodiments, the CMIP is an M-lycotoxin derivative. In some embodiments, the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. Also provided herein are methods of increasing intracellular enzymatic activity in a cell, the method including: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the cell-penetrating agent includes: (i) an enzyme capable of catalyzing the intracellular enzymatic activity; and (ii) a CMIP covalently linked to the enzyme; and (b) catalyzing the enzymatic reaction inside the cell using the enzyme portion of the cell- penetrating agent. Also provided herein are methods of restoring intracellular enzymatic activity in a cell having reduced intracellular enzymatic activity, the method including: (a) contacting a cell- penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the cell- penetrating agent includes: (i) an enzyme capable of catalyzing the intracellular enzymatic activity; and (ii) a CMIP covalently linked to the enzyme; and (b) catalyzing the enzymatic reaction inside the cell using the enzyme portion of the cell-penetrating agent. In some embodiments, the enzyme is selected from a kinase, a protease, and a reporter enzyme (or functional fragments thereof). In some embodiments, the enzyme is selected from
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT GSK3β, calpain, and luciferase. In some embodiments, the enzyme is GSK3β. In some embodiments, the enzyme is calpain. In some embodiments, the enzyme is a luciferase. Also provided herein are methods of increasing GSK3β activity in a cell, the method including: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the cell-penetrating agent includes: (i) a GSK3β enzyme; and (ii) a CMIP covalently linked to the GSK3β enzyme; and (b) phosphorylating or dephosphorylating a downstream target of GSK3β using the GSK3β enzyme of the cell-penetrating agent. Also provided herein are methods of restoring intracellular GSK3β activity in a cell having reduced intracellular GSK3β activity, the method including: (a) contacting a cell- penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the cell- penetrating agent includes: (i) a GSK3β enzyme; and (ii) a CMIP covalently linked to the GSK3β enzyme; and (b) phosphorylating or dephosphorylating a downstream target of GSK3β using the GSK3β enzyme of the cell-penetrating agent. Also provided herein are methods of increasing calpain activity in a cell, the method including: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the cell-penetrating agent includes: (i) a calpain enzyme; and (ii) a CMIP covalently linked to the calpain enzyme; and (b) cleaving a downstream target of Calpain using the calpain enzyme of the cell-penetrating agent. Also provided herein are methods of restoring intracellular calpain activity in a cell having reduced intracellular calpain activity, the method including: (a) contacting a cell- penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the cell- penetrating agent includes: (i) a calpain enzyme; and (ii) a CMIP covalently linked to the calpain enzyme; and (b) cleaving a downstream target of calpain using the calpain enzyme of the cell- penetrating agent. Also provided herein are methods of increasing luciferase activity in a cell, the method including: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT to the cytosol; where the cell-penetrating agent includes: (i) a luciferase enzyme; and (ii) a CMIP covalently linked to the luciferase enzyme; and (b) oxidizing a downstream target of luciferase using the luciferase enzyme of the cell-penetrating agent. In some embodiments, the CMIP is covalently linked to the enzyme. In some embodiments, the CMIP is covalently linked to the enzyme via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a polypeptide linker. In some embodiments, the polypeptide linker includes an amino acid sequence selected from Table 4. In some embodiments, the CMIP is an M-lycotoxin derivative. In some embodiments, the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP comprises an amino acid sequence selected from Table 3. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is in a subject. Also provided herein are polypeptides including a CMIP and a light chain of an antibody or a fragment thereof, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the light chain of the antibody or a fragment thereof and the CMIP are connected via a polypeptide linker. In some embodiments, the light chain or fragment thereof is connected to the C-terminus of the CMIP. In some embodiments, the light chain or fragment thereof is connected to the N-terminus of the CMIP. Also provided herein are polypeptides including a CMIP and a heavy chain of an antibody or a fragment thereof, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the heavy chain of the antibody or the fragment thereof and the CMIP are connected via a polypeptide linker. In some embodiments, the heavy chain or the fragment thereof is connected to the C-terminus of the CMIP. In some
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT embodiments, the heavy chain or the fragment thereof is connected to the N-terminus of the CMIP. Also provided herein are polypeptides including a CMIP and a payload protein sequence, where the payload protein sequence is about 100 to about 1500 amino acids, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a payload protein sequence, where the payload protein sequence has a molecular weight of about 5 kD to about 1000 kD, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and an antigen-binding domain, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and an antibody, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a protein inhibitor of a kinase, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and at least a portion of a protein binder of a target protein, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the protein binder is an antibody or at least a portion thereof. In some embodiments, at least a portion of the antibody is a heavy chain of an antibody or a fragment thereof. In some embodiments, at least a portion of the antibody is a light chain of an antibody or a fragment thereof. In some embodiments, the target protein is selected from beta-actin, alpha tubulin, ERO1L, TOMM20, and Nesprin-1. In some embodiments, the target protein is selected from beta-actin, alpha tubulin, ERO1L, and Nesprin-1.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Also provided herein are polypeptides including a CMIP and an enzyme, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a kinase, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a GSK3β enzyme, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a protease, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a calpain protease, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a reporter enzyme, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a luciferase enzyme, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a payload protein sequence, where the CMIP is linked to the C-terminus of the payload protein sequence, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a payload protein sequence, where the CMIP is linked to the N-terminus of the payload protein sequence, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP is an M-lycotoxin derivative. In some embodiments, the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB),
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP comprises an amino acid sequence selected from Table 3. In some embodiments, the CMIP consists essentially of an amino acid sequence selected from Table 3. In some embodiments, the CMIP consists of an amino acid sequence selected from Table 3. Also provided herein are compositions including any of the polypeptides described herein. In some embodiments, the composition is a pharmaceutical composition. Also provided herein are kits including any of the compositions described herein or any of the pharmaceutical compositions described herein. Also provided herein are methods including contacting a mammalian cell with any of the cell-penetrating agents described herein, any of the polypeptides described herein, and/or any of the compositions described herein. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A is a schematic diagram showing a cell-penetrating agent including a cell- penetrating peptide and a payload molecule. FIG. 1B shows internalization and transfer to the cytosol of a cell-penetrating agent. FIG. 2A provides a helical wheel projection of a CMIP of the present disclosure. FIG. 2B provides calculated biophysical calculations for a CMIP of the present disclosure. FIG. 3A provides a schematic of a CPA comprising an antibody connected to a CIM via the light chain of the antibody. FIG. 3B provides a schematic of a CPA comprising an antibody connected to a CIM via the heavy chain of the antibody. FIG. 3C provides a schematic of a CPA comprising a Fab antibody fragment connected to a CIM.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 4A provides the EpiQuest predicted immunogenicity profile for CMIP39 (SEQ ID NO: 97), which has an IU score of 0. FIG. 4B provides the EpiQuest predicted immunogenicity profile for CMIP34 (SEQ ID NO: 92), which has an IU score of 0.4. FIG. 4C provides the EpiQuest predicted immunogenicity profile for CMIP48 (SEQ ID NO: 106), which has an IU score of 10. FIG. 4D provides the EpiQuest predicted immunogenicity profile for CMIP30 (SEQ ID NO: 88), which has an IU score of 1.2. FIG. 5A demonstrates pTDP-43 aggregates formed in cells following transient transfection with a GFP-2a-TDP-43 [mNLS DCS] construct. FIG. 5B shows cell count for HEK cells transiently transfected with either GFP-2a- TDP43 [mNLS DCS] construct or GFP only. FIG. 5C shows staining of pTDP-43 foci in HEK cells transiently transfected with a GFP-2a-TDP43 [mNLS DCS] construct using a panel of anti-TDP-43 antibodies. FIG. 6A shows the percentage of CPA-positive cells following incubation of GFP-2a- TDP43 transfected HEK cells with anti-TDP-43 CPAs of the present disclosure. FIG. 6B shows the number of CPA-positive spots per cell following incubation of GFP- 2a-TDP43 transfected HEK cells with anti-TDP-43 CPAs of the present disclosure. FIG. 7A shows the sum of p-TDP-43 foci pixels per well area following incubation of GFP-2a-TDP43 transfected HEK cells with anti-TDP-43 CPAs of the present disclosure. FIG. 7B shows the mean focus intensity of p-TDP-43 in GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 7C shows consistent cell count per well for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 7D shows the number of p-TDP-43 foci normalized by cell count for GFP-2a- TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 7E shows the mean focus area for p-TDP-43 foci for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 8A shows the sum of p-TDP-43 foci pixels per well area for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 8B shows consistent cell count per well across all GFP-2a-TDP43 transfected HEK cell populations tested (treated and control). FIG. 8C shows the number of p-TDP-43 foci normalized by cell count for GFP-2a- TDP43 transfected HEK cells after incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 8D shows the mean focus area for p-TDP-43 foci for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 9A shows the sum of p-TDP-43 foci pixels per well area, demonstrating a concentration-dependent reduction in total foci area for cells for GFP-2a-TDP43 transfected HEK cells following incubation with different concentrations of anti-TDP-43 CPAs of the present disclosure. FIG. 9B shows consistent cell count per well across all cell populations and concentrations tested (treatment and control). FIG. 9C shows the p-TDP-43 foci number, demonstrating a concentration-dependent increase in foci count for GFP-2a-TDP43 transfected HEK cells following incubation with different concentrations of anti-TDP-43 CPAs of the present disclosure. FIG. 9D shows the p-TDP-43 mean focus size, demonstrating a concentration-dependent decrease in mean focus size for GFP-2a-TDP43 transfected HEK cells following incubation with different concentrations of anti-TDP-43 CPAs of the present disclosure. FIG. 10 shows percentage of cell death with no significant difference in cell death across all populations tested. FIG. 11A shows the sum of p-TDP-43 foci pixels per well area for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 11B shows the mean focus intensity for p-TDP-43 foci for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 11C shows consistent cell count per well across all cell populations tested. FIG. 11D shows the number of p-TDP-43 foci (normalized by cell count) for GFP-2a- TDP43 transfected HEK cells after incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 11E shows the mean focus area for p-TDP-43 foci for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 12A shows the sum of p-TDP-43 foci pixels per well area, demonstrating a reduction in total foci area for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 12B shows consistent cell count per well across all cell populations and concentrations tested. FIG. 12C shows the p-TDP-43 foci number normalized by cell count, demonstrating a concentration-dependent increase in foci count for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 12D shows the p-TDP-43 mean focus area, demonstrating a concentration- dependent decrease in mean focus area for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 13A shows the sum of p-TDP-43 foci pixels per well area for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure in either PBS or acetate buffer. FIG. 13B shows the mean focus intensity for p-TDP-43 foci for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure in either PBS or acetate buffer. FIG. 13C shows consistent cell count per well across all cell populations tested. FIG. 13D shows the number of p-TDP-43 foci normalized by cell count for GFP-2a- TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure in either PBS or acetate buffer. FIG. 13E shows the mean focus area for p-TDP-43 foci for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure in either PBS or acetate buffer. FIG. 14A shows confocal microscopy images of GFP-2a-TDP43 transfected HEK cells incubated with anti-TDP-43 CPAs of the present disclosure in either PBS or acetate buffer. FIG. 14B shows the percentage of pTDP43 colocalized with an anti-TDP-43 CPA, demonstrating significantly greater co-localization of pTDP43 with the anti-TDP-43 CPA as compared to the control antibody. FIG. 15A shows the percentage of pTDP43 colocalized with the CPAs, demonstrating significantly co-localization of pTDP43 with anti-TDP-43 CPAs of the present disclosure.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 15B shows the average number of CPA-labeled spots per cell for GFP-2a-TDP43 transfected HEK cells incubated with anti-TDP-43 CPAs of the present disclosure. FIG. 15C shows the average spot size for CPA-labeled spots for GFP-2a-TDP43 transfected HEK cells incubated with anti-TDP-43 CPAs of the present disclosure. FIG. 15D shows the average spot intensity for CPA-labeled spots for GFP-2a-TDP43 transfected HEK cells incubated with anti-TDP-43 CPAs of the present disclosure. FIG. 16A shows the percentage of pTDP43 colocalized with humanized anti-TDP-43 CPAs of the present disclosure, demonstrating co-localization of the humanized anti-TDP-43 CPAs with cytosolic p-TDP-43. FIG. 16B shows consistent cell count per well across all cell populations tested. FIG. 16C shows the CPA-labeled spot area per well for GFP-2a-TDP43 transfected HEK cells incubated with anti-TDP-43 CPAs of the present disclosure. FIG. 16D shows the number of CPA-labeled spots per cell for GFP-2a-TDP43 transfected HEK cells incubated with anti-TDP-43 CPAs of the present disclosure, demonstrating significant internalization of the anti-TDP-43 CPAs. FIG. 17A shows the total foci area for U251 glioblastoma cells treated with an anti-TDP- 43 CPA of the present disclosure, demonstrating a reduction in total foci area for the glioblastoma cells treated with the anti-TDP-43 CPA. FIG. 17B shows the mean focus size for U251 glioblastoma cells treated with an anti- TDP-43 CPA of the present disclosure, demonstrating a reduction in mean foci size for the glioblastoma cells treated with the anti-TDP-43 CPA. FIG. 17C shows the total foci count for U251 glioblastoma cells treated an anti-TDP-43 CPA of the present disclosure, demonstrating total foci count was significantly increased in glioblastoma cells treated with the anti-TDP-43 CPA. FIG. 18A shows images of primary rat cortical neuronal cells treated with anti-TDP-43 CPA of the present disclosure, demonstrating internalization and cytosolic accumulation of the anti-TDP43 CPAs. FIG. 18B shows the total foci area per well treated with an anti-TDP-43 CPA of the present disclosure, demonstrating substantial internalization of the anti-TDP-43 CPAs. FIG. 19A shows the total number of CPA-labeled spots per cell for DIV15 rat neuronal cells incubated with CPAs of the present disclosure.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 19B shows the total area of CPA-labeled spots per well for DIV15 rat neuronal cells incubated with anti-TDP-43 CPAs of the present disclosure, demonstrating higher total spot area per well for cells treated with the anti-TDP-43 CPAs compared to those treated with standard antibodies. FIG. 19C shows the mean spot size for CPA-labeled spots in DIV15 rat neuronal cells incubated with anti-TDP-43 CPAs of the present disclosure. FIG. 19D shows spot integrated intensity for CPA-labeled spots in DIV15 rat neuronal cells incubated with anti-TDP-43 CPAs of the present disclosure. FIG. 19E shows the percentage of CPA-labeled spots colocalized with EEA1 (Early Endosome Antigen 1) in DIV15 rat neuronal cells incubated with anti-TDP-43 CPAs of the present disclosure, thereby, demonstrating ~10-20% colocalization of the anti-TDP-43 CPAs with EEA1. FIG. 19F shows consistent cell count per well across all cell populations tested for the DIV15 rat neuronal cells. FIG. 20A shows high content images of untransfected HEK cells (left) and HEK cells transfected with APP (right). FIG. 20B demonstrates a large number of Aβ aggregates in the APP transfected HEK cells and no signal from the untransfected HEK cells. FIG. 21A shows the total labeled APP spot area for APP transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure. FIG. 21B shows the mean area for each labeled APP spot in APP transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure. FIG. 21C shows the number of labeled APP spots normalized by cell count for APP transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure. FIG. 22A shows the total labeled subcellular APP spot area for APP transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure, demonstrating that all cell populations tested had a similar total labeled subcellular APP spot area. FIG. 22B shows the mean area for each labeled Aβ spot for APP transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure. FIG. 22C shows the number of labeled Aβ spots normalized by cell count for APP transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 23A shows the total Aβ foci area for HEK cells transfected with APP770sw [K670N-M671L_Swedish] incubated with an anti-Aβ CPA of the present disclosure, demonstrating that anti-Aβ CPA incubation resulted in a lower total Aβ foci area in a concentration dependent manner compared to the control. FIG. 23B shows the Aβ focus count for APP770sw transfected HEK cells incubated with an anti-Aβ CPA of the present disclosure, demonstrating the anti-Aβ CPA resulted in a lower number of Aβ foci in a concentration dependent manner compared to the control. FIG. 23C shows the mean focus area for Aβ foci in APP770sw transfected HEK cells incubated with an anti-Aβ CPA of the present disclosure. FIG. 24A shows the total Aβ foci area for APP770sw transfected HEK cells incubated with an anti-Aβ CPA of the present disclosure. FIG. 24B shows the Aβ focus count for APP770sw transfected HEK cells incubated with an anti-Aβ CPA of the present disclosure. FIG. 24C shows the mean focus area for Aβ foci for APP770sw transfected HEK cells incubated with an anti-Aβ CPA of the present disclosure. FIG. 25A shows the total Aβ foci area for APP770sw transfected HEK cells incubated with different concentrations of an anti-Aβ CPA of the present disclosure, demonstrating a concentration-dependent effect of the anti-Aβ CPA. FIG. 25B shows showing the Aβ focus count for APP770sw transfected HEK cells incubated with different concentrations of an anti-Aβ CPA of the present disclosure, demonstrating a concentration-dependent effect of the anti-Aβ CPA. FIG. 25C shows the mean focus area for Aβ foci in APP770sw transfected HEK cells incubated with different concentrations of an anti-Aβ CPA of the present disclosure, demonstrating a concentration-dependent effect of the anti-Aβ CPA. FIG. 26A shows the total Aβ foci area per cell in APP770sw transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure comprising different mutations that affect Fcγ or FcRn function. FIG. 26B shows the number of Aβ foci area per cell in APP770sw transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure comprising different mutations that affect Fcγ or FcRn function.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 26C demonstrates internalization of anti-Aβ CPAs of the present disclosure into APP770sw transfected HEK cells regardless of Fcγ or FcRn functionality for the CPA. FIG. 27A shows total Aβ foci area per cell in APP770sw transfected HEK cells incubated with a murinized anti-Aβ CPA of the present disclosure. FIG. 27B shows the number of Aβ foci per cell in APP770sw transfected HEK cells incubated with a murinized anti-Aβ CPA of the present disclosure. FIG. 27C demonstrates internalization of a murinized anti-Aβ CPA of the present disclosure into APP770sw transfected HEK cells. FIG. 28A shows the total Aβ foci area per cell in APP770sw transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure, demonstrating robust activity across the panel of anti-Aβ CPAs. FIG. 28B shows the average number of Aβ foci area per cell in APP770sw transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure, demonstrating robust activity across the panel of anti-Aβ CPAs. FIG. 28C demonstrates significant internalization by APP770sw transfected HEK cells across the panel of anti-Aβ CPAs. FIG. 29A provides an image of untreated HEK-293 cells expressing APPsw following lentiviral transfection (lenti-APPsw transduced HEK-293 cells). FIG. 29B provides an image of HEK-293 cells expressing APPsw following lentiviral transfection and incubation with h2931 antibody. FIG. 29C provides an image of HEK-293 cells expressing APPsw following lentiviral transfection and incubation with an anti-Aβ CPA of the present disclosure. FIG. 30A shows the total Aβ foci area per cell in lenti-APPsw transduced HEK-293 cells incubated with an anti-Aβ CPA of the present disclosure. FIG. 30B provides a graph showing the number of Aβ foci per cell in lenti-APPsw transduced HEK-293 cells incubated with an anti-Aβ CPA of the present disclosure. FIG. 30C demonstrates internalization of an anti-Aβ CPA by lenti-APPsw transduced HEK-293 cells. FIG. 31A provides high content images HEK cells subjected to lentiviral transfection with APP770sw [K670N-M671L_Swedish] at a MOI (multiplicity of infection) of 10 followed by puromycin selection (stable APPsw-HEK cells).
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 31B shows the percentage of stable APPsw-HEK cells having Aβ aggregates compared to untransfected HEK cells (control). FIG. 32A shows the total Aβ foci area per cell for stable APPsw-HEK cells incubated with an anti-Aβ CPA of the present disclosure. FIG. 32B provides a graph showing the number of Aβ foci per cell for stable APPsw- HEK cells incubated with an anti-Aβ CPA of the present disclosure. FIG. 32C demonstrates internalization of the anti-Aβ CPA by for stable APPsw-HEK cells. FIG. 33A provides high content images of HEK cells transfected with α-tubulin and incubated with an anti-α-tubulin CPA (top) and anti-α-tubulin antibody (bottom), demonstrating internalization of the anti-α-tubulin CPA. FIG. 33B provides high content images of HEK cells transfected with α-tubulin and incubated with an anti-α-tubulin CPA (top) and anti-α-tubulin antibody (bottom), demonstrating internalization of the anti-α-tubulin CPA and co-localization of the CPA with α-tubulin. FIG. 34A provides high content images of HEK cells transfected with TOMM20 and incubated with an anti-TOMM20 CPA (top) and anti-TOMM20 antibody (bottom), demonstrating internalization of the anti-TOMM20 CPA. FIG. 34B provides high content images of HEK cells transfected with TOMM20 and incubated with an anti-TOMM20 CPA (top) and anti-TOMM20 antibody (bottom), demonstrating internalization of the anti-TOMM20 CPA and co-localization of the CPA with TOMM20. FIG. 35A provides high content images of HEK cells transfected with Nesprin-1 and incubated with an anti-Nesprin-1 CPA (top) and anti-Nesprin-1 antibody (bottom), demonstrating internalization of the anti-Nesprin-1 CPA. FIG. 35B provides high content images of HEK cells transfected with Nesprin-1 and incubated with an anti-Nesprin-1 CPA (top) and anti-Nesprin-1 antibody (bottom), demonstrating internalization of the anti-Nesprin-1 CPA and co-localization of the CPA with Nesprin-1. FIG. 36A provides high content images of HEK cells transfected with anti-β-actin and incubated with an anti-β-actin CPA (top) and anti-β-actin antibody (bottom), demonstrating internalization of the anti-anti-β-actin CPA.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 36B provides high content images of HEK cells transfected with anti-β-actin and incubated with an anti-β-actin CPA (top) and anti-β-actin antibody (bottom), demonstrating internalization of the anti-β-actin CPA and co-localization of the CPA with anti-β-actin. FIG. 37A provides high content images of HEK cells transfected with EROL1L and incubated with an anti- EROL1L CPA (top) and anti- EROL1L antibody (bottom), demonstrating internalization of the anti- EROL1L CPA. FIG. 37B provides high content images of HEK cells transfected with EROL1L and incubated with an anti- EROL1L CPA (top) and anti- EROL1L antibody (bottom), demonstrating internalization of the anti- EROL1L CPA and co-localization of the CPA with EROL1L. DETAILED DESCRIPTION The present disclosure provides systems and methods for the delivery of payload molecules into a cell. Systems and methods of the present disclosure facilitate the intracellular delivery of a variety of payload molecules. The specific examples and embodiments disclosed herein are not limiting and are merely illustrative of the breadth of the approach. In some aspects, the present disclosure provides a cell-penetrating agent (“CPA”) comprising (i) Cell Internalizing Module (“CIM”) and (ii) a payload molecule. The CIM facilitates the internalization of the cell- penetrating agent into the cell. In some embodiments, the CIM further facilitates the transfer of the cell-penetrating agent (or a functional portion thereof) from the internalization vesicle (e.g., endosome) to the cytosol. In some embodiments, the CIM disrupts the membrane of the internalization vesicle, leading to vesicle rupture and facilitating transfer of the cell-penetrating agent out of the vesicle and into the cytosol. FIG. 1A provides a scheme showing an exemplary CPA 101. Exemplary CPA 101 comprises cell internalizing module 102, payload 103, connected via optional linker 104. As discussed in more detail herein, many different variants and combinations of each of these components are within the scope of the present disclosure. FIG. 1B provides a scheme demonstrating internalization of exemplary CPA 101 from FIG. 1A by cell 111. In this exemplary, non-limiting example, one or CPA 101 interacts with cell wall 112 of cell 111 (e.g., via the interaction of CIM 102 with cell wall 112). Following interaction of CPA 101 with cell wall 112, early internalization vesicle 115 (e.g., early
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT endosome) is formed. As internalization progresses, internalization vesicle 116 (e.g., endosome) becomes fully formed, thereby fully encapsulating CPA 101. In the exemplary embodiment in FIG. 1B, CPA 101 further interacts with the membrane of internalization vesicle 116, leading to rupture of the internalization vesicle 116. CPA 101 is thereafter transferred out of ruptured vesicle 117 into cytosol 113, where the payload (or a functional fragment thereof) of the CPA 101 interacts with one or more intracellular target 118. The examples provided in FIG. 1A and FIG. 1B are non-limiting and illustrative in nature only. As set forth below, there are many other cell-penetrating agents within the scope of the present disclosure, including those that operate using different biochemical processes. Definitions The term “antibody” includes intact antibodies and antigen-binding fragments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to the target including separate heavy chains, light chains Fab, Fab', F(ab')
2, F(ab)c, Dabs, nanobodies, and Fv. Fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins. The term “antibody” also includes a bispecific or multispecific antibody and/or a humanized antibody. A bispecific or bifunctional or multifunctional antibody is an artificial hybrid antibody having two or more different heavy/light chain pairs and two or more different binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). As used herein, the term “biomolecule” means an organic molecule. A biomolecule can be produced by a living organism (e.g., including production using a mammalian host cell) or can be produced using chemical synthesis in a laboratory. “Biomolecule”, as used in the present disclosure includes synthetic derivatives of peptides, proteins, nucleic acids, and carbohydrates. Further, biomolecules includes both macromolecules (e.g., polymers and oligomers) as well as small molecules (e.g., monomers, dimers, trimers, etc.). Additional non-limiting aspects and examples of biomolecules are described herein. The term “biological sample” refers to a sample of biological material within or obtainable from a biological source, for example a human or mammalian subject. Such samples
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT can be organs, organelles, tissues, sections of tissues, bodily fluids, peripheral blood, blood plasma, blood serum, cells, molecules such as proteins and peptides, and any parts or combinations derived therefrom. The term biological sample can also encompass any material derived by processing the sample. Derived material can include cells or their progeny. Processing of the biological sample may involve one or more of filtration, distillation, extraction, concentration, fixation, inactivation of interfering components, and the like. As used herein a “Cell-Penetrating Agent (CPA)” refers to an agent (e.g., a molecule and/or molecular complex) capable of entering a cell (e.g., a mammalian cell in vitro and/or in vivo). In some embodiments, the CPA enters the cell and is transferred to the cytosol following internalization by the cell. In some embodiments, the CPA comprises a Cell Internalizing Module (CIM) that facilitates the internalization of the CPA by the cell. In some cases, the CPA comprises a CIM that facilitates the internalization of the CPA, and the transfer of the CPA or a functional portion thereof to the cytosol. In some embodiments, the CPA further comprises a payload that is linked (e.g., covalently or non-covalently) to the CIM. In some embodiments, the CPA comprises a CIM covalently linked to the payload (e.g., via a linker between the CIM and the payload). In other embodiments, the CPA comprises a CIM non-covalently linked to the payload (e.g., via a streptavidin-biotin interaction), such that the CIM remains linked to the payload in relevant conditions (e.g., blood plasma). In some embodiments, the CPA further comprises a linker connecting the CIM to the payload. In various cases, linkers can be cleavable or non-cleavable and/or can connect the CIM to the payload covalently or non-covalently. In some embodiments, the CPA comprising the payload has enhanced cell penetration compared to a reference payload molecule that is not part of a CPA. In some embodiments, the CPA comprises two or more payloads and/or two or more CIMs (e.g., the CPA comprising a dendrimer linked to a plurality of CIMs and/or payloads). Non-limiting features and examples of cell-penetrating agents are described herein. As used herein a “Cell Internalizing Module (CIM)” refers to a portion of the CPA that facilitates the internalization of the CPA (and by extension, the payload) by the cell. In various embodiments, the CIM may utilize one or more cellular internalization processes, including both active and passive cellular internalization, to effect internalization. Exemplary processes include, without limitation, endocytosis (e.g., Receptor Mediated Endocytosis (RME), phagocytosis, pinocytosis), and membrane translocation (e.g., direct penetration and/or energy-independent
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT internalization). In various non-limiting embodiments, the CIM comprises, for example, a Cell- Membrane Internalizing Peptide (CMIP), a small molecule ligand (e.g., vitamin, fatty acids, integrin binding ligand, etc.), a portion of an antibody (e.g., an scFv portion that binds an internalizing cell surface target), or a decoy receptor ligand (e.g., a cytokine or derivative thereof). In some embodiments, the CIM comprises a Cell-Membrane Internalizing Peptide (CMIP). Non-limiting features and examples of cell internalizing modules are described herein. As used herein a “Cell Membrane Internalizing Peptide (CMIP)”, is a sequence of three or more naturally occurring or non-naturally-occurring amino acids that, when covalently or non- covalently linked to a payload molecule, results in the internalization into a mammalian cell of, at a minimum, a payload molecule or a functional fragment of a payload molecule. In various embodiments, the CMIP may utilize one or more cellular internalization processes, including both active and passive cellular internalization, to effect internalization. Exemplary processes include, without limitation, endocytosis and membrane translocation. In some embodiments, the CMIP interacts with the cell membrane, and/or a cell surface antigen, leading to internalization of the CPA or a portion thereof (e.g., a portion comprising the payload or a functional portion thereof) via an endocytic vesicle. In some embodiments, the CMIP further facilitates the transfer of the CPA or portion thereof out of the endocytic vesicle and into the cytoplasm of the cell. In some cases, the CMIP further interacts with the membrane of the endocytic vesicle, leading to vesicle rupture and endosomal escape of the CPA or portion thereof into the cytosol. Thus, in some embodiments, the CMIP facilitates both the internalization of the payload or functional portion thereof into the cell, as well as transfer of the payload or functional portion thereof into the cytosol. Non-limiting examples of CMIPs include, for example, cationic peptides (including, for example, M-lycotoxin, TAT peptides, pentetratin, and polyarginine peptides, as well as derivatives thereof), amphipathic peptides (including, for example, MPG peptides, Pep-1 peptides, transportan peptides, as well as derivative thereof), and proline-rich peptides (including, for example, Bac7 peptides and derivatives thereof). In some embodiments, CMIPs include cell-penetrating peptides and derivatives thereof, including, for example, those described in I. Ruseska and A. Zimmer, Internalization mechanisms of cell-penetrating peptides, Beilstein J. Nanotechol. 202; 11:101-123 (2020). In some embodiments, a CMIP comprises an amino acid sequence selected from Formulas (IA), (IB), (IC), (ID), (IE), (IF), (IG), and (IH). Non-limiting features and examples of CMIPs are described herein.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT The term “epitope” refers to a site on an antigen to which an antibody binds. An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). The term “humanized immunoglobulin” or “humanized antibody” refers to an immunoglobulin or antibody that includes at least one humanized immunoglobulin or antibody chain (i.e., at least one humanized light or heavy chain). The term “humanized immunoglobulin chain” or “humanized antibody chain” (i.e., a “humanized immunoglobulin light chain” or “humanized immunoglobulin heavy chain”) refers to an immunoglobulin or antibody chain (i.e., a light or heavy chain, respectively) having a variable region that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs) substantially from a non-human immunoglobulin or antibody, and further includes constant regions (e.g., at least one constant region or portion thereof, in the case of a light chain, and preferably three constant regions in the case of a heavy chain). The term “humanized variable region” (e.g., “humanized light chain variable region” or “humanized heavy chain variable region”) refers to a variable region that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) substantially from a non-human immunoglobulin or antibody. Competition between antibodies is determined by an assay in which an antibody under test inhibits specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50: 1495, 1990). A test antibody competes with a reference antibody if an excess of a test antibody (e.g., at least 2x, 5x, l0x, 20x, or l00x) inhibits binding of the reference antibody by at least 50% as measured in a competitive binding assay. Some test antibodies inhibit binding of the reference antibody by at least 75%, 90%, or 99%. Antibodies identified by
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. As used herein a “linker” refers to a chemical moiety that connects two portions of a cell- penetrating agent (e.g., a linker that connects a cell internalizing moiety to a payload). In various embodiments, a linker can connect two portions of the cell-penetrating agent covalently or non- covalently, and the linker can be cleavable or non-cleavable. For example, in some embodiments, a covalent linker can be cleaved during internalization by the cell, thereby separating the CIM from the payload or functional fragment thereof. In other embodiments, the linker covalently connecting the CIM to the payload is not cleavable and the CIM remains covalently linked to the payload (or functional fragment thereof) throughout internalization. In some exemplary embodiments, a linker can be a peptide of about 3 amino acids to about 25 amino acids (e.g., about 3 amino acids to about 20, or about 3 amino acids to about 12 amino acids). In other examples, a linker can be a bond (e.g., an amide bond, an ester bond, an ether bond, and a disulfide bond). In some embodiments, the linker can comprise an organic polymer (e.g., a polyethylene glycol) and/or an organic chain (e.g., a hydrocarbon chain). In some examples, a linker can comprise a pair of affinity domains (e.g., a first domain of the pair of affinity domains can be interleukin-15 and a second domain of the pair of affinity domains can be a sushi domain of interleukin-15 receptor alpha). Non-limiting exemplary methods for covalently and non-covalently linking a payload molecule to a cell-penetrating peptide (e.g., any of the exemplary cell-penetrating peptides described herein) are described herein. Additional methods for covalently and non-covalently linking a payload molecule to a cell-penetrating peptide are also known in the art. As used herein a “payload” or a “payload molecule” as used herein refers to a molecule and/or molecular complex that is internalized when part of a CPA. In some embodiment, the payload performs one or more functions when in the cell, including, for example, modulating (e.g., increasing or decreasing) at least one activity of an intracellular target protein, catalyzing a chemical reaction, and/or facilitating detection from outside the cell (e.g., via fluorescence). In some embodiments, the payload is a molecule that has one or more activities that are desired in a cell. Some payloads are capable of being internalized into a cell upon covalent or non-covalent linkage to a cell internalizing module (e.g., any of the exemplary CMIPs described herein). In
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT some embodiments, the payload is a molecule has limited capacity for internalization, unless it is linked to a cell internalizing module. In some embodiments, the payload is a molecule that has its own capacity for internalization, but is more readily internalized when it is linked to a cell internalizing module. Payload molecules include but are not limited to biologic molecules (e.g., proteins, peptides, nucleic acids, carbohydrates), macromolecules (e.g., polymers, oligomers), proteinaceous molecules (e.g., polypeptides, enzymes, antibodies and antigen-binding fragments thereof), inorganic molecules, organo-metallic complexes (e.g., complexed metals, including, for example, chelates of gallium and/or gadolinium), or any combination thereof. In some embodiments, the payload molecule comprises a biomolecule (including those produced by a living organism and those produced synthetically) and derivatives thereof, including, but not limited to a polypeptide (e.g., a single chain or multi-chain polypeptide), a nucleic acid (e.g., a single-stranded or double-stranded nucleic acid), a lipid (e.g., cholesterol derivatives), a carbohydrate, an inorganic molecule, or any combination thereof. Additional non-limiting aspects and examples of payloads are described herein. The term “patient” or “subject” includes human and other mammalian subjects (e.g., human) that receive either prophylactic or therapeutic treatment. An individual is at increased risk of a disease if the subject has at least one known risk factor (e.g., genetic, biochemical, family history, and situational exposure) placing individuals with that risk factor at a statistically significant greater risk of developing the disease than individuals without the risk factor. The term “pharmaceutically acceptable” means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof. As used herein a “M-lycotoxin derivative” is a peptide comprising three or more amino acids (naturally-occurring or non-naturally-occurring) that was designed based on a starting M- lycotoxin peptide. In some embodiments, the “M-lycotoxin derivative” is a polypeptide having, e.g., between 80% and 99% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to wild-type M-lycotoxin. Additional non-limiting aspects and examples of M-lycotoxin derivatives are described herein.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT As used herein a “spacer” or a “spacer region” refers to an amino acid that does not have catalytic or therapeutic activity in a mammalian cell. For example, a spacer can be a peptide of 1 amino acid to about 10 amino acids (e.g., 1 to about 8 amino acids, 1 to about 6 amino acids, or 1 to about 4 amino acids). In some examples, one or more spacer regions are included in the cell internalizing module. For example, a spacer region can separate amino acids within a cell internalizing module, e.g., a spacer can be disposed after the first amino acid of a CIM. In some examples, a spacer can be disposed before the final amino acid of a CIM. In some examples, a CIM can have two or more spacer regions (e.g., two spacer regions, three spacer regions, four spacer regions, five spacer regions or more). In some examples, a spacer region is a single glycine residue. In some examples, a spacer region is a pair of glycine residues. In some examples, a spacer region is three glycine residues. In some examples, a spacer region is four glycine residues. In some examples, a spacer region is four glycine residues followed by a serine residue. As used herein, the term “macromolecule” refers to a molecule having either a molecular weight of at least 5 kDa and/or a hydrodynamic radius of at least 1.0 nm. As used herein, the term “macromolecules” includes biomolecules, organic polymers, and organometallic complexes. Unless otherwise apparent from the context, the term “about” encompasses insubstantial variations, such as values within a standard margin of error of measurement (e.g., SEM) of a stated value. For purposes of classifying amino acids substitutions as conservative or nonconservative, amino acids are grouped as follows: Group I (hydrophobic side chains): Met, Ala, Val, Leu, Ile; Group II (neutral hydrophilic side chains): Cys, Ser, Thr; Group III (acidic side chains): Asp, Glu; Group IV (basic side chains): Asn, Gln, His, Lys, Arg; Group V (residues influencing chain orientation): Gly, Pro; and Group VI (aromatic side chains): Trp, Tyr, Phe. Conservative substitutions involve substitutions between amino acids in the same class. Non-conservative substitutions constitute exchanging a member of one of these classes for a member of another. Percentage sequence identities are determined with antibody sequences maximally aligned by the Kabat numbering convention. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage. I. Cell-Penetrating Agents In some aspects, the present disclosure provides a cell-penetrating agent (CPA) comprising: (i) a Cell Internalizing Module (CIM); and (ii) a payload. In some embodiments, the CIM is covalently linked to the payload. In some embodiments, the CIM is non-covalently linked to the payload. In some embodiments, the CIM comprises a Cell Membrane Internalizing Peptide (CMIP). The CPA is a molecule and/or molecular complex that capable of entering a cell (e.g., a mammalian cell in vitro and/or in vivo) via interaction of the CIM with the cell surface. In some embodiments, the CPA enters the cell (or at least a functional portion thereof enters the cell) and is transferred (or at least a functional portion thereof is transferred) to the cytosol following internalization by the cell. In this way, the CPA facilitates transfer of the payload (or at least a functional portion of the payload) into the cell, and in some instances, into the cytosol. In some embodiments, the CPA may be internalized and/or transferred to the cytosol in an intact form. In some embodiments, the during internalization and/or cytosolic transfer, the CPA may be partially degraded and/or modified (e.g., partial hydrolysis, oxidation, reduction, etc.) leading to cytosolic transfer of a functional fragment of the CPA (e.g., a fragment comprising a substantially intact payload and/or a fragment comprising a functional fragment of the payload). Following cellular internalization of the CPA (or functional fragment thereof), the payload and/or functional fragment thereof, performs at least one function in the cell (e.g., modulating activity of an enzyme, binding to a target biomolecule, facilitating detection). In this manner, the CPA facilitates cellular internalization of the payload (which, in some embodiments, might otherwise have limited ability to enter the cell). Throughout the present disclosure, references to the CPA and/or payload will be made in the context of cell internalization, transfer, and/or intracellular function, but one of ordinary skill will understand such references to include an intact CPA and/or payload, a chemically-modified (e.g., oxidized, reduced) CPA, a precursor payload, and/or payload (e.g., chemically-modified,
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT functional CPA, payload, or functional fragments thereof), as well as partially degraded functional fragments thereof (intact CPA, CPA fragment, intact payload, and/or payload fragment, etc.). Thus, references to internalization of, cytosolic transfer of, and target engagement by either the CPA or the payload will include references to intact CPA and/or payload, chemically-modified CPA and/or payload, and/or partially degraded functional fragments thereof. For example, methods of the present disclosure may refer to internalization of and/or transfer to the cytosol of a payload, but it will be understood that such a reference encompasses the transfer of an intact CPA and/or a functional fragment of the CPA comprising the payload or a functional portion thereof, as well as chemically-modified derivatives thereof. Cell-penetrating agents of the present disclosure may utilize one or more of several biochemical processes to achieve internalization of the payload. In various embodiments, the CPAs of the present disclosure may utilize passive internalization, active internalization, or a combination thereof. In some embodiments, the CPA utilizes active internalization (e.g., endocytosis) to achieve intracellular delivery of the payload. In various embodiments, the CPA is internalized into the cell via endocytosis. Following internalization, the CPA achieves endosomal escape. In such embodiments, the CPA is thereby transferred to the cytosol, where the payload can perform one or more desired functions. In some embodiments, endosomal escape of the CPA is achieved by interaction of the CIM with the membrane of the endosome, thereby leading to disruption of the endosomal membrane. The CIM may utilize one or more methods to achieve endosomal escape. For example, the CIM may comprise a cationic portion (e.g., positively charged amino acids, a cationic polymer and/or oligomer, cationic lipid). In some embodiments, the cationic portion can interact with the negatively charged phospholipids that comprise the endosomal membrane, thereby disrupting the endosomal membrane, and achieving endosomal escape of the CPA. CPAs of the present disclosure may also comprise a CIM having an amphiphilic portion (e.g., an amphiphilic peptide). In some embodiments, the amphiphilic portion can interact with the endosomal membrane via hydrophobic interactions with the membrane lipids, thereby disrupting the endosomal membrane, and achieving endosomal escape of the CPA. Following endosomal escape of the payload, the payload is then transferred to the cytosol, where it can perform at least one desired function. Further examples of internalization mechanisms that can be utilized by CPAs are described in detail herein.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Various aspects of cell-penetrating agents of the present disclosure are described below, including examples of Cell Internalizing Modules (CIMs), payloads, and/or optional linkers. One of ordinary skill will understand how to combine the features of various CIMs, payloads, and optional linkers and spacer regions disclosed herein. Such examples are merely illustrative of the scope of the present disclosure and are non-limiting. a. Cell Internalizing Modules (CIMs) and Cell Membrane Internalizing Peptides (CMIPs) Cell Internalizing Modules As used herein a “cell internalizing module” or “CIM” is a composition that, when covalently or non-covalently linked to a payload molecule, results in the internalization into a cell of, at a minimum, the payload or a functional fragment of the payload. Non-limiting features and examples of cell internalizing modules (CIMs) are described herein. In some embodiments, a CIM is a peptide (e.g., an amino acid sequence). In such examples, the CIM can also be referred to as a cell membrane internalizing peptide or “CMIP” as further defined herein. Exemplary cell membrane internalizing peptides (CMIPs) include naturally-occurring peptides, and derivatives thereof, as well as, synthetic peptides. Non- limiting examples of CMIPs include M-lycotoxin and derivatives thereof, TAT and derivatives thereof, PEPTH, polyarginine sequences, Penetratin, DPT-C9h, DPT-C9, Transportan, Xentry, Pep-1, Pep-7, Aurein 1.2, MTS, GFWFG, DPV1047, MPG, pVEC, ARF(1_22), BPrPr, MAP, p28, VT5, Bac7, C105Y, PFVYLI, and BR2. In some embodiments, a CIM is a non-peptide moiety (e.g., a ligand) that is internalized by a cell (e.g., a mammalian cell). In such examples, a ligand can induce receptor-mediated internalization of a payload molecule (as defined herein). Generally, ligand internalization is a receptor-mediated endocytic process in which cells intake extracellular molecules (including therapeutics) if the ligand binds to its cognate receptor protein on the cell’s surface. Receptor- mediated internalization also includes transcytosis. CIMs can effectuate the internalization of payload molecules into a mammalian cell. In general, the process of cellular internalization is broadly classified as endocytosis. Typically, endocytosis pathways can be subdivided into two broader categories, phagocytosis and
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT pinocytosis. During pinocytosis the plasma membrane absorbs solutes, while during phagocytosis the cell internalizes much larger vesicles. Pinocytosis is generally further subdivided into macropinocytosis, clathrin-dependent endocytosis (e.g., receptor-mediated endocytosis), caveolin-dependent endocytosis, and clathrin/caveolin-independent endocytosis. (See e.g., Marsh, M. Endocytosis, Oxford University Press (2001); Doherty, G.J., and McMahon, H.T., Mechanisms of Endocytosis, Annu. Rev. Biochem., 78:31.1-31.46 (2009); and Xu, Y., et al., Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom, Front Biosci, 22: 1439-1457 (2017), each of which is incorporated herein by reference in their entireties). Ligand-mediated endocytosis is the mechanism by which cells internalize specific macromolecules. In some examples, the cell membrane (e.g., plasma membrane) includes clathrin pits which protrude from the cell membrane to form small vesicles called clathrin-coated vesicles. These clathrin-coated vesicles contain the receptors and the bound macromolecules, i.e., ligands. Then the clathrin-coated vesicles fuse with the early endosomes (vesicles consisting of tubular extensions residing at the periphery of the cell). The endosomes have an acidic environment (pH 6.0-6.2) that facilitates the dissociation of receptors from the ligands. Then, the ingested content is sorted out for either recycling to the plasma membrane or transport to lysosomes for degradation. Peptide-based CIMs (e.g., CMIPs) internalize payload molecules through a variety of mechanisms. In general, CMIPs have been shown to use either endocytosis (e.g., energy- dependent internalization) as described above or direct penetration (e.g., translocation) (energy- independent internalization) as the two major internalization mechanisms. For direct penetration, various mechanisms have been described including the carpet-like model (membrane destabilization) and the pore formation model (barrel-stave). Positively-charged CMIPs can interact with negatively-charged membrane components such as the phospholipid bilayer, followed by destabilization of the membrane, and crossing of the CMIP and payload molecule through the lipid bilayer. Studies have shown that several CMIPs are able to induce and shift between different uptake mechanisms depending on their concentration, cargo, and/or the cell line used (See e.g., Ruseska, I. and Zimmer, A., Internalization mechanisms of cell- penetrating peptides, Beilstein J Nanotechnol, 11: 101-123, (2020)).
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Once internalized into a cell, payload molecules typically need to escape the endosomal pathway. In general, the endocytic pathway of mammalian cells consists of distinct membrane compartments, which internalize molecules (e.g., cell-penetrating agents) from the plasma membrane and recycle membrane-bound receptors back to the surface or sort internalized molecules to various degradation pathways. The main components of the endocytic pathway include early endosomes which are the first compartment of the endocytic pathway. Early endosomes are usually located in the periphery of the cell, and receive most types of vesicles coming from the cell surface. They have a characteristic tubulo-vesicular structure and a mildly acidic pH. Early endosomes are principally sorting organelles where many endocytosed ligands dissociate from their receptors in the acid pH of the compartment and are recycled to the cell surface. Early endosomes also sort into transcytotic pathway to later compartments (e.g., late endosomes or lysosomes) via transvesicular compartments. Late endosomes generally receive endocytosed material en route to lysosomes, usually from early endosomes in the endocytic pathway, from trans-Golgi network (TGN) in the biosynthetic pathway, and from phagosomes in the phagocytic pathway. They are acidic (approx. pH 5.5) and are generally thought to mediate a final sorting prior the delivery of material to lysosomes. Lysosomes are the last compartment of the endocytic pathway. Lysosomes break down cellular waste products, fats, carbohydrates, proteins, and other macromolecules into simple compounds which are returned to the cytoplasm as new cell-building materials. Lysosomes include many different types of hydrolytic enzymes which function in an acidic environment (e.g., pH of approximately 4.8). In some embodiments, internalized molecules, including CPAs, need to escape the endosomal pathway to deliver their payload molecule to the target. Escaping the endosomal pathway has proven to be challenging, however, various strategies have been described. In general, molecular ferries, leakage-inducing molecules, and physicochemical techniques are most commonly employed to escape the endosomal pathway. For example, molecular ferries are generally molecules that are either part of a therapeutic or bind to the therapeutic and are able to translocate through membranes (e.g., endosomal membranes) including a payload molecule. Typical examples are viral membrane fusion proteins and other cell-penetrating peptides (CMIPs described herein). Alternatively, leakage-inducing molecules include compounds that destabilize the endosomal membrane, such as for example, pore-forming substances, compounds with
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT solubilizing effects, and molecules that affect the endosomal pH. Another strategy includes techniques that work through a direct physical effect, e.g., endosomal membrane disruption by light-induced effects (See e.g., Varkouhi, A.K., et al., Endosomal escape pathways for delivery of biologicals, Journal of Controlled Release, 10220-228 (2011) and Fuchs, H., et al., Driving through Membranes: Molecular Cunning to Enforce the Endosomal Escape of Antibody- Targeted Anti-Tumor Toxins, Antibodies, 2, 209-235 (2013), each of which are incorporated herein by reference in their entireties). Cell Internalizing Membrane Peptides Cell membrane internalizing peptides (“CMIPs”) are peptides that are capable of internalizing a payload molecule (as defined herein) across a cell membrane. In some embodiments, CMIPs comprise short amino acid sequences (e.g., about 5 amino acids to about 35 amino acids, about 5 amino acids to about 30 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 15 amino acids, or about 5 amino acids to about 10 amino acids). Typically, CMIPs are water- soluble, cationic, and/or amphipathic peptides which when coupled to payloads (e.g., therapeutics or vectors) facilitate intracellular delivery of such payloads. CMIPs can be cationic CMIPs which typically include short amino acid sequences including amino acid residues such as arginine, lysine, and/or histidine. Arginine, lysine, and/or histidine give the cationic charge to the CMIP and interact with anionic motifs on the plasma membrane. Cationic CMIPs are able to deliver payload molecules across a cell membrane by receptor-independent mechanisms and without significant membrane damage. Alternatively, CMIPs can be amphipathic peptides (e.g., Pep-1, MPG, etc.), which include lipophilic and hydrophilic tails capable for direct CMIP translocation mechanism across the plasma membrane via receptor-independent mechanisms and without significant membrane damage. In some examples, a CMIP can also be referred to as a cell-penetrating peptide. Exemplary cell-penetrating peptides are described in Guidotti, G, et al. Cell-Penetrating Peptides: From Basic Research to Clinics, Trends Pharmacol Sci., 38(4): 406-424 (2017) and Aroui, S. and Kenani, A., Cell-Penetrating Peptides: A Challenge for Drug Delivery, Cheminformatics and its Applications (2020), each of which are incorporated herein by reference in their entireties.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Non-limiting examples of CMIPs include M-lycotoxin and derivatives thereof, TAT and derivatives thereof, PEPTH, polyarginine sequences, Penetratin, DPT-C9h, DPT-C9, Transportan, Xentry, Pep-1, Pep-7, Aurein 1.2, MTS, GFWFG, DPV1047, MPG, pVEC, ARF(1_22), BPrPr, MAP, p28, VT5, Bac7, C105Y, PFVYLI, and BR2 (See e.g., Sakamoto K, et al. (2020), Optimizing charge switching in membrane lytic peptides for endosomal release of biomarcomolecules, Angew. Chem. Int. Ed. 59: 2-11; Takeuchi, T. and Futaki, S., Current Understanding of Direct Translocation of Arginine-Rich Cell-Penetrating Peptides and Its Internalization Mechanisms, Chem Pharm Bull 64(10):1431-1437 (2016); Arrouss I. et al. (2013), Specific Targeting of Caspase-9/PP2A Interaction as Potential New Anti-Cancer Therapy, PLoS ONE 8(4):e60816; Patel et al. (2019), Cell-penetrating peptide sequence and modification dependent uptake and subcellular distribution of green florescent protein in different cell lines, Scientific Reports 9:6298; Gaston J. et al. (2019), Intracellular delivery of therapeutic antibodies into specific cells using antibody-peptide fusions, Scientific Reports 9:18688; De Coupade, C. et al. (2005), Novel human-derived cell-penetrating peptides for specific subcellular delivery of therapeutic biomolecules, Biochem. J. 390:407–418; Morris, M.C. et al. (2008), Cell-penetrating peptides: from molecular mechanisms to therapeutics. Biol. Cell 100:201–217; Sadler, K. et al. (2002), Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7, Biochemistry 41:14150–14157, each of which is incorporated herein by reference in their entireties). Additionally, the CMIP described herein can comprise additional moieties such as additional residues (e.g., spacer regions as defined herein) or a cyclized structure. (See e.g., Reichart et al., Cyclization of a cell-penetrating peptide via click-chemistry increases proteolytic resistance and improves drug delivery, J. Pept. Sci. 22(6):421-6 (2016)). Lycotoxins were first identified from the venom of the wolf spider Lycosa carolinensis based their abilities to reduce ion (e.g., calcium) and voltage gradients across membranes. As a result, M-lycotoxin forms pores that permeabilize the cell membrane which can cause hemolysis and dissipates voltage gradients across muscle membrane. M-lycotoxins also potently inhibit the growth of bacteria, yeast, and parasitic Leishmania. M-lycotoxin may function both in the prey capture strategy as well as protection from infectious organisms arising from prey ingestion. (See e.g., Yan, L. and Adams, M.E., Lycotoxins, Antimicrobial Peptides from Venom of the Wolf Spider Lycosa carolinensis, J. Biol. Chem. 273(4):2059-2066 (1998) and UniProt Entry P61507
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT LYT1_HOGCA). Subsequently, various mutations of M-lycotoxin have been generated including M-lycotoxin L17E. The L17E mutation was first described in Akishiba, M., et al. Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide, Nature Chemistry 9:751- 761 (2017). Akishiba et al. demonstrated an approach to deliver proteins, including antibodies, into cells by using endosomolytic peptides (i.e., M-lycotoxin derivatives) derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. In particular, M-lycotoxin derivatives were generated by introducing one or two glutamic acid residues into the hydrophobic face. Akishiba et al. demonstrated that substitution of leucine for glutamic acid (M- lycotoxin L17E) enabled cytosolic liberation of antibodies from endosomes. Akishiba et al. demonstrated the predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively-charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. Table 1 below shows a non-limiting, exemplary list of CMIPs including M-lycotoxin, TAT, PEPTH, polyarginine sequences, Pep1, Pep-7, and derivatives thereof and others, including derivatives thereof, that can be included in any of the CIMs. Table 1 SEQ ID NO: 1 WT M-Lyco IWLTALKFLGKHAAKHLAKQQLSKL
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: 13 L17EΔ(23–25) _ IWLTALKFLGKHAAKHEAKQQL M-Lyco G
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: 38 MPG GALFLGFLGAAGSTMGAWSQPKKKRKV SEQ ID NO: 39 pVEC LLIILRRRIRKQAHAHSK
In some embodiments, the CIM comprises a spacer. In some embodiments, the spacer is a spacer in Table 2. Table 2 SEQ ID NO: 50 G
The present disclosure further provides CMIPs for use, among other things, in the context of developing novel cell-penetrating agents. In some embodiments, the cell-internalizing effects of CMIPs can be improved by designing CMIPs having an α-helical conformation with the right degree of amphipathicity. When folded to their native, three-dimensional structure, α-helical conformation is the most common structural motif reportedly adopted by amphipathic cell-penetrating peptides. In some embodiments, CMIPs are generated by mutagenesis to generate amphipathic peptides that adopt a α-helical conformation and have distinct regions of hydrophobic and hydrophilic amino
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT acid clusters along their surface. In some embodiments, the opposing hydrophilic and hydrophobic faces of the amphipathic helix promote partitioning between an aqueous and lipophilic environment such as a cell membrane. For example, it may be desirable to have CMIPs that fold into an α-Helical conformation with a hydrophobic face on one side of the helix and a positively-charged face on the other side of the helix, thereby allowing interaction with both the hydrophobic and hydrophilic portions of the cell membrane. Helical wheels are an important tool for visualizing the structural amphipathicity in helical peptides. By way of example, Figure 2A provides a helical wheel projection of CMIP4 (SEQ ID NO: 57). Figure 2A was generated using software that predicts the helical structure based on the peptide’s primary sequence and approximates its secondary structure to idealized α- helices. In Figure 2A, the helix is viewed down its axis, and the two-dimensional projection results in a circle, where the amino acid side chains protrude outward every 100°, forming a helical wheel. The present disclosure provides guidance regarding structural features that lead to the enhanced internalization of CMIPs of the present disclosure. For example, the present disclosure provides CMIPs that fold into an α-helical conformation with a hydrophobic face on one side of the helix and a positively charged face on the other side of the helix. As a full-turn of an alpha helix occurs with every 3 to 4 amino acid residues, the hydrophobic- and hydrophilic-sides of the alpha helix can be achieved, for example, by spacing positively charged amino acid residues every 3 to 4 amino acid positions, with a balanced placement of hydrophobic amino acids between these positively-charged residues. For example, positions 7, 10, and 16 (the L6/G10/L17 triad in CMIP 4) are part of a hydrophobic face on the alpha helix that facilitates internalization, and a balanced level of hydrophobicity at these positions may improve internalization. The amino acids at positions 8, 12, and 13 similarly comprise a portion of the hydrophobic face. The hydrophilic and/or positively charged face of the helix comprises the lysine residues at positions 7, 11, 15, and 19. For CMIPs to remain active, substitutions at these positions should not substantially disrupt either the hydrophobic face (e.g., by adding too many hydrophilic residues as these positions) or the hydrophilic face (e.g., be replacing three or more of these lysine residues with hydrophobic residues). Further, introducing significantly increased hydrophobicity at two or more of positions 4, 11, and 18 (TKA in CMIP4) may negatively affect internalization. Finally, some truncation of the CMIP may be tolerated (e.g., truncation of amino acid residues
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 22-25). These structural guidelines are not meant to limit the scope of the present disclosure, but rather provide, in conjunction with further guidance herein, one of ordinary skill in the art with guidance correlating structure of the CMIP and the functionality of internalization. Software used to generate the helical wheel projection of Figure 2A also generates projections also provides predicted physicochemical features, shown in Figure 2B, that include hydrophobicity (“H”), hydrophobic moment (“μH”), and net charge (“z”), among others. The amphipathic character of the helix can be cast in quantitative terms by calculating its hydrophobic moment, as shown in Figure 2B for CMIP4 (SEQ ID NO: 57). Hydrophobic moments of helical segments are commonly derived from their two- dimensional helical wheel projections. Hydrophobic moment of a molecule is determined by evaluating the surface distribution of all hydrophilic and lipophilic regions over any given helix shape. The electrostatic potential on the molecular surface is calculated based on the atomic point charges. The resulting hydrophobic moment vector is specific for the instantaneous conformation, and it takes into account all structural characteristics of the molecule (e.g., partial unfolding, bending, and side chains torsion angles). To each residue i is assigned a vector H→i, whose direction is from the center of the circle to the α-carbon and whose magnitude is given by a hydrophobicity scale (e.g., one based on the free energy of transfer to water). In such embodiments, the values are positive for hydrophobic residues and negative for hydrophilic ones. The hydrophobic moment is the magnitude of the vector sum of the hydrophobicities, divided by the number of residues (N). For example, the net hydrophobicity of CMIP4, provided in Figure 2B, is 0.426 and the hydrophobic moment is only 0.048, even though the net charge of the peptide is 4. The present disclosure further provides guidance on how the parameters of net charge, hydrophobicity, and hydrophobic moment affect internalization. These three parameters work in concert, and no single attribute is dominating factor above the others. As described herein, for each of these parameters, values that are either too high or too low may negatively impact internalization of the CMIP. In some embodiments, the CMIP comprises a net-charge of 2, 3, 4, or 5. In some embodiments, the CMIP comprises a net-charge of 3, 4, or 5. In some embodiments, the CMIP comprises a net-charge of 2, 3, or 4. In some embodiments, the CMIP comprises a net-charge of 2 or 3. In some embodiments, the CMIP comprises a net-charge of 3 or 4. In some embodiments,
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT the CMIP comprises a net-charge of 4 or 5. In some embodiments, the CMIP comprises a net- charge of 2. In some embodiments, the CMIP comprises a net-charge of 3. In some embodiments, the CMIP comprises a net-charge of 4. In some embodiments, the CMIP comprises a net-charge of 5. In some embodiments, the CMIP comprises a hydrophobicity from about 0.25 to about 0.63 (e.g., about 0.35 to about 0.63, about 0.40 to about 0.63, about 0.45 to about 0.63, or about 0.50 to about 0.63). In some embodiments, the CMIP comprises a hydrophobicity from about 0.25 to about 0.60 (e.g., about 0.35 to about 0.60, about 0.40 to about 0.60, about 0.45 to about 0.60, or about 0.50 to about 0.60). In some embodiments, the CMIP comprises a hydrophobicity from about 0.25 to about 0.55 (e.g., about 0.30 to about 0.55, about 0.35 to about 0.55, about 0.40 to about 0.55, or about 0.45 to about 0.55). In some embodiments, the CMIP comprises a hydrophobicity from about 0.25 to about 0.50 (e.g., about 0.30 to about 0.50, about 0.35 to about 0.50, about 0.40 to about 0.50, or about 0.45 to about 0.50). In some embodiments, the CMIP comprises a hydrophobic moment from about 0.035 to about 0.16 (e.g., about 0.035 to about 0.15, about 0.035 to about 0.125, about 0.035 to about 0.10, or about 0.035 to about 0.75). In some embodiments, the CMIP comprises a hydrophobic moment from about 0.040 to about 0.16 (e.g., about 0.040 to about 0.15, about 0.040 to about 0.125, about 0.040 to about 0.10, or about 0.040 to about 0.75). In some embodiments, the CMIP comprises a hydrophobic moment from about 0.045 to about 0.16 (e.g., about 0.045 to about 0.15, about 0.045 to about 0.125, about 0.045 to about 0.10, or about 0.045 to about 0.75). In some embodiments, the CMIP comprises a hydrophobic moment from about 0.050 to about 0.16 (e.g., about 0.050 to about 0.15, about 0.050 to about 0.125, about 0.050 to about 0.10, or about 0.050 to about 0.75). In some embodiments, the CMIP comprises a hydrophobic moment from about 0.035 to about 0.080 (e.g., about 0.035 to about 0.075, about 0.035 to about 0.070, about 0.035 to about 0.065, about 0.035 to about 0.060, or about 0.035 to about 0.055). In some embodiments, the CMIP comprises a hydrophobic moment from about 0.45 to about 0.080. In some embodiments, the CMIP comprises a hydrophobicity of from about 0.25 to about 0.5, a net-charge of 3, 4, or 5, and a hydrophobic moment about 0.035 to about 0.16. In some embodiments, the CMIP comprises a hydrophobicity of between 0.25 and 0.5, a net-charge of 3, 4, or 5, and a hydrophobic moment from about 0.45 to about 0.080. In some embodiments, the CMIP comprises a hydrophobicity of between 0.25 and 0.5, a net-charge of 5, and about 0.035 to
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT about 0.16. In some embodiments, the CMIP comprises a hydrophobicity of from about 0.25 to about 0.35, a net-charge of 3, 4, or 5, and a hydrophobic moment about 0.035 to about 0.16. In some embodiments, the CMIP comprises a hydrophobicity of between 0.25 and 0.35, a net- charge of 3, 4, or 5, and a hydrophobic moment from about 0.45 to about 0.080. In some embodiments, the CMIP comprises a hydrophobicity of between 0.25 and 0.35, a net-charge of 5, and a hydrophobic moment from about 0.45 to about 0.080. In some embodiments, the CMIP is not a CMIP of Table 1. The present disclosure also provides CMIPs having particular polypeptide sequences useful for internalizing payload molecules. For example, amino acid sequences of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), or (IIIC), are set forth below. Each of these formulas describes a polypeptide having up to 25 amino acids in the and makes references to different positions X
1 through X
25 along the generic amino acid sequence: X
1-X
2-X
3-X
4-X
5-X
6-X
7-X
8-X
9-X
10-X
11-X
12-X
13-X
14-X
15-X
16-X
17-X
18-X
19-X
20-X
21-X
22-X
23-X
24-X
25
may at various positions along the sequence. However, one of ordinary skill will understand these specified amino acid residues to occupy the corresponding position (e.g., X
1, X
2, etc.) along the amino acid sequence, even if that variable is not recited in the formula. Certain embodiments herein require a particular number of amino acid residues having a certain feature (e.g., positively-charged and/or hydrophobic amino acids) at certain positions in the amino acid sequence. When such disclosures make reference to a formula having particular amino acids specified at these positions in the sequence, these specified amino acids should be taken into account. For example, for a formula requiring a lysine at a particular position, this lysine residue should be counted as one of the required number of positively-charged amino acids if it is located in a recited position. In some aspects, the present disclosure provides a cell membrane internalizing peptide (CMIP) comprising an amino acid sequence selected from Formulas (IA) to (IH): X
1-X
2-X
3-X
4-X
5-X
6-X
7-F-S-G-K-A-A-A-K-X
16-E-A-K-X
20-X
21-X
22-X
23-X
24-X
25 (IA); I-W-L-T-A-L-X
7-F-X
9-X
10-X
11-X
12-X
13-X
14-X
15-A-X
17-X
18-X
19-Q-F-X
22-X
23-X
24-X
25 (IB); I-W-X
3-X
4-X
5-X
6-K-X
8-S-X
10-X
11-H-X
13-X
14-X
15-A-E-X
18-X
19-X
20-X
21-L-S-K-L (IC);
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT X
1-W-X
3-T-A-X
6-X
7-X
8-X
9-G-K-A-X
13-A-K-A-X
17-A-K-Q-X
21-X
22-S-K-X
25 (ID); I-X
2-X
3-X
4-X
5-L-K-F-X
9-X
10-X
11-X
12-A-A-K-X
16-E-X
18-K-Q-F-X
22-X
23-X
24-L (IE); X
1-W-L-T-A-X
6-X
7-X
8-S-G-X
11-X
12-A-A-X
15-A-X
17-X
18-K-X
20-F-X
22-X
23-X
24-X
25 (IF); S-X
2-L-T-A-X
6-X
7-X
8-X
9-X
10-K-H-X
13-I-T-Q-X
17-X
18-K-R-R-X
22-X
23-X
24-X
25 (IG); R-R-L-T-X
5-L-F-K-X
9-G-X
11-X
12-X
13-X
14-E- X
16-I-X
18-X
19-X
20-F (IH); wherein X
1 X
2, X
3, X
4, X
5, X
6, X
7, X
8, X
9, X
10, X
11, X
12, X
13, X
14, X
15, X
16, X
17, X
18, X
19, X
20, and X
21 are each independently a natural or unnatural amino acid residue; and X
22, X
23, X
24, and X
25 are each independently absent or are each independently a natural or unnatural amino acid residue; or a pharmaceutically acceptable salt thereof. In some aspects, the present disclosure provides a cell membrane internalizing peptide (CMIP) comprising an amino acid sequence selected from Formulas (IA) to (IH): X
1-X
2-X
3-X
4-X
5-X
6-X
7-F-S-G-K-A-A-A-K-X
16-E-A-K-X
20-X
21-X
22-X
23-X
24-X
25 (IA); I-W-L-T-A-L-X
7-F-X
9-X
10-X
11-X
12-X
13-X
14-X
15-A-X
17-X
18-X
19-Q-F-X
22-X
23-X
24-X
25 (IB); I-W-X
3-X
4-X
5-X
6-K-X
8-S-X
10-X
11-H-X
13-X
14-X
15-A-E-X
18-X
19-X
20-X
21-L-S-K-L (IC); X
1-W-X
3-T-A-X
6-X
7-X
8-X
9-G-K-A-X
13-A-K-A-X
17-A-K-Q-X
21-X
22-S-K-X
25 (ID); I-X
2-X
3-X
4-X
5-L-K-F-X
9-X
10-X
11-X
12-A-A-K-X
16-E-X
18-K-Q-F-X
22-X
23-X
24-L (IE); X
1-W-L-T-A-X
6-X
7-X
8-S-G-X
11-X
12-A-A-X
15-A-X
17-X
18-K-X
20-F-X
22-X
23-X
24-X
25 (IF); S-X
2-L-T-A-X
6-X
7-X
8-X
9-X
10-K-H-X
13-I-T-Q-X
17-X
18-K-R-R-X
22-X
23-X
24-X
25 (IG); R-R-L-T-X
5-L-F-K-X
9-G-X
11-X
12-X
13-X
14-E- X
16-I-X
18-X
19-X
20-F (IH); wherein each of X
1 through X
25 is independently an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; or a pharmaceutically acceptable salt thereof. In some embodiments, at least one of (a) to (c) is present in the CMIP sequence: (a) X
21 is selected from alanine, arginine, lysine, phenylalanine, and tryptophan; (b) X
9 is serine and X
12 is selected from alanine, arginine, glutamic acid, glutamine, phenylalanine, and tryptophan; or (c) X
5 is histidine and X
3 is selected from alanine, glutamic acid, leucine, lysine, phenylalanine, and tryptophan.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In some embodiments, the CMIP comprises Formula (IA): X
1-X
2-X
3-X
4-X
5-X
6-X
7-F-S-G-K-A-A-A-K-X
16-E-A-K-X
20-X
21-X
22-X
23-X
24-X
25 (IA) or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IB): I-W-L-T-A-L-X
7-F-X
9-X
10-X
11-X
12-X
13-X
14-X
15-A-X
17-X
18-X
19-Q-F-X
22-X
23-X
24-X
25 (IB); or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IC): I-W-X
3-X
4-X
5-X
6-K-X
8-S-X
10-X
11-H-X
13-X
14-X
15-A-E-X
18-X
19-X
20-X
21-L-S-K-L (IC); or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (ID): X
1-W-X
3-T-A-X
6-X
7-X
8-X
9-G-K-A-X
13-A-K-A-X
17-A-K-Q-X
21-X
22-S-K-X
25 (ID); or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IE): I-X
2-X
3-X
4-X
5-L-K-F-X
9-X
10-X
11-X
12-A-A-K-X
16-E-X
18-K-Q-F-X
22-X
23-X
24-L (IE); or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IF): X
1-W-L-T-A-X
6-X
7-X
8-S-G-X
11-X
12-A-A-X
15-A-X
17-X
18-K-X
20-F-X
22-X
23-X
24-X
25 (IF); or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IG): S-X
2-L-T-A-X
6-X
7-X
8-X
9-X
10-K-H-X
13-I-T-Q-X
17-X
18-K-R-R-X
22-X
23-X
24-X
25 (IG); or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IH): R-R-L-T-X
5-L-F-K-X
9-G-X
11-X
12-X
13-X
14-E- X
16-I-X
18-X
19-X
20-F (IH); or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP consists essentially of an amino acid sequence selected from Formula (IA) to (IH). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IA). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IB). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IC). In some embodiments, the CMIP consists essentially
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT of the amino acid sequence of Formula (ID). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IE). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IF). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IG). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IH). In some embodiments, the CMIP consists of an amino acid sequence selected from Formula (IA) to (IH). In some embodiments, the CMIP consists of the amino acid sequence of Formula (IA). In some embodiments, the CMIP consists of the amino acid sequence of Formula (IB). In some embodiments, the CMIP consists of the amino acid sequence of Formula (IC). In some embodiments, the CMIP consists of the amino acid sequence of Formula (ID). In some embodiments, the CMIP consists of the amino acid sequence of Formula (IE). In some embodiments, the CMIP consists of the amino acid sequence of Formula (IF). In some embodiments, the CMIP consists of the amino acid sequence of Formula (IG). In some embodiments, the CMIP consists of the amino acid sequence of Formula (IH). In another aspect, the present disclosure provides a CMIP comprising an amino acid sequence selected from Formulas (IIA) to (IID): I-X
2-X
3-T-A-L-X
7-F-X
9-G-X
11-A-A-X
14-K-X
16-E-A-X
19-Q-F-L-X
23-X
24-L (II-A); I-W-X
3-X
4-A-L-X
7-F-X
9-G-X
11-X
12-X
13-A-X
15-A-E-A-X
19-X
20-F-X
22-S-X
24-L (II-B); X
1-X
2-L-T-X
5-L-K-X
8-S-X
10-K-A-A-A-X
15-A-E-A-K-Q-X
21-L-S-X
24-L (II-C); I-W-L-T-X
5-X
6-K-F-S-X
10-K-X
12-A-A-K-A-X
17-X
18-K-Q-F-L-X
23-X
24-X
25 (II-D); wherein X
1 X
2, X
3, X
4, X
5, X
6, X
7, X
8, X
9, X
10, X
11, X
12, X
13, X
14, X
15, X
16, X
17, X
18, X
19, X
20, X
21 and X
22 are each independently a natural or unnatural amino acid residue; and X
23, X
24, and X
25 are either absent or each independently a natural or unnatural amino acid residue; or a pharmaceutically acceptable salt thereof. In another aspect, the present disclosure provides a polypeptide of a formula selected from Formula (IIA) to Formula (ID): I-X
2-X
3-T-A-L-X
7-F-X
9-G-X
11-A-A-X
14-K-X
16-E-A-X
19-Q-F-L-X
23-X
24-L (II-A); I-W-X
3-X
4-A-L-X
7-F-X
9-G-X
11-X
12-X
13-A-X
15-A-E-A-X
19-X
20-F-X
22-S-X
24-L (II-B); X
1-X
2-L-T-X
5-L-K-X
8-S-X
10-K-A-A-A-X
15-A-E-A-K-Q-X
21-L-S-X
24-L (II-C); I-W-L-T-X
5-X
6-K-F-S-X
10-K-X
12-A-A-K-A-X
17-X
18-K-Q-F-L-X
23-X
24-X
25 (II-D);
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT wherein: X
1 is a hydrophobic amino acid residue or a positively-charged amino acid residue; X
2 is an aromatic amino acid residue, a positively-charged amino acid residue, or a negatively- charged amino acid residue; X
3 is an aromatic amino acid residue, a hydrophobic amino acid residue, a positively-charged amino acid residue, or a negatively-charged amino acid residue; X
4 is a neutral hydrophilic amino acid residue or an aromatic amino acid residue; X
5 is a hydrophobic amino acid residue or an aromatic amino acid residue; X
6 is a hydrophobic amino acid residue; X
7 is a hydrophobic amino acid residue or a positively-charged amino acid; X
8 is an aromatic amino acid residue; X
9 is a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively- charged amino acid residue; X
10 is a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively- charged amino acid residue; X
11 is a neutral hydrophilic amino acid residue, a hydrophobic amino acid residue, or a positively-charged amino acid residue; X
12 is a neutral hydrophilic amino acid residue, a hydrophobic amino acid residue, or a positively-charged amino acid residue; X
13 is a neutral hydrophilic amino acid residue or a hydrophobic amino acid residue; X
14 is a hydrophobic amino acid residue or a positively-charged amino acid; X
15 is an aromatic amino acid residue, a positively-charged amino acid residue, or a negatively-charged amino acid residue; X
16 is a hydrophobic amino acid residue, an aromatic amino acid residue, a positively-charged amino acid residue, or a negatively-charged amino acid residue; X
17 is a positively-charged amino acid residue or a negatively-charged amino acid residue; X
18 is a hydrophobic amino acid residue, an aromatic amino acid residue, or a positively- charged amino acid residue; X
19 is a hydrophobic amino acid residue or a positively-charged amino acid residue; X
20 is a neutral hydrophilic amino acid residue or a negative-charged amino acid residue; X
21 is a hydrophobic amino acid residue or an aromatic amino acid residue;
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT X
22 is absent, a hydrophobic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue; X
23 is absent, a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue; X
24 is absent, an aromatic amino acid residue, or a positively-charged amino acid residue; and X
25 is absent or a hydrophobic amino acid; or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IIA): I-X
2-X
3-T-A-L-X
7-F-X
9-G-X
11-A-A-X
14-K-X
16-E-A-X
19-Q-F-L-X
23-X
24-L (IIA) or pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IIB): I-W-X
3-X
4-A-L-X
7-F-X
9-G-X
11-X
12-X
13-A-X
15-A-E-A-X
19-X
20-F-X
22-S-X
24-L (IIB) or pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IIC): X
1-X
2-L-T-X
5-L-K-X
8-S-X
10-K-A-A-A-X
15-A-E-A-K-Q-X
21-L-S-X
24-L (IIC) or pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IID): I-W-L-T-X
5-X
6-K-F-S-X
10-K-X
12-A-A-K-A-X
17-X
18-K-Q-F-L-X
23-X
24-X
25 (IID) or pharmaceutically acceptable salt thereof. In some embodiments, the CMIP consists essentially of an amino acid sequence selected from Formula (IIA) to (IID). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IIA). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IIB). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IIC). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IID). In some embodiments, the CMIP consists of an amino acid sequence selected from Formulas (IIA) to (IID). In some embodiments, the CMIP consists of the amino acid sequence of Formulas (IIA). In some embodiments, the CMIP consists of the amino acid sequence of Formulas (IIB). In some embodiments, the CMIP consists of the amino acid sequence of Formulas (IIC). In some embodiments, the CMIP consists of the amino acid sequence of Formulas (IID).
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), the polypeptide comprises at least three positively-charged amino acid residues, each of which occupies a position corresponding to X
7, X
11, X
15, X
19, or X
24. In some embodiments, the polypeptide comprises at least four positively-charged amino acid residues, each of which occupies a position corresponding to X
7, X
11, X
15, X
19, or X
24. In some embodiments, the polypeptide comprises five positively-charged amino acid residues, each of which occupies a position corresponding to X
7, X
11, X
15, X
19, or X
24. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), the polypeptide comprises at least four hydrophobic amino acid residues, each of which occupies a position corresponding to X
3, X
5, X
6, X
8, X
13, X
14, X
16, X
18, or X
22. In some embodiments, the polypeptide comprises at least five hydrophobic amino acid residues, each of which occupies a position corresponding to X
3, X
5, X
6, X
8, X
13, X
14, X
16, X
18, or X
22. In some embodiments, the polypeptide comprises at least six hydrophobic amino acid residues, each of which occupies a position corresponding to X
3, X
5, X
6, X
8, X
13, X
14, X
16, X
18, or X
22. In some embodiments, the polypeptide comprises at least seven hydrophobic amino acid residues, each of which occupies a position corresponding to X
3, X
5, X
6, X
8, X
13, X
14, X
16, X
18, or X
22. In some embodiments, the polypeptide comprises at least eight hydrophobic amino acid residues, each of which occupies a position corresponding to X
3, X
5, X
6, X
8, X
13, X
14, X
16, X
18, or X
22. In some embodiments, the polypeptide comprises nine hydrophobic amino acid residues, each of which occupies a position corresponding to X
3, X
5, X
6, X
8, X
13, X
14, X
16, X
18, or X
22. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
1 is a hydrophobic amino acid residue or a positively- charged amino acid residue. In some embodiments, X
1 is a hydrophobic amino acid residue. In some embodiments, X
1 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
1 is an amino acid residue selected from alanine, arginine, aspartic acid, isoleucine, and lysine. In some embodiments, X
1 is isoleucine or lysine. In some embodiments, X
1 is isoleucine.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
2 is an aromatic amino acid residue, a positively-charged amino acid residue, or a negatively-charged amino acid residue. In some embodiments, X
2 is an aromatic amino acid residue. In some embodiments, X
2 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
2 is an amino acid residue selected from alanine, arginine, aspartic acid, phenylalanine, tryptophan, and tyrosine. In some embodiments, X
2 is arginine, aspartic acid, phenylalanine, tryptophan, or tyrosine. In some embodiments, X
2 is tryptophan. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
3 is an aromatic amino acid residue, a hydrophobic amino acid residue, a positively-charged amino acid residue, or a negatively-charged amino acid residue. In some embodiments, X
3 is a hydrophobic amino acid residue. In some embodiments, X
3 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
3 is an amino acid residue selected from alanine, arginine, glutamic acid, leucine, lysine, phenylalanine, and tryptophan. In some embodiments, arginine, glutamic acid, leucine, phenylalanine, or tryptophan. In some embodiments, X
3 is leucine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
4 is a neutral hydrophilic amino acid residue or an aromatic amino acid residue. In some embodiments, X
4 is a neutral hydrophilic amino acid residue. In some embodiments, X
4 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
4 is an amino acid residue selected from alanine, histidine, phenylalanine, threonine, and tryptophan. In some embodiments, X
4 is threonine or tryptophan. In some embodiments, X
4 is threonine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
5 is a hydrophobic amino acid residue or an aromatic amino
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT acid residue. In some embodiments, X
5 is a hydrophobic amino acid residue. In some embodiments, X
5 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
5 is an amino acid residue selected from alanine, arginine, histidine, phenylalanine, and tryptophan. In some embodiments, X
5 is alanine or phenylalanine. In some embodiments, X
5 is alanine. In some embodiments, X
5 is histidine. In some embodiments, X
5 is histidine; and X
3 is an amino acid residue selected from alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
5 is histidine; and X
3 is an amino acid residue selected from alanine, glutamic acid, leucine, lysine, phenylalanine, and tryptophan. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
6 is a hydrophobic amino acid residue. In some embodiments, X
6 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
6 is an amino acid residue selected from alanine, arginine, glutamic acid, isoleucine, and leucine. In some embodiments, X
6 is leucine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
7 is a hydrophobic amino acid residue or a positively charged amino acid. In some embodiments, X
7 is a positively charged amino acid. In some embodiments, X
7 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
7 is an amino acid residue selected from alanine, arginine, aspartic acid, isoleucine, leucine, lysine, and phenylalanine. In some embodiments, X
7 is arginine, isoleucine, leucine, or lysine. In some embodiments, X
7 is lysine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
8 is an aromatic amino acid residue. In some embodiments,
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT X
8 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
8 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, glutamine, histidine, lysine, phenylalanine, serine, and tryptophan. In some embodiments, X
8 is phenylalanine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
9 is a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively charged amino acid residue. In some embodiments, X
9 is a neutral hydrophilic amino acid residue. In some embodiments, X
9 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
9 is an amino acid residue selected from alanine, arginine, histidine, leucine, phenylalanine, serine, and tryptophan. In some embodiments, X
9 is arginine, histidine, phenylalanine, or serine. In some embodiments, X
9 is serine. In some embodiments, X
9 is serine; and X
12 is not histidine. In some embodiments, X
9 is serine; and X
12 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
9 is serine; and X
12 is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, phenylalanine, and tryptophan. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
10 is a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue. In some embodiments, X
10 is a neutral, hydrophilic amino acid residue. In some embodiments, X
10 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
10 is an amino acid residue selected from alanine, arginine, glycine, tryptophan, and tyrosine. In some embodiments, X
10 is arginine, glycine, or tyrosine. In some embodiments, X
10 is glycine.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
11 is a neutral hydrophilic amino acid residue, a hydrophobic amino acid residue, or a positively-charged amino acid residue. In some embodiments, X
11 is a positively-charged amino acid residue. In some embodiments, X
11 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
11 is an amino acid residue selected from alanine, arginine, asparagine, glutamic acid, isoleucine, lysine, threonine, and tryptophan. In some embodiments, X
11 is arginine, isoleucine, lysine, or threonine. In some embodiments, X
11 is lysine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
12 is a neutral hydrophilic amino acid residue, a hydrophobic amino acid residue, or a positively-charged amino acid residue. In some embodiments, X
12 is a hydrophobic amino acid residue. In some embodiments, X
12 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
12 is an amino acid residue selected from alanine, glutamic acid, glutamine, histidine, phenylalanine, and tryptophan. In some embodiments, X
12 is alanine or glutamine. In some embodiments, X
12 is alanine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
13 is a neutral hydrophilic amino acid residue or a hydrophobic amino acid residue. In some embodiments, X
13 is a hydrophobic amino acid residue. In some embodiments, X
13 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
13 is an amino acid residue selected from alanine, arginine, aspartic acid, glutamic acid, glycine, leucine, phenylalanine, serine, and tryptophan. In some embodiments, X
13 is alanine or serine. In some embodiments, X
13 is alanine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
14 is a hydrophobic amino acid residue or a positively- charged amino acid. In some embodiments, X
14 is a hydrophobic amino acid residue. In some
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT embodiments, X
14 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
14 is an amino acid residue selected from alanine, arginine, glutamic acid, histidine, isoleucine, leucine, phenylalanine, tryptophan, tyrosine, and valine. In some embodiments, X
14 is alanine, histidine, leucine, or valine. In some embodiments, X
14 is alanine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
15 is an aromatic amino acid residue, a positively-charged amino acid residue, or a negatively0charged amino acid residue. In some embodiments, X
15 is a positively-charged amino acid residue. In some embodiments, X
15 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
15 is an amino acid residue selected from alanine, arginine, glutamic acid, lysine, threonine, and tryptophan. In some embodiments, X
15 is arginine, glutamic acid, or lysine. In some embodiments, X
15 is lysine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
16 is a hydrophobic amino acid residue, an aromatic amino acid residue, a positively-charged amino acid residue, or a negatively-charged amino acid residue. In some embodiments, X
16 is a hydrophobic amino acid residue. In some embodiments, X
16 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
16 is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, histidine, lysine, serine, and tryptophan. In some embodiments, X
16 is not histidine. In some embodiments, X
16 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
16 is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, lysine, serine, and tryptophan. In some embodiments, X
16 is alanine, glutamic acid, or lysine. In some embodiments, X
16 is alanine.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In some embodiments, for the CMIP of for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
17 is a positively-charged amino acid residue or a negatively-charged amino acid residue. In some embodiments, X
17 is a negatively-charged amino acid residue. In some embodiments, X
17 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
17 is an amino acid residue selected from alanine, arginine, aspartic acid, glutamic acid, leucine, phenylalanine, and tryptophan. In some embodiments, X
17 is arginine or glutamic acid. In some embodiments, X
17 is glutamic acid. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
18 is a hydrophobic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue. In some embodiments, X
18 is a hydrophobic amino acid residue. In some embodiments, X
18 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
18 is an amino acid residue selected from alanine, arginine, glutamic acid, leucine, methionine, and tryptophan. In some embodiments, X
18 is alanine, glutamic acid, methionine, or tryptophan. In some embodiments, X
18 is alanine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
19 is a hydrophobic amino acid residue or a positively- charged amino acid residue. In some embodiments, X
19 is a positively-charged amino acid residue. In some embodiments, X
19 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine
. In some embodiments, X
19 is an amino acid residue selected from alanine, arginine, leucine, lysine, threonine, and tryptophan. In some embodiments, X
19 is arginine, leucine, or lysine. In some embodiments, X
19 is lysine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
20 is a neutral hydrophilic amino acid residue or a negative- charged amino acid residue. In some embodiments, X
20 is a neutral hydrophilic amino acid
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT residue. In some embodiments, X
20 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
20 is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, methionine, serine, and tryptophan. In some embodiments, X
20 is glutamic acid or glutamine. In some embodiments, X
20 is glutamine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
21 is a hydrophobic amino acid residue or an aromatic amino acid residue. In some embodiments, X
21 is an aromatic amino acid residue. In some embodiments, X
21 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
21 is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, lysine, phenylalanine, and tryptophan. In some embodiments, X
21 is an amino acid residue selected from alanine, arginine, lysine, phenylalanine, and tryptophan. In some embodiments, X
21 is arginine, phenylalanine, or tryptophan. In some embodiments, X
21 is phenylalanine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), is absent, a hydrophobic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue. In some embodiments, X
22 is a hydrophobic amino acid residue. In some embodiments, X
22 is either absent or an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
22 is either absent or an amino acid residue selected from alanine, arginine, aspartic acid, leucine, and lysine. In some embodiments, X
22 is either absent or is leucine. In some embodiments, X
22 is absent. In some embodiments, X
22 is Arginine, leucine, or tryptophan. In some embodiments, X
22 is leucine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
23 is absent, a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue. In some embodiments, wherein X
23 is a neutral hydrophilic amino acid residue. In some embodiments, X
23 is either
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT absent or an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
23 is either absent or an amino acid residue selected from alanine, arginine, aspartic acid, glutamic acid, leucine, lysine, threonine, tryptophan, and valine. In some embodiments, X
23 is either absent or is serine. In some embodiments, X
23 is absent. In some embodiments, X
23 is absent, or is arginine, methionine, serine, or tryptophan. In some embodiments, X
23 is serine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
24 is absent, an aromatic amino acid residue, or a positively- charged amino acid residue. In some embodiments, X
24 is a positively-charged amino acid residue. In some embodiments, X
24 is either absent or an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
24 is either absent or an amino acid residue selected from alanine, arginine, and lysine. In some embodiments, X
24 is either absent or is lysine. In some embodiments, X
24 is absent. In some embodiments, X
24 is absent, or is arginine, lysine, or tryptophan. In some embodiments, X
24 is lysine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
25 is absent or a hydrophobic amino acid. In some embodiments, X
25 is a hydrophobic amino acid. In some embodiments, X
25 is either absent or is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X
25 is either absent or an amino acid residue selected from alanine, arginine, leucine, and lysine. In some embodiments, X
25 is either absent or is leucine. In some embodiments, X
25 is absent. In some embodiments, X
25 is leucine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X
22, X
23, X
24, and X
25 are each absent. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), the CMIP comprises a net-charge of 2, 3, 4, or 5. In some embodiments, the CMIP comprises a hydrophobicity from about 0.25 to about 0.63 (e.g., as
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT determined using the HELIQUEST described herein). In some embodiments, the CMIP comprises a hydrophobicity from about 0.25 to about 0.50. In some embodiments, the CMIP comprises a hydrophobic moment from about 0.035 to about 0.16. In some embodiments, the CMIP comprises a hydrophobic moment from about 0.050 to about 0.16. In some embodiments, the CMIP comprises a hydrophobicity of from about 0.25 to about 0.5, a net-charge of 3, 4, or 5, and a hydrophobic moment about 0.035 to about 0.16. In some embodiments, the CMIP comprises a hydrophobicity of between 0.25 and 0.5, a net-charge of 3, 4, or 5, and a hydrophobic moment from about 0.45 to about 0.080. In some embodiments, the CMIP comprises a hydrophobicity of between 0.25 and 0.5, a net-charge of 5, and about 0.035 to about 0.16. In some embodiments, the CMIP comprises a hydrophobicity of from about 0.25 to about 0.35, a net-charge of 3, 4, or 5, and a hydrophobic moment about 0.035 to about 0.16. Table 3 below shows a non-limiting, exemplary list of CMIPs of the present disclosure. In some embodiments, the CMIP is selected from a CMIP of Table 3. In some embodiments, the CMIP comprises an amino acid sequence selected from Table 3. In some embodiments, the CMIP consists essentially of an amino acid sequence selected from Table 3. In some embodiments, the CMIP consists of an amino acid sequence selected from Table 3. Table 3 SEQ ID NO: 54 CMIP1 IWLTALKFLGKAAAKAEAKQQLSKL
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: 67 CMIP4-9 IWLTALKFAGKAAAKAEAKQFLSKL SEQ ID NO: 68 CMIP4-10 IWLTALKFSAKAAAKAEAKQFLSKL
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: 97 CMIP39 IWLTALRFSGKAAAKAEAKQFLSRL SEQ ID NO: 98 CMIP40 IWLTALKFSGKAAAKAEAKQFLRRL
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: 127 CMIP]\ IWLTALKFHGKHAAKAEAKQFLSKL SEQ ID NO: 128 CMIP]Z IWLTALKFSGKAAAKAEAKQFRSKL
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: 157 CMIPa`\ RRLTRLFKHGWRFAEHIWKSF SEQ ID NO: 158 CMIPa`Z RRLTRLFKHGARFAEHIWKSF
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: 187 CMIPac\ IWLTALKFSYKEAYKAEAKQFLSKL SEQ ID NO: 188 CMIPacZ IWLTALKFSYEALAKAEAKQFLSKL
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: 217 CMIPaZ\ IWLTALKFSGIAAAKWERKQFLSKL SEQ ID NO: 218 CMIPaZZ IWLTALKFFGIWAAKAEAKQFRSKL
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: 247 CMIPa_\ IWLTALKFSWIAAFKAEAKQFLSRL SEQ ID NO: 248 CMIPa Z DWETADKFSGKAAAKAEAKQFLSKL
In some embodiments, CMIPs of the present disclosure provide the additional benefit of having reduced predicted immunogenicity compared to other cell-penetrating peptides. One method of calculating immunogenicity of CMIP sequences is to assign each sequence an Immunogenicity Units (IU), as explained in Example 2, below. In some embodiments, the CMIP comprises an immunogenicity value of 2.0 IU or less (e.g., 1.8 IU or less, 1.6 IU or less, 1.4 IU or less, 1.2 IU or less, or 1.0 IU or less). In some embodiments, the CMIP comprises an immunogenicity value of 1.6 IU or less. In some embodiments, the CMIP comprises an immunogenicity value of from 0 to 2.0 IU (e.g., from 0 to 1.8 IU, from 0 to 1.6 IU, from 0 to 1.4 IU, from 0 to 1.2 IU, from 0 to 1.0 IU, from 0 to 0.8 IU, from 0 to 0.6 IU, from 0 to 0.4 IU, from 0 to 0.2 IU). In some embodiments, the CMIP comprises an immunogenicity value of from 0 to 1.6 IU. In another aspect, the present disclosure provides a CMIP comprising an amino acid sequence having a formula selected from Formula (IIIA) to Formula (IIIC): I-X
2-X
3-T-A-L-X
7-F-X
9-G-X
11-A-A-X
14-K-X
16-E-A-X
19-Q-F-L-X
23-X
24-L (IIIA); I-W-X
3-X
4-A-L-X
7-F-X
9-G-X
11-X
12-X
13-A-X
15-A-E-A-X
19-Q-F-X
22-S-X
24-L (IIIB); X
1-X
2-L-T-A-L-K-F-S-X
10-K-A-A-A-X
15-A-E-A-K-Q-X
21-L-S-X
24-L (IIIC); or pharmaceutically acceptable salt thereof; wherein X
1 is isoleucine or lysine; X
2 is arginine or tryptophan; X
3 is arginine, glutamic acid, leucine, or tryptophan; X
4 is threonine or tryptophan; X
7 is arginine, isoleucine, or lysine; X
9 is arginine, histidine, or serine; X
10 is glycine or tyrosine; X
11 is arginine, isoleucine, lysine, or threonine; X
12 is alanine or glutamine; X
13 is alanine or serine; X
14 is alanine, histidine, leucine, or valine; X
15 is arginine, glutamic acid, or lysine; X
16 is alanine or lysine; X
18 is alanine, methionine, or tryptophan; X
19 is arginine or lysine; X
21 is
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT arginine or phenylalanine; X
22 is arginine, leucine, or tryptophan; X
23 is arginine or serine; and X
24 is arginine, lysine, or tryptophan. In some embodiments, the CMIP comprises the amino acid sequence of Formulas (IIIA). In some embodiments, the CMIP comprises the amino acid sequence of Formulas (IIIB). In some embodiments, the CMIP comprises the amino acid sequence of Formulas (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from Formulas (IIIA) to (IIIC). In some embodiments, the CMIP consists of the amino acid sequence of Formulas (IIIA). In some embodiments, the CMIP consists of the amino acid sequence of Formulas (IIIB). In some embodiments, the CMIP consists of the amino acid sequence of Formulas (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from Formulas (IIIA) to (IIIC). In some embodiments, the CMIP consists of the amino acid sequence of Formulas (IIIA). In some embodiments, the CMIP consists of the amino acid sequence of Formulas (IIIB). In some embodiments, the CMIP consists of the amino acid sequence of Formulas (IIIC). In some embodiments, for the CMIP of Formula (IIIA), (IIIB), and (IIIC), X
1 is Isoleucine. In some embodiments, X
2 is tryptophan. In some embodiments, X
3 is leucine. In some embodiments, X
4 is threonine. In some embodiments, X
7 is lysine. In some embodiments, X
9 is serine. In some embodiments, X
10 is glycine. In some embodiments, X
11 is lysine. In some embodiments, X
12 is alanine. In some embodiments, X
13 is alanine. In some embodiments, X
14 is alanine. In some embodiments, X
15 is lysine. In some embodiments, X
16 is alanine. In some embodiments, X
18 is alanine. In some embodiments, X
19 is lysine. In some embodiments, X
21 is phenylalanine. In some embodiments, X
22 is leucine. In some embodiments, X
23 is serine. In some embodiments, X
24 is lysine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC), the CMIP comprises a free carboxylate at the C-terminus of the peptide. In some embodiments, the CMIP comprises an amide at the C- terminus of the peptide. In some embodiments, the CMIP is a retro-inverso peptide (i.e., a peptide comprising all D-amino acids with the reverse order of the amino acid sequence). b. Linkers
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In various aspects of the present disclosure, the cell-penetrating agent further comprises a linker connecting the CIM to the payload. Linkers of the present disclosure may connect the CIM to the payload via either a covalent linkage or a non-covalent linkage. In some embodiments, the CIM is covalently linked to the payload via a linker (i.e., a covalent linker). In some embodiments, the CIM is non-covalently linked to the payload via the linker (i.e., a non-covalent linker). In some embodiments, the CIM is covalently linked to the linker. In some embodiments, the payload is covalently linked to the linker. In some embodiments, the linker is covalently linked to both the CIM and the payload molecule. In some embodiments, the linker is a non-covalent linker. A non-covalent linkage can be achieved using an affinity pair that interact strongly in a noncovalent manner (e.g., by hydrogen bonding, ionic bonding, van Der Waals interactions, or any combination thereof). Numerous examples of non-covalent linkages are known in the art. For example, biotin and a biotin-binding agent (e.g., streptavidin) are one example of an affinity pair. For example, by connecting biotin to one side of the CPA (e.g., the CIM), and connecting the biotin-binding agent to the other side of the CPA (e.g., the payload), a non-covalent linkage can be achieved between the CIM and the payload. In some embodiments, a linker comprises a pair of affinity domains (e.g., a first domain of the pair of affinity domains can be interleukin-15 and a second domain of the pair of affinity domains can be a sushi domain of interleukin-15 receptor alpha). In some embodiments, the linker is a covalent linker. Many covalent linkers are known in the art. For example, in some embodiments, the covalent linker comprises an organic linker (e.g., an alkylene chain, a polyethylene glycol chain, a polyacrylamide, a polyacrylic acid, a polyvinyl alcohol, or a polyethyleneimine chain). In some cases, the covalent linker comprises an unsubstituted or substituted alkylene chain (including, for example, a polyvinyl alcohol chain, a polyacrylamide chain, or a polyacrylic acid chain). In some cases, the covalent linker comprises an unsubstituted or substituted heteroalkylene chain (e.g., a polyethylene glycol chain or a polyethyleneimine chain). In various embodiments, the linker is a straight-chain linker or a branched linker. In some such embodiments, the branched linker allows incorporation of two or more CIMs and/or payloads into a CPA (e.g., a dendrimer linker structure). In some embodiments, the linker comprises an amino acid residue. In some embodiments, the linker comprises a polypeptide. In some exemplary embodiments, a linker can be a peptide of about 1 amino acid to about 50 amino acids (e.g., about 1 amino acid to about 40,
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT or about 1 amino acid to about 30 amino acids). In some exemplary embodiments, a linker can be a peptide of about 1 amino acid to about 25 amino acids (e.g., about 1 amino acid to about 20, or about 1 amino acid to about 12 amino acids). In some exemplary embodiments, a linker can be a peptide of about 1 amino acid to about 10 amino acids (e.g., about 1 amino acid to about 6, or about 1 amino acid to about 7 amino acids). In some exemplary embodiments, a linker can be a peptide of about 1 amino acid to about 5 amino acids (e.g., about 1 amino acid to about 4, or about 1 amino acid to about 3 amino acids). In some exemplary embodiments, a linker can be a peptide of about 3 amino acids to about 20 amino acids (e.g., about 3 amino acids to about 15, or about 3 amino acids to about 12 amino acids). In some exemplary embodiments, a linker can be a peptide of about 3 amino acids to about 10 amino acids (e.g., about 3 amino acids to about 8, or about 3 amino acids to about 6 amino acids). In some embodiments, the linker comprises a glycine residue. In some embodiments, the linker comprises two or more glycine residues. In some embodiments, the linker comprises two or more consecutive glycine residues (e.g., two to three consecutive glycine residues, two to four consecutive glycine residues, two to five consecutive glycine residues, or two to six consecutive glycine residues. In some embodiments, the linker comprises two, three, four, five, or six consecutive glycine residues. In some embodiments, the linker comprises a serine residue. In some embodiments, the linker comprises an amino acid sequence selected from GS, GGG, GGGGS (SEQ ID NO: 255), and GGGGSGGGGS (SEQ ID NO: 258). In some embodiments, the linker comprises an amino acid sequence of GGGGS (SEQ ID NO: 255). In some embodiments, the linker comprises an amino acid sequence selected from the amino acid sequences in Table 4. Table 4 SEQ ID NO: 253 GS
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In other examples, a linker can be a bond, including, for example, a carbon-heteroatom bond (e.g., a carbon-carbon bond, a carbon-nitrogen bond, a carbon-oxygen bond, a carbon- sulfur bond) or a heteroatom-heteroatom bond (e.g., a nitrogen-nitrogen bond, a nitrogen-oxygen bond, a nitrogen-sulfur bond, oxygen-oxygen bond, sulfur-sulfur bond). In additional examples, the linker can be a functional group (e.g., an amide bond, an ester bond, an ether bond, or a disulfide bond). Further, linkers comprise saturated groups (e.g., alkylene, heteroalkylene, cycloalkylene, heterocycloalkylene), unsaturated groups (e.g., groups comprising double or triple bonds), aromatic groups (e.g., arylenes or heteroarylenes), or any combination thereof. In various embodiments, CPAs of the present disclosure comprise a cleavable linker and/or a non-cleavable linker. In some embodiments, the linker is a noncleavable linker. In some embodiments, the linker is a cleavable linker. Cleavable linkers are known in the art and include valine-cysteine, phenylalanine-lysine, glycine-phenylalanine-leucine-glycine, alanine-leucine- alanine-leucine, and the like. Many cleavable linkers are known in the art, including, but not limited to, those cleaved by proteolysis, acid-catalyzed hydrolysis, base-catalyzed hydrolysis, reduction, and/or oxidation, including self-immolative linkers that further degrade after chemical reaction (e.g., via cyclization or elimination), thereby separating the CIM from the payload. Linkers can be attached to the CIM and/or the payload using a number of methods known in the art including, for example, genetic fusion (expression as a single polypeptide) and chemical ligation (e.g., chemical conjugation using click chemistry and/or biorthogonal chemistry). For example, in some embodiments, the linker comprises a binding unit formed by a 1,3-dipolar cycloaddition reaction, hetero-Diels-Alder reaction, nucleophilic substitution reaction, non-aldol type carbonyl reaction, addition to carbon-carbon multiple bond, oxidation reaction, or click reaction. The binding unit may be formed by a reaction between acetylene and azide, or a reaction between an aldehyde or ketone group and a hydrazine or alkoxyamine. In some examples, linkers can be attached using a SpyTag/SpyCatcher system. c. Payload Molecules Systems and methods of the present disclosure facilitate the cellular internalization and/or cytosolic release of a wide range of payload molecules. The specific examples and embodiments disclosed herein are not limiting and are merely illustrative of the breadth of the approach.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In some embodiments, the payload molecule is a biomolecule. In some embodiments, the biomolecule comprises a carbohydrate, a nucleic acid, a protein, or a peptide. In some embodiments, the payload is a macromolecule. In some embodiments, the payload molecule has a molecular weight of about 5 kD to about 1000 kD (e.g., about 50 kD to about 1000 kD, about 100 kD to about 1000 kD, about 50 kD to about 600 kD, about 100 kD to about 600 kD, about 50 kD to about 400 kD, about 100 kD to about 400 kD, about 50 kD to about 200 kD, or about 100 kD to about 200 kD). In some embodiments, the payload molecule has a molecular weight of about 5 kD to about 500 kD (e.g., about 5 kD to about 100 kD, about 5 kD to about 200 kD, about 5 kD to about 300 kD, or about 5 kD to about 400 kD). In some embodiments, the payload molecule has a molecular weight of about 50 kD to about 500 kD (e.g., about 50 kD to about 100 kD, about 50 kD to about 200 kD, about 50 kD to about 300 kD, or about 50 kD to about 400 kD). In some embodiments, the payload molecule has a molecular weight of at least about 5 kD (e.g., at least about 10 kD, at least about 20 kD, at least about 30 kD, at least about 50 kD, at least about 70 kD, at least about 100 kD, at least about 140 kD, or at least about 200 kD). In some embodiments, the payload has a hydrodynamic radius (Stokes Radius) of about 1.0 nm to about 20 nm (e.g., the payload has a hydrodynamic radius of about 1.0 nm to about 20 nm, 2.0 nm to about 20 nm, 3.0 nm to about 20 nm, 4.0 nm to about 20 nm, or 5.0 nm to about 20 nm). In some embodiments, the payload has a hydrodynamic radius of about 1.0 nm to about 10 nm (the payload has a hydrodynamic radius of about 1.0 nm to about 10 nm, 2.0 nm to about 10 nm, 3.0 nm to about 10 nm, 4.0 nm to about 10 nm, 5.0 nm to about 10 nm). In some embodiments, the payload has a hydrodynamic radius of about 1.0 nm to about 5.0 nm (the payload has a hydrodynamic radius of about 1.0 nm to about 5.0 nm, 2.0 nm to about 5.0 nm, 3.0 nm to about 5.0 nm, or 4.0 nm to about 5.0 nm). In some embodiments, the payload has a hydrodynamic radius of at least 1.0 nm (e.g., the payload has a hydrodynamic radius of at least about 1.0 nm, at least about 2.0 nm, at least about 3.0 nm, at least about 4.0 nm, at least about 5.0 nm, or at least about 6.0 nm). In some embodiments, the payload comprises a proteinaceous molecule. In some embodiments, the proteinaceous molecule is an antibody, an enzyme, a protein, or a peptide. In some embodiments, the proteinaceous molecule is about 50 to about 2000 amino acids In some embodiments, the proteinaceous molecule is about 50 to about 1500 amino acids (e.g., the
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT proteinaceous molecule is about 100 to about 1500 amino acids, 200 to about 1500 amino acids, 300 to about 1500 amino acids, 400 to about 1500 amino acids, 500 to about 1500 amino acids, 600 to about 1500 amino acids, 700 to about 1500 amino acids, 800 to about 1500 amino acids, 900 to about 1500 amino acids, or 1000 to about 1500 amino acids). In some embodiments, the proteinaceous molecule is about 500 to about 1400 amino acids (e.g., the proteinaceous molecule is about 600 to about 1400 amino acids, about 700 to about 1400 amino acids, about 800 to about 1400 amino acids, about 900 to about 1400 amino acids, about 1000 to about 1400 amino acids, about 1100 to about 1400 amino acids, about 1200 to about 1400 amino acids, or about 1300 to about 1400 amino acids). In some embodiments, the proteinaceous molecule is at least about 50 amino acids (e.g., the proteinaceous molecule is at least about 100 amino acids, at least about 200 amino acids, at least about 300 amino acids, at least about 400 amino acids, at least about 500 amino acids, at least about 600 amino acids, at least about 700 amino acids, at least about 800 amino acids, at least about 900 amino acids, at least about 1000 amino acids, at least about 1100 amino acids, or at least about 1200 amino acids). In some embodiments, the proteinaceous molecule is linked to the CMIP via a polypeptide linker. In some embodiments, the cell-penetrating agent is a fusion protein. In some embodiments, the cell-penetrating agent is encoded by a nucleic acid encoding a single polypeptide comprising the CMIP and the payload, optionally linked by any of the peptide linkers described herein. In some embodiments, the payload molecule or the portion thereof is linked to the C-terminus of the CMIP. In some embodiments, the payload molecule or the portion thereof is linked to the N-terminus of the CMIP. Antibody Payloads In some embodiments, the payload molecule is an antibody or an antigen-binding portion thereof. In some embodiments, the antibody or antigen-binding portion thereof comprises a light chain. In some embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain. Figures 3A-B provide two exemplary schematics for the connection of the CIM to the antibody payload. As shown in Figure 3A, cell-penetrating agent 300 is comprised of an antibody comprising of heavy chain 301 and light chain 302. CIM 303 (e.g., a CMIP of the present disclosure) is connected to the C-terminus of light chain 302 (e.g., via optional linker 304). In
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT some embodiments, the CMIP 303 is covalently linked to the light chain 302. In some embodiments, the CMIP 303 is covalently linked to a C-terminus of the light chain 302. In some embodiments, the CMIP 303 is covalently linked to a N-terminus of the light chain 302. In some embodiments, the light chain 302 of the antibody or a portion thereof and the CMIP 303 are expressed as a single polypeptide. In some embodiments, the light chain 302 of the antibody or a portion thereof, the linker 304, and the CMIP 303 are expressed as a single polypeptide. As shown in Figure 3B, cell-penetrating agent 310 is comprised of an antibody comprising of heavy chain 311 and light chain 312. CIM 313 (e.g., a CMIP of the present disclosure) is connected to the C-terminus of heavy chain 311 (e.g., via optional linker 314). In some embodiments, the CMIP 313 is covalently linked to the heavy chain 311. In some embodiments, the CMIP 313 is covalently linked to a C-terminus of the heavy chain 311. In some embodiments, the CMIP 313 is covalently linked to a N-terminus of the heavy chain 311. In some embodiments, the heavy chain 311 of the antibody or a portion thereof and the CMIP 313 are expressed as a single polypeptide. In some embodiments, the heavy chain 311 of the antibody or a portion thereof, the linker 314, and the CMIP 313 are expressed as a single polypeptide. As shown in Figure 3C, cell-penetrating agent 320 is comprised of a Fab antibody fragment comprising a heavy chain 321 and light chain 322. CIM 323 (e.g., a CMIP of the present disclosure) is connected to the C-terminus of heavy chain 321 (e.g., via optional linker 324). In some embodiments, the CMIP 323 is linked to a C-terminus of the heavy chain 321. In some embodiments the CMIP 323 is linked to a N-terminus of the heavy chain 321. In some embodiments, the heavy chain 321 of the Fab antibody fragment and the CMIP 323 are expressed as a single polypeptide. In some embodiments, the heavy chain 321, the linker 324, and the CMIP 323 are expressed as a single polypeptide. Although Figure 3C shows cell-penetrating agent 320 comprised of a Fab antibody fragment connected to CIM 323 via the heavy chain 321, CIM 323 (e.g., a CMIP of the present disclosure) can be connected to the C-terminus of light chain 322 (e.g., via optional linker 324) instead. In some embodiments, the CMIP 323 is linked to a C-terminus of the light chain 322. In some embodiments the CMIP 323 is linked to a N-terminus of the light chain 322. In some embodiments, the light chain 322 of the Fab antibody fragment and the CMIP 323 are expressed
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT as a single polypeptide. In some embodiments, the light chain 322, the linker 324, and the CMIP 323 are expressed as a single polypeptide. In some embodiments, the antibody or antigen-binding fragment thereof specifically binds to a target protein. In some embodiments, the target protein is a cytosolic protein. In some embodiments, the target protein is a membrane-associated protein. In some embodiments, the target protein is an enzyme. In some embodiments, the target protein is a cytoskeletal protein. In some embodiments, the target protein comprises a beta-actin protein. In some embodiments, the target protein is a beta-actin protein. In some embodiments, the payload comprises an anti- beta-actin antibody (e.g., antibodies or antigen-binding antibody fragments that selectively bind to the beta-actin protein). In some embodiments, the payload is an anti-beta-actin antibody. Additional exemplary anti-beta actin antibodies useful as payloads in CPAs of the present disclosure include those disclosed in Gimona, et al., Beta-actin specific monoclonal antibody. Cell Motil Cytoskeleton. 1994; 27(2):108-16. In some embodiments, the target protein is an alpha-tubulin protein. In some embodiments, the payload comprises an anti-α-tubulin antibody (e.g., antibodies and antigen- binding antibody fragments that selectively bind to the α-tubulin protein). In some embodiments, the payload is an anti-α tubulin antibody or an antigen-binding fragment thereof. Additional exemplary anti-α tubulin antibodies useful as payloads in CPAs of the present disclosure include those disclosed in Amin et al., Anti-tubulin-alpha-1c antibody as a marker of value in Behçet syndrome, Clin. Rheumatol. 2022 Jun; 41(6):1759-1767; Banerjee, et al., A monoclonal antibody to alpha-tubulin: purification of functionally active alpha-tubulin isoforms, Biochemistry 1999 Apr 27;38(17):5438-46; and Rüdiger, et al., Monoclonal antibody ID5: epitope characterization and minimal requirements for the recognition of polyglutamylated alpha- and beta-tubulin, Eur. J. Cell. Biol. 1999 Jan;78(1):15-20. In some embodiments, the target protein is an oxidoreductase. In some embodiments, the target protein is an endoplasmic reticulum oxidoreductin-1-like (EROL1L) protein. In some embodiments, the payload comprises an anti-EROL1L antibody (e.g., antibodies or antigen- binding antibody fragments that selectively bind to the EROL1L protein). In some embodiments, the payload is an anti-EROL1L antibody or an antigen-binding fragment thereof.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In some embodiments, the target protein is a mitochondrial protein. In some embodiments, the target protein is a mitochondrial transport protein. In some embodiments, the target protein is a translocase of outer mitochondrial membrane 20 (TOMM20) protein. In some embodiments, the payload comprises an anti-TOMM20 antibody (e.g., antibodies or antigen- binding antibody fragments that selectively bind to the TOMM20 protein). In some embodiments, the payload is an anti-TOMM20 antibody or an antigen-binding fragment thereof. In some embodiments, the target protein is a nuclear membrane protein. In some embodiments, the target protein is an outer nuclear membrane protein. In some embodiments, the target protein is a Nesprin-1 protein. In some embodiments, the payload comprises an anti- Nesprin-1 antibody (e.g., antibodies or antigen-binding antibody fragments that selectively bind to the Nesprin-1 protein). In some embodiments, the payload is an anti-Nesprin-1 antibody or an antigen-binding fragment thereof. Additional exemplary anti-Nesprin-1 antibodies useful as payloads in CPAs of the present disclosure include those disclosed in J. Zhang., Nesprin 1 is critical for nuclear positioning and anchorage, Human Molecular Genetics, 2010 Jan 15, 19(2):329-341; I. Holt, et al., Specific localization of nesprin-1-α2, the short isoform of nesprin-1 with a KASH domain, in developing, fetal and regenerating muscle, using a new monoclonal antibody, BMC Cell Biology, 2016 Jun 27, 17(2):26; and A. Espigat-Georger et al., Nuclear alignment in myotubes requires centrosome proteins recruited by nesprin-1, J. Cell Sci., 2016 Nov 15, 129(22):4227-4237. Enzyme Payloads In some embodiments, the payload molecule is an enzyme (or a functional portion thereof). In some embodiments, the CMIP is linked to the C-terminus of the enzyme or functional portion thereof. In some embodiments, the CMIP is linked to the N-terminus of the enzyme or the functional portion thereof. In some embodiments, the enzyme is a kinase. In some embodiments, the enzyme is a serine/threonine kinase. In some embodiments, the serine/threonine kinase is GSK3β. Methods of detecting kinase activity within a cell are well known in the art (e.g., direct or indirect detection of a phosphorylated substrate in the cell or a corresponding cell lysate). In some embodiments, the enzyme is a protease. In some embodiments, the protease is a cysteine protease. In some embodiments, the cysteine protease is a non-lysosomal cysteine
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT protease. In some embodiments, the non-lysosomal cysteine protease is calpain. Methods for detecting protease activity within a cell are well known in the art (e.g., direct or indirect detection of the products of the proteolytic activity of the protease in the cell or a corresponding cell lysate). In some embodiments, the enzyme is a bioluminescent protein. In some embodiments, the bioluminescent protein is a luciferase. Methods for detecting activity of a bioluminescent protein in a cell are well known in the art (e.g., detection using a luminometer). II. Nucleic Acids, Vectors, and Host Cells The present disclosure provides nucleic acids encoding at least a portion of any of the CMIPs described herein. In some embodiments, the nucleic acids encode any of the CMIPs described herein. In some embodiments, the nucleic acids encode any of the CMIPs described herein and at least a portion of a linker (e.g., a linker comprising a polypeptide). The present disclosure also provides nucleic acids encoding at least a portion of any of the cell-penetrating agents described herein. For example, the nucleic acids can encode any of the CIMs, linkers, and/or spacers described herein in addition to any of the heavy chains and/or light chains described herein. In some embodiments, the nucleic acid encodes for the CMIP. In some embodiments, the nucleic acid encodes for the CMIP and at least a portion of the payload. In some embodiments, the nucleic acid encodes for the CMIP and the payload. In some embodiments, the nucleic acid encodes for the CMIP and at least a portion of the linker. In some embodiments, the nucleic acid encodes for the CMIP and the linker. In some embodiments, the nucleic acid encodes for the CMIP and at least a portion of the linker and/or the payload. In some embodiments, the nucleic acid encodes for the CMIP and the linker and/or the payload. In some embodiments, the nucleic acid encodes for the CMIP, the polypeptide linker, and at least a portion of the payload molecule. In some embodiments, the nucleic acid encodes for the CMIP, the polypeptide linker, and the payload molecule. The present disclosure further provides nucleic acids encoding a CMIP and any of the heavy chain variable domains and/or light variable chain domains of any of the antibodies described herein. In some embodiments, the present disclosure provides nucleic acids encoding a CMIP, at least a portion of a linker, and any of the heavy chain variable domains and/or light variable chain domains of any of the antibodies described herein. In some embodiments, the
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT present disclosure provides nucleic acids encoding at least a portion of the CMIP and any of the heavy chain variable domains and/or light variable chain domains of any of the antibodies described herein. In some embodiments, the present disclosure provides nucleic acids encoding at least a portion of the CMIP, at least a portion of a linker, and any of the heavy chain variable domains and/or light variable chain domains of any of the antibodies described herein. Optionally, such nucleic acids further encode a signal peptide and can be expressed with the signal peptide linked to any of the exemplary polypeptides described herein. Coding sequences of nucleic acids can be operably linked with regulatory sequences to ensure expression of the coding sequences, such as a promoter, enhancer, ribosome binding site, transcription termination signal, and the like. The nucleic acids encoding any of the polypeptides described herein (e.g., any of the cell-penetrating agents described herein) can occur in isolated form or can be cloned into one or more vectors. The nucleic acids can be synthesized by, for example, solid state synthesis or PCR of overlapping oligonucleotides. Nucleic acids encoding any of the polypeptides described herein (e.g., any of the cell-penetrating agents described herein) can be joined as one contiguous nucleic acid, e.g., within an expression vector, or can be separate, e.g., each cloned into its own expression vector. In some embodiments, the nucleic acid is codon-optimized for expression in a host cell. A number of methods are known for producing chimeric and humanized antibodies using an antibody-expressing cell line (e.g., hybridoma), and such methods can be used to produce CPAs of the present disclosure. For example, the immunoglobulin variable regions of antibodies can be cloned and sequenced using well known methods. In one method, the heavy chain variable VH region is cloned by RT-PCR using mRNA prepared from hybridoma cells. Consensus primers are employed to the VH region leader peptide encompassing the translation initiation codon as the 5’ primer and a g2b constant regions specific 3’ primer. Exemplary primers are described in U.S. patent publication US 2005/0009150 by Schenk et al. (hereinafter “Schenk”). The sequences from multiple, independently derive clones can be compared to ensure no changes are introduced during amplification. The sequence of the VH region can also be determined or confirmed by sequencing a VH fragment obtained by 5’ RACE RT-PCR methodology and the 3’ g2b specific primer. The light chain variable VL region can be cloned in an analogous manner. In one approach, a consensus primer set is designed for amplification of VL regions using a 5’ primer
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT designed to hybridize to the VL region encompassing the translation initiation codon and a 3’ primer specific for the Ck region downstream of the V-J joining region. In a second approach, 5’RACE RT-PCR methodology is employed to clone a VL encoding cDNA. Exemplary primers are described in Schenk, supra. The cloned sequences are then combined with sequences encoding human (or other non-human species) constant regions. Such methods can be used in the production of CPAs of the present disclosure, for example, by utilizing (1) nucleic acids that encode for both a CMIP and a VL region and/or a VH region, or (2) nucleic acids that encode for a CMIP, a polypeptide linker, and a VL region and/or a VH region. Also provided herein are vectors including any of the nucleic acids described herein operably linked to one or more regulatory sequences to effect expression in a mammalian cell of any of the cell-penetrating agents described herein. Also provided herein are vectors including a nucleic acid encoding any of the exemplary polypeptides described herein (e.g., any of the cell-penetrating agents described herein) operably linked to one or more regulatory sequences to effect expression in a mammalian cell of any of the polypeptides described herein. One example of such vectors includes, for example, a nucleic acid encoding a CMIP, a mature heavy chain variable domain and/.or a light chain variable domain operably linked to one or more regulatory sequences to effect expression in a mammalian cell. Another example of such vectors includes, a nucleic acid encoding a CMIP, a polypeptide linker, a mature heavy chain variable domain and/or a light chain variable domain operably linked to one or more regulatory sequences to effect expression in a mammalian cell. In various embodiments, the nucleic acid will encode for a CMIP connected, for example, to at least one of the following: (1) the C-terminus of the light chain or a fragment thereof; (2) the N- terminus of the light chain or a fragment thereof; (3) the C-terminus of the heavy chain or a fragment thereof; and (4) the N-terminus of the heavy chain or a fragment thereof. In one exemplary approach, the heavy and light chain variable regions are re-engineered to encode splice donor sequences downstream of the respective VDJ or VJ junctions and are cloned into a mammalian expression vector, such as pCMV-hyl for the heavy chain and pCMV- Mcl for the light chain. These vectors encode human Kl and Ck constant regions as exonic fragments downstream of the inserted variable region cassette. Following sequence verification, the heavy chain and light chain expression vectors can be co-transfected into CHO cells to produce chimeric antibodies. Conditioned media is collected 48 hours post-transfection and
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT assayed by western blot analysis for antibody production or ELISA for antigen binding. This approach can be used to produce cell-penetrating agents by utilizing nucleic acids encoding for CMIP (and in some cases, an optional linker) that is genetically fused to the heavy and/or light chain (or fragment thereof) of the antibody. Cell-penetrating agents comprising chimeric, veneered, humanized, and human antibodies (including portions thereof) can be produced by recombinant expression. Recombinant polynucleotide constructs typically include an expression control sequence operably linked to sequence encoding any of the exemplary polypeptides described herein, including naturally associated or heterologous expression control elements, such as a promoter. The expression control sequences can be promoter systems in vectors capable of transforming or transfecting eukaryotic or prokaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences and the collection and purification of the polypeptide. Thus, provided herein are host cells transformed with any of the vectors described herein. Also provided herein are host cells including any of the nucleic acids described herein. Expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. Commonly, expression vectors contain selection markers, e.g., ampicillin resistance or hygromycin resistance, to permit detection of those cells transformed with the desired DNA sequences. E. coli is one prokaryotic host useful for expressing antibodies, particularly antibody fragments. Microbes, such as yeast, are also useful for expression. Saccharomyces is a yeast host with suitable vectors having expression control sequences, an origin of replication, termination sequences, and the like as desired. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization. Mammalian cells can be used for expressing nucleotide segments encoding any of the polypeptides described herein (e.g., cell-penetrating agents). See, Winnacker, From Genes to Clones, (VCH Publishers, NY, 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed, and include CHO cell lines, various COS cell lines, HeLa cells, HEK293 cells, L cells, and non-antibody-producing myelomas including Sp2/0
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT and NS0. The cells can be non-human. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Expression control sequences can include promoters derived from endogenous genes, cytomegalovirus, SV 40, adenovirus, bovine papillomavirus, and the like. See Co et al., J. Immunol. 148: 1149 (1992). In some embodiments, the promoter is a eukaryotic promoter. Alternatively, polypeptide-encoding sequences can be incorporated in transgenes for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., U.S. Pat. No. 5,741,957; U.S. Pat. No. 5,304,489; and U.S. Pat. No. 5,849,992). Suitable transgenes encoding any of the polypeptides described herein can be operably linked with a promoter and enhancer from a mammary gland specific gene, such as casein or beta lactoglobulin. Such methods can be utilized for the production of CPAs of the present disclosure, for example, by generating transgenes that include coding sequences for CMIPs and light and/or heavy chains (optionally further encoding for a polypeptide linker between the CMIP and the light and/or heavy chain) operably linked with a promoter and enhancer from a mammary gland specific gene. The vectors containing the DNA segments of interest can be transferred into the host cell by methods depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics, or viral-based transfection can be used for other cellular hosts. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and micro injection. For production of transgenic animals, trans genes can be microinjected into fertilized oocytes or can be incorporated into the genome of embryonic stem cells, and the nuclei of such cells transferred into enucleated oocytes. Having introduced vector(s) encoding the any of the polypeptides described herein (e.g., any of the exemplary cell-penetrating agents described herein) into cell culture, cell pools can be screened for growth productivity and product quality in serum-free media. Top-producing cell pools can then be subjected of FACS-based single-cell cloning to generate cell lines. Specific productivities above 50 pg or 100 pg per cell per day, which correspond to product titers of greater than 7.5 g/L culture, can be used. Cell-penetrating agents produced by single cell clones
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT can also be tested for turbidity, filtration properties, PAGE, IEF, UV scan, HPSEC, carbohydrate-oligosaccharide mapping, mass spectrometry, and binding assay, such as ELISA or Biacore. A selected clone can then be banked in multiple vials and stored frozen for subsequent use. Once expressed, CMIPs, CPAs, and antibodies can be purified according to standard procedures of the art, including protein A capture, HPLC purification, column chromatography, gel electrophoresis and the like (See generally, Scopes, Protein Purification (Springer-Verlag, NY, 1982)). Methods for commercial production of antibodies can be employed for the production of CPAs comprising antibodies, including codon optimization, selection of promoters, selection of transcription elements, selection of terminators, serum-free single cell cloning, cell banking, use of selection markers for amplification of copy number, CHO terminator, or improvement of protein titers (see, e.g., U.S. Patent No. 5,786,464; U.S. Patent No. 6,114,148; US 6,063,598; U.S. Patent No. 7,569,339; W02004/050884; W02008/012142; W02008/012142; W02005/019442; W02008/107388; W02009/027471; and U.S. Patent No. 5,888,809). The DNA can be delivered in naked form (i.e., without colloidal or encapsulating materials). Alternatively, a number of viral vector systems can be used including retro viral systems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenoviral vectors (see, e.g., Bett et al, J. Virol. 67, 5911 (1993)); adeno-associated virus vectors (see, e.g., Zhou et al., J. Exp. Med. 179, 1867 (1994)), viral vectors from the pox family including vaccinia virus and the avian pox viruses, viral vectors from the alpha virus genus such as those derived from Sindbis and Semliki Forest Viruses (see, e.g., Dubensky et al., J. Virol. 70, 508-519 (1996)), Venezuelan equine encephalitis virus (see U.S. Patent No. 5,643,576) and rhabdoviruses, such as vesicular stomatitis virus (see WO 96/34625) and papillomaviruses (Ohe et al., Human Gene Therapy 6:325-333 (1995); Woo et al., WO 94/12629 and Xiao & Brandsma, Nucleic Acids. Res.24:2630-2622 (1996)). Thus, in another aspect, the present disclosure provides methods of producing the cell- penetrating agent of the present disclosure, the method comprising: culturing a cell comprising a nucleic acid encoding the CMIP and at least a portion of the payload molecule in a liquid culture medium under conditions that allow the cell to produce a cell-penetrating agent comprising the
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT CMIP and at least a portion of the payload molecule; and harvesting the cell-penetrating agent from the cell or the liquid culture medium. III. Additional Conjugates The cell-penetrating agents described herein, can be further conjugated with other moieties, such as, for example radioisotopes and/or detectable labels. For examples, some such cell-penetrating agents can be linked to radioisotopes. Examples of radioisotopes include, for example, yttrium
90 (90Y), indium
111 (111In),
1311,
99mTc, radiosilver-111, radiosilver-199, and Bismuth
213. Linkage of radioisotopes to cell-penetrating agents or payloads may be performed with conventional bifunction chelates. For radiosilver-111 and radiosilver-199 linkage, sulfur- based linkers may be used. See Hazra et al., Cell Biophys. 24-25:1-7 (1994). Linkage of silver radioisotopes may involve reducing the immunoglobulin with ascorbic acid. For radioisotopes such as 111In and 90Y, ibritumomab tiuxetan can be used and will react with such isotopes to form 111In -ibritumomab tiuxetan and 90Y-ibritumomab tiuxetan, respectively. See Witzig, Cancer Chemother. Pharmacol., 48(Suppl l):S91-S95 (2001). Cell-penetrating agents or payloads can also be coupled with a detectable label. Such cell-penetrating agents and payloads can be used, for example, for diagnosing a disease or condition. Representative detectable labels that may be coupled or linked to a cell-penetrating agent or payload include various enzymes, such as horseradish peroxidase, alkaline phosphatase, betagalactosidase, or acetylcholinesterase; prosthetic groups, such streptavidin/biotin and avidin/biotin; fluorescent materials, such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as luminol; bioluminescent materials, such as luciferase, luciferin, and aequorin; radioactive materials, such as radiosilver-111, radiosilver-199, Bismuth
213, iodine (
131I,
125I,
123I,
121I), carbon (
14C), sulfur (
5S), tritium (
3H), indium (
115In
113In
112In
111In), technetium (
99Tc), thallium (
201Ti), gallium (
68Ga,
67Ga), palladium (
103Pd), molybdenum (
99Mo ), xenon (
133Xe), fluorine (
18F),
153Sm,
177Lu,
159Gd,
149Pm,
140La,
175Yb,
166Ho,
90Y,
47Sc,
186Re,
188Re,
142Pr,
105Rh,
97Ru,
68Ge,
57Co,
65ZN,
85SR,
32P,
153Gd,
169Yb,
51CR,
54Mn,
75Se,
113Sn, and
117Sn; positron emitting metals using various positron emission tomographies; nonradioactive
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT paramagnetic metal ions; and molecules that are radiolabelled or conjugated to specific radioisotopes. IV. Pharmaceutical Compositions and Products The present disclosure also provides pharmaceutical compositions and products. Thus, provided herein are pharmaceutical compositions including any of the cell-penetrating agents described herein and a pharmaceutically acceptable carrier. In prophylactic applications, a cell-penetrating agent (e.g., or a nucleic acid or a vector encoding any of the cell-penetrating agent) or a pharmaceutical composition of the same is administered to a patient susceptible to, or otherwise at risk of a disease. In therapeutic applications, a cell-penetrating agent is administered to a patient suspected of, or already suffering from a disease in a regime (dose, frequency and route of administration) effective to ameliorate or at least inhibit further deterioration of at least one sign or symptom of the disease. A regime is considered therapeutically or prophylactically effective if an individual treated patient achieves an outcome more favorable than the mean outcome in a control population of comparable patients not treated by methods disclosed herein. Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and manufactured under GMP conditions. Administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Some cell-penetrating agents can be administered into the systemic circulation by intravenous or subcutaneous administration. Pharmaceutical compositions can be provided in unit dosage form (i.e., the dosage for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries. The formulation depends on the route of administration chosen. For injection, cell-penetrating agents can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline or acetate buffer (to reduce discomfort at the site of injection). The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, cell-penetrating agents can be in lyophilized form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In some embodiments, it can be desirable to use a pharmaceutical composition comprising any of the cell-penetrating agents described herein in an ex vivo or in vitro method. For example, the method can be for a non-diagnostic and/or non-therapeutic purposes. In such instances, samples such as cells, tissues, and/or organs that have been removed from the patient are exposed to a pharmaceutical composition comprising any of the cell-penetrating agents described herein. V. Methods of Delivering Payloads and/or Binding a Target Protein In another aspect, the present disclosure provides a method of delivering a payload molecule into a mammalian cell, comprising contacting any of the cell-penetrating agents described herein with the cell, thereby resulting in the internalization of the payload molecule into the cell and transfer of the payload molecule to the cytosol. In some embodiments, the payload molecule is an antibody (e.g., an intact antibody or a functional antigen-binding portion thereof). In another aspect, the present disclosure provides a method of delivering an antibody into a mammalian cell, comprising contacting a cell-penetrating agent of the present disclosure comprising an antibody as the payload with the cell, thereby resulting in the internalization of the antibody into the cell and transfer of the antibody to the cytosol. In another aspect, the present disclosure provides a method of binding an intracellular target protein in a mammalian cell, the method comprising: contacting a cell-penetrating agent of the present disclosure comprising a payload molecule that specifically binds to the intracellular target protein with the cell thereby resulting in the internalization of the payload molecule into the cell, transfer of the payload molecule to the cytosol, and binding of the payload molecule to the intracellular target protein. In another aspect, the present disclosure provided methods of modulating at least one activity of an intracellular target protein in a cell, the method comprising: contacting a cell- penetrating agent of the present disclosure comprising a payload molecule that specifically binds to the intracellular target protein with the cell thereby resulting in the internalization of the payload molecule into the cell, transfer of the payload molecule to the cytosol, and binding of the payload molecule to the intracellular target protein, thereby modulating at least one activity
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT of the intracellular target protein. In some embodiments, modulating the at least one activity of the intracellular target protein includes increasing the activity of the intracellular protein. In some embodiments, modulating the at least one activity of the intracellular target protein includes decreasing the activity of the intracellular protein. In another aspect, the present disclosure provides methods of inhibiting at least one activity of an intracellular target protein in a cell, the method comprising: contacting a cell- penetrating agent of the present disclosure comprising a payload molecule that specifically binds to the intracellular target protein with the cell thereby resulting in the internalization of the payload molecule into the cell, transfer of the payload molecule to the cytosol, and binding of the payload molecule to the intracellular target protein, thereby inhibiting at least one activity of the intracellular target protein. In another aspect, the present disclosure provides methods of delivering an enzyme into a mammalian cell, comprising contacting a cell-penetrating agent of the present disclosure comprising an enzyme as the payload with the cell, thereby resulting in the internalization of the enzyme into the cell and transfer of the enzyme to the cytosol. In another aspect, the present disclosure provides methods of increasing intracellular enzyme activity in a mammalian cell, the method comprising contacting a cell-penetrating agent of the present disclosure comprising an enzyme as the payload with the cell, thereby resulting in the internalization of the enzyme into the cell and transfer of the enzyme to the cytosol; wherein the enzyme is active in the cytosol of the mammalian cell. In another aspect, the present disclosure provides methods of restoring intracellular enzyme activity in a mammalian cell having reduced activity of an enzyme, the method comprising contacting a cell-penetrating agent of the present disclosure comprising an enzyme having the same or similar enzymatic activity as the intracellular enzyme as the payload with the cell, thereby resulting in the internalization of the enzyme into the cell and transfer of the enzyme to the cytosol; wherein the enzyme is active in the cytosol of the mammalian cell. In another aspect, the present disclosure provides a method of delivering a payload protein sequence to the cytosol of a mammalian cell, the method comprising contacting the cell with a polypeptide comprising a CMIP and the payload protein sequence, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. In another aspect, the present disclosure provides a method of delivering a payload protein sequence to the nucleus of a mammalian cell, the method comprising contacting the cell with a cell-penetrating agent comprising a CMIP and the payload protein sequence, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. In another aspect, the present disclosure provides a method of degrading a target protein in the cytosol of a mammalian cell, the method comprising contacting the cell with a cell- penetrating agent comprising a CMIP and a protease capable of cleaving the target protein, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. In some embodiments, the payload protein sequence has a molecular weight of about 5 kD to about 1000 kD (e.g., about 50 kD to about 1000 kD, about 100 kD to about 1000 kD, about 50 kD to about 600 kD, about 100 kD to about 600 kD, about 50 kD to about 400 kD, about 100 kD to about 400 kD, about 50 kD to about 200 kD, or about 100 kD to about 200 kD).
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In some embodiments, payload protein sequence has a molecular weight of about 5 kD to about 500 kD (e.g., about 5 kD to about 100 kD, about 5 kD to about 200 kD, about 5 kD to about 300 kD, or about 5 kD to about 400 kD). In some embodiments, payload protein sequence has a molecular weight of about 50 kD to about 500 kD (e.g., about 50 kD to about 100 kD, about 50 kD to about 200 kD, about 50 kD to about 300 kD, or about 50 kD to about 400 kD). In some embodiments, payload protein sequence has a molecular weight of at least about 5 kD (e.g., at least about 10 kD, at least about 20 kD, at least about 30 kD, at least about 50 kD, at least about 70 kD, at least about 100 kD, at least about 140 kD, or at least about 200 kD). In some embodiments, the payload protein sequence comprises an antibody light chain or a fragment thereof. In some embodiments, the payload protein sequence comprises an antibody light chain or a fragment thereof and a linker. In some embodiments, the payload protein sequence comprises an antibody heavy chain or a fragment thereof. In some embodiments, the payload protein sequence comprises an antibody heavy chain or a fragment thereof and a linker. In some embodiments, the payload protein sequence is about 50 to about 2000 amino acids. In some embodiments, the payload protein sequence is about 50 to about 1500 amino acids (e.g., the payload protein sequence is about 100 to about 1500 amino acids, about 200 to about 1500 amino acids, 300 to about 1500 amino acids, 400 to about 1500 amino acids, 500 to about 1500 amino acids, 600 to about 1500 amino acids, 700 to about 1500 amino acids, 800 to about 1500 amino acids, 900 to about 1500 amino acids, or 1000 to about 1500 amino acids). In some embodiments, the payload protein sequence about 100 to about 500 amino acids (e.g., the payload protein sequence about 150 to about 500 amino acids, about 200 to about 500 amino acids, about 250 to about 500 amino acids, about 300 to about 500 amino acids, about 350 to about 500 amino acids, or about 400 to about 500 amino acids). In some embodiments, the payload protein sequence about 50 to about 300 amino acids (e.g., the payload protein sequence about 100 to about 300 amino acids, about 150 to about 300 amino acids, about 200 to about 300 amino acids, or about 250 to about 300 amino acids). In some embodiments the payload protein sequence is at least about 50 amino acids (e.g., the payload protein sequence is at least about 100 amino acids, at least about 200 amino acids, at least about 300 amino acids, at least about 400 amino acids, or at least about 500 amino acids). In another aspect, the present disclosure provides a method of delivering an antigen- binding domain to the cytosol of a mammalian cell, the method comprising contacting the cell
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT with a cell-penetrating agent comprising a CMIP and the antigen-binding domain as the payload, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. In another aspect, the present disclosure provides a method of delivering an antibody to the cytosol of a mammalian cell, the method comprising contacting the cell with a cell- penetrating agent comprising a CMIP and the antibody as the payload, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. In another aspect, the present disclosure provides methods of inhibiting an intracellular target protein in a cell, the method comprising: contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; wherein the cell-penetrating agent comprises: (i) an antibody that specifically binds the intracellular target protein; and (ii) a CMIP covalently linked to the antibody. In another aspect, the present disclosure provides methods of inhibiting an intracellular target protein in a cell, the method comprising: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the cell-penetrating agent comprises: (i) an antibody that specifically binds the intracellular target protein; and (ii) a CMIP covalently linked
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT to the antibody; and (b) binding of the cell-penetrating agent or antibody to the intracellular target protein. In some embodiments, the target protein is selected from a cytosolic protein, a mitochondrial protein, an endoplasmic reticulum lumen protein, and a nuclear membrane protein. In some embodiments, the target protein is selected from a protease, a cytoskeletal protein, an oxidoreductase, a mitochondrial transport protein, and an outer nuclear membrane protein. In some embodiments, the target protein is selected from beta-actin, alpha tubulin, ERO1L, TOMM20, and Nesprin-1. In some embodiments, the target protein is a beta-actin protein. In some embodiments, the target protein is an alpha tubulin protein. In some embodiments, the target protein is an endoplasmic reticulum oxidoreductin-1-like (EROL1L) protein. In some embodiments, the target protein is a translocase of outer mitochondrial membrane 20 (TOMM20) protein. In some embodiments, the target protein is a Nesprin-1 protein. In another aspect, the present disclosure provides methods for inhibiting at least one activity of an intracellular beta-actin protein in a mammalian cell comprising: (a) contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises (i) an antibody that specifically binds to the beta-actin protein, and (ii) a CMIP derived from M-lycotoxin, thereby resulting in the internalization of the cell-penetrating agent or fragment thereof (e.g., antibody) into the cell and transfer of the cell-penetrating agent or fragment thereof (e.g., antibody) to the cytosol; and (b) binding the antibody of the cell-penetrating agent to the intracellular beta-actin protein, thereby inhibiting at least one activity of the intracellular beta- actin protein. In another aspect, the present disclosure provides methods for at least one activity of an intracellular alpha-tubulin protein in a mammalian cell comprising: (a) contacting a cell- penetrating agent with the cell, wherein the cell-penetrating peptide comprises: (i) an antibody that specifically binds to the alpha-tubulin protein, and (ii) a CMIP derived from M-lycotoxin, thereby resulting in the internalization of the cell-penetrating agent or fragment thereof (e.g., the antibody) into the cell and transfer of the cell-penetrating agent or fragment thereof (e.g., the antibody) to the cytosol; and (b) binding the antibody of the cell-penetrating agent to the intracellular alpha-tubulin protein, thereby inhibiting at least one activity of the intracellular alpha-tubulin protein.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In another aspect, the present disclosure provides methods for modulating (e.g., inhibiting) an intracellular endoplasmic reticulum oxidoreductin-1-like protein (ERO1L) protein in a mammalian cell comprising: (a) contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises (i) an antibody that specifically binds to the ERO1L protein, and (ii) a CMIP derived from M-lycotoxin, thereby resulting in the internalization of the cell- penetrating agent or fragment thereof (e.g., the antibody) into the cell and transfer of the cell- penetrating agent or fragment thereof (e.g., the antibody) to the cytosol; and (b) binding the antibody of the cell-penetrating agent to the intracellular ERO1L protein, thereby inhibiting at least one activity of the intracellular ERO1L protein. In another aspect, the present disclosure provides methods for inhibiting an intracellular translocase of outer mitochondrial membrane 20 (TOMM20) protein in a mammalian cell comprising: (a) contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises: (i) an antibody that specifically binds to the TOMM20 protein, and (ii) a CMIP derived from M-lycotoxin; thereby resulting in the internalization of the cell-penetrating agent or fragment thereof (e.g., the antibody) into the cell and transfer of the cell-penetrating agent or fragment thereof (e.g., the antibody) to the cytosol; and (b) binding of the antibody of the cell- penetrating agent to the intracellular TOMM20 protein, thereby inhibiting at least one activity of the intracellular TOMM20 protein. In another aspect, the present disclosure provides methods for inhibiting at least one activity of an intracellular Nesprin-1 protein in a mammalian cell comprising: (a) contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises: (i) an antibody that specifically binds to the Nesprin-1 protein; and (ii) a CMIP derived from M-lycotoxin, thereby resulting in the internalization of the cell-penetrating agent or fragment thereof (e.g., the antibody) and transfer of the cell-penetrating agent or fragment thereof (e.g., the antibody) to the cytosol; and (b) binding the antibody of the cell-penetrating agent to the intracellular Nesprin-1 protein, thereby inhibiting at least one activity of the intracellular Nesprin-1 protein. In some embodiments, the CMIP is covalently linked to the antibody. In some embodiments, the CMIP is covalently linked to the antibody via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In various embodiments, the CMIPs used in these methods wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF),
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. In another aspect, the present disclosure provides a method of delivering an enzyme to the cytosol of a mammalian cell, the method comprising contacting the cell with a cell- penetrating agent comprising a CMIP and the enzyme. In some embodiments, the CMIP is an M- lycotoxin derivative. In some embodiments, the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. In another aspect, the present disclosure provides methods of increasing intracellular enzymatic activity in a mammalian cell, the method comprising: (a) contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises (i) an enzyme capable of catalyzing the intracellular enzymatic activity, and (ii) a CMIP covalently or non-covalently linked to the enzyme, thereby resulting in the internalization of the cell-penetrating agent or fragment thereof (e.g., the enzyme) into the cell and transfer of the cell-penetrating agent or fragment thereof (e.g., the enzyme) to the cytosol; and (b) the enzyme of the cell-penetrating agent performs the enzymatic activity inside the cell. In another aspect, the present disclosure provides methods of restoring intracellular enzymatic activity in a mammalian cell having reduced intracellular enzymatic activity, the method comprising: (a) contacting a cell-penetrating agent with the cell, wherein the cell- penetrating agent comprises (i) an enzyme capable of catalyzing the intracellular enzyme activity and (ii) a CMIP covalently or non-covalently linked to the enzyme, thereby resulting in the internalization of the cell-penetrating agent or fragment thereof (e.g., the enzyme) into the cell
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT and transfer of the cell-penetrating agent or fragment thereof (e.g., the enzyme) to the cytosol; and (b) restoring the enzymatic activity inside the cell. Restoring intracellular enzymatic activity includes restoring 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of intracellular enzymatic activity. In some examples, restoring enzymatic activity includes restoring greater than 100% activity (e.g., greater than wild-type activity). For example, restoring intracellular enzymatic activity includes restoring 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, 200% or more of intracellular enzymatic activity. In some embodiments, the enzyme is selected from a kinase, a protease, and a reporter enzyme. In some embodiments, the enzyme is selected from GSK3β, calpain, and luciferase. In some embodiments, the enzyme is GSK3β. In some embodiments, the enzyme is calpain. In some embodiments, the enzyme is a luciferase. In another aspect, the present disclosure provides a method of increasing at least one GSK3β activity in a mammalian cell, the method comprising: contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises (i) a GSK3β enzyme or a functional fragment thereof and (ii) a CMIP covalently or non-covalently linked to the GSK3β enzyme, thereby resulting in the internalization of the cell-penetrating agent or fragment thereof (e.g., the GSK3β enzyme) into the cell and transfer of the cell-penetrating agent of fragment thereof (e.g., the GSK3β enzyme) to the cytosol, thereby increasing at least one GSK3β activity in the mammalian cell. In another aspect, the present disclosure provides a method of restoring at least one intracellular GSK3β activity in a mammalian cell having reduced intracellular GSK3β activity, the method comprising: contacting a cell-penetrating agent with the cell, wherein the cell- penetrating agent comprises (i) a GSK3β enzyme or a functional fragment thereof and (ii) a CMIP covalently linked to the GSK3β enzyme, thereby resulting in the internalization of the cell-penetrating agent or fragment thereof (e.g., the GSK3β enzyme) into the cell and transfer of the cell-penetrating agent or fragment thereof (e.g., the GSK3β enzyme) to the cytosol; thereby restoring at least one intracellular GSK3β activity in the cell. In another aspect, the present disclosure provides a method of increasing at least one activity of Calpain in a mammalian cell, the method comprising: contacting a cell-penetrating
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT agent with the cell, wherein the cell-penetrating agent comprises: (i) a calpain enzyme or a functional fragment thereof and (ii) a CMIP covalently or non-covalently linked to the calpain enzyme, thereby resulting in the internalization of the cell-penetrating agent or fragment thereof (e.g., the calpain enzyme) into the cell and transfer of the cell-penetrating agent or fragment thereof (e.g., the calpain enzyme) to the cytosol; thereby increasing at least one activity of Calpain in the cell. In another aspect, the present disclosure provides a method of restoring at least one intracellular calpain activity in a cell having reduced intracellular calpain activity, the method comprising: contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises (i) a calpain enzyme or a functional fragment thereof and (ii) a CMIP covalently or non-covalently linked to the calpain enzyme, thereby resulting in the internalization of the cell- penetrating agent or fragment thereof (e.g., the calpain enzyme) into the cell, transfer of the cell- penetrating agent or fragment thereof (e.g., the calpain enzyme) to the cytosol, and restoration of at least one intracellular Calpain activity in the cell. In another aspect, the present disclosure provides a method of increasing luciferase activity in a cell, the method comprising: contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises (i) a luciferase enzyme or a functional fragment thereof and (ii) a CMIP covalently or non-covalently linked to the luciferase enzyme, thereby resulting in the internalization of the cell-penetrating agent or fragment thereof (e.g., the luciferase enzyme) into the cell, transfer of the cell-penetrating agent or fragment thereof (e.g., the luciferase enzyme) to the cytosol, and an increase in luciferase activity in the cell. In some embodiments, the CMIP is covalently linked to the enzyme. In some embodiments, the CMIP is covalently linked to the enzyme via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a polypeptide linker. In some embodiments, the polypeptide linker comprises an amino acid sequence selected from Table 4. In some embodiments, the CMIP comprises an M-lycotoxin derivative. In some embodiments, the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA),
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP comprises an amino acid sequence selected from Table 1 or Table 3. In some embodiments, the CMIP comprises an amino acid sequence selected from Table 3. In some embodiments, the cell is a mammalian cell. In some embodiments, wherein the cell is in vitro. In some embodiments, the cell is in a subject. VI. Treatment Regimens As used herein, the terms “treat” and “treatment” refer to the alleviation or amelioration of one or more symptoms or effects associated with the disease, prevention, inhibition or delay of the onset of one or more symptoms or effects of the disease, lessening of the severity or frequency of one or more symptoms or effects of the disease, and/or increasing or trending toward desired outcomes as described herein. Provided herein are methods of delivering an antibody that specifically binds to a target into a cell, the methods comprising contacting any of the cell-penetrating agents described herein that include the antibody as a payload with the cell, thereby resulting in the internalization of, at a minimum, an antigen-binding fragment of the antibody, into the cell. In some embodiments, the method includes transferring, at a minimum, an antigen-binding fragment of the antibody, to the cytosol of the cell. In some embodiments, the cell-penetrating agent is administered by intravenous injection into the body of the subject. In some embodiments, the cell-penetrating agent or the antibody in the cell-penetrating agent is labeled. In some embodiments, the cell-penetrating agent is labeled with a fluorescent label, a paramagnetic label, or a radioactive label. In some embodiments, the radioactive label is detected using positron emission tomography (PET) or single-photon emission computed tomography (SPECT). The cell-penetrating agents are administered in an effective regime meaning a dosage, route of administration and frequency of administration that delays the onset, reduces the severity, inhibits further deterioration, and/or ameliorates at least one sign or symptom of a disorder being treated. If a patient is already suffering from a disorder, the regime can be referred to as a therapeutically effective regime. If the patient is at elevated risk of the disorder relative to the general population but is not yet experiencing symptoms, the regime can be
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT referred to as a prophylactically effective regime. In some instances, therapeutic or prophylactic efficacy can be observed in an individual patient relative to historical controls or past experience in the same patient. In other instances, therapeutic or prophylactic efficacy can be demonstrated in a preclinical or clinical trial in a population of treated patients relative to a control population of untreated patients. Administration can be parenteral, intravenous, oral, subcutaneous, intra-arterial, intracranial, intrathecal, intraperitoneal, topical, intranasal or intramuscular. Some cell- penetrating agents can be administered into the systemic circulation by intravenous or subcutaneous administration. Cell-penetrating agents are usually administered on multiple occasions. The frequency of administration depends on the half-life of the cell-penetrating agent in circulation, the condition of the patient and the route of administration among other factors. The frequency can be daily, weekly, monthly, quarterly, or at irregular intervals in response to changes in the patient’s condition or progression of the disorder being treated. An exemplary treatment regimen entails administration once per every two weeks, once a month, or once every 3 to 6 months. The number of dosages administered depends on whether the disorder is acute or chronic and the response of the disorder to the treatment. The dosing frequency can be adjusted depending on the pharmacokinetic profile of the antibody formulation in the patient. For example, the half-life of the cell-penetrating agent may warrant a two week frequency of dosing. In some embodiments disclosed herein, the cell-penetrating agent is administered to the patient for at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, 5 years, 10 years, or for the life of the patient. VII. Kits The present disclosure further provides kits (e.g., containers) comprising any of the cell- penetrating agents described herein and related materials, such as instructions for use (e.g., package insert). The instructions for use can contain, for example, instructions for administration of the cell-penetrating agent and optionally one or more additional agents. The containers of cell-penetrating agents may be unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT EXAMPLES The following examples have been included to illustrate modes disclosed herein. Certain aspects of the following examples are described in terms of techniques and procedures found or contemplated to work well in the practice disclosed herein. In light of the present disclosure and the general level of skill in the art, those of skill appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications, and alterations may be employed without departing from the scope of the disclosure. Example 1: Cellular Internalization of Anti-GFP Cell-Penetrating Agents Comprising Selected CMIPs To demonstrate the capacity for CMIPs of the present disclosure to be internalized by cells, a panel of exemplary CMIPs (shown in Table 5, below) was assessed for internalization. A panel of anti-GFP cell-penetrating agents was expressed, with each CPA comprising an anti-GFP antibody having a CMIP of Table 5 attached to the C-terminus of the light chain via a polypeptide linker having a GGSSS sequence (SEQ ID NO: 256). The anti-GFP antibody comprised the heavy chain variable region and light chain variable regions of the ScFv disclosed in WO 2022/053642 A1. HEK cells were transiently transfected with Htt-exon1-Q103-eGFP, to express GFP within the cytosol of the cell. After 24 hours, the cells were incubated with 11 ug/ml of anti-GFP CPA for another 24 hours (n=2 per CPA). Cells were then washed, fixed/permeabilized, and stained with AF647-conjugated anti-human secondary antibodies to stain the intracellular CPAs. Stained cells were imaged by high content imaging (Operetta system) with 40x water objective (30 fields/well). The amount of intracellular CPA detected via secondary antibody was quantified and the number normalized to the value observed for the CPA comprising CMIP4 (SEQ ID NO: 57). Quantitative analyses were carried out with Harmony software. Table 5 provides relative internalization efficiency (normalized to CMIP4) for exemplary CMIPs for the present disclosure. This internalization efficiency was assessed in the manner described above. Table 5
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: Name Internalization SEQ ID NO: Name Internalization (Normalized) (Normalized) M
IP4 MIP11
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 130 CMIP78 117.9 213 CMIP161
173.7 131 CMIP79 171.4 214 CMIP162
--
Example 2: Calculated Biophysical Characteristics of Selected CMIPs
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT To understand the correlation between CMIP internalization and biophysical parameters, net charge (z), hydrophobic moment (<µH>), and hydrophobicity (<H>) for exemplary CMIPs of the present disclosure. These parameters were calculated using the HELIQUEST online program from the Institutde Pharmacologie Moléculaireet Cellulaire, Universitéde Nice Sophia Antipolisand CNRS, 660 routedes Lucioles, 06560 Valbonne, France. Table 6 provides calculated values for net charge (z), hydrophobic moment (<µH>), and hydrophobicity (<H>) for exemplary CMIPs of the present disclosure. These values were calculated in the manner described above. Table 6 SEQ ID Name z <µH> <H> SEQ ID Name z <µH> <H> NO: NO: 6 5 3 3 1 1 7 7 1 8 6 7 9 5 3 4 7 5
4 4 4 9 5 7
9
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT C
MIP65 4 0.138 0.516 200 CMIP148 1 0.198 0.338 C
MIP66 4 0.079 0.463 201 CMIP149 4 0.094 0.452 8 8 4 8 8 6 8 2
8 5 4
3 1 3 3 6 6
8 3 8 4 4 6 2 2
2 8 8 9 8 7 6 2
5 1 9
2
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 1
62 CMIP110 4 0.657 0.378 245 CMIP193 5 0.162 0.511 1
63 CMIP111 5 0.159 0.315 246 CMIP194 5 0.107 0.43 6 1
2 8 8 4
xamp e : mmunogen c y o s
To understand the therapeutic potential for cell-penetrating agents of the present disclosure, the immunogenicity of exemplary CMIPs was assessed. Qualitative immunogenicity profiles for CMIP sequences were generated using the EpiQuest program, which provides a matrix of predicted probability of CTL epitopes for each CMIP sequence analyzed. Exemplary EpiQuest immunogenicity profiles are provided in FIGs. 4A through 4D. As shown in Figures 4A through 4D, each amino acid residue of the sequence is given a position along the X-axis, and each amino acid residue is provided a predicted immunogenicity (PI) value as an integer along the Y-axis (shown as shaded tiles). A positive PI value indicates a probability for an immunogenic epitope comprising the amino acid residue at that position, and a negative PI value indicates a low probability for an immunogenic epitope comprising that the amino acid residue at that position. These values across all amino acid residues in the CMIP sequence are combined resulting in an immunogenicity profile for each CMIP sequence analyzed. To compare potential immunogenicity across the CMIPs, the immunogenicity profiles generated by the EpiQuest program were converted to an Immunogenicity Unit (IU) scale of 0 through 10 IU. The EpiQuest immunogenicity profiles were generated using the default EpiQuest threshold, and were converted to an IU value in the following manner: First, each residue is assigned an IU value based on the PI value provided by the EpiQuest program. For each residue having a positive PI value, a score of 0.2 Immunogenicity Units (IU) is assigned a for each shaded tile between 0 to 5 (for example, if a residue has all five vertical tiles shaded from 0 to 5, an IU of 1 is assigned to that residue). In addition to the vertical tiles from 1 to 5, any vertical tile that is shaded from 6 to 10 is assigned an additional IU score of 0.4 for each shaded tile. Similarly, any shaded vertical tile from 11 to15 is assigned a IU score of 0.6 for each shaded tile. For example, if an amino acid residue has a PI value of 12 (indicated by
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 12 vertical shaded tiles ), then it would be assigned a IU score of 4.2 IU (add 0.2 for each of the five shaded tiles from 1 to 5 for a total of 1.0; add 0.4 for each of the five shaded tiles from 6 to 10 for a total of 2.0; and add 0.6 for each of the two shaded tiles from 11 to 12 for a total of 1.2). Second, a penalty score is added to the IU score based on the number of continuous amino acid residues assessed as having a positive PI value. A penalty of 1 IU is imposed for three or more contiguous amino acid residues having PI values from 1 to 5 (indicated by positive shaded tiles for three or more continuous residues). No additional penalty is imposed even if all contiguous tiles have PI values of five. A penalty score of 2 is imposed for three or more contiguous amino acid residues having PI values from 6 to 10. Additionally, A penalty score of 3 is imposed for three or more contiguous amino acid residues having PI values from 11 to 15. A molecule will be assigned a maximum score 10 even if calculated score is higher. For example, Figure 4A provides the EpiQuest predicted immunogenicity profile for CMIP39 (SEQ ID NO: 97), which has an IU score of 0, as there are no amino acid residues having a positive PI value in the EpiQuest predicted immunogenicity profile. Figure 4B provides the EpiQuest predicted immunogenicity profile for CMIP34 (SEQ ID NO: 92), which has an IU score of 0.4, as there is only one amino acid residues having a positive PI value in the EpiQuest predicted immunogenicity profile. The residue has a PI value of 2, which leads to the IU score of 0.4. Figure 4C provides the EpiQuest predicted immunogenicity profile for CMIP48 (SEQ ID NO: 106), which has an IU score of 10. For this sequence, there are six amino acid residues having a positive PI value in the EpiQuest predicted immunogenicity profile. In addition to the individual penalty score for each of these residues, further penalty scores are imposed for the three contiguous residues (1-2) having values greater than 5 and three continuous amino acid residues (4-6) having values between 1 and 5. As the total IU score for this sequence exceeds 10, a value of 10 IU is assigned to this sequence. Figure 4D provides the EpiQuest predicted immunogenicity profile for CMIP30 (SEQ ID NO: 88), which has an IU score of 1.2. For this sequence, there are three amino acid residues having a positive PI value in the EpiQuest predicted immunogenicity profile. Amino acid residue 21 has a PI value of 3, adding IU penalty of 0.6. Amino acid residue 22 has a PI value of 2, adding IU penalty of 0.4. Amino acid residue 26 has a PI value of 1, adding IU penalty of 0.2,
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT for a total IU score of 1.2. No additional penalty is imposed, as the three immunogenic residues are not contiguous. Table 7 provides IU scores for exemplary CMIPs for the present disclosure. These IU scores were calculated in the manner described above. Table 7 SEQ ID NO: Name IU Score SEQ ID NO: Name IU Score MIP4 16 MIP11 75
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT CMIP71
10 206 CMIP154
1.2 CMIP72
1.6 207 CMIP155
1.2
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 168 CMIP116
0.5 251 CMIP199
0.4 169
CMIP117 10 252 CMIP200 0.2
. ibod
y y tes of TDP-43 and Clearance of TDP-43 Aggregates in Transfected HEK Cells Figure 5A shows confocal microscopy images of HEK cells transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)] or GFP only (2a is a self-cleaving peptide that releases the TDP-43 upon expression). The top panels show staining for GFP, nuclei (grey), and pTDP-43 (white). The bottom panels show only pTDP-43 staining which is not present in the GFP alone control transfection. Figure 5B (left) is a graph showing cell count for HEK cells transfected with either GFP-2a-TDP43 or GFP alone. The data demonstrate approximately equivalent cell counts between the two populations. Further, Figure 5B (right) shows pTDP-43 foci counts for HEK cells transfected with GFP-2a-TDP43 or GFP alone. The data demonstrate TDP-43 foci are formed only in the HEK cells transfected with GFP-2a- TDP43. Figure 5C shows TDP-43 staining with either commercial antibodies or antibodies of the present disclosure including 13D3, 13C13, and 2D4. Cells were stained with antibodies for 24 hours and 24 hours later, cells were incubated again with 100 ug/ml antibodies for another 24 hours. Cells were then washed, fixed/permeabilized, and stained with anti-pTDP-43 antibodies, followed by AF647-conjugated anti-mouse secondary antibodies. Stained cells were imaged by high content imaging (Operetta system) with 40x water objective. Quantitative analyses were carried out with Harmony software. Antibodies 13D3, 13C13, and 2D4 detect overexpressed mislocalized TDP-43 in HEK cells. The top row shows transfection with GFP-2a-TDP-43 where the phosphorylated TDP43 does not include a nuclear localization signal (i.e., phosphorylated TDP43 remains in the cytoplasm) and the bottom row shows transfection with a GFP only construct. The data demonstrate that TDP-43 foci are formed only in HEK cell transfected with GFP-2a-TDP-43. Various control antibodies were included in the assay including pTDP-43 (Cosmo)+, pTDP-43 (1D3)+, Total TDP-43 (PT), and 3B12 (ED)+. The control antibodies verify TDP-43 aggregation within the cytoplasm. Antibodies 13D3, 13C13, and 2D4 were similarly tested and demonstrated similar binding to cytoplasmic aggregates of phosphorylated TDP-43. The 13D3 antibody was identified as an appropriate antibody to utilize for CPA-mediated targeting of cytosolic pTDP-43 aggregates.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Example 5. Internalization and Clearance of TDP-43 Aggregates in Transfected Cells with Anti-TDP-43 Cell-Penetrating Agents Figures 6A-B through 19A-E demonstrate internalization of and/or target engagement by anti-TDP-43 cell-penetrating agents of the present disclosure. Throughout Figures 6A-B to 19A- E, references to “M-Lyco,” Lycotoxin,” “ML” and the like refer to CPAs comprising L17E_M- lycotoxin linked to a 13D3 antibody (murine, chimeric, or humanized). Throughout Figures 6A- B to 19A-E, references to other CIMs and/or CMIPs (e.g., “CMIP”, “CMIP4”, “cTAT”, “PEPTH”, etc.) refer to CPAs comprising the corresponding CIM linked to a 13D3 antibody (murine, chimeric, or humanized). Unless otherwise specified (e.g., by HC), the CIM is linked to the 13D3 antibody via the c-terminus of the light chain. HEK cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)]. 24 hours later, cells were incubated with 100 µg/ml cell-penetrating agents including a cell internalization module and an anti-TDP-43 antibody for 4 hours. The cells were then washed, fixed/permeabilized, and followed by AF647-conjugated anti-mouse secondary antibodies. Stained cells were imaged by high content imaging (Operetta system) with a 40x water objective. Quantitative analyses were carried out with Harmony software. Specifically, Figure 6A shows the percentage of CPA-positive cells following incubation with m13D3 CPAs including different CIMs (i.e., TAT, M-Lycotoxin_L17E (LC), M-Lycotoxin_L17E (HC), PEPTH (HC)) or under various control conditions (i.e., untagged, isotype, and vehicle). HC (heavy chain) and LC (light chain) indicate the location of the CIM. Labels to “M-Lycotoxin” in Figure 6A refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of either the light chain (LC) or heavy chain (HC) of the m13D3 antibody. The data demonstrate increased internalization of the m13D3 CPAs compared to both the naked antibody (“untagged”) and the vehicle controls. Figure 6B shows the number of CPA- positive spots per cell following incubation with the m13D3 CPAs. The data demonstrate increased pTDP-43 foci detection for the m13D3 CPAs compared to both the naked antibody and vehicle controls. Figures 7A-E are graphs showing the results of transfected HEK cells with various m13D3 CPAs. Briefly, HEK cells were transiently transfected with either GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)] or no plasmid (e.g., untransfected) as a negative
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT control. 24 hours later the cells were incubated with 100 µg/ml of antibodies (e.g., CPAs) for another 24 hours. Cells were then washed, fixed/permeabilized, and stained with anti-pTDP-43 antibodies, followed by AF647-conjugated anti-mouse secondary antibodies. Stained cells were imaged by high content imaging (Operetta system) with 40x water objective. Quantitative analyses were carried out with Harmony software. Figure 7A shows the number of pTDP-43 foci per well area. Labels to “M-Lycotoxin” in Figures7A-E refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of the light chain of the m13D3 antibody. The data demonstrate that cells treated with different m13D3 CPAs had a lower number of foci per well area as compared to cells treated with untagged m13D3 (e.g., no cell internalizing module). As expected, no foci were observed in untransfected cells. Figure 7B shows the mean focus intensity for pTDP-43 foci. The data demonstrate that cells treated with different m13D3 CPAs had a lower mean focus intensity as compared to cells treated with untagged m13D3 antibody. As expected, very low focus intensity was observed with untransfected cells. Figure 7C shows consistent cell count per well across all cell populations tested (i.e., transfected with different m13D3 CPAs or untransfected cells). Figure 7D shows the number of pTDP-43 foci normalized by cell count. The data demonstrate that cells treated with different m13D3 CPAs had a lower number of foci per cell as compared to those treated with untagged m13D3. As expected, no foci were observed with untransfected cells. Figure 7E shows the mean focus area for p-TDP-43 foci, demonstrating that cells treated with different m13D3 CPAs had a lower mean area intensity compared to those treated with the untagged m13D3; very low focus intensity was observed with untransfected cells Figure 8A shows the number of pTDP-43 foci per well area. The data demonstrate that cells treated with different m13D3 CPAs (i.e., TAT (HC), L17E M-Lycotoxin (LC), L17E M- lycotoxin (HC), and PEPTH (HC)) had a lower number of foci per well area as compared to those treated with the untagged m13D3. Labels to “M-Lycotoxin” in Figures 8A-D refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of either the light chain (LC) or heavy chain (HC) of the m13D3 antibody. Figure 8B shows consistent cell count per well across all cell populations tested. Figure 8C shows the number of pTDP-43 foci normalized by cell count which demonstrates that cells treated with different m13D3 CPAs had a lower number of foci per cell as compared to cells treated with the untagged m13D3. As expected, no foci were observed in untransfected cells. Figure 8D shows the mean focus area for pTDP-43 foci. The
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT data demonstrate that cells treated with different m13D3 CPAs had a lower mean area intensity compared to those treated with the untagged m13D3; very low focus intensity was observed with untransfected cells Figures 9A-D are graphs showing the results of cells incubated with m13D3 M- Lycotoxin [17E] CPAs at different concentrations under the same experimental conditions described in Figures 8A-D. Labels to “M-Lycotoxin” in Figures 8A-D refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of either the light chain (LC) or heavy chain (HC) of the m13D3 antibody. In addition, the cell internalizing module was located on either the heavy chain or light chain of the m13D3 antibody. Cells were also incubated with untagged m13D3, IgG isotype control, and a PBS control. Untransfected HEK cells were used as a negative control. Figure 9A shows pTDP-43 total foci area per well area. The data show a concentration- dependent reduction in total foci area for cells treated with the m13D3 M-Lycotoxin_L17E CPAs. Labels to “M-Lycotoxin” in Figures 9A-D refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of either the light chain (LC) or heavy chain (HC) of the m13D3 antibody. In contrast, the cells treated with the 13D3 antibody, IgG isotype control, and PBS showed a higher levels of foci as compared to cells treated with the m13D3 m-Lycotoxin [L17E] CPAs. Untransfected cells had no foci (data not shown). Figure 9B shows consistent cell count per well across all cell populations tested and no concentration dependent reduction in cell viability for cells transfected with the m13D3 M-Lycotoxin CPAs. Figure 9C shows the pTDP- 43 foci count which demonstrates a concentration-dependent increase in foci count for the m13D3 M-Lycotoxin CPAs, with significantly higher number of foci compared to control. Figure 9D shows the pTDP-43 mean focus size, demonstrating a concentration-dependent decrease in mean focus size for the m13D3 M-Lycotoxin CPAs, with significantly lower mean focus size compared to control. Collectively, Figures 9A-D demonstrate that the m13D3 M- Lycotoxin CPAs interfere with foci aggregation in a concentration-dependent manner. Figure 10 shows graphs of untransfected HEK cells (left) or transfected with GFP-2a- TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)] (right) incubated with either m13D3 antibody or an M-Lycotoxin m13D3 CPA for 24 hours and subjected to the XTT metabolic assay to assess cell viability. Labels to “M-Lycotoxin” in Figure 10 refer to CPAs comprising M- Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody. The
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT data show no significant difference in cell death across all populations tested (as measured by percent cell death), demonstrating that the CPA (m13D3 M-Lycotoxin [L17E]) does not induce significant cytotoxicity. Collectively, Figures 7A-E through 10 demonstrate that anti-TDP-43 cell-penetrating agents of the present disclosure are internalized by cells and can effectively engage intracellular TDP-43. Example 6. Internalization and Clearance of Phosphorylated Cytoplasmic Aggregates of TDP-43 with Anti-TDP-43 Cell-Penetrating Agents Figures 11A-E are graphs showing cells incubated with different m13D3 CPAs and untagged m13D3 antibody. Untransfected HEK cells were used as a negative control. The data in Figures 11A-E were generated with the same experimental conditions described in Figures 7A-D above. More specially, Figure 11A shows the sum of pTDP-43 foci per well area. Labels to “ML-13D3” in Figures 11A-E refer to CPAs comprising M-Lycotoxin_L17E linked at the C- terminus of the light chain (LC) of the m13D3 antibody. Labels to “CMIP1-5” in Figures 11A-E refer to CPAs comprising CMIP1, CMIP2, CMIP3, CMIP4, or CMIP5 linked at the C-terminus of the light chain (LC) of the m13D3 antibody. The data demonstrate that cells treated with different m13D3 CPAs (e.g., M-Lyco_L17E, CMIP1, CMIP2, CMIP3, CMIP4, CMIP5) had a lower number of foci per well area as compared to cells treated with the untagged m13D3. As expected, no foci were observed with untransfected cells. Figure 11B shows the mean focus intensity for pTDP-43 foci. The data demonstrate that cells treated with different m13D3 CPAs had a lower mean focus intensity as compared to cells treated with untagged m13D3 antibody and very low focus intensity was observed with untransfected cells. Figure 11C shows consistent cell count per well across all cell populations tested. Figure 11D shows the number of p-TDP-43 foci (normalized by cell count). The data demonstrates that cells treated with different m13D3 CPAs had a higher number of foci per cell as compared to cells treated with untagged m13D3 antibody. As expected, no foci were observed with untransfected cells. Last, Figure 11E shows the mean focus area for pTDP-43 foci. The data demonstrate that cells treated with different m13D3 CPAs had a lower mean area intensity compared to those treated with the untagged m13D3; very low focus intensity was observed with untransfected cells.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Figures 12A-D are graphs showing the results of cells were incubated with either m13D3 m-Lycotoxin [L17E] CPAs or m13D3 CMIP4 CPA at different concentrations; cells were alternatively incubated with the untagged m13D3 or an IgG isotype control; untransfected HEK cells were used as a negative control. The data in Figures 12A-D was generated with the same experimental conditions described in Figures 7A-D above. Labels to “13D3-ML” in Figures 12A-D refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody. Labels to “13D3-CMIP4” in Figures 12A-D and Figures 11A-E refer to CPAs comprising CMIP4 linked at the C-terminus of the light chain (LC) of the m13D3 antibody. Figure 12A shows the total number of foci p-TDP-43, demonstrating a concentration- dependent reduction in total foci area for cells treated with the 13D3 m-Lyco and 13D3 CMIP4 CPAs. In contrast, the cells treated with the 13D3 antibody and isotype showed a higher levels of foci. Untransfected cells showed no foci. Figure 12B shows consistent cell count per well across all cell populations tested, and no concentration dependent reduction in cell viability for the m13D3 m-Lycotoxin CPAs. Figure 12C shows the p-TDP-43 foci number normalized by cell count, demonstrating a concentration-dependent increase in foci count for the 13D3 m-Lycotoxin and 13D3 CMIP4 CPAs, with significantly higher number of foci compared to control. Figure 12D shows the p-TDP-43 mean focus area, demonstrating a concentration-dependent decrease in mean focus area for the 13D3 m-Lycotoxin and 13D3 CMIP4 CPAs, with significantly lower mean focus area compared to control. Thus, Figures 12A-D demonstrates that the 13D3 m- Lycotoxin and 13D3 CMIP4 CPAs interfere with foci aggregation in a concentration-dependent manner. To obtain the data provided in Figures 13A-E, cells were incubated with the m13D3 CMIP4 CPA and the untagged m13D3 antibody in either acetate buffer or PBS. The data in Figures 13A-E were generated with the same experimental conditions described in Figures 7A-D above. More specifically, Figure 13A shows the pTDP-43 foci per well area, demonstrating that cells treated with the m13D3 CMIP4 CPA had a lower number of foci per well area compared to those treated with the untagged m13D3; no change in cell-penetrating activity was observed between PBS and acetate. Figure 13B shows the mean focus intensity for pTDP-43 foci, demonstrating that cells treated with the m13D3 CMIP4 CPA had a lower mean focus intensity
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT compared to those treated with the untagged m13D3; no change in cell-penetrating agent activity was observed between PBS and acetate. Figure 13C shows consistent cell count per well across all cell populations tested. Figure 13D shows the number of p-TDP-43 foci normalized by cell count, demonstrating that cells treated with the m13D3 CMIP4 CPA had a higher number of foci per cell compared to those treated with the untagged m13D3; no foci were observed with untransfected cells. Figure 13E shows the mean focus area for p-TDP-43 foci, demonstrating that cells treated with the m13D3 CMIP4 CPA had a lower mean area intensity compared to those treated with the untagged m13D3 Example 7. Internalization and Clearance of Phosphorylated Cytoplasmic Aggregates of TDP-43 with Novel Cell-Internalizing Modules and Chimeric anti-TDP-43 Antibody HEK cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)] (2a is a self-cleaving peptide that releases the TDP-43 upon expression). 24 hours later the cells were incubated with 100 ug/ml antibodies (e.g., CPAs) for another 24 hours. The cells were then washed, fixed/permeabilized, and stained with anti-pTDP-43 antibodies, followed by AF647-conjugated anti-mouse and AF594-conjugated anti-human secondary antibodies. Stained cells were imaged by high content imaging (Operetta system) with 40x water objective. Quantitative analyses were carried out with Harmony software. Figure 14A shows confocal microscopy images of cells treated with the untagged ch13D3 antibody (left) and the ch13D3 m-Lycotoxin_L17E CPA (right). Foci are shown in white, the 13D3 antibody is shown in light grey, and nuclei are shown in dark grey. White arrows indicate illustrative foci co-localized with the 13D3 antibody. Figure 14A demonstrates significant colocalization between the pTDP43 foci and the 13D3 antibody for cells treated with the ch13D3 m-Lycotoxin CPA, with little colocalization observed for cells treated with the ch13D3 antibody. Figure 14B is a graph showing the percentage of pTDP43 colocalized with the ch13D3 antibody. The data demonstrate significantly greater co-localization for cells treated with the ch13D3 m- Lycotoxin CPA (~50-60%) as compared to the cells treated with the ch13D3 antibody (~15- 20%). Collectively, Figures 14A-B demonstrates that anti-TDP-43 CPAs of the present disclosure are internalized by cells and bind to intracellular p-TDP-43. Figures 15A-D are graphs showing anti-TDP-43 CPAs of the present disclosure ((i.e., chimeric 13D3 CPAs (ch13D3 m-Lyco CPA, ch13D3 CMIP4 with LALA mutation, ch13D3
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT CMIP4 with H310-H435Q mutations), the untagged ch13D3 antibody, and an hIgG isotype control) are internalized by cells and bind to intracellular p-TDP-43 (i.e., cells transfected with GFP-2a-TDP43 as described herein). Labels to “M-Lyco” in Figures 15A-D refer to CPAs comprising M-Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody. Figure 15A provides a graph showing the percentage of p-TDP-43 colocalized with the ch13D3 antibody for ch13D3 CPAs as well as ch13D3 antibody, Isotype, and PBS controls using both confocal (left) and non-confocal (right) microscopy. Figure 15A demonstrates significantly greater co-localization for cells treated with the various ch13D3 CPAs (~50-60%) compared to the cells treated with the ch13D3 antibody (~15-20%). Cells treated with isotype control and PBS demonstrated no co-localization. Figure 15B provides a graph showing the average number of 13D3 antibody spots per cell for various ch13D3 CPAs, as well as ch13D3 antibody, isotype, and PBS controls. Figure 15B demonstrates that the ch13D3 CPAs result in significantly more 13D3 spots per cell (~2.5 – 3 spots per cell) than the controls. Figure 15C provides a graph showing the average spot size for the 13D3 antibody spots for various ch13D3 CPAs, as well as ch13D3 antibody, isotype, and PBS controls. Figure 15C demonstrates that the ch13D3 CPAs result in significantly smaller 13D3 spots per cell than the control. Figure 15D provides a graph showing the average spot size for the 13D3 antibody spots for various ch13D3 CPAs, as well as ch13D3 antibody, isotype, and PBS controls. Figure 15D demonstrates that the ch13D3 CPAs result in significantly smaller (corrected for spot intensity) 13D3 spots per cell than the control. Thus, Figures 15A-D demonstrates that anti-TDP-43 CPAs of the present disclosure are internalized by cells and bind to intracellular p-TDP-43. Example 8. Internalization and Clearance of Phosphorylated Cytoplasmic Aggregates of TDP-43 with Anti-TDP-43 Cell-penetrating Agents Figures 16A-D are graphs showing internalization and colocalization of phosphorylated cytoplasmic aggregates of pTDP-43 (i.e., cells transfected with GFP-2a-TDP43 as described herein) with novel cell internalizing modules and humanized anti-TDP-43 antibodies. The data shown in Figures 16A-D was generated under the same experimental conditions as described in Figures 7A-D above. Labels to “ch13D3-ML” in Figures 16A-D refer to CPAs comprising M- Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Figure 16A provides a graph showing the percentage of pTDP43 colocalized with the humanized 13D3 antibody for humanized 13D3 CPAs as well as ch13D3 and h13D3 antibody controls. Figure 16A demonstrates significantly greater co-localization for cells treated with the various h13D3 CPAs (~60-70%) compared to the cells treated with the h13D3 antibody (~40%). Figure 16B shows consistent cell count per well across all cell populations tested. Figure 16C is a graph showing the 13D3 spot area per well for pTDP43 colocalized with the humanized 13D3 antibody observed in cells treated with humanized 13D3 CPAs, as well as IgG isotype, ch13D3, and h13D3 antibodies controls. Figure 16C demonstrates significant increases in total co- localization area for cell treated with humanized 13D3 CPAs of the present disclosure. Figure 16D provides a graph showing the number of 13D3 co-localization spots per cell observed in cells treated with humanized 13D3 CPAs, as well as IgG isotype, ch13D3, and h13D3 antibodies controls. Figure 16D demonstrates significant increases in number of co-localization spots for cells treated with humanized 13D3 CPAs of the present disclosure. Collectively, the data in Figures 16A-D show internalization and colocalization of phosphorylated cytoplasmic aggregates of pTDP-43 with novel cell internalizing modules and humanized anti-TDP-43 antibodies. Example 9. Internalization and Clearance of Phosphorylated Cytoplasmic Aggregates of TDP-43 in Glioblastoma Cells with Anti-TDP-43 Cell-penetrating Agents Figures 17A-C are graphs showing internalization and colocalization of phosphorylated cytoplasmic aggregates of TDP-43 (i.e., cells transfected with GFP-2a-TDP43 as described herein) in glioblastoma cells with CPAs including a cell internalizing module and an anti-TDP- 43 antibody. Labels to “M-lycotoxin” in Figures 17A-C refer to CPAs comprising M- Lycotoxin_L17E linked at the C-terminus of the light chain (LC) of the m13D3 antibody. Briefly, U251 cells were transiently transfected with GFP-2a-TDP43 [mNLS (R82L/K83Q) DCS (C173S/C175S)]. 24 hours later the cells were incubated with 100 µg/ml antibodies (e.g., CPAs) for another 24 hours. The cells were then washed, fixed/permeabilized, and stained with anti- pTDP-43 antibodies, followed by AF647-conjugated anti-mouse secondary antibodies. The stained cells were imaged by high content imaging (Operetta system) with a 40x water objective. Quantitative analyses were carried out with Harmony software.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT More specifically, Figure 17A is a graph showing the total foci area for U251 glioblastoma cells treated with either m13D3 m-Lycotoxin CPA or m13D3 antibody alone (e.g., untagged). The data demonstrate a reduction in total foci area which was significantly reduced in glioblastoma cells treated with m13D3 m-Lycotoxin CPA as compared to cells treated with the m13D3 antibody. Figure 17B is a graph showing the mean foci size for U251 glioblastoma cells treated with either m13D3 m-Lycotoxin CPA or m13D3 antibody alone (e.g., untagged). The data demonstrate a reduction in mean foci size which was significantly reduced in glioblastoma cells treated with m13D3 m-Lycotoxin CPA compared to those cells treated with the m13D3 antibody. Figure 17C is a graph showing the total foci count for U251 glioblastoma cells treated with either m13D3 m-Lycotoxin CPA or m13D3 antibody. The data demonstrate an increase in total foci count which was significantly increased in glioblastoma cells treated with m13D3 m-Lycotoxin CPA as compared to those cells treated with the m13D3 antibody. Thus, Figures 17A-C demonstrates that anti-TDP-43 CPAs of the present disclosure are internalized by glioblastoma cells that bind to intracellular p-TDP-43 and therefore disrupt TDP- 43 aggregation. Example 10. Internalization and Clearance of Phosphorylated Cytoplasmic Aggregates of TDP-43 in Rat Cortical Neuron Cells with Anti-TDP-43 Cell-penetrating Agents Figures 18A-B and 19A-F are graphs showing that CPAs including a cell internalizing module and an anti-TDP-43 antibody are internalized and colocalization of phosphorylated cytoplasmic aggregates in primary rat cortical neurons. Briefly, primary rat cortical neurons cells (DIV15) were incubated with 50 µg/ml antibodies for 2 hours. The cells were then washed, fixed/permeabilized, and stained with anti-EEA1 antibodies, followed by AF596-conjugated anti-rabbit and AF647-conjugated anti-mouse secondary antibodies. The stained cells were imaged by high content imaging (Operetta system) with a 40x water objective. Quantitative analyses were carried out with Harmony software. Specifically, Figure 18A shows images of primary rat cortical neuronal cells treated with IgG isotype control (top, left), m13D3 (top, right), m13D3 m-Lycotoxin CPA (bottom, left), and m13D3CMIP4 CPA (bottom, right). Figure 18A demonstrates significant internalization of the m13D3 CPAs (depicted by white spots inside the cells, bottom panels) and little to no internalization of the isotype controls (top panels). Figure 18B is a graph showing the total of foci area per well which demonstrates little to no internalization of the isotype control and the
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 13D3 antibody and substantial internalization by the m13D3 m-Lycotoxin_L17E CPA and m13D3 CMIP4 CPA (labeled as 13D3-ML). Figure 19A is a graph showing the total number of 13D3 spots per cell for the DIV15 rat neuronal cells incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA. Figure 19A demonstrates higher numbers of 13D3 spots per cell for cells treated with m13D3-Cterm-LC-M-Lycotoxin_L17E CPA (labeled as “M-Lyco” in Figures 19A-F) and m13D3-Cterm-LC-M CMIP4 CPA (labeled as “CMIP” in Figures 19A-F), than for cells treated with either the IgG isotype control or the m13D3 antibody. Figure 19B is a graph showing the total area of 13D3 spots per well for the DIV15 rat neuronal cells incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lyco CPA, or m13D3 CMIP4 CPA. Figure 19B demonstrates higher total spot area per well for cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPAs, than for cells treated with either the IgG isotype control or the m13D3 antibody. Figure 19C is a graph showing the mean spot size for 13D3 spots in the DIV15 rat neuronal cells incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA. Figure 19C demonstrates higher mean spot size for cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPAs, than for cells treated with either the IgG isotype control or the m13D3 antibody. Figure 19D is a graph showing spot integrated intensity for 13D3 spots in the DIV15 rat neuronal cells incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA. Figure 19D demonstrates higher spot integrated intensity for cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPAs, than for cells treated with either the IgG isotype control or the m13D3 antibody. Figure 19E is a graph showing the percentage of 13D3 spots colocalized with EEA1 (Early Endosome Antigen 1) in the DIV15 rat neuronal cells incubated with hIgG isotype control, m13D3 antibody, m13D3 m-Lycotoxin CPA, or m13D3 CMIP4 CPA. Figure 19E demonstrates ~10-20% of 13D3 spots colocalized with EEA1 in cells treated with m13D3 m-Lycotoxin CPA and m13D3 CMIP4 CPAs, with no co- localization observed for cells treated with either the IgG isotype control or the m13D3 antibody. Figure 19F shows consistent cell count per well across all cell populations tested. Collectively, Figures 18A-B and 19A-F show that cell internalizing modules of the present disclosure are internalized by primary rat cortical neurons.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Example 11: APP Expression in APP770-Transfected HEK Cells Method: HEK cells were initially transfected with APP770. After 24 hours, the cells were washed, fixed, permeabilized, and then stained using an anti-APP antibody (Invitrogen REF13- 0200) and AF647-conjugated anti-mouse secondary antibodies. Stained cells were imaged by high content imaging (Operetta system) with 40x water objective. Quantitative analyses were carried out with Harmony software. Figure 20A shows high content images of untransfected HEK cells (left) transfected with APP (right). Cell bodies are shown in grey, and Aβ aggregates are shown as white spots with exemplary aggregates identified with white arrows. Comparison of left and right shows Abeta aggregates identified forming in cells transfected APP (right) but not control cells (left). Figure 20B shows the number of subcellular signal spots for labeled with an anti-Aβ antibody, thereby demonstrating a large number of Aβ aggregates in the APP transfected cells and no signal from the untransfected cells. Example 12: Target Engagement by Anti-Aβ CPAs in APP770-Transfected HEK Cells Method: HEK cells were transiently transfected with APP770. After the initial 24 hours, the transfected HEK cells were exposed to different treatments for an additional 24 hours. These treatments included IgG1, untagged h2931 antibody, h2931-ML, h2931_LC-cTAT, h2931_LC- cR8, h2931-LC-TAT and h2931_LC-R8, each at a concentration of 100 µg/ml concentration. Following this, the cells were fixed, permeabilized, and stained using anti-APP antibody (Invitrogen REF13-0200) and AF647-conjugated anti-mouse secondary antibodies. The stained cells were then imaged using the high content imaging Operetta system with a 40x water objective. Quantitative analyses were performed using Harmony software. Figure 21A provides a graph of the total labeled APP spot area, demonstrating that all cell populations tested had a similar total labeled subcellular APP spot area. Figure 21B provides a graph of the mean area for each labeled APP spot, demonstrating that cells treated with h2931 cell-penetrating agents had a lower mean area for each labeled spot compared to those treated with the untagged h2931 or IgG1 isotype. Figure 21C shows the number of labeled APP spots normalized by cell count, demonstrating that cells treated with different h2931 cell-
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT penetrating agents had a higher number of labeled APP spots per cell compared to those treated with the untagged h2931 or IgG1 isotype. Thus, Figures 21A-C demonstrates that the cell-penetrating agents of the present disclosure interfere with APP aggregation. Example 13: Target Engagement by Anti-Aβ CPAs in APP770-Transfected HEK Cells Method: HEK cells were transiently transfected with APP770. After the initial 24 hours, the transfected HEK cells were exposed to different treatments for an additional 24 hours. These treatments included IgG1 and untagged h2931 antibody, as well as h2931_LC-CMIP1 to h2931_LC-CMIP5, each at a concentration of 100 ug/ml concentration. Following this, the cells were fixed, permeabilized, and stained using anti-APP antibody (Invitrogen REF13-0200) and AF647-conjugated anti-mouse secondary antibodies. The stained cells were then imaged using the high content imaging Operetta system with a 40x water objective. Quantitative analyses were performed using Harmony software. Figure 22A provides a graph of the total labeled subcellular APP spot area, demonstrating that all cell populations tested had a similar total labeled subcellular APP spot area. Figure 22B provides a graph of the mean area for each labeled Aβ spot, demonstrating that cells treated with h2931 cell-penetrating agents had a lower mean area for each labeled spot compared to those treated with the untagged h2931 or IgG1 isotype. Figure 22C shows the number of labeled Aβ spots normalized by cell count, demonstrating that cells treated with different h2931 cell- penetrating agents had a higher number of labeled APP spots per cell compared to those treated with the untagged h2931 or IgG1 isotype. Thus, Figures 22A-C demonstrates that the cell-penetrating agents of the present disclosure interfere with APP aggregation. Examples 14: Concentration-Dependent Effect of Aβ CPA on Aβ Aggregation in Transiently Transfected APP770sw-HEK Cells HEK cells were initially transfected with APP770sw [K670N-M671L_Swedish] which is a model for analyzing Aβ-related diseases. After 24 hours, the cells were subjected to separate 24-hour incubations with cell-penetrating agent (h2931_ML (L17E linked to C-terminus of the light chain)) at concentrations of 1, 10, and 100 μg/ml, respectively. Subsequently, the cells were washed, fixed, permeabilized, and stained using m3D6 anti-Aβ antibody and AF647-conjugated
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT anti-mouse secondary antibodies. Stained cells were imaged by high content imaging (Operetta system) with a 40x water objective. Quantitative analyses were carried out with Harmony software. Figure 23A is a graph showing the total Aβ foci area (px2). The data demonstrate that incubation with h2931-M-Lycotoxin cell-penetrating agent (e.g., an anti-Aβ cell-penetrating agent) resulted in a lower total Aβ foci area in a concentration dependent manner. The data also showed a reduction in total Aβ foci area at each concentration as compared to the control sample. Figure 23B is a graph showing Aβ focus count which shows that incubation with the h2931-M- Lycotoxin cell-penetrating agent resulted in a lower number of Aβ foci in a concentration dependent manner compared to the control. Figure 23C is a graph showing the mean focus area for Aβ foci in cells treated with h2931 or h2931 cell-penetrating agent. Thus, Figures 23A-C demonstrate that the h2931 cell-penetrating agents (i.e., cell-penetrating agents) interfere with Aβ aggregation in a concentration-dependent manner. Example 15: Concentration-Dependent Effect of Aβ CPA on Aβ Aggregation in Transiently Transfected APP770sw-HEK Cells HEK cells were transiently transfected with APP770sw [K670N-M671L_Swedish]. 24 hours later, cells were incubated with either 10 µg/ml or 100 µg/ml h2931_ML (L17E linked to the C-terminus of the light chain), or with 100 ug/ml h2931 untagged antibody for 24 hours, respectively. The cells were then washed, fixed, permeabilized, and stained with m3D6 anti-Aβ antibody and AF647-conjugated anti-mouse secondary antibodies. The stained cells were imaged by high content imaging (Operetta system) with a 40x water objective. Quantitative analyses were carried out with Harmony software. Figure 24A is a graph showing total Aβ foci area. The data demonstrate that incubation with the h2931-M-Lycotoxin (L17E linked to the C-terminus of the light chain) cell-penetrating agent (e.g., an anti-Aβ cell-penetrating agent) resulted in reduced total Aβ foci area in a concentration dependent manner as compared to the untreated and IgG isotype controls. Figure 24B is a graph showing Aβ foci count demonstrating that incubation with the h2931-M- Lycotoxin cell-penetrating agent resulted in a lower number of Aβ foci in a concentration dependent manner as compared to the untreated and IgG isotype controls. Figure 24C is a graph showing the mean focus area for Aβ foci in cells treated with h2931 or h2931 cell-penetrating agent. Collectively, Figures 24A-C demonstrate that h2931 cell-penetrating agents interfere with Aβ aggregation in a concentration-dependent manner.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Examples 16: Concentration-Dependent Effect of Aβ CPAs on Aβ Aggregation in Transiently Transfected APP770sw-HEK Cells HEK cells were transiently transfected with APP770sw [K670N-M671L_Swedish]. After 24 hours, transfected HEK cells were incubated with five different concentrations of a h2931-M- Lycotoxin (L17E) cell-penetrating agent (e.g., an anti-Aβ cell-penetrating agent) at 1, 2.5, 5, 10 and 100 µg/ml. Cells treated with 100 ug/ml untagged h2931 were used as controls. The cells were then fixed, permeabilized, and stained with m3D6 anti-Aβ antibody and AF647-conjugated anti-mouse secondary antibodies. The stained cells were imaged by high content imaging (Operetta system) with a 40x water objective. Quantitative analyses were carried out with Harmony software. Figure 25A is a graph showing the total Aβ foci area. The data demonstrate that incubation with the h2931-M-Lycotoxin cell-penetrating agent (e.g., an anti-Aβ cell-penetrating agent) resulted in a reduced total Aβ foci area in a concentration dependent manner as compared to the untreated and IgG isotype controls. Figure 25B is a graph showing the Aβ focus count, demonstrating that incubation with the h2931-M-Lycotoxin cell-penetrating agent resulted in a lower number of Aβ foci in a concentration dependent manner compared to the untreated and IgG isotype controls. Figure 25C is a graph showing the mean focus area for Aβ foci in cells treated with either h2931 (i.e., untagged) or h2931 cell-penetrating agents. Thus, Figures 25A-C demonstrate that h2931 cell-penetrating agents interfere with Aβ aggregation in a concentration- dependent manner. Example 17: Internalization and Target Engagement of Anti-Aβ CPAs having Reduced FcRn and/or Antibody Effector Function HEK cells were transiently transfected with APP770sw [K670N-M671L_Swedish]. After 24 hours, transfected HEK cells were incubated with various h2931-CMIP4 cell-penetrating agents including h2931 (i.e., untagged antibody), h2931 with a disrupted Fcγ function (L234A/L325A mutation), or an h2931 antibody having a mutation in the FcRn region (H310A/H435Q mutation) for another 24 hours, each at a concentration of 100 ug/ml. For each of h2931_CMIP4, h2931_CMIP4_LALA, and h2931_CMIP4_ H310A_H435Q CMIP4 is linked to the C-terminus of the light chain. Subsequently, the cells were fixed, permeabilized, and stained using the primary m3D6 anti-Aβ antibody, followed by the secondary antibodies AF647- conjugated anti-mouse and AF568-conjugated anti-human. Stained cells were imaged by high
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT content imaging (Operetta system) with a 40x water objective. Quantitative analyses were carried out with Harmony software. Figure 26A is a graph showing the total Aβ foci area per cell. The data demonstrate that incubation with the h2931 cell-penetrating agents resulted in a reduced total Aβ foci area per cell as compared to the untreated and IgG isotype controls, regardless of Fcγ or FcRn function. That is, the h2931 cell-penetrating agents (e.g., anti-Aβ cell-penetrating agents) are able to disrupt Aβ deposits intracellularly without the function of either a Fcγ domain or a FcRn region. Figure 26B is a graph showing the number of Aβ foci area per cell, demonstrating that incubation with the h2931 cell-penetrating agents resulted in a lower number of Aβ foci per cell compared to the untreated and IgG isotype controls, regardless of Fcγ or FcRn function. Figure 26C is a graph showing the antibody (i.e., anti-Aβ cell-penetrating agents) internalization per cell, demonstrating that incubation with the h2931 cell-penetrating agents resulted in increased antibody internalization per cell as compared to the untagged h2931 antibody, regardless of Fcγ or FcRn function. Thus, Figures 26A-C demonstrate that the h2931 cell-penetrating agents interfere with intracellular Aβ deposits even when Fcγ or FcRn function is abrogated through mutation. Example 18: Clearance of Murinized Anti-Aβ CPA in Transiently Transfected APP770sw- HEK Cells HEK cells were transiently transfected with APP770sw [K670N-M671L_Swedish]. After 24 hours, the transfected HEK cells were incubated with either 100 µg/ml untagged murinized 2931 antibody (mzd-2931) or 100 µg/ml mzd-2931-CMIP4 cell-penetrating agent (i.e., an anti- Aβ cell-penetrating agent) for an additional 24 hours. For mzd-2931_CMIP4 CMIP4 linked to the C-terminus of the light chain. The cells were fixed, permeabilized, stained using the primary m3D6 anti-Aβ antibody, followed by secondary antibodies AF647-conjugated anti-mouse and AF568-conjugated anti-human. The stained cells were imaged by high content imaging (Operetta system) with a 40x water objective. Quantitative analyses were carried out with Harmony software. Figure 27A is a graph showing the total Aβ foci area per cell, demonstrating that incubation with the mzd-2931-CMIP4 cell-penetrating agent resulted in a reduced total Aβ foci area per cell as compared to cells treated with untagged mzd-2931 antibody (i.e., no cell internalizing module). Figure 27B is a graph showing the number of Aβ foci per cell,
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT demonstrating that incubation with the mzd-2931-CMIP4 cell-penetrating agent resulted in a lower number of Aβ foci area per cell as compared to cells treated with untagged mzd-2931 antibody. Figure 27C is a graph showing the antibody or cell-penetrating agent internalization per cell. The data demonstrate that incubation with the mzd-2931-CMIP4 cell-penetrating agent resulted in a greater internalization into these cells compared to the cells treated with untagged mzd-2931. Thus, Figures II- 8A-C demonstrates that cell-penetrating agents of the present disclosure are internalized by disrupt Aβ aggregation, even when the cell-penetrating agents comprise murinized antibodies. Example 19: Internalization and Target Engagement Assessment in Transiently Transfected APP770sw-HEK Cells for Panel of Anti-Aβ CPAs HEK cells were transiently transfected with APP770sw [K670N-M671L_Swedish]. After the initial 24 hours, the transfected HEK cells were exposed to different treatments for an additional 24 hours. These treatments included untagged h2931 antibody, h2931-CMIP4, and h2931 conjugated with CMIP4-derived peptides, each at a concentration of 100 µg/ml. For each of h2931_CMIP4-1 through h2931-CMIP4-25, CMIP4 is linked at the C-terminus of the light chain. Following this, the cells were fixed, permeabilized, and stained using m3D6 anti-Aβ antibody along with AF647-conjugated anti-mouse and AF568-conjugated anti-human secondary antibodies. The stained cells were then imaged using the high content imaging Operetta system with a 40x water objective. Quantitative analyses were performed using Harmony software. Figure 28A is a graph showing the total Aβ foci area per cell. The data demonstrate that cell- penetrating agents of the present disclosure significantly reduced the total area of Aβ foci per cell as compared to untreated cells and cells treated with either IgG isotype or untagged h2931 antibody (i.e., no cell internalizing module). Figure 28B is a graph showing the average number of Aβ foci area per cell which shows that cell-penetrating agents (e.g., cell-penetrating agents) of the present disclosure significantly reduced the number of Aβ foci per cell as compared to untreated cells and cells treated with either IgG isotype or untagged h2931 antibody. Figure 28C is a graph showing antibody or cell-penetrating agent internalization per cell. The data demonstrates that cell-penetrating agents of the present disclosure were readily internalized into transfected HEK cells, leading to higher signals as compared to untreated cells and cells treated with either IgG isotype or untagged h2931 antibody. Thus, Figures 28A-C demonstrates that
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT cell-penetrating agents comprising anti-Aβ antibodies effectively internalize and bind to Aβ peptides, effectively disrupting intracellular Aβ aggregates. Figures 28A-C further shows that this effect is observed in cell-penetrating agents comprising a variety of CMIPs. Example 20: Clearance of Anti-Aβ CPA in Lentivirus-Transfected APP770sw-HEK Cells HEK cells were transduced with lentivirus vectors comprising APP770sw [K670N- M671L_Swedish]. After 48 hours the transduced HEK cells were subjected to a further 24 hour incubation with various treatments as follow: either untagged h2931 antibody or h2931-CMIP4 antibody each at a concentration of 100 µg/ml. Following incubation, the cells were fixed, permeabilized, and stained using m3D6 anti-Aβ antibody along with AF647-conjugated anti- mouse and AF568-conjugated anti-human secondary antibodies. The stained cells were then imaged using the high content imaging Operetta system with a 40x water objective. Quantitative analyses were performed using Harmony software. Figure 29A is an image of untreated HEK cells expressing APPsw following lentiviral transfection. Cells are shown in grey and Aβ aggregates are shown in white. Figure 29A demonstrates substantial Aβ production and aggregation for untreated HEK cells. Figure 29B is an image of HEK cells expressing APPsw following lentiviral transfection and incubation with untagged h2931 antibody. Cells are shown in grey and Aβ aggregates are shown in white. Figure 29B demonstrates reduced Aβ aggregation for h2931 treated HEK cells. Figure 29C is an image of HEK cells expressing APPsw following lentiviral transfection and incubation with h2931-CMIP4 cell-penetrating agent. Cells are shown in grey, with Aβ aggregates shown in white. CMIP4 is linked to the C-terminus of the light chain. Figure 29C demonstrates substantially reduced Aβ aggregation for h2931-CMIP4 cell- penetrating agent treated HEK cells. Example 21: Internalization and Target Engagement of Anti-Aβ CPA in Lentivirus- Transfected APP770sw-HEK Cells HEK cells were transduced with lentivirus vectors comprising APP770sw [K670N- M671L_Swedish] at MOI (multiplicity of infection) = 10. After 48 hours, the transduced HEK cells were subjected to a further 24 hour incubation with either untagged h2931 antibody or h2931-CMIP4 antibody each at a concentration of 100 µg/ml. CMIP4 is linked to the C- terminus of the light chain. Following incubation, the cells were fixed, permeabilized, and
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT stained using m3D6 anti-Aβ antibody along with AF647-conjugated anti-mouse and AF568- conjugated anti-human secondary antibodies. The stained cells were then imaged using the high content imaging Operetta system with a 40x water objective. Quantitative analyses were performed using Harmony software. Figure 30A is a graph showing the total Aβ foci area per cell, demonstrating that incubation with the h2931-CMIP4 cell-penetrating agent resulted in a lower total Aβ foci area per cell in lenti-APPsw transduced HEK-293 cells compared to untreated cells and cells treated with untagged h2931 antibody. Figure 30B is a graph showing the number of Aβ foci per cell, demonstrating that incubation with the h2931-CMIP4 cell-penetrating agent resulted in a lower number of Aβ foci area per cell compared to untreated cells and cells treated with untagged h2931 antibody. Figure 30C is a graph showing antibody or cell-penetrating agent internalization per cell, demonstrating that incubation with the h2931-CMIP4 cell-penetrating agent resulted in increased internalization as compared to untreated cells and cells treated with untagged h2931 antibody alone. Example 22: Aβ Expression in Stably Transfected APP770sw-HEK Cells HEK cells were initially transduced with lentivirus vectors containing APP770sw [K670N-M671L_Swedish] at a MOI (multiplicity of infection) of 10. Following transduction, a dose-dependent puromycin selection process was employed to eliminate non-transduced cells and enrich for those that successfully integrated the lentiviral construct. To achieve stable expression of APPsw, the surviving cells underwent two additional rounds of lentiviral transduction and puromycin selection. Subsequently, the cells were fixed, permeabilized, and stained using primary antibodies m3D6 anti-Aβ, CT695, and LN27 anti-APP. Secondary antibodies AF647-conjugated anti-mouse and AF568-conjugated anti-rabbit were utilized to bind the primary antibody. High content imaging was performed using the Operetta system with a 40x water objective, and quantitative analyses were conducted using Harmony software. Figure 31A shows high content images of stable APPsw-HEK cells stained with either anti-Aβ antibody m3D6 (left) or anti-Aβ antibody CT695 (right). Cell bodies are shown as grey rounded structures and Aβ aggregates are shown as diffuse grey structures with exemplary aggregates identified with white arrows. Figure 31A shows that stable APPsw-HEK cells are expressing substantial amounts of Aβ, leading to aggregation. Figure 31B is a graph showing the percentage of stable APPsw-HEK cells having Aβ aggregates compared to untransfected HEK cells
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT (control), as measured by either anti-Aβ antibody m3D6 (left) or anti-Aβ antibody CT695 (right). Under both measures, about 80% of stable APPsw-HEK cells include Aβ1-42 aggregates. Thus, Figures 31A-B demonstrates that stable APPsw-HEK cells express Aβ and lead to substantial Aβ
1-42 aggregation. Example 23: Internalization and Target Engagement of Anti-Aβ CPAs in Stably Transfected APP770sw-HEK Cells HEK cells were initially transduced with lentivirus vectors containing APP770sw [K670N-M671L_Swedish] at a MOI (multiplicity of infection) of 10. Following transduction, a dose-dependent puromycin selection process was employed to eliminate non-transduced cells and enrich for those that successfully integrated the lentiviral construct. To achieve stable expression of APPsw, the surviving cells underwent two additional rounds of lentiviral transduction and puromycin selection. Subsequently, the APPsw stable cells were exposed to untagged mzd-2931 and mzd-2931-CMIP4 cell-penetrating agents separately for a 24-hour incubation each at a concentration of 100 µg/ml. Cells were then fixed, permeabilized, and stained using h2931 anti-Aβ antibody along with AF647-conjugated anti-mouse and AF568- conjugated anti-human secondary antibodies. The stained cells were imaged by high content imaging (Operetta system) with a 40x water objective. Quantitative analyses were carried out with Harmony software. Figure 32A is a graph showing the total Aβ foci area per cell demonstrating that incubation with the mzd2931-CMIP4 cell-penetrating agent (e.g., an anti-Aβ cell-penetrating agent) resulted in a lower total Aβ foci area per cell in lenti-APPsw transduced HEK-293 cells as compared to untreated cells and cells treated with untagged mzd2931 antibody. Figure 32B is a graph showing the number of Aβ foci per cell, demonstrating that incubation with the mzd2931- CMIP4 cell-penetrating agent resulted in a lower number of Aβ foci area per cell as compared to untreated cells and cells treated with untagged mzd2931 antibody. Figure 32C is a graph showing cell-penetrating agent internalization per cell, demonstrating that incubation with the mzd2931-CMIP4 cell-penetrating agent resulted in greater cell-penetrating agent internalization compared to untreated cells and cells treated with untagged mzd2931 antibody CMIP4 is linked to the C-terminus of the light chain. Example 24: Internalization and Colocalization of Anti-α-Tubulin CPA in HEK Cells
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Methods: An anti-α-tubulin CPA was generated by expressing a commercially available anti-α-tubulin antibody with CMIP4 (SEQ ID NO: 57) fused to the C-terminus of the light chain via a polypeptide linker having an amino acid sequence of GGGGSGGGGS SEQ ID NO: 258. HEK cells were incubated with 100 ug/ml of either the anti-α-tubulin CPA or the corresponding anti-α-tubulin antibody. Cells were then washed, fixed/permeabilized, and incubation with a commercially available murine anti-α-tubulin antibody to facilitate target protein detection. This incubation was followed by staining with an AF647-conjugated anti-human IgG antibody (for detection of the CPA), and an AF594-conjugated anti-mouse IgG antibody (for detection of the α-tubulin). For all co-localization experiments, nuclear staining using 4',6-Diamidino-2- Phenylindole, Dihydrochloride (DAPI) was performed. Stained cells were imaged by high content imaging (Operetta system) with 40x objective. Figure 33A provides images of cells incubated with either the anti-α-tubulin CPA (top two panels) or the anti-α-tubulin antibody (bottom panels). As seen in Figure 33A, significant intracellular accumulation of the anti-α-tubulin CPA was observed (top right panel), but little to no accumulation of the anti-α-tubulin antibody was observed (bottom right panel). Figure 33Bprovides images of cells incubated with either the anti-α-tubulin CPA (top two panels) or the anti-α-tubulin antibody (bottom panels). Figure 33Bshows labeling for α-tubulin (right panels), labeling for IgG using secondary antibodies (center panels), and composite images of the two (left panels). White arrows highlight exemplary portions of the image having substantial accumulation of the anti-α-tubulin CPA. Cell nuclei, stained by DAPI, are shown as lighter grey oblong structures in the images, and the anti-α-tubulin CPA is shown as dark grey diffuse structures (top center panel). Significant staining for the CPA was observed (via the AF647- conjugated anti-human IgG antibody) (top, center panel), demonstrating internalization of the anti-α-tubulin CPA. Further, the composite image shows significant spatial overlap between the anti-α-tubulin CPA and the α-tubulin staining, including exemplary clusters of CPAs shown by white arrows (top left panel). In contrast, no significant internalization of the anti-α-tubulin antibody was observed (bottom center panel). Thus, Figures 33A and 33B demonstrate internalization of the anti-α-tubulin CPA and colocalization of the anti-α-tubulin CPA with the target protein. Example 25: Internalization and Colocalization of Anti-α-TOMM20 CPA in HEK Cells
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Methods: An anti-TOMM20 CPA was generated by expressing a commercially available anti-TOMM20antibody with CMIP4 (SEQ ID NO: 57) fused to the C-terminus of the light chain via a polypeptide linker having an amino acid sequence of GGGGSGGGGS SEQ ID NO: 258. HEK cells were incubated with 100 ug/ml of either the anti-TOMM20 CPA or the corresponding anti-TOMM20 antibody. Cells were then washed, fixed/permeabilized, and incubation with a commercially available murine anti-TOMM20 antibody to facilitate target protein detection. This incubation was followed by staining with an AF647-conjugated anti-human IgG antibody (for detection of the CPA), and an AF594-conjugated anti-mouse IgG antibody (for detection of the TOMM20). For all co-localization experiments, nuclear staining using DAPI was performed. Stained cells were imaged by high content imaging (Operetta system) with 40x objective. Figure 34A provides images of cells incubated with either the anti-TOMM20 CPA (top two panels) or the anti-TOMM20 antibody (bottom panels). As seen in Figure 34A, significant intracellular accumulation of the anti-TOMM20 CPA was observed (top right panel), but little to no accumulation of the anti-TOMM20 antibody was observed (bottom right panel). Figure 34Bprovides images of cells incubated with either the anti-TOMM20 CPA (top two panels) or the anti-TOMM20 antibody (bottom panels). Figure 34B shows labeling for TOMM20 (right panels), labeling for IgG using secondary antibodies (center panels), and composite images of the two (left panels). White arrows highlight exemplary portions of the image having substantial accumulation of the anti-TOMM20 CPA. Cell nuclei, stained by DAPI, are shown as lighter grey oblong structures in the images, and the anti-TOMM20 CPA is shown as dark grey diffuse structures (top center panel). Significant staining for the CPA was observed (via the AF647- conjugated anti-human IgG antibody) (top, center panel), demonstrating internalization of the anti-TOMM20 CPA. Further, the composite image shows significant spatial overlap between the anti-TOMM20 CPA and the TOMM20 staining, including exemplary clusters of CPAs shown by white arrows (top left panel). In contrast, no significant internalization of the anti-TOMM20 antibody was observed (bottom center panel). Thus, Figures 34A and 34B demonstrate internalization of the anti-TOMM20 CPA and colocalization of the anti-TOMM20 CPA with the target protein. Example 26: Internalization and Colocalization of Anti-Nesprin-1 CPA in HEK Cells
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Methods: An anti-Nesprin-1 CPA was generated by expressing a commercially available anti-Nesprin-1 antibody with CMIP4 (SEQ ID NO: 57) fused to the C-terminus of the light chain via a polypeptide linker having an amino acid sequence of GGGGSGGGGS SEQ ID NO: 258. HEK cells were incubated with 100 ug/ml of either the anti-Nesprin-1 CPA or the corresponding anti-Nesprin-1 antibody. Cells were then washed, fixed/permeabilized, and incubation with a commercially available murine anti-Nesprin-1 antibody to facilitate target protein detection. This incubation was followed by staining with an AF647-conjugated anti-human IgG antibody (for detection of the CPA), and an AF594-conjugated anti-mouse IgG antibody (for detection of the Nesprin-1). For all co-localization experiments, nuclear staining using DAPI was performed. Stained cells were imaged by high content imaging (Operetta system) with 40x objective. Figure 35A provides images of cells incubated with either the anti-Nesprin-1 CPA (top two panels) or the anti-Nesprin-1 antibody (bottom panels). As seen in Figure 35A , significant intracellular accumulation of the anti-Nesprin-1 CPA was observed (top right panel), but little to no accumulation of the anti-Nesprin-1 antibody was observed (bottom right panel). Figure 35B provides images of cells incubated with either the anti-Nesprin-1 CPA (top two panels) or the anti-Nesprin-1 antibody (bottom panels). Figure 35B shows labeling for Nesprin-1 (right panels), labeling for IgG using secondary antibodies (center panels), and composite images of the two (left panels). White arrows highlight exemplary portions of the image having substantial accumulation of the anti-Nesprin-1 CPA. Cell nuclei, stained by DAPI, are shown as lighter grey oblong structures in the images, and the anti-Nesprin-1 CPA is shown as dark grey diffuse structures (top center panel). Significant staining for the CPA was observed (via the AF647- conjugated anti-human IgG antibody) (top, center panel), demonstrating internalization of the anti-Nesprin-1 CPA. Further, the composite image shows significant spatial overlap between the anti-Nesprin-1 CPA and the Nesprin-1 staining, including exemplary clusters of CPAs shown by white arrows (top left panel). In contrast, no significant internalization of the anti-Nesprin-1 antibody was observed (bottom center panel). Thus, Figures 35A and 35B demonstrate internalization of the anti-Nesprin-1 CPA and colocalization of the anti-Nesprin-1 CPA with the target protein. Example 27: Internalization and Colocalization of Anti-β-Actin CPA in HEK Cells Methods: An anti-β-actin CPA was generated by expressing a commercially available anti-β-actin antibody with CMIP4 (SEQ ID NO: 57) fused to the C-terminus of the light chain
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT via a polypeptide linker having an amino acid sequence of GGGGSGGGGS SEQ ID NO: 258. HEK cells were incubated with 100 ug/ml of either the anti-β-actin CPA or the corresponding anti-β-actin antibody. Cells were then washed, fixed/permeabilized, and incubation with a commercially available murine anti-β-actin antibody to facilitate target protein detection. This incubation was followed by staining with an AF647-conjugated anti-human IgG antibody (for detection of the CPA), and an AF594-conjugated anti-mouse IgG antibody (for detection of the β-actin). For all co-localization experiments, nuclear staining using DAPI was performed. Stained cells were imaged by high content imaging (Operetta system) with 40x objective. Figure 36Aprovides images of cells incubated with either the anti-β-actin CPA (top two panels) or the anti-β-actin antibody (bottom panels). As seen in Figure 36A, significant intracellular accumulation of the anti-β-actin CPA was observed (top right panel), but little to no accumulation of the anti-β-actin antibody was observed (bottom right panel). Figure 36Bprovides images of cells incubated with either the anti-β-actin CPA (top two panels) or the anti-β-actin antibody (bottom panels). Figure 36Bshows labeling for β-actin (right panels), labeling for IgG using secondary antibodies (center panels), and composite images of the two (left panels). White arrows highlight exemplary portions of the image having substantial accumulation of the anti-β-actin CPA. Cell nuclei, stained by DAPI, are shown as lighter grey oblong structures in the images, and the anti-β-actin CPA is shown as dark grey diffuse structures (top center panel). Significant staining for the CPA was observed (via the AF647- conjugated anti-human IgG antibody) (top, center panel), demonstrating internalization of the anti-β-actin CPA. Further, the composite image shows significant spatial overlap between the anti-β-actin CPA and the β-actin staining, including exemplary clusters of CPAs shown by white arrows (top left panel). In contrast, no significant internalization of the anti-β-actin antibody was observed (bottom center panel). Thus, Figures 36A and 36B demonstrate internalization of the anti-β-actin CPA and colocalization of the anti-β-actin CPA with the target protein. Example 28: Internalization and Colocalization of Anti-ERO1L CPA in HEK Cells Methods: An anti-ERO1L CPA was generated by expressing a commercially available anti-ERO1L antibody with CMIP4 (SEQ ID NO: 57) fused to the C-terminus of the light chain via a polypeptide linker having an amino acid sequence of GGGGSGGGGS SEQ ID NO: 258.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT HEK cells were incubated with 100 ug/ml of either the anti-ERO1L CPA or the corresponding anti-ERO1L antibody. Cells were then washed, fixed/permeabilized, and incubation with a commercially available rabbit anti-ERO1L antibody to facilitate target protein detection. This incubation was followed by staining with an AF647-conjugated anti-human IgG antibody (for detection of the CPA), and an AF594-conjugated anti-rabbit IgG antibody (for detection of the ERO1L). For all co-localization experiments, nuclear staining using DAPI was performed. Stained cells were imaged by high content imaging (Operetta system) with 40x objective. Figure 37A provides images of cells incubated with either the anti- ERO1L CPA (top two panels) or the anti-ERO1L antibody (bottom panels). As seen in Figure 37A, significant intracellular accumulation of the anti-ERO1L CPA was observed (top right panel), but little to no accumulation of the anti-ERO1L antibody was observed (bottom right panel). Figure 37B provides images of cells incubated with either the anti-ERO1L CPA (top two panels) or the anti- ERO1L antibody (bottom panels). Figure 37B shows labeling for ERO1L (right panels), labeling for IgG using secondary antibodies (center panels), and composite images of the two (left panels). White arrows highlight exemplary portions of the image having substantial accumulation of the anti-ERO1L CPA. Cell nuclei, stained by DAPI, are shown as lighter grey oblong structures in the images, and the anti-ERO1L CPA is shown as dark grey diffuse structures (top center panel). Significant staining for the CPA was observed (via the AF647- conjugated anti-human IgG antibody) (top, center panel), demonstrating internalization of the anti-ERO1L CPA. Further, the composite image shows significant spatial overlap between the anti-ERO1L CPA and the ERO1L staining, including exemplary clusters of CPAs shown by white arrows (top left panel). In contrast, no significant internalization of the anti-ERO1L antibody was observed (bottom center panel). Thus, Figures 37A and 37B demonstrate internalization of the anti-ERO1L CPA and colocalization of the anti-ERO1L CPA with the target protein.
X25 are either absent or each independently a natural or unnatural amino acid residue; or a pharmaceutically acceptable salt thereof. In another aspect, the present disclosure provides a CMIP comprising an amino acid sequence having a formula selected from Formula (IIIA) to Formula (IIIC): I-X2-X3-T-A-L-X7-F-X9-G-X11-A-A-X14-K-X16-E-A-X19-Q-F-L-X23-X24-L (IIIA); I-W-X3-X4-A-L-X7-F-X9-G-X11-X12-X13-A-X15-A-E-A-X19-Q-F-X22-S-X24-L (IIIB); X1-X2-L-T-A-L-K-F-S-X10-K-A-A-A-X15-A-E-A-K-Q-X21-L-S-X24-L (IIIC); wherein X1 is Isoleucine or lysine; X2 is arginine or tryptophan; X3 is arginine, glutamic acid, leucine, or tryptophan; X4 is threonine or tryptophan; X7 is arginine, isoleucine, or lysine; X9 is arginine, histidine, or serine; X10 is glycine or tyrosine; X11 is arginine, isoleucine, lysine, or
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT threonine; X12 is alanine or glutamine; X13 is alanine or serine; X14 is alanine, histidine, leucine, or valine; X15 is arginine, glutamic acid, or lysine; X16 is alanine or lysine; X18 is alanine, methionine, or tryptophan; X19 is arginine or lysine; X21 is arginine or phenylalanine; X22 is arginine, leucine, or tryptophan; X23 is arginine or serine; and X24 is arginine, lysine, or tryptophan; or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP consists essentially of an amino acid sequence selected from Formulas (IA) to (IH), Formulas (IIA) to (IID), and Formulas (IIIA) to (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from Formulas (IA) to (IH), Formulas (IIA) to (IID), and Formulas (IIIA) to (IIIC). Also provided herein are nucleic acids encoding any of the CMIPs described herein. Also provided herein are vectors including any of the nucleic acids described herein. Also provided herein are cells including any of the CMIPs described herein, any of the nucleic acids described herein, and/or any of the vectors described herein. In some embodiments, the cell is a mammalian cell. Also provided herein are methods of producing any of the CMIPs described herein, the methods including culturing a cell including a nucleic acid encoding the CMIP in a liquid culture medium under conditions that allow the cell to produce the CMIP; and harvesting the CMIP from the cell or the liquid culture medium. Also provided herein are pharmaceutical compositions including any of the CMIPs described herein and a pharmaceutically acceptable excipient. Also provided herein are cell-penetrating agents (CPA) including (i) a CMIP and (ii) a payload molecule. In some embodiments, the CMIP is an M-lycotoxin derivative. In some embodiments, the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In some embodiments, the CMIP is covalently linked to the payload molecule. In some embodiments, the CMIP is non-covalently linked to the payload molecule. In some embodiments, the cell-penetrating agent includes a linker connecting the CMIP to the payload molecule. In some embodiments, the linker is covalently linked to both the CMIP and the payload molecule. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker includes a polypeptide. In some embodiments, the linker includes an amino acid sequence selected from Table 4. In some embodiments, the payload molecule is a biomolecule. In some embodiments, the biomolecule including a carbohydrate, a lipid, a nucleic acid, a peptide nucleic acid, a peptidomimetic, a protein, or a peptide. In some embodiments, the payload molecule is a macromolecule. In some embodiments, the payload molecule has a molecular weight of about 5 kD to about 1000 kD. In some embodiments, the payload includes a proteinaceous molecule. In some embodiments, the proteinaceous molecule is about 100 to about 1500 amino acids. In some embodiments, the proteinaceous molecule is an is an antibody, an enzyme, a protein, or a peptide. In some embodiments, the proteinaceous molecule is linked to the CMIP via a polypeptide linker. In some embodiments, the cell-penetrating agent is a fusion protein. In some embodiments, the cell-penetrating agent is encoded by a nucleic acid encoding a CMIP and a payload molecule or a portion thereof. In some embodiments, the payload molecule or the portion thereof is linked to the C-terminus of the CMIP. In some embodiments, the payload molecule or the portion thereof is linked to the N-terminus of the CMIP. In some embodiments, the payload molecule is an antibody or a portion thereof. In some embodiments, the antibody or a portion thereof includes a heavy chain. In some embodiments, the CMIP is covalently linked to a C-terminus of the heavy chain or a functional portion thereof. In some embodiments, the CMIP is covalently linked to a N-terminus of the heavy chain or a functional portion thereof. In some embodiments, the antibody or portion thereof includes a light chain. In some embodiments, the CMIP is covalently linked to a C-terminus of the light chain or a functional portion thereof. In some embodiments, the CMIP is covalently linked to a N- terminus of the light chain or a functional portion thereof. In some embodiments, the heavy chain of the antibody or a portion thereof and the CMIP are expressed as a single polypeptide. In
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT some embodiments, the light chain of the antibody or a portion thereof and the CMIP are expressed as a single polypeptide. In some embodiments, the antibody or fragment thereof specifically binds to a target protein. In some embodiments, the target protein is a cytosolic protein. In some embodiments, the target protein is a membrane-associated protein. In some embodiments, the target protein is an enzyme. In some embodiments, the target protein is a cytoskeletal protein. In some embodiments, the target protein is a beta-actin protein. In some embodiments, the target protein is an alpha tubulin protein. In some embodiments, the target protein is an oxidoreductase. In some embodiments, the target protein is an endoplasmic reticulum oxidoreductin-1-like (EROL1L) protein. In some embodiments, the target protein is a mitochondrial protein. In some embodiments, the target protein is a mitochondrial transport protein. In some embodiments, the target protein is a translocase of outer mitochondrial membrane 20 (TOMM20) protein. In some embodiments, the target protein is a nuclear membrane protein. In some embodiments, the target protein is an outer nuclear membrane protein. In some embodiments, the target protein is a nuclear envelope spectrin repeat protein-1 (Nesprin-1) protein. In some embodiments, the payload molecule is an enzyme or a functional portion thereof. In some embodiments, the enzyme is a kinase. In some embodiments, the enzyme is a serine/threonine kinase. In some embodiments, the serine/threonine kinase is GSK3β. In some embodiments, the enzyme is a protease. In some embodiments, the protease is a cysteine protease. In some embodiments, the cysteine protease is a non-lysosomal cysteine protease. In some embodiments, the non-lysosomal cysteine protease is calpain. In some embodiments, the enzyme is a bioluminescent protein. In some embodiments, the bioluminescent protein is a luciferase. In some embodiments, the CMIP is linked to the C- terminus of the enzyme or a functional portion thereof. In some embodiments, the CMIP is linked to the N-terminus of the enzyme or a functional portion thereof. In some embodiments, the cell-penetrating agent is a fusion protein. In some embodiments, the CMIP and the enzyme are expressed as a single polypeptide. Also provided herein are pharmaceutical compositions including any of the cell- penetrating agents described herein and a pharmaceutically acceptable excipient. Also provided herein are kits including any of the pharmaceutical compositions described herein.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Also provided herein are nucleic acids encoding at least a portion of any of the cell- penetrating agents described herein. In some embodiments, the nucleic acid encodes the CMIP. In some embodiments, the nucleic acid encodes the CMIP and at least a portion of the payload molecule. In some embodiments, the nucleic acid encodes the CMIP, a polypeptide linker, and at least a portion of the payload molecule. In some embodiments, the nucleic acid encodes the CMIP, the polypeptide linker, and the payload molecule. Also provided herein are vectors including any of the nucleic acids described herein. Also provided herein are cells including any of the cell-penetrating agents described herein, any of the nucleic acids described herein, or any of the vectors described herein. In some embodiments, the cell is a mammalian cell. Also provided herein are methods of producing any of the cell-penetrating agents described herein, the method including: culturing a cell including a nucleic acid encoding the CMIP and at least a portion of the payload molecule in a liquid culture medium under conditions that allow the cell to produce a polypeptide including the CMIP and at least a portion of the payload molecule; and harvesting the polypeptide from the cell or the liquid culture medium. Also provided herein are methods of delivering a payload molecule into a cell, including contacting any of the cell-penetrating agents described herein with the cell, thereby resulting in the internalization of the payload molecule into the cell and transfer of the payload molecule to the cytosol. Also provided herein are methods of delivering an antibody into a cell, including contacting any of the cell-penetrating agents described herein with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol. Also provided herein are methods of binding an intracellular target protein in a cell, the method including: (a) contacting any of the cell-penetrating agents described herein with the cell thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; and (b) binding of the cell-penetrating agent to the intracellular target protein. Also provided herein are methods of modulating at least one activity of an intracellular target (a) contacting any of the cell-penetrating agents described herein with the cell thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT penetrating agent to the cytosol; and (b) binding of the cell-penetrating agent to the intracellular target protein, thereby directly or indirectly modulating at least one activity of an intracellular target protein in the cell. In some embodiments, modulating includes reducing an activity of the intracellular target protein. In some embodiments, modulating includes increasing an activity of the intracellular target. In some embodiments, modulating includes altering the site of activity of the intracellular target. In some embodiments, modulating includes altering the turnover of the intracellular target. Also provided herein are methods of inhibiting at least one activity of an intracellular target protein in a cell, the method including: (a) contacting any of the cell-penetrating agents described herein with the cell thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; and (b) binding of the cell- penetrating agent to the intracellular target protein, thereby inhibiting at least one activity of an intracellular target protein in the cell. Also provided herein are methods of delivering an enzyme into a cell, including contacting any of the cell-penetrating agents described herein with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol. Also provided herein are methods of increasing intracellular enzyme activity in a cell, the method including contacting any of the cell-penetrating agents described herein with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the enzyme portion of the cell-penetrating agent is enzymatically active in the cytosol. Also provided herein are methods of restoring intracellular enzyme activity in a cell having reduced activity of an intracellular enzyme, the method including contacting any of the cell-penetrating agents described herein with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the enzyme portion of the cell-penetrating agent performs the same or a similar enzymatic reaction as the intracellular enzyme and the enzyme portion of the cell-penetrating agent performs is active in the cytosol. Also provided herein are methods of delivering a payload protein sequence to the cytosol of a cell, the method including contacting the cell with a polypeptide including a CMIP and the
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT payload protein sequence, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. Also provided herein are methods of delivering a payload protein sequence to the nucleus of a cell, the method including contacting the cell with a polypeptide including a CMIP and the payload protein sequence. In some embodiments, the CMIP is an M-lycotoxin derivative. In some embodiments, the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. In some embodiments, the payload protein sequence has a molecular weight of about 5 kD to about 1000 kD. In some embodiments, the payload protein sequence is about 100 to about 1500 amino acids. Also provided herein are methods of delivering an antigen-binding domain to the cytosol of a cell, the method including contacting the cell with a polypeptide including a CMIP and the antigen-binding domain, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG),
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. Also provided herein are methods of delivering an antibody to the cytosol of a cell, the method including contacting the cell with a polypeptide including a CMIP and the antibody, wherein the CMIP comprises an amino acid sequence selected the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. Also provided herein are methods of inhibiting an intracellular target protein in a cell, the method including: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the cell-penetrating agent includes: (i) an antibody that specifically binds the intracellular target protein; and (ii) a CMIP covalently linked to the antibody; and (b) binding of the cell-penetrating agent to the intracellular target protein. In some embodiments, the target protein is selected from a cytosolic protein, a mitochondrial protein, an endoplasmic reticulum lumen protein, and a nuclear membrane protein. In some embodiments, the target protein is selected from a protease, a cytoskeletal protein, an oxidoreductase, a mitochondrial transport protein, and an outer nuclear membrane protein. In some embodiments, the target protein is selected from beta-actin, alpha tubulin, ERO1L, TOMM20, and Nesprin-1. In some embodiments, the target protein is selected from beta-actin, alpha tubulin, ERO1L, and TOMM20. In some embodiments, the target protein is a beta-actin protein. In some embodiments, the target protein is an alpha tubulin protein. In some embodiments, the target protein is an EROL1L protein. In some embodiments, the target protein is a TOMM20 protein. In some embodiments, the target protein is a Nesprin-1 protein. Also provided herein are method for inhibiting an intracellular TOMM20 protein in a cell including: (a) contacting a cell-penetrating agent with the cell thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer to the cytosol, the cell-
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT penetrating agent including: (i) an antibody that specifically binds to the TOMM20 protein; and (ii) a CMIP derived from M-lycotoxin; and (b) binding the antibody of the cell-penetrating agent to the intracellular TOMM20 protein, thereby inhibiting the intracellular TOMM20 protein. Also provided herein are methods for inhibiting an intracellular beta-actin protein in a cell including: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer to the cytosol, the cell- penetrating agent including: (i) an antibody that specifically binds to the beta-actin protein; and (ii) a CMIP derived from M-lycotoxin; and (b) binding the antibody of the cell-penetrating agent to the intracellular beta-actin protein. Also provided herein are methods for inhibiting polymerization of an intracellular alpha- tubulin protein in a cell including: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer to the cytosol, the cell-penetrating agent including: (i) an antibody that specifically binds to the alpha- tubulin protein; and (ii) a CMIP derived from M-lycotoxin; and (b) binding the antibody of the cell-penetrating agent to the intracellular alpha-tubulin protein. Also provided herein are methods for inhibiting an intracellular endoplasmic reticulum oxidoreductin-1-like protein (ERO1L) protein in a cell including: (a) contacting a cell- penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer to the cytosol, the cell-penetrating agent including: (i) an antibody that specifically binds to the ERO1L protein; and (ii) a CMIP derived from M-lycotoxin; and (b) binding the antibody of the cell-penetrating agent to the intracellular ERO1L protein. Also provided herein are methods for inhibiting an intracellular translocase of outer mitochondrial membrane 20 (TOMM20) protein in a cell including: (a) contacting a cell- penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer to the cytosol, the cell-penetrating agent including: (i) an antibody that specifically binds to the TOMM20 protein; and (ii) a CMIP derived from M-lycotoxin; and (b) binding of the cell-penetrating agent to the intracellular TOMM20 protein. Also provided herein are methods for inhibiting an intracellular Nesprin-1 protein in a cell including: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer to the cytosol, the cell- penetrating agent including: (i) an antibody that specifically binds to the Nesprin-1 protein; and
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT (ii) a CMIP derived from M-lycotoxin; and (b) binding of the cell-penetrating agent to the intracellular Nesprin-1 protein. In some embodiments, the CMIP is covalently linked to the antibody. In some embodiments, the CMIP is covalently linked to the antibody via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. Also provided herein are methods of delivering an enzyme to the cytosol of a cell, the method including contacting the cell with a polypeptide including a CMIP and the enzyme. In some embodiments, the CMIP is an M-lycotoxin derivative. In some embodiments, the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP includes an amino acid sequence selected from Table 3. Also provided herein are methods of increasing intracellular enzymatic activity in a cell, the method including: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the cell-penetrating agent includes: (i) an enzyme capable of catalyzing the intracellular enzymatic activity; and (ii) a CMIP covalently linked to the enzyme; and (b) catalyzing the enzymatic reaction inside the cell using the enzyme portion of the cell- penetrating agent. Also provided herein are methods of restoring intracellular enzymatic activity in a cell having reduced intracellular enzymatic activity, the method including: (a) contacting a cell- penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the cell- penetrating agent includes: (i) an enzyme capable of catalyzing the intracellular enzymatic activity; and (ii) a CMIP covalently linked to the enzyme; and (b) catalyzing the enzymatic reaction inside the cell using the enzyme portion of the cell-penetrating agent. In some embodiments, the enzyme is selected from a kinase, a protease, and a reporter enzyme (or functional fragments thereof). In some embodiments, the enzyme is selected from
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT GSK3β, calpain, and luciferase. In some embodiments, the enzyme is GSK3β. In some embodiments, the enzyme is calpain. In some embodiments, the enzyme is a luciferase. Also provided herein are methods of increasing GSK3β activity in a cell, the method including: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the cell-penetrating agent includes: (i) a GSK3β enzyme; and (ii) a CMIP covalently linked to the GSK3β enzyme; and (b) phosphorylating or dephosphorylating a downstream target of GSK3β using the GSK3β enzyme of the cell-penetrating agent. Also provided herein are methods of restoring intracellular GSK3β activity in a cell having reduced intracellular GSK3β activity, the method including: (a) contacting a cell- penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the cell- penetrating agent includes: (i) a GSK3β enzyme; and (ii) a CMIP covalently linked to the GSK3β enzyme; and (b) phosphorylating or dephosphorylating a downstream target of GSK3β using the GSK3β enzyme of the cell-penetrating agent. Also provided herein are methods of increasing calpain activity in a cell, the method including: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the cell-penetrating agent includes: (i) a calpain enzyme; and (ii) a CMIP covalently linked to the calpain enzyme; and (b) cleaving a downstream target of Calpain using the calpain enzyme of the cell-penetrating agent. Also provided herein are methods of restoring intracellular calpain activity in a cell having reduced intracellular calpain activity, the method including: (a) contacting a cell- penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent to the cytosol; where the cell- penetrating agent includes: (i) a calpain enzyme; and (ii) a CMIP covalently linked to the calpain enzyme; and (b) cleaving a downstream target of calpain using the calpain enzyme of the cell- penetrating agent. Also provided herein are methods of increasing luciferase activity in a cell, the method including: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell-penetrating agent
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT to the cytosol; where the cell-penetrating agent includes: (i) a luciferase enzyme; and (ii) a CMIP covalently linked to the luciferase enzyme; and (b) oxidizing a downstream target of luciferase using the luciferase enzyme of the cell-penetrating agent. In some embodiments, the CMIP is covalently linked to the enzyme. In some embodiments, the CMIP is covalently linked to the enzyme via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the linker is a polypeptide linker. In some embodiments, the polypeptide linker includes an amino acid sequence selected from Table 4. In some embodiments, the CMIP is an M-lycotoxin derivative. In some embodiments, the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP comprises an amino acid sequence selected from Table 3. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is in a subject. Also provided herein are polypeptides including a CMIP and a light chain of an antibody or a fragment thereof, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the light chain of the antibody or a fragment thereof and the CMIP are connected via a polypeptide linker. In some embodiments, the light chain or fragment thereof is connected to the C-terminus of the CMIP. In some embodiments, the light chain or fragment thereof is connected to the N-terminus of the CMIP. Also provided herein are polypeptides including a CMIP and a heavy chain of an antibody or a fragment thereof, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the heavy chain of the antibody or the fragment thereof and the CMIP are connected via a polypeptide linker. In some embodiments, the heavy chain or the fragment thereof is connected to the C-terminus of the CMIP. In some
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT embodiments, the heavy chain or the fragment thereof is connected to the N-terminus of the CMIP. Also provided herein are polypeptides including a CMIP and a payload protein sequence, where the payload protein sequence is about 100 to about 1500 amino acids, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a payload protein sequence, where the payload protein sequence has a molecular weight of about 5 kD to about 1000 kD, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and an antigen-binding domain, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and an antibody, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a protein inhibitor of a kinase, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and at least a portion of a protein binder of a target protein, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the protein binder is an antibody or at least a portion thereof. In some embodiments, at least a portion of the antibody is a heavy chain of an antibody or a fragment thereof. In some embodiments, at least a portion of the antibody is a light chain of an antibody or a fragment thereof. In some embodiments, the target protein is selected from beta-actin, alpha tubulin, ERO1L, TOMM20, and Nesprin-1. In some embodiments, the target protein is selected from beta-actin, alpha tubulin, ERO1L, and Nesprin-1.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Also provided herein are polypeptides including a CMIP and an enzyme, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a kinase, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a GSK3β enzyme, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a protease, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a calpain protease, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a reporter enzyme, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a luciferase enzyme, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a payload protein sequence, where the CMIP is linked to the C-terminus of the payload protein sequence, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). Also provided herein are polypeptides including a CMIP and a payload protein sequence, where the CMIP is linked to the N-terminus of the payload protein sequence, wherein the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP is an M-lycotoxin derivative. In some embodiments, the CMIP comprises an amino acid sequence selected from the CMIP of Formula (IA), (IB),
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists essentially of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP consists of an amino acid sequence selected from the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), (IID), (IIIA), (IIIB), and (IIIC). In some embodiments, the CMIP comprises an amino acid sequence selected from Table 3. In some embodiments, the CMIP consists essentially of an amino acid sequence selected from Table 3. In some embodiments, the CMIP consists of an amino acid sequence selected from Table 3. Also provided herein are compositions including any of the polypeptides described herein. In some embodiments, the composition is a pharmaceutical composition. Also provided herein are kits including any of the compositions described herein or any of the pharmaceutical compositions described herein. Also provided herein are methods including contacting a mammalian cell with any of the cell-penetrating agents described herein, any of the polypeptides described herein, and/or any of the compositions described herein. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1A is a schematic diagram showing a cell-penetrating agent including a cell- penetrating peptide and a payload molecule. FIG. 1B shows internalization and transfer to the cytosol of a cell-penetrating agent. FIG. 2A provides a helical wheel projection of a CMIP of the present disclosure. FIG. 2B provides calculated biophysical calculations for a CMIP of the present disclosure. FIG. 3A provides a schematic of a CPA comprising an antibody connected to a CIM via the light chain of the antibody. FIG. 3B provides a schematic of a CPA comprising an antibody connected to a CIM via the heavy chain of the antibody. FIG. 3C provides a schematic of a CPA comprising a Fab antibody fragment connected to a CIM.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 4A provides the EpiQuest predicted immunogenicity profile for CMIP39 (SEQ ID NO: 97), which has an IU score of 0. FIG. 4B provides the EpiQuest predicted immunogenicity profile for CMIP34 (SEQ ID NO: 92), which has an IU score of 0.4. FIG. 4C provides the EpiQuest predicted immunogenicity profile for CMIP48 (SEQ ID NO: 106), which has an IU score of 10. FIG. 4D provides the EpiQuest predicted immunogenicity profile for CMIP30 (SEQ ID NO: 88), which has an IU score of 1.2. FIG. 5A demonstrates pTDP-43 aggregates formed in cells following transient transfection with a GFP-2a-TDP-43 [mNLS DCS] construct. FIG. 5B shows cell count for HEK cells transiently transfected with either GFP-2a- TDP43 [mNLS DCS] construct or GFP only. FIG. 5C shows staining of pTDP-43 foci in HEK cells transiently transfected with a GFP-2a-TDP43 [mNLS DCS] construct using a panel of anti-TDP-43 antibodies. FIG. 6A shows the percentage of CPA-positive cells following incubation of GFP-2a- TDP43 transfected HEK cells with anti-TDP-43 CPAs of the present disclosure. FIG. 6B shows the number of CPA-positive spots per cell following incubation of GFP- 2a-TDP43 transfected HEK cells with anti-TDP-43 CPAs of the present disclosure. FIG. 7A shows the sum of p-TDP-43 foci pixels per well area following incubation of GFP-2a-TDP43 transfected HEK cells with anti-TDP-43 CPAs of the present disclosure. FIG. 7B shows the mean focus intensity of p-TDP-43 in GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 7C shows consistent cell count per well for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 7D shows the number of p-TDP-43 foci normalized by cell count for GFP-2a- TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 7E shows the mean focus area for p-TDP-43 foci for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 8A shows the sum of p-TDP-43 foci pixels per well area for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 8B shows consistent cell count per well across all GFP-2a-TDP43 transfected HEK cell populations tested (treated and control). FIG. 8C shows the number of p-TDP-43 foci normalized by cell count for GFP-2a- TDP43 transfected HEK cells after incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 8D shows the mean focus area for p-TDP-43 foci for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 9A shows the sum of p-TDP-43 foci pixels per well area, demonstrating a concentration-dependent reduction in total foci area for cells for GFP-2a-TDP43 transfected HEK cells following incubation with different concentrations of anti-TDP-43 CPAs of the present disclosure. FIG. 9B shows consistent cell count per well across all cell populations and concentrations tested (treatment and control). FIG. 9C shows the p-TDP-43 foci number, demonstrating a concentration-dependent increase in foci count for GFP-2a-TDP43 transfected HEK cells following incubation with different concentrations of anti-TDP-43 CPAs of the present disclosure. FIG. 9D shows the p-TDP-43 mean focus size, demonstrating a concentration-dependent decrease in mean focus size for GFP-2a-TDP43 transfected HEK cells following incubation with different concentrations of anti-TDP-43 CPAs of the present disclosure. FIG. 10 shows percentage of cell death with no significant difference in cell death across all populations tested. FIG. 11A shows the sum of p-TDP-43 foci pixels per well area for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 11B shows the mean focus intensity for p-TDP-43 foci for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 11C shows consistent cell count per well across all cell populations tested. FIG. 11D shows the number of p-TDP-43 foci (normalized by cell count) for GFP-2a- TDP43 transfected HEK cells after incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 11E shows the mean focus area for p-TDP-43 foci for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 12A shows the sum of p-TDP-43 foci pixels per well area, demonstrating a reduction in total foci area for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 12B shows consistent cell count per well across all cell populations and concentrations tested. FIG. 12C shows the p-TDP-43 foci number normalized by cell count, demonstrating a concentration-dependent increase in foci count for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 12D shows the p-TDP-43 mean focus area, demonstrating a concentration- dependent decrease in mean focus area for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure. FIG. 13A shows the sum of p-TDP-43 foci pixels per well area for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure in either PBS or acetate buffer. FIG. 13B shows the mean focus intensity for p-TDP-43 foci for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure in either PBS or acetate buffer. FIG. 13C shows consistent cell count per well across all cell populations tested. FIG. 13D shows the number of p-TDP-43 foci normalized by cell count for GFP-2a- TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure in either PBS or acetate buffer. FIG. 13E shows the mean focus area for p-TDP-43 foci for GFP-2a-TDP43 transfected HEK cells following incubation with anti-TDP-43 CPAs of the present disclosure in either PBS or acetate buffer. FIG. 14A shows confocal microscopy images of GFP-2a-TDP43 transfected HEK cells incubated with anti-TDP-43 CPAs of the present disclosure in either PBS or acetate buffer. FIG. 14B shows the percentage of pTDP43 colocalized with an anti-TDP-43 CPA, demonstrating significantly greater co-localization of pTDP43 with the anti-TDP-43 CPA as compared to the control antibody. FIG. 15A shows the percentage of pTDP43 colocalized with the CPAs, demonstrating significantly co-localization of pTDP43 with anti-TDP-43 CPAs of the present disclosure.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 15B shows the average number of CPA-labeled spots per cell for GFP-2a-TDP43 transfected HEK cells incubated with anti-TDP-43 CPAs of the present disclosure. FIG. 15C shows the average spot size for CPA-labeled spots for GFP-2a-TDP43 transfected HEK cells incubated with anti-TDP-43 CPAs of the present disclosure. FIG. 15D shows the average spot intensity for CPA-labeled spots for GFP-2a-TDP43 transfected HEK cells incubated with anti-TDP-43 CPAs of the present disclosure. FIG. 16A shows the percentage of pTDP43 colocalized with humanized anti-TDP-43 CPAs of the present disclosure, demonstrating co-localization of the humanized anti-TDP-43 CPAs with cytosolic p-TDP-43. FIG. 16B shows consistent cell count per well across all cell populations tested. FIG. 16C shows the CPA-labeled spot area per well for GFP-2a-TDP43 transfected HEK cells incubated with anti-TDP-43 CPAs of the present disclosure. FIG. 16D shows the number of CPA-labeled spots per cell for GFP-2a-TDP43 transfected HEK cells incubated with anti-TDP-43 CPAs of the present disclosure, demonstrating significant internalization of the anti-TDP-43 CPAs. FIG. 17A shows the total foci area for U251 glioblastoma cells treated with an anti-TDP- 43 CPA of the present disclosure, demonstrating a reduction in total foci area for the glioblastoma cells treated with the anti-TDP-43 CPA. FIG. 17B shows the mean focus size for U251 glioblastoma cells treated with an anti- TDP-43 CPA of the present disclosure, demonstrating a reduction in mean foci size for the glioblastoma cells treated with the anti-TDP-43 CPA. FIG. 17C shows the total foci count for U251 glioblastoma cells treated an anti-TDP-43 CPA of the present disclosure, demonstrating total foci count was significantly increased in glioblastoma cells treated with the anti-TDP-43 CPA. FIG. 18A shows images of primary rat cortical neuronal cells treated with anti-TDP-43 CPA of the present disclosure, demonstrating internalization and cytosolic accumulation of the anti-TDP43 CPAs. FIG. 18B shows the total foci area per well treated with an anti-TDP-43 CPA of the present disclosure, demonstrating substantial internalization of the anti-TDP-43 CPAs. FIG. 19A shows the total number of CPA-labeled spots per cell for DIV15 rat neuronal cells incubated with CPAs of the present disclosure.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 19B shows the total area of CPA-labeled spots per well for DIV15 rat neuronal cells incubated with anti-TDP-43 CPAs of the present disclosure, demonstrating higher total spot area per well for cells treated with the anti-TDP-43 CPAs compared to those treated with standard antibodies. FIG. 19C shows the mean spot size for CPA-labeled spots in DIV15 rat neuronal cells incubated with anti-TDP-43 CPAs of the present disclosure. FIG. 19D shows spot integrated intensity for CPA-labeled spots in DIV15 rat neuronal cells incubated with anti-TDP-43 CPAs of the present disclosure. FIG. 19E shows the percentage of CPA-labeled spots colocalized with EEA1 (Early Endosome Antigen 1) in DIV15 rat neuronal cells incubated with anti-TDP-43 CPAs of the present disclosure, thereby, demonstrating ~10-20% colocalization of the anti-TDP-43 CPAs with EEA1. FIG. 19F shows consistent cell count per well across all cell populations tested for the DIV15 rat neuronal cells. FIG. 20A shows high content images of untransfected HEK cells (left) and HEK cells transfected with APP (right). FIG. 20B demonstrates a large number of Aβ aggregates in the APP transfected HEK cells and no signal from the untransfected HEK cells. FIG. 21A shows the total labeled APP spot area for APP transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure. FIG. 21B shows the mean area for each labeled APP spot in APP transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure. FIG. 21C shows the number of labeled APP spots normalized by cell count for APP transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure. FIG. 22A shows the total labeled subcellular APP spot area for APP transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure, demonstrating that all cell populations tested had a similar total labeled subcellular APP spot area. FIG. 22B shows the mean area for each labeled Aβ spot for APP transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure. FIG. 22C shows the number of labeled Aβ spots normalized by cell count for APP transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 23A shows the total Aβ foci area for HEK cells transfected with APP770sw [K670N-M671L_Swedish] incubated with an anti-Aβ CPA of the present disclosure, demonstrating that anti-Aβ CPA incubation resulted in a lower total Aβ foci area in a concentration dependent manner compared to the control. FIG. 23B shows the Aβ focus count for APP770sw transfected HEK cells incubated with an anti-Aβ CPA of the present disclosure, demonstrating the anti-Aβ CPA resulted in a lower number of Aβ foci in a concentration dependent manner compared to the control. FIG. 23C shows the mean focus area for Aβ foci in APP770sw transfected HEK cells incubated with an anti-Aβ CPA of the present disclosure. FIG. 24A shows the total Aβ foci area for APP770sw transfected HEK cells incubated with an anti-Aβ CPA of the present disclosure. FIG. 24B shows the Aβ focus count for APP770sw transfected HEK cells incubated with an anti-Aβ CPA of the present disclosure. FIG. 24C shows the mean focus area for Aβ foci for APP770sw transfected HEK cells incubated with an anti-Aβ CPA of the present disclosure. FIG. 25A shows the total Aβ foci area for APP770sw transfected HEK cells incubated with different concentrations of an anti-Aβ CPA of the present disclosure, demonstrating a concentration-dependent effect of the anti-Aβ CPA. FIG. 25B shows showing the Aβ focus count for APP770sw transfected HEK cells incubated with different concentrations of an anti-Aβ CPA of the present disclosure, demonstrating a concentration-dependent effect of the anti-Aβ CPA. FIG. 25C shows the mean focus area for Aβ foci in APP770sw transfected HEK cells incubated with different concentrations of an anti-Aβ CPA of the present disclosure, demonstrating a concentration-dependent effect of the anti-Aβ CPA. FIG. 26A shows the total Aβ foci area per cell in APP770sw transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure comprising different mutations that affect Fcγ or FcRn function. FIG. 26B shows the number of Aβ foci area per cell in APP770sw transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure comprising different mutations that affect Fcγ or FcRn function.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 26C demonstrates internalization of anti-Aβ CPAs of the present disclosure into APP770sw transfected HEK cells regardless of Fcγ or FcRn functionality for the CPA. FIG. 27A shows total Aβ foci area per cell in APP770sw transfected HEK cells incubated with a murinized anti-Aβ CPA of the present disclosure. FIG. 27B shows the number of Aβ foci per cell in APP770sw transfected HEK cells incubated with a murinized anti-Aβ CPA of the present disclosure. FIG. 27C demonstrates internalization of a murinized anti-Aβ CPA of the present disclosure into APP770sw transfected HEK cells. FIG. 28A shows the total Aβ foci area per cell in APP770sw transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure, demonstrating robust activity across the panel of anti-Aβ CPAs. FIG. 28B shows the average number of Aβ foci area per cell in APP770sw transfected HEK cells incubated with anti-Aβ CPAs of the present disclosure, demonstrating robust activity across the panel of anti-Aβ CPAs. FIG. 28C demonstrates significant internalization by APP770sw transfected HEK cells across the panel of anti-Aβ CPAs. FIG. 29A provides an image of untreated HEK-293 cells expressing APPsw following lentiviral transfection (lenti-APPsw transduced HEK-293 cells). FIG. 29B provides an image of HEK-293 cells expressing APPsw following lentiviral transfection and incubation with h2931 antibody. FIG. 29C provides an image of HEK-293 cells expressing APPsw following lentiviral transfection and incubation with an anti-Aβ CPA of the present disclosure. FIG. 30A shows the total Aβ foci area per cell in lenti-APPsw transduced HEK-293 cells incubated with an anti-Aβ CPA of the present disclosure. FIG. 30B provides a graph showing the number of Aβ foci per cell in lenti-APPsw transduced HEK-293 cells incubated with an anti-Aβ CPA of the present disclosure. FIG. 30C demonstrates internalization of an anti-Aβ CPA by lenti-APPsw transduced HEK-293 cells. FIG. 31A provides high content images HEK cells subjected to lentiviral transfection with APP770sw [K670N-M671L_Swedish] at a MOI (multiplicity of infection) of 10 followed by puromycin selection (stable APPsw-HEK cells).
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 31B shows the percentage of stable APPsw-HEK cells having Aβ aggregates compared to untransfected HEK cells (control). FIG. 32A shows the total Aβ foci area per cell for stable APPsw-HEK cells incubated with an anti-Aβ CPA of the present disclosure. FIG. 32B provides a graph showing the number of Aβ foci per cell for stable APPsw- HEK cells incubated with an anti-Aβ CPA of the present disclosure. FIG. 32C demonstrates internalization of the anti-Aβ CPA by for stable APPsw-HEK cells. FIG. 33A provides high content images of HEK cells transfected with α-tubulin and incubated with an anti-α-tubulin CPA (top) and anti-α-tubulin antibody (bottom), demonstrating internalization of the anti-α-tubulin CPA. FIG. 33B provides high content images of HEK cells transfected with α-tubulin and incubated with an anti-α-tubulin CPA (top) and anti-α-tubulin antibody (bottom), demonstrating internalization of the anti-α-tubulin CPA and co-localization of the CPA with α-tubulin. FIG. 34A provides high content images of HEK cells transfected with TOMM20 and incubated with an anti-TOMM20 CPA (top) and anti-TOMM20 antibody (bottom), demonstrating internalization of the anti-TOMM20 CPA. FIG. 34B provides high content images of HEK cells transfected with TOMM20 and incubated with an anti-TOMM20 CPA (top) and anti-TOMM20 antibody (bottom), demonstrating internalization of the anti-TOMM20 CPA and co-localization of the CPA with TOMM20. FIG. 35A provides high content images of HEK cells transfected with Nesprin-1 and incubated with an anti-Nesprin-1 CPA (top) and anti-Nesprin-1 antibody (bottom), demonstrating internalization of the anti-Nesprin-1 CPA. FIG. 35B provides high content images of HEK cells transfected with Nesprin-1 and incubated with an anti-Nesprin-1 CPA (top) and anti-Nesprin-1 antibody (bottom), demonstrating internalization of the anti-Nesprin-1 CPA and co-localization of the CPA with Nesprin-1. FIG. 36A provides high content images of HEK cells transfected with anti-β-actin and incubated with an anti-β-actin CPA (top) and anti-β-actin antibody (bottom), demonstrating internalization of the anti-anti-β-actin CPA.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT FIG. 36B provides high content images of HEK cells transfected with anti-β-actin and incubated with an anti-β-actin CPA (top) and anti-β-actin antibody (bottom), demonstrating internalization of the anti-β-actin CPA and co-localization of the CPA with anti-β-actin. FIG. 37A provides high content images of HEK cells transfected with EROL1L and incubated with an anti- EROL1L CPA (top) and anti- EROL1L antibody (bottom), demonstrating internalization of the anti- EROL1L CPA. FIG. 37B provides high content images of HEK cells transfected with EROL1L and incubated with an anti- EROL1L CPA (top) and anti- EROL1L antibody (bottom), demonstrating internalization of the anti- EROL1L CPA and co-localization of the CPA with EROL1L. DETAILED DESCRIPTION The present disclosure provides systems and methods for the delivery of payload molecules into a cell. Systems and methods of the present disclosure facilitate the intracellular delivery of a variety of payload molecules. The specific examples and embodiments disclosed herein are not limiting and are merely illustrative of the breadth of the approach. In some aspects, the present disclosure provides a cell-penetrating agent (“CPA”) comprising (i) Cell Internalizing Module (“CIM”) and (ii) a payload molecule. The CIM facilitates the internalization of the cell- penetrating agent into the cell. In some embodiments, the CIM further facilitates the transfer of the cell-penetrating agent (or a functional portion thereof) from the internalization vesicle (e.g., endosome) to the cytosol. In some embodiments, the CIM disrupts the membrane of the internalization vesicle, leading to vesicle rupture and facilitating transfer of the cell-penetrating agent out of the vesicle and into the cytosol. FIG. 1A provides a scheme showing an exemplary CPA 101. Exemplary CPA 101 comprises cell internalizing module 102, payload 103, connected via optional linker 104. As discussed in more detail herein, many different variants and combinations of each of these components are within the scope of the present disclosure. FIG. 1B provides a scheme demonstrating internalization of exemplary CPA 101 from FIG. 1A by cell 111. In this exemplary, non-limiting example, one or CPA 101 interacts with cell wall 112 of cell 111 (e.g., via the interaction of CIM 102 with cell wall 112). Following interaction of CPA 101 with cell wall 112, early internalization vesicle 115 (e.g., early
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT endosome) is formed. As internalization progresses, internalization vesicle 116 (e.g., endosome) becomes fully formed, thereby fully encapsulating CPA 101. In the exemplary embodiment in FIG. 1B, CPA 101 further interacts with the membrane of internalization vesicle 116, leading to rupture of the internalization vesicle 116. CPA 101 is thereafter transferred out of ruptured vesicle 117 into cytosol 113, where the payload (or a functional fragment thereof) of the CPA 101 interacts with one or more intracellular target 118. The examples provided in FIG. 1A and FIG. 1B are non-limiting and illustrative in nature only. As set forth below, there are many other cell-penetrating agents within the scope of the present disclosure, including those that operate using different biochemical processes. Definitions The term “antibody” includes intact antibodies and antigen-binding fragments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to the target including separate heavy chains, light chains Fab, Fab', F(ab')2, F(ab)c, Dabs, nanobodies, and Fv. Fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins. The term “antibody” also includes a bispecific or multispecific antibody and/or a humanized antibody. A bispecific or bifunctional or multifunctional antibody is an artificial hybrid antibody having two or more different heavy/light chain pairs and two or more different binding sites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). As used herein, the term “biomolecule” means an organic molecule. A biomolecule can be produced by a living organism (e.g., including production using a mammalian host cell) or can be produced using chemical synthesis in a laboratory. “Biomolecule”, as used in the present disclosure includes synthetic derivatives of peptides, proteins, nucleic acids, and carbohydrates. Further, biomolecules includes both macromolecules (e.g., polymers and oligomers) as well as small molecules (e.g., monomers, dimers, trimers, etc.). Additional non-limiting aspects and examples of biomolecules are described herein. The term “biological sample” refers to a sample of biological material within or obtainable from a biological source, for example a human or mammalian subject. Such samples
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT can be organs, organelles, tissues, sections of tissues, bodily fluids, peripheral blood, blood plasma, blood serum, cells, molecules such as proteins and peptides, and any parts or combinations derived therefrom. The term biological sample can also encompass any material derived by processing the sample. Derived material can include cells or their progeny. Processing of the biological sample may involve one or more of filtration, distillation, extraction, concentration, fixation, inactivation of interfering components, and the like. As used herein a “Cell-Penetrating Agent (CPA)” refers to an agent (e.g., a molecule and/or molecular complex) capable of entering a cell (e.g., a mammalian cell in vitro and/or in vivo). In some embodiments, the CPA enters the cell and is transferred to the cytosol following internalization by the cell. In some embodiments, the CPA comprises a Cell Internalizing Module (CIM) that facilitates the internalization of the CPA by the cell. In some cases, the CPA comprises a CIM that facilitates the internalization of the CPA, and the transfer of the CPA or a functional portion thereof to the cytosol. In some embodiments, the CPA further comprises a payload that is linked (e.g., covalently or non-covalently) to the CIM. In some embodiments, the CPA comprises a CIM covalently linked to the payload (e.g., via a linker between the CIM and the payload). In other embodiments, the CPA comprises a CIM non-covalently linked to the payload (e.g., via a streptavidin-biotin interaction), such that the CIM remains linked to the payload in relevant conditions (e.g., blood plasma). In some embodiments, the CPA further comprises a linker connecting the CIM to the payload. In various cases, linkers can be cleavable or non-cleavable and/or can connect the CIM to the payload covalently or non-covalently. In some embodiments, the CPA comprising the payload has enhanced cell penetration compared to a reference payload molecule that is not part of a CPA. In some embodiments, the CPA comprises two or more payloads and/or two or more CIMs (e.g., the CPA comprising a dendrimer linked to a plurality of CIMs and/or payloads). Non-limiting features and examples of cell-penetrating agents are described herein. As used herein a “Cell Internalizing Module (CIM)” refers to a portion of the CPA that facilitates the internalization of the CPA (and by extension, the payload) by the cell. In various embodiments, the CIM may utilize one or more cellular internalization processes, including both active and passive cellular internalization, to effect internalization. Exemplary processes include, without limitation, endocytosis (e.g., Receptor Mediated Endocytosis (RME), phagocytosis, pinocytosis), and membrane translocation (e.g., direct penetration and/or energy-independent
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT internalization). In various non-limiting embodiments, the CIM comprises, for example, a Cell- Membrane Internalizing Peptide (CMIP), a small molecule ligand (e.g., vitamin, fatty acids, integrin binding ligand, etc.), a portion of an antibody (e.g., an scFv portion that binds an internalizing cell surface target), or a decoy receptor ligand (e.g., a cytokine or derivative thereof). In some embodiments, the CIM comprises a Cell-Membrane Internalizing Peptide (CMIP). Non-limiting features and examples of cell internalizing modules are described herein. As used herein a “Cell Membrane Internalizing Peptide (CMIP)”, is a sequence of three or more naturally occurring or non-naturally-occurring amino acids that, when covalently or non- covalently linked to a payload molecule, results in the internalization into a mammalian cell of, at a minimum, a payload molecule or a functional fragment of a payload molecule. In various embodiments, the CMIP may utilize one or more cellular internalization processes, including both active and passive cellular internalization, to effect internalization. Exemplary processes include, without limitation, endocytosis and membrane translocation. In some embodiments, the CMIP interacts with the cell membrane, and/or a cell surface antigen, leading to internalization of the CPA or a portion thereof (e.g., a portion comprising the payload or a functional portion thereof) via an endocytic vesicle. In some embodiments, the CMIP further facilitates the transfer of the CPA or portion thereof out of the endocytic vesicle and into the cytoplasm of the cell. In some cases, the CMIP further interacts with the membrane of the endocytic vesicle, leading to vesicle rupture and endosomal escape of the CPA or portion thereof into the cytosol. Thus, in some embodiments, the CMIP facilitates both the internalization of the payload or functional portion thereof into the cell, as well as transfer of the payload or functional portion thereof into the cytosol. Non-limiting examples of CMIPs include, for example, cationic peptides (including, for example, M-lycotoxin, TAT peptides, pentetratin, and polyarginine peptides, as well as derivatives thereof), amphipathic peptides (including, for example, MPG peptides, Pep-1 peptides, transportan peptides, as well as derivative thereof), and proline-rich peptides (including, for example, Bac7 peptides and derivatives thereof). In some embodiments, CMIPs include cell-penetrating peptides and derivatives thereof, including, for example, those described in I. Ruseska and A. Zimmer, Internalization mechanisms of cell-penetrating peptides, Beilstein J. Nanotechol. 202; 11:101-123 (2020). In some embodiments, a CMIP comprises an amino acid sequence selected from Formulas (IA), (IB), (IC), (ID), (IE), (IF), (IG), and (IH). Non-limiting features and examples of CMIPs are described herein.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT The term “epitope” refers to a site on an antigen to which an antibody binds. An epitope can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of one or more proteins. Epitopes formed from contiguous amino acids (also known as linear epitopes) are typically retained on exposure to denaturing solvents whereas epitopes formed by tertiary folding (also known as conformational epitopes) are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, and more usually, at least 5 or 8-10 amino acids in a unique spatial conformation. Methods of determining spatial conformation of epitopes include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996). The term “humanized immunoglobulin” or “humanized antibody” refers to an immunoglobulin or antibody that includes at least one humanized immunoglobulin or antibody chain (i.e., at least one humanized light or heavy chain). The term “humanized immunoglobulin chain” or “humanized antibody chain” (i.e., a “humanized immunoglobulin light chain” or “humanized immunoglobulin heavy chain”) refers to an immunoglobulin or antibody chain (i.e., a light or heavy chain, respectively) having a variable region that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) (e.g., at least one CDR, preferably two CDRs, more preferably three CDRs) substantially from a non-human immunoglobulin or antibody, and further includes constant regions (e.g., at least one constant region or portion thereof, in the case of a light chain, and preferably three constant regions in the case of a heavy chain). The term “humanized variable region” (e.g., “humanized light chain variable region” or “humanized heavy chain variable region”) refers to a variable region that includes a variable framework region substantially from a human immunoglobulin or antibody and complementarity determining regions (CDRs) substantially from a non-human immunoglobulin or antibody. Competition between antibodies is determined by an assay in which an antibody under test inhibits specific binding of a reference antibody to a common antigen (see, e.g., Junghans et al., Cancer Res. 50: 1495, 1990). A test antibody competes with a reference antibody if an excess of a test antibody (e.g., at least 2x, 5x, l0x, 20x, or l00x) inhibits binding of the reference antibody by at least 50% as measured in a competitive binding assay. Some test antibodies inhibit binding of the reference antibody by at least 75%, 90%, or 99%. Antibodies identified by
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibody and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. As used herein a “linker” refers to a chemical moiety that connects two portions of a cell- penetrating agent (e.g., a linker that connects a cell internalizing moiety to a payload). In various embodiments, a linker can connect two portions of the cell-penetrating agent covalently or non- covalently, and the linker can be cleavable or non-cleavable. For example, in some embodiments, a covalent linker can be cleaved during internalization by the cell, thereby separating the CIM from the payload or functional fragment thereof. In other embodiments, the linker covalently connecting the CIM to the payload is not cleavable and the CIM remains covalently linked to the payload (or functional fragment thereof) throughout internalization. In some exemplary embodiments, a linker can be a peptide of about 3 amino acids to about 25 amino acids (e.g., about 3 amino acids to about 20, or about 3 amino acids to about 12 amino acids). In other examples, a linker can be a bond (e.g., an amide bond, an ester bond, an ether bond, and a disulfide bond). In some embodiments, the linker can comprise an organic polymer (e.g., a polyethylene glycol) and/or an organic chain (e.g., a hydrocarbon chain). In some examples, a linker can comprise a pair of affinity domains (e.g., a first domain of the pair of affinity domains can be interleukin-15 and a second domain of the pair of affinity domains can be a sushi domain of interleukin-15 receptor alpha). Non-limiting exemplary methods for covalently and non-covalently linking a payload molecule to a cell-penetrating peptide (e.g., any of the exemplary cell-penetrating peptides described herein) are described herein. Additional methods for covalently and non-covalently linking a payload molecule to a cell-penetrating peptide are also known in the art. As used herein a “payload” or a “payload molecule” as used herein refers to a molecule and/or molecular complex that is internalized when part of a CPA. In some embodiment, the payload performs one or more functions when in the cell, including, for example, modulating (e.g., increasing or decreasing) at least one activity of an intracellular target protein, catalyzing a chemical reaction, and/or facilitating detection from outside the cell (e.g., via fluorescence). In some embodiments, the payload is a molecule that has one or more activities that are desired in a cell. Some payloads are capable of being internalized into a cell upon covalent or non-covalent linkage to a cell internalizing module (e.g., any of the exemplary CMIPs described herein). In
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT some embodiments, the payload is a molecule has limited capacity for internalization, unless it is linked to a cell internalizing module. In some embodiments, the payload is a molecule that has its own capacity for internalization, but is more readily internalized when it is linked to a cell internalizing module. Payload molecules include but are not limited to biologic molecules (e.g., proteins, peptides, nucleic acids, carbohydrates), macromolecules (e.g., polymers, oligomers), proteinaceous molecules (e.g., polypeptides, enzymes, antibodies and antigen-binding fragments thereof), inorganic molecules, organo-metallic complexes (e.g., complexed metals, including, for example, chelates of gallium and/or gadolinium), or any combination thereof. In some embodiments, the payload molecule comprises a biomolecule (including those produced by a living organism and those produced synthetically) and derivatives thereof, including, but not limited to a polypeptide (e.g., a single chain or multi-chain polypeptide), a nucleic acid (e.g., a single-stranded or double-stranded nucleic acid), a lipid (e.g., cholesterol derivatives), a carbohydrate, an inorganic molecule, or any combination thereof. Additional non-limiting aspects and examples of payloads are described herein. The term “patient” or “subject” includes human and other mammalian subjects (e.g., human) that receive either prophylactic or therapeutic treatment. An individual is at increased risk of a disease if the subject has at least one known risk factor (e.g., genetic, biochemical, family history, and situational exposure) placing individuals with that risk factor at a statistically significant greater risk of developing the disease than individuals without the risk factor. The term “pharmaceutically acceptable” means that the carrier, diluent, excipient, or auxiliary is compatible with the other ingredients of the formulation and not substantially deleterious to the recipient thereof. As used herein a “M-lycotoxin derivative” is a peptide comprising three or more amino acids (naturally-occurring or non-naturally-occurring) that was designed based on a starting M- lycotoxin peptide. In some embodiments, the “M-lycotoxin derivative” is a polypeptide having, e.g., between 80% and 99% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to wild-type M-lycotoxin. Additional non-limiting aspects and examples of M-lycotoxin derivatives are described herein.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT As used herein a “spacer” or a “spacer region” refers to an amino acid that does not have catalytic or therapeutic activity in a mammalian cell. For example, a spacer can be a peptide of 1 amino acid to about 10 amino acids (e.g., 1 to about 8 amino acids, 1 to about 6 amino acids, or 1 to about 4 amino acids). In some examples, one or more spacer regions are included in the cell internalizing module. For example, a spacer region can separate amino acids within a cell internalizing module, e.g., a spacer can be disposed after the first amino acid of a CIM. In some examples, a spacer can be disposed before the final amino acid of a CIM. In some examples, a CIM can have two or more spacer regions (e.g., two spacer regions, three spacer regions, four spacer regions, five spacer regions or more). In some examples, a spacer region is a single glycine residue. In some examples, a spacer region is a pair of glycine residues. In some examples, a spacer region is three glycine residues. In some examples, a spacer region is four glycine residues. In some examples, a spacer region is four glycine residues followed by a serine residue. As used herein, the term “macromolecule” refers to a molecule having either a molecular weight of at least 5 kDa and/or a hydrodynamic radius of at least 1.0 nm. As used herein, the term “macromolecules” includes biomolecules, organic polymers, and organometallic complexes. Unless otherwise apparent from the context, the term “about” encompasses insubstantial variations, such as values within a standard margin of error of measurement (e.g., SEM) of a stated value. For purposes of classifying amino acids substitutions as conservative or nonconservative, amino acids are grouped as follows: Group I (hydrophobic side chains): Met, Ala, Val, Leu, Ile; Group II (neutral hydrophilic side chains): Cys, Ser, Thr; Group III (acidic side chains): Asp, Glu; Group IV (basic side chains): Asn, Gln, His, Lys, Arg; Group V (residues influencing chain orientation): Gly, Pro; and Group VI (aromatic side chains): Trp, Tyr, Phe. Conservative substitutions involve substitutions between amino acids in the same class. Non-conservative substitutions constitute exchanging a member of one of these classes for a member of another. Percentage sequence identities are determined with antibody sequences maximally aligned by the Kabat numbering convention. After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage. I. Cell-Penetrating Agents In some aspects, the present disclosure provides a cell-penetrating agent (CPA) comprising: (i) a Cell Internalizing Module (CIM); and (ii) a payload. In some embodiments, the CIM is covalently linked to the payload. In some embodiments, the CIM is non-covalently linked to the payload. In some embodiments, the CIM comprises a Cell Membrane Internalizing Peptide (CMIP). The CPA is a molecule and/or molecular complex that capable of entering a cell (e.g., a mammalian cell in vitro and/or in vivo) via interaction of the CIM with the cell surface. In some embodiments, the CPA enters the cell (or at least a functional portion thereof enters the cell) and is transferred (or at least a functional portion thereof is transferred) to the cytosol following internalization by the cell. In this way, the CPA facilitates transfer of the payload (or at least a functional portion of the payload) into the cell, and in some instances, into the cytosol. In some embodiments, the CPA may be internalized and/or transferred to the cytosol in an intact form. In some embodiments, the during internalization and/or cytosolic transfer, the CPA may be partially degraded and/or modified (e.g., partial hydrolysis, oxidation, reduction, etc.) leading to cytosolic transfer of a functional fragment of the CPA (e.g., a fragment comprising a substantially intact payload and/or a fragment comprising a functional fragment of the payload). Following cellular internalization of the CPA (or functional fragment thereof), the payload and/or functional fragment thereof, performs at least one function in the cell (e.g., modulating activity of an enzyme, binding to a target biomolecule, facilitating detection). In this manner, the CPA facilitates cellular internalization of the payload (which, in some embodiments, might otherwise have limited ability to enter the cell). Throughout the present disclosure, references to the CPA and/or payload will be made in the context of cell internalization, transfer, and/or intracellular function, but one of ordinary skill will understand such references to include an intact CPA and/or payload, a chemically-modified (e.g., oxidized, reduced) CPA, a precursor payload, and/or payload (e.g., chemically-modified,
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT functional CPA, payload, or functional fragments thereof), as well as partially degraded functional fragments thereof (intact CPA, CPA fragment, intact payload, and/or payload fragment, etc.). Thus, references to internalization of, cytosolic transfer of, and target engagement by either the CPA or the payload will include references to intact CPA and/or payload, chemically-modified CPA and/or payload, and/or partially degraded functional fragments thereof. For example, methods of the present disclosure may refer to internalization of and/or transfer to the cytosol of a payload, but it will be understood that such a reference encompasses the transfer of an intact CPA and/or a functional fragment of the CPA comprising the payload or a functional portion thereof, as well as chemically-modified derivatives thereof. Cell-penetrating agents of the present disclosure may utilize one or more of several biochemical processes to achieve internalization of the payload. In various embodiments, the CPAs of the present disclosure may utilize passive internalization, active internalization, or a combination thereof. In some embodiments, the CPA utilizes active internalization (e.g., endocytosis) to achieve intracellular delivery of the payload. In various embodiments, the CPA is internalized into the cell via endocytosis. Following internalization, the CPA achieves endosomal escape. In such embodiments, the CPA is thereby transferred to the cytosol, where the payload can perform one or more desired functions. In some embodiments, endosomal escape of the CPA is achieved by interaction of the CIM with the membrane of the endosome, thereby leading to disruption of the endosomal membrane. The CIM may utilize one or more methods to achieve endosomal escape. For example, the CIM may comprise a cationic portion (e.g., positively charged amino acids, a cationic polymer and/or oligomer, cationic lipid). In some embodiments, the cationic portion can interact with the negatively charged phospholipids that comprise the endosomal membrane, thereby disrupting the endosomal membrane, and achieving endosomal escape of the CPA. CPAs of the present disclosure may also comprise a CIM having an amphiphilic portion (e.g., an amphiphilic peptide). In some embodiments, the amphiphilic portion can interact with the endosomal membrane via hydrophobic interactions with the membrane lipids, thereby disrupting the endosomal membrane, and achieving endosomal escape of the CPA. Following endosomal escape of the payload, the payload is then transferred to the cytosol, where it can perform at least one desired function. Further examples of internalization mechanisms that can be utilized by CPAs are described in detail herein.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Various aspects of cell-penetrating agents of the present disclosure are described below, including examples of Cell Internalizing Modules (CIMs), payloads, and/or optional linkers. One of ordinary skill will understand how to combine the features of various CIMs, payloads, and optional linkers and spacer regions disclosed herein. Such examples are merely illustrative of the scope of the present disclosure and are non-limiting. a. Cell Internalizing Modules (CIMs) and Cell Membrane Internalizing Peptides (CMIPs) Cell Internalizing Modules As used herein a “cell internalizing module” or “CIM” is a composition that, when covalently or non-covalently linked to a payload molecule, results in the internalization into a cell of, at a minimum, the payload or a functional fragment of the payload. Non-limiting features and examples of cell internalizing modules (CIMs) are described herein. In some embodiments, a CIM is a peptide (e.g., an amino acid sequence). In such examples, the CIM can also be referred to as a cell membrane internalizing peptide or “CMIP” as further defined herein. Exemplary cell membrane internalizing peptides (CMIPs) include naturally-occurring peptides, and derivatives thereof, as well as, synthetic peptides. Non- limiting examples of CMIPs include M-lycotoxin and derivatives thereof, TAT and derivatives thereof, PEPTH, polyarginine sequences, Penetratin, DPT-C9h, DPT-C9, Transportan, Xentry, Pep-1, Pep-7, Aurein 1.2, MTS, GFWFG, DPV1047, MPG, pVEC, ARF(1_22), BPrPr, MAP, p28, VT5, Bac7, C105Y, PFVYLI, and BR2. In some embodiments, a CIM is a non-peptide moiety (e.g., a ligand) that is internalized by a cell (e.g., a mammalian cell). In such examples, a ligand can induce receptor-mediated internalization of a payload molecule (as defined herein). Generally, ligand internalization is a receptor-mediated endocytic process in which cells intake extracellular molecules (including therapeutics) if the ligand binds to its cognate receptor protein on the cell’s surface. Receptor- mediated internalization also includes transcytosis. CIMs can effectuate the internalization of payload molecules into a mammalian cell. In general, the process of cellular internalization is broadly classified as endocytosis. Typically, endocytosis pathways can be subdivided into two broader categories, phagocytosis and
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT pinocytosis. During pinocytosis the plasma membrane absorbs solutes, while during phagocytosis the cell internalizes much larger vesicles. Pinocytosis is generally further subdivided into macropinocytosis, clathrin-dependent endocytosis (e.g., receptor-mediated endocytosis), caveolin-dependent endocytosis, and clathrin/caveolin-independent endocytosis. (See e.g., Marsh, M. Endocytosis, Oxford University Press (2001); Doherty, G.J., and McMahon, H.T., Mechanisms of Endocytosis, Annu. Rev. Biochem., 78:31.1-31.46 (2009); and Xu, Y., et al., Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom, Front Biosci, 22: 1439-1457 (2017), each of which is incorporated herein by reference in their entireties). Ligand-mediated endocytosis is the mechanism by which cells internalize specific macromolecules. In some examples, the cell membrane (e.g., plasma membrane) includes clathrin pits which protrude from the cell membrane to form small vesicles called clathrin-coated vesicles. These clathrin-coated vesicles contain the receptors and the bound macromolecules, i.e., ligands. Then the clathrin-coated vesicles fuse with the early endosomes (vesicles consisting of tubular extensions residing at the periphery of the cell). The endosomes have an acidic environment (pH 6.0-6.2) that facilitates the dissociation of receptors from the ligands. Then, the ingested content is sorted out for either recycling to the plasma membrane or transport to lysosomes for degradation. Peptide-based CIMs (e.g., CMIPs) internalize payload molecules through a variety of mechanisms. In general, CMIPs have been shown to use either endocytosis (e.g., energy- dependent internalization) as described above or direct penetration (e.g., translocation) (energy- independent internalization) as the two major internalization mechanisms. For direct penetration, various mechanisms have been described including the carpet-like model (membrane destabilization) and the pore formation model (barrel-stave). Positively-charged CMIPs can interact with negatively-charged membrane components such as the phospholipid bilayer, followed by destabilization of the membrane, and crossing of the CMIP and payload molecule through the lipid bilayer. Studies have shown that several CMIPs are able to induce and shift between different uptake mechanisms depending on their concentration, cargo, and/or the cell line used (See e.g., Ruseska, I. and Zimmer, A., Internalization mechanisms of cell- penetrating peptides, Beilstein J Nanotechnol, 11: 101-123, (2020)).
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Once internalized into a cell, payload molecules typically need to escape the endosomal pathway. In general, the endocytic pathway of mammalian cells consists of distinct membrane compartments, which internalize molecules (e.g., cell-penetrating agents) from the plasma membrane and recycle membrane-bound receptors back to the surface or sort internalized molecules to various degradation pathways. The main components of the endocytic pathway include early endosomes which are the first compartment of the endocytic pathway. Early endosomes are usually located in the periphery of the cell, and receive most types of vesicles coming from the cell surface. They have a characteristic tubulo-vesicular structure and a mildly acidic pH. Early endosomes are principally sorting organelles where many endocytosed ligands dissociate from their receptors in the acid pH of the compartment and are recycled to the cell surface. Early endosomes also sort into transcytotic pathway to later compartments (e.g., late endosomes or lysosomes) via transvesicular compartments. Late endosomes generally receive endocytosed material en route to lysosomes, usually from early endosomes in the endocytic pathway, from trans-Golgi network (TGN) in the biosynthetic pathway, and from phagosomes in the phagocytic pathway. They are acidic (approx. pH 5.5) and are generally thought to mediate a final sorting prior the delivery of material to lysosomes. Lysosomes are the last compartment of the endocytic pathway. Lysosomes break down cellular waste products, fats, carbohydrates, proteins, and other macromolecules into simple compounds which are returned to the cytoplasm as new cell-building materials. Lysosomes include many different types of hydrolytic enzymes which function in an acidic environment (e.g., pH of approximately 4.8). In some embodiments, internalized molecules, including CPAs, need to escape the endosomal pathway to deliver their payload molecule to the target. Escaping the endosomal pathway has proven to be challenging, however, various strategies have been described. In general, molecular ferries, leakage-inducing molecules, and physicochemical techniques are most commonly employed to escape the endosomal pathway. For example, molecular ferries are generally molecules that are either part of a therapeutic or bind to the therapeutic and are able to translocate through membranes (e.g., endosomal membranes) including a payload molecule. Typical examples are viral membrane fusion proteins and other cell-penetrating peptides (CMIPs described herein). Alternatively, leakage-inducing molecules include compounds that destabilize the endosomal membrane, such as for example, pore-forming substances, compounds with
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT solubilizing effects, and molecules that affect the endosomal pH. Another strategy includes techniques that work through a direct physical effect, e.g., endosomal membrane disruption by light-induced effects (See e.g., Varkouhi, A.K., et al., Endosomal escape pathways for delivery of biologicals, Journal of Controlled Release, 10220-228 (2011) and Fuchs, H., et al., Driving through Membranes: Molecular Cunning to Enforce the Endosomal Escape of Antibody- Targeted Anti-Tumor Toxins, Antibodies, 2, 209-235 (2013), each of which are incorporated herein by reference in their entireties). Cell Internalizing Membrane Peptides Cell membrane internalizing peptides (“CMIPs”) are peptides that are capable of internalizing a payload molecule (as defined herein) across a cell membrane. In some embodiments, CMIPs comprise short amino acid sequences (e.g., about 5 amino acids to about 35 amino acids, about 5 amino acids to about 30 amino acids, about 5 amino acids to about 25 amino acids, about 5 amino acids to about 20 amino acids, about 5 amino acids to about 15 amino acids, or about 5 amino acids to about 10 amino acids). Typically, CMIPs are water- soluble, cationic, and/or amphipathic peptides which when coupled to payloads (e.g., therapeutics or vectors) facilitate intracellular delivery of such payloads. CMIPs can be cationic CMIPs which typically include short amino acid sequences including amino acid residues such as arginine, lysine, and/or histidine. Arginine, lysine, and/or histidine give the cationic charge to the CMIP and interact with anionic motifs on the plasma membrane. Cationic CMIPs are able to deliver payload molecules across a cell membrane by receptor-independent mechanisms and without significant membrane damage. Alternatively, CMIPs can be amphipathic peptides (e.g., Pep-1, MPG, etc.), which include lipophilic and hydrophilic tails capable for direct CMIP translocation mechanism across the plasma membrane via receptor-independent mechanisms and without significant membrane damage. In some examples, a CMIP can also be referred to as a cell-penetrating peptide. Exemplary cell-penetrating peptides are described in Guidotti, G, et al. Cell-Penetrating Peptides: From Basic Research to Clinics, Trends Pharmacol Sci., 38(4): 406-424 (2017) and Aroui, S. and Kenani, A., Cell-Penetrating Peptides: A Challenge for Drug Delivery, Cheminformatics and its Applications (2020), each of which are incorporated herein by reference in their entireties.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT Non-limiting examples of CMIPs include M-lycotoxin and derivatives thereof, TAT and derivatives thereof, PEPTH, polyarginine sequences, Penetratin, DPT-C9h, DPT-C9, Transportan, Xentry, Pep-1, Pep-7, Aurein 1.2, MTS, GFWFG, DPV1047, MPG, pVEC, ARF(1_22), BPrPr, MAP, p28, VT5, Bac7, C105Y, PFVYLI, and BR2 (See e.g., Sakamoto K, et al. (2020), Optimizing charge switching in membrane lytic peptides for endosomal release of biomarcomolecules, Angew. Chem. Int. Ed. 59: 2-11; Takeuchi, T. and Futaki, S., Current Understanding of Direct Translocation of Arginine-Rich Cell-Penetrating Peptides and Its Internalization Mechanisms, Chem Pharm Bull 64(10):1431-1437 (2016); Arrouss I. et al. (2013), Specific Targeting of Caspase-9/PP2A Interaction as Potential New Anti-Cancer Therapy, PLoS ONE 8(4):e60816; Patel et al. (2019), Cell-penetrating peptide sequence and modification dependent uptake and subcellular distribution of green florescent protein in different cell lines, Scientific Reports 9:6298; Gaston J. et al. (2019), Intracellular delivery of therapeutic antibodies into specific cells using antibody-peptide fusions, Scientific Reports 9:18688; De Coupade, C. et al. (2005), Novel human-derived cell-penetrating peptides for specific subcellular delivery of therapeutic biomolecules, Biochem. J. 390:407–418; Morris, M.C. et al. (2008), Cell-penetrating peptides: from molecular mechanisms to therapeutics. Biol. Cell 100:201–217; Sadler, K. et al. (2002), Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7, Biochemistry 41:14150–14157, each of which is incorporated herein by reference in their entireties). Additionally, the CMIP described herein can comprise additional moieties such as additional residues (e.g., spacer regions as defined herein) or a cyclized structure. (See e.g., Reichart et al., Cyclization of a cell-penetrating peptide via click-chemistry increases proteolytic resistance and improves drug delivery, J. Pept. Sci. 22(6):421-6 (2016)). Lycotoxins were first identified from the venom of the wolf spider Lycosa carolinensis based their abilities to reduce ion (e.g., calcium) and voltage gradients across membranes. As a result, M-lycotoxin forms pores that permeabilize the cell membrane which can cause hemolysis and dissipates voltage gradients across muscle membrane. M-lycotoxins also potently inhibit the growth of bacteria, yeast, and parasitic Leishmania. M-lycotoxin may function both in the prey capture strategy as well as protection from infectious organisms arising from prey ingestion. (See e.g., Yan, L. and Adams, M.E., Lycotoxins, Antimicrobial Peptides from Venom of the Wolf Spider Lycosa carolinensis, J. Biol. Chem. 273(4):2059-2066 (1998) and UniProt Entry P61507
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT LYT1_HOGCA). Subsequently, various mutations of M-lycotoxin have been generated including M-lycotoxin L17E. The L17E mutation was first described in Akishiba, M., et al. Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide, Nature Chemistry 9:751- 761 (2017). Akishiba et al. demonstrated an approach to deliver proteins, including antibodies, into cells by using endosomolytic peptides (i.e., M-lycotoxin derivatives) derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. In particular, M-lycotoxin derivatives were generated by introducing one or two glutamic acid residues into the hydrophobic face. Akishiba et al. demonstrated that substitution of leucine for glutamic acid (M- lycotoxin L17E) enabled cytosolic liberation of antibodies from endosomes. Akishiba et al. demonstrated the predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively-charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. Table 1 below shows a non-limiting, exemplary list of CMIPs including M-lycotoxin, TAT, PEPTH, polyarginine sequences, Pep1, Pep-7, and derivatives thereof and others, including derivatives thereof, that can be included in any of the CIMs. Table 1 SEQ ID NO: 1 WT M-Lyco IWLTALKFLGKHAAKHLAKQQLSKL
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: 38 MPG GALFLGFLGAAGSTMGAWSQPKKKRKV SEQ ID NO: 39 pVEC LLIILRRRIRKQAHAHSK
In some embodiments, the CIM comprises a spacer. In some embodiments, the spacer is a spacer in Table 2. Table 2 SEQ ID NO: 50 G
may at various positions along the sequence. However, one of ordinary skill will understand these specified amino acid residues to occupy the corresponding position (e.g., X1, X2, etc.) along the amino acid sequence, even if that variable is not recited in the formula. Certain embodiments herein require a particular number of amino acid residues having a certain feature (e.g., positively-charged and/or hydrophobic amino acids) at certain positions in the amino acid sequence. When such disclosures make reference to a formula having particular amino acids specified at these positions in the sequence, these specified amino acids should be taken into account. For example, for a formula requiring a lysine at a particular position, this lysine residue should be counted as one of the required number of positively-charged amino acids if it is located in a recited position. In some aspects, the present disclosure provides a cell membrane internalizing peptide (CMIP) comprising an amino acid sequence selected from Formulas (IA) to (IH): X1-X2-X3-X4-X5-X6-X7-F-S-G-K-A-A-A-K-X16-E-A-K-X20-X21-X22-X23-X24-X25 (IA); I-W-L-T-A-L-X7-F-X9-X10-X11-X12-X13-X14-X15-A-X17-X18-X19-Q-F-X22-X23-X24-X25 (IB); I-W-X3-X4-X5-X6-K-X8-S-X10-X11-H-X13-X14-X15-A-E-X18-X19-X20-X21-L-S-K-L (IC);
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT X1-W-X3-T-A-X6-X7-X8-X9-G-K-A-X13-A-K-A-X17-A-K-Q-X21-X22-S-K-X25 (ID); I-X2-X3-X4-X5-L-K-F-X9-X10-X11-X12-A-A-K-X16-E-X18-K-Q-F-X22-X23-X24-L (IE); X1-W-L-T-A-X6-X7-X8-S-G-X11-X12-A-A-X15-A-X17-X18-K-X20-F-X22-X23-X24-X25 (IF); S-X2-L-T-A-X6-X7-X8-X9-X10-K-H-X13-I-T-Q-X17-X18-K-R-R-X22-X23-X24-X25 (IG); R-R-L-T-X5-L-F-K-X9-G-X11-X12-X13-X14-E- X16-I-X18-X19-X20-F (IH); wherein X1 X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, and X21 are each independently a natural or unnatural amino acid residue; and X22, X23, X24, and X25 are each independently absent or are each independently a natural or unnatural amino acid residue; or a pharmaceutically acceptable salt thereof. In some aspects, the present disclosure provides a cell membrane internalizing peptide (CMIP) comprising an amino acid sequence selected from Formulas (IA) to (IH): X1-X2-X3-X4-X5-X6-X7-F-S-G-K-A-A-A-K-X16-E-A-K-X20-X21-X22-X23-X24-X25 (IA); I-W-L-T-A-L-X7-F-X9-X10-X11-X12-X13-X14-X15-A-X17-X18-X19-Q-F-X22-X23-X24-X25 (IB); I-W-X3-X4-X5-X6-K-X8-S-X10-X11-H-X13-X14-X15-A-E-X18-X19-X20-X21-L-S-K-L (IC); X1-W-X3-T-A-X6-X7-X8-X9-G-K-A-X13-A-K-A-X17-A-K-Q-X21-X22-S-K-X25 (ID); I-X2-X3-X4-X5-L-K-F-X9-X10-X11-X12-A-A-K-X16-E-X18-K-Q-F-X22-X23-X24-L (IE); X1-W-L-T-A-X6-X7-X8-S-G-X11-X12-A-A-X15-A-X17-X18-K-X20-F-X22-X23-X24-X25 (IF); S-X2-L-T-A-X6-X7-X8-X9-X10-K-H-X13-I-T-Q-X17-X18-K-R-R-X22-X23-X24-X25 (IG); R-R-L-T-X5-L-F-K-X9-G-X11-X12-X13-X14-E- X16-I-X18-X19-X20-F (IH); wherein each of X1 through X25 is independently an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; or a pharmaceutically acceptable salt thereof. In some embodiments, at least one of (a) to (c) is present in the CMIP sequence: (a) X21 is selected from alanine, arginine, lysine, phenylalanine, and tryptophan; (b) X9 is serine and X12 is selected from alanine, arginine, glutamic acid, glutamine, phenylalanine, and tryptophan; or (c) X5 is histidine and X3 is selected from alanine, glutamic acid, leucine, lysine, phenylalanine, and tryptophan.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In some embodiments, the CMIP comprises Formula (IA): X1-X2-X3-X4-X5-X6-X7-F-S-G-K-A-A-A-K-X16-E-A-K-X20-X21-X22-X23-X24-X25 (IA) or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IB): I-W-L-T-A-L-X7-F-X9-X10-X11-X12-X13-X14-X15-A-X17-X18-X19-Q-F-X22-X23-X24-X25 (IB); or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IC): I-W-X3-X4-X5-X6-K-X8-S-X10-X11-H-X13-X14-X15-A-E-X18-X19-X20-X21-L-S-K-L (IC); or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (ID): X1-W-X3-T-A-X6-X7-X8-X9-G-K-A-X13-A-K-A-X17-A-K-Q-X21-X22-S-K-X25 (ID); or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IE): I-X2-X3-X4-X5-L-K-F-X9-X10-X11-X12-A-A-K-X16-E-X18-K-Q-F-X22-X23-X24-L (IE); or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IF): X1-W-L-T-A-X6-X7-X8-S-G-X11-X12-A-A-X15-A-X17-X18-K-X20-F-X22-X23-X24-X25 (IF); or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IG): S-X2-L-T-A-X6-X7-X8-X9-X10-K-H-X13-I-T-Q-X17-X18-K-R-R-X22-X23-X24-X25 (IG); or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IH): R-R-L-T-X5-L-F-K-X9-G-X11-X12-X13-X14-E- X16-I-X18-X19-X20-F (IH); or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP consists essentially of an amino acid sequence selected from Formula (IA) to (IH). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IA). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IB). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IC). In some embodiments, the CMIP consists essentially
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT of the amino acid sequence of Formula (ID). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IE). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IF). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IG). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IH). In some embodiments, the CMIP consists of an amino acid sequence selected from Formula (IA) to (IH). In some embodiments, the CMIP consists of the amino acid sequence of Formula (IA). In some embodiments, the CMIP consists of the amino acid sequence of Formula (IB). In some embodiments, the CMIP consists of the amino acid sequence of Formula (IC). In some embodiments, the CMIP consists of the amino acid sequence of Formula (ID). In some embodiments, the CMIP consists of the amino acid sequence of Formula (IE). In some embodiments, the CMIP consists of the amino acid sequence of Formula (IF). In some embodiments, the CMIP consists of the amino acid sequence of Formula (IG). In some embodiments, the CMIP consists of the amino acid sequence of Formula (IH). In another aspect, the present disclosure provides a CMIP comprising an amino acid sequence selected from Formulas (IIA) to (IID): I-X2-X3-T-A-L-X7-F-X9-G-X11-A-A-X14-K-X16-E-A-X19-Q-F-L-X23-X24-L (II-A); I-W-X3-X4-A-L-X7-F-X9-G-X11-X12-X13-A-X15-A-E-A-X19-X20-F-X22-S-X24-L (II-B); X1-X2-L-T-X5-L-K-X8-S-X10-K-A-A-A-X15-A-E-A-K-Q-X21-L-S-X24-L (II-C); I-W-L-T-X5-X6-K-F-S-X10-K-X12-A-A-K-A-X17-X18-K-Q-F-L-X23-X24-X25 (II-D); wherein X1 X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21 and X22 are each independently a natural or unnatural amino acid residue; and X23, X24, and X25 are either absent or each independently a natural or unnatural amino acid residue; or a pharmaceutically acceptable salt thereof. In another aspect, the present disclosure provides a polypeptide of a formula selected from Formula (IIA) to Formula (ID): I-X2-X3-T-A-L-X7-F-X9-G-X11-A-A-X14-K-X16-E-A-X19-Q-F-L-X23-X24-L (II-A); I-W-X3-X4-A-L-X7-F-X9-G-X11-X12-X13-A-X15-A-E-A-X19-X20-F-X22-S-X24-L (II-B); X1-X2-L-T-X5-L-K-X8-S-X10-K-A-A-A-X15-A-E-A-K-Q-X21-L-S-X24-L (II-C); I-W-L-T-X5-X6-K-F-S-X10-K-X12-A-A-K-A-X17-X18-K-Q-F-L-X23-X24-X25 (II-D);
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT wherein: X1 is a hydrophobic amino acid residue or a positively-charged amino acid residue; X2 is an aromatic amino acid residue, a positively-charged amino acid residue, or a negatively- charged amino acid residue; X3 is an aromatic amino acid residue, a hydrophobic amino acid residue, a positively-charged amino acid residue, or a negatively-charged amino acid residue; X4 is a neutral hydrophilic amino acid residue or an aromatic amino acid residue; X5 is a hydrophobic amino acid residue or an aromatic amino acid residue; X6 is a hydrophobic amino acid residue; X7 is a hydrophobic amino acid residue or a positively-charged amino acid; X8 is an aromatic amino acid residue; X9 is a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively- charged amino acid residue; X10 is a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively- charged amino acid residue; X11 is a neutral hydrophilic amino acid residue, a hydrophobic amino acid residue, or a positively-charged amino acid residue; X12 is a neutral hydrophilic amino acid residue, a hydrophobic amino acid residue, or a positively-charged amino acid residue; X13 is a neutral hydrophilic amino acid residue or a hydrophobic amino acid residue; X14 is a hydrophobic amino acid residue or a positively-charged amino acid; X15 is an aromatic amino acid residue, a positively-charged amino acid residue, or a negatively-charged amino acid residue; X16 is a hydrophobic amino acid residue, an aromatic amino acid residue, a positively-charged amino acid residue, or a negatively-charged amino acid residue; X17 is a positively-charged amino acid residue or a negatively-charged amino acid residue; X18 is a hydrophobic amino acid residue, an aromatic amino acid residue, or a positively- charged amino acid residue; X19 is a hydrophobic amino acid residue or a positively-charged amino acid residue; X20 is a neutral hydrophilic amino acid residue or a negative-charged amino acid residue; X21 is a hydrophobic amino acid residue or an aromatic amino acid residue;
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT X22 is absent, a hydrophobic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue; X23 is absent, a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue; X24 is absent, an aromatic amino acid residue, or a positively-charged amino acid residue; and X25 is absent or a hydrophobic amino acid; or a pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IIA): I-X2-X3-T-A-L-X7-F-X9-G-X11-A-A-X14-K-X16-E-A-X19-Q-F-L-X23-X24-L (IIA) or pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IIB): I-W-X3-X4-A-L-X7-F-X9-G-X11-X12-X13-A-X15-A-E-A-X19-X20-F-X22-S-X24-L (IIB) or pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IIC): X1-X2-L-T-X5-L-K-X8-S-X10-K-A-A-A-X15-A-E-A-K-Q-X21-L-S-X24-L (IIC) or pharmaceutically acceptable salt thereof. In some embodiments, the CMIP comprises Formula (IID): I-W-L-T-X5-X6-K-F-S-X10-K-X12-A-A-K-A-X17-X18-K-Q-F-L-X23-X24-X25 (IID) or pharmaceutically acceptable salt thereof. In some embodiments, the CMIP consists essentially of an amino acid sequence selected from Formula (IIA) to (IID). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IIA). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IIB). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IIC). In some embodiments, the CMIP consists essentially of the amino acid sequence of Formula (IID). In some embodiments, the CMIP consists of an amino acid sequence selected from Formulas (IIA) to (IID). In some embodiments, the CMIP consists of the amino acid sequence of Formulas (IIA). In some embodiments, the CMIP consists of the amino acid sequence of Formulas (IIB). In some embodiments, the CMIP consists of the amino acid sequence of Formulas (IIC). In some embodiments, the CMIP consists of the amino acid sequence of Formulas (IID).
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), the polypeptide comprises at least three positively-charged amino acid residues, each of which occupies a position corresponding to X7, X11, X15, X19, or X24. In some embodiments, the polypeptide comprises at least four positively-charged amino acid residues, each of which occupies a position corresponding to X7, X11, X15, X19, or X24. In some embodiments, the polypeptide comprises five positively-charged amino acid residues, each of which occupies a position corresponding to X7, X11, X15, X19, or X24. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), the polypeptide comprises at least four hydrophobic amino acid residues, each of which occupies a position corresponding to X3, X5, X6, X8, X13, X14, X16, X18, or X22. In some embodiments, the polypeptide comprises at least five hydrophobic amino acid residues, each of which occupies a position corresponding to X3, X5, X6, X8, X13, X14, X16, X18, or X22. In some embodiments, the polypeptide comprises at least six hydrophobic amino acid residues, each of which occupies a position corresponding to X3, X5, X6, X8, X13, X14, X16, X18, or X22. In some embodiments, the polypeptide comprises at least seven hydrophobic amino acid residues, each of which occupies a position corresponding to X3, X5, X6, X8, X13, X14, X16, X18, or X22. In some embodiments, the polypeptide comprises at least eight hydrophobic amino acid residues, each of which occupies a position corresponding to X3, X5, X6, X8, X13, X14, X16, X18, or X22. In some embodiments, the polypeptide comprises nine hydrophobic amino acid residues, each of which occupies a position corresponding to X3, X5, X6, X8, X13, X14, X16, X18, or X22. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X1 is a hydrophobic amino acid residue or a positively- charged amino acid residue. In some embodiments, X1 is a hydrophobic amino acid residue. In some embodiments, X1 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X1 is an amino acid residue selected from alanine, arginine, aspartic acid, isoleucine, and lysine. In some embodiments, X1 is isoleucine or lysine. In some embodiments, X1 is isoleucine.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X2 is an aromatic amino acid residue, a positively-charged amino acid residue, or a negatively-charged amino acid residue. In some embodiments, X2 is an aromatic amino acid residue. In some embodiments, X2 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X2 is an amino acid residue selected from alanine, arginine, aspartic acid, phenylalanine, tryptophan, and tyrosine. In some embodiments, X2 is arginine, aspartic acid, phenylalanine, tryptophan, or tyrosine. In some embodiments, X2 is tryptophan. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X3 is an aromatic amino acid residue, a hydrophobic amino acid residue, a positively-charged amino acid residue, or a negatively-charged amino acid residue. In some embodiments, X3 is a hydrophobic amino acid residue. In some embodiments, X3 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X3 is an amino acid residue selected from alanine, arginine, glutamic acid, leucine, lysine, phenylalanine, and tryptophan. In some embodiments, arginine, glutamic acid, leucine, phenylalanine, or tryptophan. In some embodiments, X3 is leucine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X4 is a neutral hydrophilic amino acid residue or an aromatic amino acid residue. In some embodiments, X4 is a neutral hydrophilic amino acid residue. In some embodiments, X4 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X4 is an amino acid residue selected from alanine, histidine, phenylalanine, threonine, and tryptophan. In some embodiments, X4 is threonine or tryptophan. In some embodiments, X4 is threonine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X5 is a hydrophobic amino acid residue or an aromatic amino
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT acid residue. In some embodiments, X5 is a hydrophobic amino acid residue. In some embodiments, X5 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X5 is an amino acid residue selected from alanine, arginine, histidine, phenylalanine, and tryptophan. In some embodiments, X5 is alanine or phenylalanine. In some embodiments, X5 is alanine. In some embodiments, X5 is histidine. In some embodiments, X5 is histidine; and X3 is an amino acid residue selected from alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X5 is histidine; and X3 is an amino acid residue selected from alanine, glutamic acid, leucine, lysine, phenylalanine, and tryptophan. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X6 is a hydrophobic amino acid residue. In some embodiments, X6 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X6 is an amino acid residue selected from alanine, arginine, glutamic acid, isoleucine, and leucine. In some embodiments, X6 is leucine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X7 is a hydrophobic amino acid residue or a positively charged amino acid. In some embodiments, X7 is a positively charged amino acid. In some embodiments, X7 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X7 is an amino acid residue selected from alanine, arginine, aspartic acid, isoleucine, leucine, lysine, and phenylalanine. In some embodiments, X7 is arginine, isoleucine, leucine, or lysine. In some embodiments, X7 is lysine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X8 is an aromatic amino acid residue. In some embodiments,
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT X8 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X8 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, glutamine, histidine, lysine, phenylalanine, serine, and tryptophan. In some embodiments, X8 is phenylalanine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X9 is a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively charged amino acid residue. In some embodiments, X9 is a neutral hydrophilic amino acid residue. In some embodiments, X9 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X9 is an amino acid residue selected from alanine, arginine, histidine, leucine, phenylalanine, serine, and tryptophan. In some embodiments, X9 is arginine, histidine, phenylalanine, or serine. In some embodiments, X9 is serine. In some embodiments, X9 is serine; and X12 is not histidine. In some embodiments, X9 is serine; and X12 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X9 is serine; and X12 is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, phenylalanine, and tryptophan. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X10 is a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue. In some embodiments, X10 is a neutral, hydrophilic amino acid residue. In some embodiments, X10 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X10 is an amino acid residue selected from alanine, arginine, glycine, tryptophan, and tyrosine. In some embodiments, X10 is arginine, glycine, or tyrosine. In some embodiments, X10 is glycine.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X11 is a neutral hydrophilic amino acid residue, a hydrophobic amino acid residue, or a positively-charged amino acid residue. In some embodiments, X11 is a positively-charged amino acid residue. In some embodiments, X11 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X11 is an amino acid residue selected from alanine, arginine, asparagine, glutamic acid, isoleucine, lysine, threonine, and tryptophan. In some embodiments, X11 is arginine, isoleucine, lysine, or threonine. In some embodiments, X11 is lysine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X12 is a neutral hydrophilic amino acid residue, a hydrophobic amino acid residue, or a positively-charged amino acid residue. In some embodiments, X12 is a hydrophobic amino acid residue. In some embodiments, X12 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X12 is an amino acid residue selected from alanine, glutamic acid, glutamine, histidine, phenylalanine, and tryptophan. In some embodiments, X12 is alanine or glutamine. In some embodiments, X12 is alanine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X13 is a neutral hydrophilic amino acid residue or a hydrophobic amino acid residue. In some embodiments, X13 is a hydrophobic amino acid residue. In some embodiments, X13 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X13 is an amino acid residue selected from alanine, arginine, aspartic acid, glutamic acid, glycine, leucine, phenylalanine, serine, and tryptophan. In some embodiments, X13 is alanine or serine. In some embodiments, X13 is alanine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X14 is a hydrophobic amino acid residue or a positively- charged amino acid. In some embodiments, X14 is a hydrophobic amino acid residue. In some
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT embodiments, X14 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X14 is an amino acid residue selected from alanine, arginine, glutamic acid, histidine, isoleucine, leucine, phenylalanine, tryptophan, tyrosine, and valine. In some embodiments, X14 is alanine, histidine, leucine, or valine. In some embodiments, X14 is alanine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X15 is an aromatic amino acid residue, a positively-charged amino acid residue, or a negatively0charged amino acid residue. In some embodiments, X15 is a positively-charged amino acid residue. In some embodiments, X15 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X15 is an amino acid residue selected from alanine, arginine, glutamic acid, lysine, threonine, and tryptophan. In some embodiments, X15 is arginine, glutamic acid, or lysine. In some embodiments, X15 is lysine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X16 is a hydrophobic amino acid residue, an aromatic amino acid residue, a positively-charged amino acid residue, or a negatively-charged amino acid residue. In some embodiments, X16 is a hydrophobic amino acid residue. In some embodiments, X16 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X16 is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, histidine, lysine, serine, and tryptophan. In some embodiments, X16 is not histidine. In some embodiments, X16 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X16 is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, lysine, serine, and tryptophan. In some embodiments, X16 is alanine, glutamic acid, or lysine. In some embodiments, X16 is alanine.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT In some embodiments, for the CMIP of for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X17 is a positively-charged amino acid residue or a negatively-charged amino acid residue. In some embodiments, X17 is a negatively-charged amino acid residue. In some embodiments, X17 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X17 is an amino acid residue selected from alanine, arginine, aspartic acid, glutamic acid, leucine, phenylalanine, and tryptophan. In some embodiments, X17 is arginine or glutamic acid. In some embodiments, X17 is glutamic acid. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X18 is a hydrophobic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue. In some embodiments, X18 is a hydrophobic amino acid residue. In some embodiments, X18 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X18 is an amino acid residue selected from alanine, arginine, glutamic acid, leucine, methionine, and tryptophan. In some embodiments, X18 is alanine, glutamic acid, methionine, or tryptophan. In some embodiments, X18 is alanine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X19 is a hydrophobic amino acid residue or a positively- charged amino acid residue. In some embodiments, X19 is a positively-charged amino acid residue. In some embodiments, X19 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X19 is an amino acid residue selected from alanine, arginine, leucine, lysine, threonine, and tryptophan. In some embodiments, X19 is arginine, leucine, or lysine. In some embodiments, X19 is lysine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X20 is a neutral hydrophilic amino acid residue or a negative- charged amino acid residue. In some embodiments, X20 is a neutral hydrophilic amino acid
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT residue. In some embodiments, X20 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X20 is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, methionine, serine, and tryptophan. In some embodiments, X20 is glutamic acid or glutamine. In some embodiments, X20 is glutamine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X21 is a hydrophobic amino acid residue or an aromatic amino acid residue. In some embodiments, X21 is an aromatic amino acid residue. In some embodiments, X21 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X21 is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, lysine, phenylalanine, and tryptophan. In some embodiments, X21 is an amino acid residue selected from alanine, arginine, lysine, phenylalanine, and tryptophan. In some embodiments, X21 is arginine, phenylalanine, or tryptophan. In some embodiments, X21 is phenylalanine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), is absent, a hydrophobic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue. In some embodiments, X22 is a hydrophobic amino acid residue. In some embodiments, X22 is either absent or an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X22 is either absent or an amino acid residue selected from alanine, arginine, aspartic acid, leucine, and lysine. In some embodiments, X22 is either absent or is leucine. In some embodiments, X22 is absent. In some embodiments, X22 is Arginine, leucine, or tryptophan. In some embodiments, X22 is leucine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X23 is absent, a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue. In some embodiments, wherein X23 is a neutral hydrophilic amino acid residue. In some embodiments, X23 is either
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT absent or an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X23 is either absent or an amino acid residue selected from alanine, arginine, aspartic acid, glutamic acid, leucine, lysine, threonine, tryptophan, and valine. In some embodiments, X23 is either absent or is serine. In some embodiments, X23 is absent. In some embodiments, X23 is absent, or is arginine, methionine, serine, or tryptophan. In some embodiments, X23 is serine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X24 is absent, an aromatic amino acid residue, or a positively- charged amino acid residue. In some embodiments, X24 is a positively-charged amino acid residue. In some embodiments, X24 is either absent or an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X24 is either absent or an amino acid residue selected from alanine, arginine, and lysine. In some embodiments, X24 is either absent or is lysine. In some embodiments, X24 is absent. In some embodiments, X24 is absent, or is arginine, lysine, or tryptophan. In some embodiments, X24 is lysine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X25 is absent or a hydrophobic amino acid. In some embodiments, X25 is a hydrophobic amino acid. In some embodiments, X25 is either absent or is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In some embodiments, X25 is either absent or an amino acid residue selected from alanine, arginine, leucine, and lysine. In some embodiments, X25 is either absent or is leucine. In some embodiments, X25 is absent. In some embodiments, X25 is leucine. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), X22, X23, X24, and X25 are each absent. In some embodiments, for the CMIP of Formula (IA), (IB), (IC), (ID), (IE), (IF), (IG), (IH), (IIA), (IIB), (IIC), and (IID), the CMIP comprises a net-charge of 2, 3, 4, or 5. In some embodiments, the CMIP comprises a hydrophobicity from about 0.25 to about 0.63 (e.g., as
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT determined using the HELIQUEST described herein). In some embodiments, the CMIP comprises a hydrophobicity from about 0.25 to about 0.50. In some embodiments, the CMIP comprises a hydrophobic moment from about 0.035 to about 0.16. In some embodiments, the CMIP comprises a hydrophobic moment from about 0.050 to about 0.16. In some embodiments, the CMIP comprises a hydrophobicity of from about 0.25 to about 0.5, a net-charge of 3, 4, or 5, and a hydrophobic moment about 0.035 to about 0.16. In some embodiments, the CMIP comprises a hydrophobicity of between 0.25 and 0.5, a net-charge of 3, 4, or 5, and a hydrophobic moment from about 0.45 to about 0.080. In some embodiments, the CMIP comprises a hydrophobicity of between 0.25 and 0.5, a net-charge of 5, and about 0.035 to about 0.16. In some embodiments, the CMIP comprises a hydrophobicity of from about 0.25 to about 0.35, a net-charge of 3, 4, or 5, and a hydrophobic moment about 0.035 to about 0.16. Table 3 below shows a non-limiting, exemplary list of CMIPs of the present disclosure. In some embodiments, the CMIP is selected from a CMIP of Table 3. In some embodiments, the CMIP comprises an amino acid sequence selected from Table 3. In some embodiments, the CMIP consists essentially of an amino acid sequence selected from Table 3. In some embodiments, the CMIP consists of an amino acid sequence selected from Table 3. Table 3 SEQ ID NO: 54 CMIP1 IWLTALKFLGKAAAKAEAKQQLSKL
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: 97 CMIP39 IWLTALRFSGKAAAKAEAKQFLSRL SEQ ID NO: 98 CMIP40 IWLTALKFSGKAAAKAEAKQFLRRL
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: 127 CMIP]\ IWLTALKFHGKHAAKAEAKQFLSKL SEQ ID NO: 128 CMIP]Z IWLTALKFSGKAAAKAEAKQFRSKL
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: 157 CMIPa`\ RRLTRLFKHGWRFAEHIWKSF SEQ ID NO: 158 CMIPa`Z RRLTRLFKHGARFAEHIWKSF
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: 187 CMIPac\ IWLTALKFSYKEAYKAEAKQFLSKL SEQ ID NO: 188 CMIPacZ IWLTALKFSYEALAKAEAKQFLSKL
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: 217 CMIPaZ\ IWLTALKFSGIAAAKWERKQFLSKL SEQ ID NO: 218 CMIPaZZ IWLTALKFFGIWAAKAEAKQFRSKL
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT SEQ ID NO: 247 CMIPa_\ IWLTALKFSWIAAFKAEAKQFLSRL SEQ ID NO: 248 CMIPa Z DWETADKFSGKAAAKAEAKQFLSKL
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 130 CMIP78 117.9 213 CMIP161 173.7 131 CMIP79 171.4 214 CMIP162 --
Example 2: Calculated Biophysical Characteristics of Selected CMIPs
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT To understand the correlation between CMIP internalization and biophysical parameters, net charge (z), hydrophobic moment (<µH>), and hydrophobicity (<H>) for exemplary CMIPs of the present disclosure. These parameters were calculated using the HELIQUEST online program from the Institutde Pharmacologie Moléculaireet Cellulaire, Universitéde Nice Sophia Antipolisand CNRS, 660 routedes Lucioles, 06560 Valbonne, France. Table 6 provides calculated values for net charge (z), hydrophobic moment (<µH>), and hydrophobicity (<H>) for exemplary CMIPs of the present disclosure. These values were calculated in the manner described above. Table 6 SEQ ID Name z <µH> <H> SEQ ID Name z <µH> <H> NO: NO: 6 5 3 3 1 1 7 7 1 8 6 7 9 5 3 4 7 5 4 4 4 9 5 7 9
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT CMIP65 4 0.138 0.516 200 CMIP148 1 0.198 0.338 CMIP66 4 0.079 0.463 201 CMIP149 4 0.094 0.452 8 8 4 8 8 6 8 2 8 5 4 3 1 3 3 6 6 8 3 8 4 4 6 2 2 2 8 8 9 8 7 6 2 5 1 9 2
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 162 CMIP110 4 0.657 0.378 245 CMIP193 5 0.162 0.511 163 CMIP111 5 0.159 0.315 246 CMIP194 5 0.107 0.43 6 1 2 8 8 4
xamp e : mmunogen c y o s
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 168 CMIP116 0.5 251 CMIP199 0.4 169 CMIP117 10 252 CMIP200 0.2
. ibod
8. The CMIP or salt of any one of claims 1 to 4, wherein the CMIP comprises Formula (ID): X1-W-X3-T-A-X6-X7-X8-X9-G-K-A-X13-A-K-A-X17-A-K-Q-X21-X22-S-K-X25 (ID); or a pharmaceutically acceptable salt thereof. 9. The CMIP or salt of any one of claims 1 to 4, wherein the CMIP comprises Formula (IE): I-X2-X3-X4-X5-L-K-F-X9-X10-X11-X12-A-A-K-X16-E-X18-K-Q-F-X22-X23-X24-L (IE); or a pharmaceutically acceptable salt thereof. 10. The CMIP or salt of any one of claims 1 to 4, wherein the CMIP comprises Formula (IF): X1-W-L-T-A-X6-X7-X8-S-G-X11-X12-A-A-X15-A-X17-X18-K-X20-F-X22-X23-X24-X25 (IF); or a pharmaceutically acceptable salt thereof. 11. The CMIP or salt of any one of claims 1 to 4, wherein the CMIP comprises Formula (IG): S-X2-L-T-A-X6-X7-X8-X9-X10-K-H-X13-I-T-Q-X17-X18-K-R-R-X22-X23-X24-X25 (IG); or a pharmaceutically acceptable salt thereof. 12. The CMIP or salt of any one of claims 1 to 4, wherein the CMIP comprises Formula (IH): R-R-L-T-X5-L-F-K-X9-G-X11-X12-X13-X14-E- X16-I-X18-X19-X20-F (IH); or a pharmaceutically acceptable salt thereof. 13. A cell membrane internalizing peptide (CMIP) comprising an amino acid sequence selected from Formulas (IIA) to (IID): I-X2-X3-T-A-L-X7-F-X9-G-X11-A-A-X14-K-X16-E-A-X19-Q-F-L-X23-X24-L (IIA);
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT I-W-X3-X4-A-L-X7-F-X9-G-X11-X12-X13-A-X15-A-E-A-X19-X20-F-X22-S-X24-L (IIB); X1-X2-L-T-X5-L-K-X8-S-X10-K-A-A-A-X15-A-E-A-K-Q-X21-L-S-X24-L (IIC); I-W-L-T-X5-X6-K-F-S-X10-K-X12-A-A-K-A-X17-X18-K-Q-F-L-X23-X24-X25 (IID); wherein X1 X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, X14, X15, X16, X17, X18, X19, X20, X21
are or a or acid residue; or a pharmaceutically acceptable salt thereof. 14. The CMIP or salt of claim 13, wherein the CMIP consists essentially of an amino acid sequence selected from Formulas (IIA) to (IID). 15. The CMIP or salt of claim 13 or claim 14, wherein the CMIP consists of an amino acid sequence selected from Formulas (IIA) to (IID). 16. The CMIP or salt of any one of claims 13 to 15, wherein the CMIP comprises Formula (IIA): I-X2-X3-T-A-L-X7-F-X9-G-X11-A-A-X14-K-X16-E-A-X19-Q-F-L-X23-X24-L (IIA); or a pharmaceutically acceptable salt thereof. 17. The CMIP or salt of any one of claims 13 to 15, wherein the CMIP comprises Formula (IIB): I-W-X3-X4-A-L-X7-F-X9-G-X11-X12-X13-A-X15-A-E-A-X19-X20-F-X22-S-X24-L (IIB); or a pharmaceutically acceptable salt thereof. 18. The CMIP or salt of any one of claims 13 to 15, wherein the CMIP comprises Formula (IIC): X1-X2-L-T-X5-L-K-X8-S-X10-K-A-A-A-X15-A-E-A-K-Q-X21-L-S-X24-L (IIC); or a pharmaceutically acceptable salt thereof. 19. The CMIP or salt of any one of claims 13 to 15, wherein the CMIP comprises Formula (IID): I-W-L-T-X5-X6-K-F-S-X10-K-X12-A-A-K-A-X17-X18-K-Q-F-L-X23-X24-X25 (IID); or a pharmaceutically acceptable salt thereof. 20. The CMIP or salt of any one of claims 13 to 19, wherein the polypeptide comprises at least three positively charged amino acid residues, each of which occupies a position corresponding to X7, X11, X15, X19, or X24. 21. The CMIP or salt of any one of claims 13 to 19, wherein the polypeptide comprises at least four positively charged amino acid residues, each of which occupies a position corresponding to X7, X11, X15, X19, or X24.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 22. The CMIP or salt of any one of claims 13 to 21, wherein the polypeptide comprises at least five hydrophobic amino acid residues, each of which occupies a position corresponding to X3, X5, X6, X8, X13, X14, X16, X18, or X22. 23. The CMIP or salt of any one of claims 13 to 21, wherein the polypeptide comprises at least seven hydrophobic amino acid residues, each of which occupies a position corresponding to X3, X5, X6, X8, X13, X14, X16, X18, or X22. 24. The CMIP or salt of any one of claims 1 to 23, wherein X1 is a hydrophobic amino acid residue or a positively-charged amino acid residue. 25. The CMIP or salt of any one of claims 1 to 23, wherein X1 is a hydrophobic amino acid residue. 26. The CMIP or salt of any one of claims 1 to 23, wherein X1 is an amino acid residue selected from Alanine, Arginine, Aspartic acid, Isoleucine, and Lysine. 27. The CMIP or salt of any one of claims 1 to 23, wherein X1 is Isoleucine or Lysine. 28. The CMIP or salt of any one of claims 1 to 23, wherein X1 is Isoleucine. 29. The CMIP or salt of any one of claims 1 to 28, wherein X2 is an aromatic amino acid residue, a positively-charged amino acid residue, or a negatively-charged amino acid residue; 30. The CMIP or salt of any one of claims 1 to 28, wherein X2 is an aromatic amino acid residue. 31. The CMIP or salt of any one of claims 1 to 28, wherein X2 is an amino acid residue selected from alanine, arginine, aspartic acid, phenylalanine, tryptophan, and tyrosine. 32. The CMIP or salt of any one of claims 1 to 28, wherein X2 is arginine, aspartic acid, phenylalanine, tryptophan, or tyrosine. 33. The CMIP or salt of any one of claims 1 to 28, wherein X2 is tryptophan. 34. The CMIP or salt of any one of claims 1 to 33, wherein X3 is an aromatic amino acid residue, a hydrophobic amino acid residue, a positively-charged amino acid residue, or a negatively- charged amino acid residue. 35. The CMIP or salt of any one of claims 1 to 33, wherein X3 is a hydrophobic amino acid residue. 36. The CMIP or salt of any one of claims 1 to 33, wherein X3 is an amino acid residue selected from alanine, arginine, glutamic acid, leucine, lysine, phenylalanine, and tryptophan. 37. The CMIP or salt of any one of claims 1 to 33, wherein X3 is arginine, glutamic acid, leucine, phenylalanine, or tryptophan.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 38. The CMIP or salt of any one of claims 1 to 33, wherein X3 is leucine. 39. The CMIP or salt of any one of claims 1 to 38, wherein X4 is a neutral hydrophilic amino acid residue or an aromatic amino acid residue. 40. The CMIP or salt of any one of claims 1 to 38, wherein X4 is a neutral hydrophilic amino acid residue. 41. The CMIP or salt of any one of claims 1 to 38, wherein X4 is an amino acid residue selected from alanine, histidine, phenylalanine, threonine, and tryptophan. 42. The CMIP or salt of any one of claims 1 to 38, wherein X4 is threonine or tryptophan. 43. The CMIP or salt of any one of claims 1 to 38, wherein X4 is threonine. 44. The CMIP or salt of any one of claims 1 to 43, wherein X5 is a hydrophobic amino acid residue or an aromatic amino acid residue. 45. The CMIP or salt of any one of claims 1 to 43, wherein X5 is a hydrophobic amino acid residue. 46. The CMIP or salt of any one of claims 1 to 43, wherein X5 is an amino acid residue selected from alanine, arginine, histidine, phenylalanine, and tryptophan. 47. The CMIP or salt of any one of claims 1 to 43, wherein X5 is alanine or phenylalanine. 48. The CMIP or salt of any one of claims 1 to 43, wherein X5 is alanine. 49. The CMIP or salt of any one of claims 1 to 43, wherein X5 is histidine; and X3 is an amino acid residue selected from alanine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. 50. The CMIP or salt of any one of claims 1 to 43, wherein X5 is histidine; and X3 is an amino acid residue selected from alanine, glutamic acid, leucine, lysine, phenylalanine, and tryptophan. 51. The CMIP or salt of any one of claims 1 to 50, wherein X6 is a hydrophobic amino acid residue. 52. The CMIP or salt of any one of claims 1 to 50, wherein X6 is an amino acid residue selected from alanine, arginine, glutamic acid, isoleucine, and leucine. 53. The CMIP or salt of any one of claims 1 to 50, wherein X6 is Leucine. 54. The CMIP or salt of any one of claims 1 to 53, wherein X7 is a hydrophobic amino acid residue or a positively-charged amino acid.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 55. The CMIP or salt of any one of claims 1 to 53, wherein X7 is a positively-charged amino acid. 56. The CMIP or salt of any one of claims 1 to 53, wherein X7 is an amino acid residue selected from alanine, arginine, aspartic acid, isoleucine, leucine, lysine, and phenylalanine. 57. The CMIP or salt of any one of claims 1 to 53, wherein X7 is arginine, isoleucine, leucine, or lysine. 58. The CMIP or salt of any one of claims 1 to 53, wherein X7 is lysine. 59. The CMIP or salt of any one of claims 1 to 58, wherein X8 is an aromatic amino acid residue. 60. The CMIP or salt of any one of claims 1 to 58, wherein X8 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, glutamine, histidine, lysine, phenylalanine, serine, and tryptophan. 61. The CMIP or salt of any one of claims 1 to 58, wherein X8 is phenylalanine. 62. The CMIP or salt of any one of claims 1 to 61, wherein X9 is a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue. 63. The CMIP or salt of any one of claims 1 to 61, wherein X9 is a neutral hydrophilic amino acid residue. 64. The CMIP or salt of any one of claims 1 to 61, wherein X9 is an amino acid residue selected from alanine, arginine, histidine, leucine, phenylalanine, serine, and tryptophan. 65. The CMIP or salt of any one of claims 1 to 61, wherein X9 is arginine, histidine, phenylalanine, or serine. 66. The CMIP or salt of any one of claims 1 to 61, wherein X9 is serine. 67. The CMIP or salt of any one of claims 1 to 61, wherein X9 is serine; and X12 is an amino acid residue selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. 68. The CMIP or salt of any one of claims 1 to 61, wherein X9 is serine; and X12 is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, phenylalanine, and tryptophan. 69. The CMIP or salt of any one of claims 1 to 68, wherein X10 is a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 70. The CMIP or salt of any one of claims 1 to 68, wherein X10 is a neutral hydrophilic amino acid residue. 71. The CMIP or salt of any one of claims 1 to 68, wherein X10 is an amino acid residue selected from alanine, arginine, glycine, tryptophan, and tyrosine. 72. The CMIP or salt of any one of claims 1 to 68, wherein X10 is arginine, glycine, or tyrosine. 73. The CMIP or salt of any one of claims 1 to 68, wherein X10 is glycine. 74. The CMIP or salt of any one of claims 1 to 73, wherein X11 is a neutral hydrophilic amino acid residue, a hydrophobic amino acid residue, or a positively-charged amino acid residue. 75. The CMIP or salt of any one of claims 1 to 73, wherein X11 is a positively-charged amino acid residue. 76. The CMIP or salt of any one of claims 1 to 73, wherein X11 is an amino acid residue selected from alanine, arginine, asparagine, glutamic acid, isoleucine, lysine, threonine, and tryptophan. 77. The CMIP or salt of any one of claims 1 to 73, wherein X11 is arginine, isoleucine, lysine, or threonine. 78. The CMIP or salt of any one of claims 1 to 73, wherein X11 is lysine. 79. The CMIP or salt of any one of claims 1 to 78, wherein X12 is a neutral hydrophilic amino acid residue, a hydrophobic amino acid residue, or a positively-charged amino acid residue. 80. The CMIP or salt of any one of claims 1 to 78, wherein X12 is a hydrophobic amino acid residue. 81. The CMIP or salt of any one of claims 1 to 78, wherein X12 is an amino acid residue selected from alanine, glutamic acid, glutamine, histidine, phenylalanine, and tryptophan. 82. The CMIP or salt of any one of claims 1 to 78, wherein X12 is alanine or glutamine. 83. The CMIP or salt of any one of claims 1 to 78, wherein X12 is alanine. 84. The CMIP or salt of any one of claims 1 to 83, wherein X13 is a neutral hydrophilic amino acid residue or a hydrophobic amino acid residue. 85. The CMIP or salt of any one of claims 1 to 83, wherein X13 is a hydrophobic amino acid residue. 86. The CMIP or salt of any one of claims 1 to 83, wherein X13 is an amino acid residue selected from alanine, arginine, aspartic acid, glutamic acid, glycine, leucine, phenylalanine, serine, and tryptophan.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 87. The CMIP or salt of any one of claims 1 to 83, wherein X13 is alanine or serine. 88. The CMIP or salt of any one of claims 1 to 83, wherein X13 is alanine. 89. The CMIP or salt of any one of claims 1 to 88, X14 is a hydrophobic amino acid residue or a positively-charged amino acid. 90. The CMIP or salt of any one of claims 1 to 88, X14 is a hydrophobic amino acid residue. 91. The CMIP or salt of any one of claims 1 to 88, wherein X14 is an amino acid residue selected from alanine, arginine, glutamic acid, histidine, isoleucine, leucine, phenylalanine, tryptophan, tyrosine, and valine. 92. The CMIP or salt of any one of claims 1 to 88, wherein X14 is alanine, histidine, leucine, or valine. 93. The CMIP or salt of any one of claims 1 to 88, wherein X14 is alanine. 94. The CMIP or salt of any one of claims 1 to 93, wherein X15 is an aromatic amino acid residue, a positively-charged amino acid residue, or a negatively-charged amino acid residue. 95. The CMIP or salt of any one of claims 1 to 93, wherein X15 is a positively-charged amino acid residue. 96. The CMIP or salt of any one of claims 1 to 93, wherein X15 is an amino acid residue selected from alanine, arginine, glutamic acid, lysine, threonine, and tryptophan. 97. The CMIP or salt of any one of claims 1 to 93, wherein X15 is arginine, glutamic acid, or lysine. 98. The CMIP or salt of any one of claims 1 to 93, wherein X15 is lysine. 99. The CMIP or salt of any one of claims 1 to 98, wherein X16 is a hydrophobic amino acid residue, an aromatic amino acid residue, a positively-charged amino acid residue, or a negatively-charged amino acid residue. 100. The CMIP or salt of any one of claims 1 to 98, wherein X16 is a hydrophobic amino acid residue. 101. The CMIP or salt of any one of claims 1 to 98, wherein X16 is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, histidine, lysine, serine, and tryptophan. 102. The CMIP or salt of any one of claims 1 to 98, wherein X16 is alanine, glutamic acid, or lysine. 103. The CMIP or salt of any one of claims 1 to 98, wherein X16 is alanine.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 104. The CMIP or salt of any one of claims 1 to 103, wherein X17 is a positively-charged amino acid residue or a negatively-charged amino acid residue. 105. The CMIP or salt of any one of claims 1 to 103, wherein X17 is a negatively-charged amino acid residue. 106. The CMIP or salt of any one of claims 1 to 103, wherein X17 is an amino acid residue selected from alanine, arginine, aspartic acid, glutamic acid, leucine, phenylalanine, and tryptophan. 107. The CMIP or salt of any one of claims 1 to 103, wherein X17 is arginine or glutamic acid. 108. The CMIP or salt of any one of claims 1 to 103, wherein X17 is glutamic acid. 109. The CMIP or salt of any one of claims 1 to 108, wherein X18 is a hydrophobic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue. 110. The CMIP or salt of any one of claims 1 to 108, wherein X18 is a hydrophobic amino acid residue. 111. The CMIP or salt of any one of claims 1 to 108, wherein X18 is an amino acid residue selected from alanine, arginine, glutamic acid, leucine, methionine, and tryptophan. 112. The CMIP or salt of any one of claims 1 to 108, wherein X18 is alanine, glutamic acid, methionine, or tryptophan. 113. The CMIP or salt of any one of claims 1 to 108, wherein X18 is alanine. 114. The CMIP or salt of any one of claims 1 to 113, wherein X19 is a hydrophobic amino acid residue or a positively-charged amino acid residue. 115. The CMIP or salt of any one of claims 1 to 113, wherein X19 is a positively-charged amino acid residue. 116. The CMIP or salt of any one of claims 1 to 113, wherein X19 is an amino acid residue selected from alanine, arginine, leucine, lysine, threonine, and tryptophan. 117. The CMIP or salt of any one of claims 1 to 113, wherein X19 is arginine, leucine, or lysine. 118. The CMIP or salt of any one of claims 1 to 113, wherein X19 is lysine. 119. The CMIP or salt of any one of claims 1 to 118, wherein X20 is a neutral hydrophilic amino acid residue or a negative-charged amino acid residue. 120. The CMIP or salt of any one of claims 1 to 118, wherein X20 is a neutral hydrophilic amino acid residue.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 121. The CMIP or salt of any one of claims 1 to 118, wherein X20 is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, methionine, serine, and tryptophan. 122. The CMIP or salt of any one of claims 1 to 118, wherein X20 is glutamic acid or glutamine. 123. The CMIP or salt of any one of claims 1 to 118, wherein X20 is glutamine. 124. The CMIP or salt of any one of claims 1 to 123, wherein X21 is a hydrophobic amino acid residue or an aromatic amino acid residue. 125. The CMIP or salt of any one of claims 1 to 123, wherein X21 is an aromatic amino acid residue. 126. The CMIP or salt of any one of claims 1 to 123, wherein X21 is an amino acid residue selected from alanine, arginine, glutamic acid, glutamine, lysine, phenylalanine, and tryptophan. 127. The CMIP or salt of any one of claims 1 to 123 wherein X21 is an amino acid residue selected from alanine, arginine, lysine, phenylalanine, and tryptophan. 128. The CMIP or salt of any one of claims 1 to 123 wherein X21 is arginine, phenylalanine, or tryptophan. 129. The CMIP or salt of any one of claims 1 to 123, wherein X21 is phenylalanine. 130. The CMIP or salt of any one of claims 1 to 129, wherein X22 is absent, a hydrophobic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue. 131. The CMIP or salt of any one of claims 1 to 129, wherein X22 is a hydrophobic amino acid residue. 132. The CMIP or salt of any one of claims 1 to 129, wherein X22 is either absent or an amino acid residue selected from alanine, arginine, aspartic acid, leucine, and lysine. 133. The CMIP or salt of any one of claims 1 to 129, wherein X22 is arginine, leucine, or tryptophan. 134. The CMIP or salt of any one of claims 1 to 129, wherein X22 is either absent or leucine. 135. The CMIP or salt of any one of claims 1 to 129, wherein X22 is absent. 136. The CMIP or salt of any one of claims 1 to 129, wherein X22 is leucine.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 137. The CMIP or salt of any one of claims 1 to 136, wherein X23 is absent, a neutral hydrophilic amino acid residue, an aromatic amino acid residue, or a positively-charged amino acid residue. 138. The CMIP or salt of any one of claims 1 to 136, wherein X23 is a neutral hydrophilic amino acid residue. 139. The CMIP or salt of any one of claims 1 to 136, wherein X23 is either absent or an amino acid residue selected from alanine, arginine, aspartic acid, glutamic acid, leucine, lysine, threonine, tryptophan, and valine. 140. The CMIP or salt of any one of claims 1 to 136, wherein X23 is absent, arginine, methionine, serine, or tryptophan. 141. The CMIP or salt of any one of claims 1 to 136, wherein X23 is either absent or serine. 142. The CMIP or salt of any one of claims 1 to 136, wherein X23 is absent. 143. The CMIP or salt of any one of claims 1 to 136, wherein X23 is serine. 144. The CMIP or salt of any one of claims 1 to 143, wherein X24 is absent, an aromatic amino acid residue, or a positively-charged amino acid residue. 145. The CMIP or salt of any one of claims 1 to 143, wherein X24 is a positively-charged amino acid residue. 146. The CMIP or salt of any one of claims 1 to 143, wherein X24 is either absent or an amino acid residue selected from alanine, arginine, and lysine. 147. The CMIP or salt of any one of claims 1 to 143, wherein X24 is absent, arginine, lysine, or tryptophan. 148. The CMIP or salt of any one of claims 1 to 143, wherein X24 is either absent or lysine. 149. The CMIP or salt of any one of claims 1 to 143, wherein X24 is absent. 150. The CMIP or salt of any one of claims 1 to 143, wherein X24 is lysine. 151. The CMIP or salt of any one of claims 1 to 150, wherein X25 is absent or a hydrophobic amino acid. 152. The CMIP or salt of any one of claims 1 to 150, wherein X25 is a hydrophobic amino acid. 153. The CMIP or salt of any one of claims 1 to 150, wherein X25 is either absent or is an amino acid residue selected from alanine, arginine, leucine, and lysine. 154. The CMIP or salt of any one of claims 1 to 150, wherein X25 is either absent or is leucine.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 155. The CMIP or salt of any one of claims 1 to 150, wherein X25 is absent. 156. The CMIP or salt of any one of claims 1 to 150, wherein X25 is leucine. 157. The CMIP or salt of any one of claims 1 to 123, wherein X21, X22, X23, X24, and X25 are each absent. 158. A CMIP comprising an amino acid sequence selected from the amino acid sequences of Table 3, or a pharmaceutically acceptable salt thereof. 159. A CMIP consisting of an amino acid sequence selected from the amino acid sequences of Table 3, or a pharmaceutically acceptable salt thereof. 160. A nucleic acid encoding the CMIP of any one of claims 1 to 159. 161. A vector comprising the nucleic acid of claim 160. 162. A cell comprising the CMIP of any one of claims 1 to 159, the nucleic acid of claim 160, or the vector of claim 161. 163. The cell of claim 162, wherein the cell is a mammalian cell. 164. A method of producing the cell-penetrating peptide of any one of claims 1 to 159, the method comprising: culturing a cell comprising a nucleic acid encoding the cell-penetrating peptide in a liquid culture medium under conditions that allow the cell to produce the cell-penetrating peptide; and harvesting the cell-penetrating peptide from the cell or the liquid culture medium. 165. A pharmaceutical composition comprising the cell-penetrating peptide of any one of claims 1 to 159 and a pharmaceutically acceptable excipient. 166. A cell-penetrating agent (CPA) comprising (i) a cell membrane internalizing peptide (CMIP) of any one of claims 1 to 159 and (ii) a payload. 167. The cell-penetrating agent of claim 166 , wherein the CMIP is covalently linked to the payload. 168. The cell-penetrating agent of claim 166 or 167, wherein the CMIP is non-covalently linked to the payload. 169. The cell-penetrating agent of any one of claims 166 to 168, wherein the cell-penetrating agent further comprises a linker connecting the CMIP to the payload. 170. The cell-penetrating agent of claim 169, wherein the linker is covalently linked to both the CMIP and the payload.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 171. The cell-penetrating agent of claim 169 or 170, wherein the linker is a cleavable linker. 172. The cell-penetrating agent of claim 169 or 170, wherein the linker is a non-cleavable linker. 173. The cell-penetrating agent of any one of claims 169 to 172, wherein the linker comprises a polypeptide. 174. The cell-penetrating agent of any one of claims 169 to 173, wherein the linker comprises a polypeptide comprising an amino acid sequence selected from Table 4. 175. The cell-penetrating agent of any one of claims 166 to 174, wherein the payload is a biomolecule. 176. The cell-penetrating agent of any one of claims 166 to 175, wherein the biomolecule comprises a carbohydrate, a nucleic acid, a protein, or a peptide. 177. The cell-penetrating agent of any one of claims 166 to 176, wherein the payload is a macromolecule. 178. The cell-penetrating agent of any one of claims 166 to 177, wherein the payload has a molecular weight of about 5 kD to about 1000 kD, 179. The cell-penetrating agent of any one of claims 166 to 178, wherein the payload comprises a proteinaceous molecule. 180. The cell-penetrating agent of claim 179, wherein the proteinaceous molecule is about 100 to about 2000 amino acids. 181. The cell-penetrating agent of claim 179 or 180, wherein the proteinaceous molecule is an antibody, an enzyme, a protein, or a peptide. 182. The cell-penetrating agent of any one of claims 179 to 181, wherein the proteinaceous molecule is linked to the CMIP via a polypeptide linker. 183. The cell-penetrating agent of claim 182, wherein the cell-penetrating agent is a fusion protein. 184. The cell-penetrating agent of claim 182, wherein the CMIP is genetically fused to the payload or a portion thereof. 185. The cell-penetrating agent of any one of claims 166 to 184, wherein the payload or the portion thereof is linked to the C-terminus of the CMIP. 186. The cell-penetrating agent of any one of claims 166 to 184, wherein the payload or the portion thereof is linked to the N-terminus of the CMIP.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 187. The cell-penetrating agent of any one of claims 166 to 186, wherein the payload is an antibody or a portion thereof. 188. The cell-penetrating agent of claim 187, wherein the antibody or a portion thereof comprises a heavy chain. 189. The cell-penetrating agent of claim 188, wherein the CMIP is covalently linked to a C- terminus of the heavy chain. 190. The cell-penetrating agent of claim 188, wherein the CMIP is covalently linked to a N- terminus of the heavy chain. 191. The cell-penetrating agent of any one of claims 187 to 190, wherein the antibody or portion thereof comprises a light chain. 192. The cell-penetrating agent of claim 191, wherein the CMIP is covalently linked to a C- terminus of the light chain. 193. The cell-penetrating agent of claim 191, wherein the CMIP is covalently linked to a N- terminus of the light chain. 194. The cell-penetrating agent of any one of claims 188 to 193, wherein the heavy chain of the antibody or a portion thereof and the CMIP are expressed as a single polypeptide. 195. The cell-penetrating agent of any one of claims 191 to 193, wherein the light chain of the antibody or a portion thereof and the CMIP are expressed as a single polypeptide. 196. The cell-penetrating agent of any one of claims 187 to 195, wherein the antibody or fragment thereof specifically binds to a target protein. 197. The cell-penetrating agent of claim 196, wherein the target protein is a cytosolic protein. 198. The cell-penetrating agent of claim 196, wherein the target protein is a membrane- associated protein. 199. The cell-penetrating agent of any one of claims 196 to 198, wherein the target protein is an enzyme. 200. The cell-penetrating agent of any one of claims 196, wherein the target protein is a Translocase of outer mitochondrial membrane 20 (TOMM20) protein. 201. The cell-penetrating agent of any one of claims 196 to 198, wherein the target protein is a cytoskeletal protein. 202. The cell-penetrating agent of claim 201, wherein the target protein is a beta-actin protein.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 203. The cell-penetrating agent of claim 201, wherein the target protein is an alpha tubulin protein. 204. The cell-penetrating agent of any one of claims 196 to 198, wherein the target protein is an oxidoreductase. 205. The cell-penetrating agent of claim 204, wherein the target protein is an endoplasmic reticulum oxidoreductin-1-like (EROL1L) protein. 206. The cell-penetrating agent of claim 196, wherein the target protein is a mitochondrial protein. 207. The cell-penetrating agent of claim 206, wherein the target protein is a mitochondrial transport protein. 208. The cell-penetrating agent of claim 207, wherein the target protein is a nuclear envelope spectrin repeat protein-1 (Nesprin-1) protein. 209. The cell-penetrating agent of any one of claims 166 to 186, wherein the payload is an enzyme or a functional portion thereof. 210. The cell-penetrating agent of claim 209, wherein the enzyme is a kinase. 211. The cell-penetrating agent of claim 210, wherein the enzyme is a serine/threonine kinase. 212. The cell-penetrating agent of claim 211, wherein the serine/threonine kinase is GSK3β. 213. The cell-penetrating agent of claim 209, wherein the enzyme is a protease. 214. The cell-penetrating agent of claim 213, wherein the protease is a cysteine protease. 215. The cell-penetrating agent of claim 214, wherein the cysteine protease is a non-lysosomal cysteine protease. 216. The cell-penetrating agent of claim 215, wherein the non-lysosomal cysteine protease is calpain. 217. The cell-penetrating agent of claim 209, wherein the enzyme is a bioluminescent protein. 218. The cell-penetrating agent of claim 217, wherein the bioluminescent protein is a luciferase. 219. The cell-penetrating agent of any one of claims 209 to 218, wherein the CMIP is linked to the C-terminus of the enzyme. 220. The cell-penetrating agent of any one of claims 209 to 218, wherein the CMIP is linked to the N-terminus of the enzyme.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 221. The cell-penetrating agent of any one of claims 209 to 220, wherein the cell-penetrating agent is a fusion protein. 222. The cell-penetrating agent of any one of claims 209 to 221, wherein the CMIP and the enzyme are expressed as a single polypeptide. 223. A pharmaceutical composition comprising the cell-penetrating agent of any one of claims 166 to 222 and a pharmaceutically acceptable excipient. 224. A kit comprising the pharmaceutical composition of claim 223. 225. A nucleic acid encoding at least a portion of the cell-penetrating agent of any one of claims 166 to 222. 226. The nucleic acid of claim 225, wherein the nucleic acid encodes the CMIP. 227. The nucleic acid of claim 225, wherein the nucleic acid encodes the CMIP and at least a portion of the payload. 228. The nucleic acid of claim 225, wherein the nucleic acid encodes the CMIP, the polypeptide linker, and at least a portion of the payload molecule. 229. The nucleic acid of claim 225, wherein the nucleic acid encodes the CMIP, the polypeptide linker, and the payload. 230. A vector comprising the nucleic acid of any one of claims 225 to 229. 231. A cell comprising the cell-penetrating agent of any one of claims 166 to 222, the nucleic acid of any one of claims 225 to 229, or the vector of claim 230. 232. The cell of claim 231, wherein the cell is a mammalian cell. 233. A method of producing the cell-penetrating agent of any one of claims 166 to 222, the method comprising: culturing a cell comprising a nucleic acid encoding the CMIP and at least a portion of the payload in a liquid culture medium under conditions that allow the cell to produce the CMIP and at least a portion of the payload molecule; and harvesting the CMIP and at least the portion of the payload molecule from the cell or the liquid culture medium. 234. A method of delivering a payload into a mammalian cell, comprising contacting a cell- penetrating agent of any one of claims 166 to 222 with the cell, thereby resulting in the internalization of the payload into the cell and transfer of the payload to the cytosol.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 235. A method of delivering an antibody into a mammalian cell, comprising contacting a cell- penetrating agent of any one of claims 187 to 208 with the cell, thereby resulting in the internalization of the antibody or a functional portion thereof into the cell and transfer of the antibody or a functional portion thereof to the cytosol. 236. A method of binding an intracellular target protein in a mammalian cell, the method comprising: contacting a cell-penetrating agent of any one of claims 166 to 208 with the cell thereby resulting in the internalization of the payload into the cell, transfer of the payload to the cytosol, and binding of the payload to the intracellular target protein. 237. A method of inhibiting at least one activity of an intracellular target protein in a cell, the method comprising: contacting a cell-penetrating agent of any one of claims 166 to 208 with the cell thereby resulting in the internalization of the payload into the cell, transfer of the payload to the cytosol, and binding of the payload to the intracellular target protein, thereby inhibiting at least one activity of the intracellular target protein. 238. A method of delivering an enzyme into a mammalian cell, comprising contacting a cell- penetrating agent of any one of claims 209 to 222 with the cell, thereby resulting in the internalization of the enzyme into the cell and transfer of the enzyme to the cytosol. 239. A method of increasing intracellular enzyme activity in a mammalian cell, the method comprising contacting a cell-penetrating agent of any one of claims 209 to 222 with the cell, thereby resulting in the internalization of the enzyme into the cell and transfer of the enzyme to the cytosol; wherein the enzyme is active in the cytosol of the mammalian cell. 240. A method of restoring intracellular enzyme activity in a mammalian cell having reduced activity of an enzyme, the method comprising contacting a cell-penetrating agent of any one of claims 209 to 222 with the cell, thereby resulting in the internalization of the enzyme into the cell and transfer of the enzyme to the cytosol; wherein the enzyme performs the same or a similar enzymatic reaction as the intracellular enzyme and the enzyme is active in the cytosol of the mammalian cell.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 241. A method of delivering a payload protein sequence to the cytosol of a mammalian cell, the method comprising contacting the cell with a polypeptide comprising the payload protein sequence and a CMIP of any one of claims 1 to 159. 242. A method of delivering a payload protein sequence to the nucleus of a mammalian cell, the method comprising contacting the cell with a polypeptide comprising the payload protein sequence and a CMIP of any one of claims 1 to 159. 243. A method of degrading a target protein in the cytosol of a mammalian cell, the method comprising contacting the cell with a polypeptide comprising a CMIP of any one of claims 1 to 159 and a protease capable of cleaving the target protein. 244. The method of any one of claims 241 to 243, wherein the payload protein sequence has a molecular weight of about 50 kD to about 500 kD. 245. The method of any one of claims 241 to 244, wherein the payload protein sequence is about 100 to about 500 amino acids. 246. A method of delivering an antigen-binding domain to the cytosol of a mammalian cell, the method comprising contacting the cell with a polypeptide comprising the antigen-binding domain and a CMIP of any one of claims 1 to 159. 247. A method of delivering an antibody to the cytosol of a mammalian cell, the method comprising contacting the cell with a polypeptide comprising the antibody and a CMIP of any one of claims 1 to 159. 248. A method of delivering a protein inhibitor of a target protein to the cytosol of a mammalian cell, the method comprising contacting the cell with a polypeptide comprising the protein inhibitor of the target protein and a CMIP of any one of claims 1 to 159. 249. A method of inhibiting an intracellular target protein in a cell, the method comprising: (a) contacting a cell-penetrating agent with the cell, thereby resulting in the internalization of the cell-penetrating agent into the cell and transfer of the cell- penetrating agent to the cytosol; wherein the cell-penetrating agent comprises: (i) an antibody that specifically binds the intracellular target protein; and (ii) a CMIP covalently linked to the antibody; and (b) binding of the antibody to the intracellular target protein.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 250. The method of claim 249, wherein the target protein is selected from a cytosolic protein, a mitochondrial protein, and an endoplasmic reticulum lumen protein. 251. The method of any one of claims 248 or 250, wherein the target protein is selected from a protease, a cytoskeletal protein, an oxidoreductase, and a mitochondrial transport protein. 252. The method of any one of claims 248 to 251, wherein the target protein is selected from beta-actin, alpha tubulin, ERO1L, TOMM20, and Nesprin-1. 253. The method of claim 105, wherein the target protein is a translocase of outer mitochondrial membrane 20 (TOMM20) protein. 254. The method of claim 249, wherein the target protein is a beta-actin protein. 255. The method of claim 249, wherein the target protein is an alpha-tubulin protein. 256. The method of claim 249, wherein the target protein is an endoplasmic reticulum oxidoreductin-1-like (EROL1L) protein. 257. The method of claim 249, wherein the target protein is a Nesprin-1 protein. 258. A method for inhibiting at least one activity of an intracellular TOMM20 protein in a mammalian cell comprising: (a) contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises: (i) an antibody that specifically binds to the TOMM20 protein; and (ii) a CMIP derived from M-lycotoxin, thereby resulting in the internalization of the antibody and transfer of the antibody to the cytosol; and (b) binding the antibody of the cell-penetrating agent to the intracellular TOMM20 protein, thereby inhibiting at least one activity of the intracellular TOMM20 protein. 259. A method for inhibiting at least one activity of an intracellular beta-actin protein in a mammalian cell comprising: (a) contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises (i) an antibody that specifically binds to the beta-actin protein, and (ii) a CMIP derived from M-lycotoxin, thereby resulting in the internalization of the antibody into the cell and transfer of the antibody to the cytosol; and (b) binding the antibody of the cell-penetrating agent to the intracellular beta-actin protein, thereby inhibiting at least one activity of the intracellular beta-actin protein.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 260. A method for at least one activity of an intracellular alpha-tubulin protein in a mammalian cell comprising: (a) contacting a cell-penetrating agent with the cell, wherein the cell-penetrating peptide comprises (i) an antibody that specifically binds to the alpha-tubulin protein, and (ii) a CMIP derived from M-lycotoxin, thereby resulting in the internalization of the antibody into the cell and transfer of the antibody to the cytosol; and (b) binding the antibody of the cell-penetrating agent to the intracellular alpha-tubulin protein, thereby inhibiting at least one activity of the intracellular alpha-tubulin protein. 261. A method for inhibiting an intracellular endoplasmic reticulum oxidoreductin-1-like protein (ERO1L) protein in a mammalian cell comprising: (a) contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises (i) an antibody that specifically binds to the ERO1L protein, and (ii) a CMIP derived from M-lycotoxin, thereby resulting in the internalization of the antibody into the cell and transfer of the antibody to the cytosol; and (b) binding the antibody of the cell-penetrating agent to the intracellular ERO1L protein, thereby inhibiting at least one activity of the intracellular ERO1L protein. 262. A method for inhibiting an intracellular Nesprin-1 protein in a mammalian cell comprising: (a) contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises (i) an antibody that specifically binds to the Nesprin-1 protein, and (ii) a CMIP derived from M-lycotoxin; thereby resulting in the internalization of the antibody into the cell and transfer of the antibody to the cytosol; and (b) binding of the antibody of the cell-penetrating agent to the intracellular Nesprin-1 protein, thereby inhibiting at least one activity of the intracellular Nesprin-1 protein. 263. The method of any one of claims 246 and 249 to 262, wherein the CMIP is covalently linked to the antibody. 264. The method of any one of claims 246 and 249 to 262, wherein the CMIP is covalently linked to the antibody via a linker.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 265. The method of claim 264, wherein the linker is a cleavable linker. 266. The method of claim 264, wherein the linker is a non-cleavable linker. 267. A method of delivering an enzyme to the cytosol of a mammalian cell, the method comprising contacting the cell with a cell-penetrating agent comprising the enzyme and a CMIP of any one of claims 1 to 159. 268. A method of increasing intracellular enzymatic activity in a mammalian cell, the method comprising: (a) contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises (i) an enzyme capable of catalyzing the intracellular enzymatic activity, and (ii) a CMIP covalently or non-covalently linked to the enzyme, thereby resulting in the internalization of the enzyme into the cell and transfer of the enzyme to the cytosol; and the enzyme performs the enzymatic activity inside the cell. 269. A method of restoring intracellular enzymatic activity in a mammalian cell having reduced intracellular enzymatic activity, the method comprising: (a) contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises (i) an enzyme capable of catalyzing the intracellular enzyme activity and (ii) a CMIP covalently or non-covalently linked to the enzyme, thereby resulting in the internalization of the enzyme into the cell and transfer of the enzyme to the cytosol; and restoration of the enzymatic activity inside the cell. 270. The method of any one of claims 267 to 269, wherein the enzyme is selected from a kinase, a protease, and a reporter enzyme. 271. The method of any one of claims 267 to 269, wherein the enzyme is selected from GSK3β, calpain, and luciferase. 272. The method of claim 271, wherein the enzyme is GSK3β. 273. The method of claim 271, wherein the enzyme is calpain. 274. The method of claim 271, wherein the enzyme is a luciferase. 275. A method of increasing at least one GSK3β activity in a mammalian cell, the method comprising:
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises (i) a GSK3β enzyme and (ii) a CMIP covalently or non-covalently linked to the GSK3β enzyme, thereby resulting in the internalization of the GSK3β enzyme into the cell and transfer of the GSK3β enzyme to the cytosol, thereby increasing at least one GSK3β activity in the mammalian cell. 276. A method of restoring at least one intracellular GSK3β activity in a mammalian cell having reduced intracellular GSK3β activity, the method comprising: contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises (i) a GSK3β enzyme and (ii) a CMIP covalently linked to the GSK3β enzyme, thereby resulting in the internalization of the GSK3β enzyme into the cell and transfer of the GSK3β enzyme to the cytosol; thereby restoring at least one intracellular GSK3β activity in the cell. 277. A method of increasing at least one activity of calpain in a mammalian cell, the method comprising: contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises (i) a calpain enzyme and (ii) a CMIP covalently or non-covalently linked to the calpain enzyme, thereby resulting in the internalization of the calpain enzyme into the cell and transfer of the calpain enzyme to the cytosol; thereby increasing at least one activity of calpain in the cell. 278. A method of restoring at least one intracellular calpain activity in a cell having reduced intracellular calpain activity, the method comprising: contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises (i) a calpain enzyme and (ii) a CMIP covalently or non-covalently linked to the calpain enzyme, thereby resulting in the internalization of the calpain enzyme into the cell, transfer of the calpain enzyme to the cytosol, and restoration of at least one intracellular calpain activity in the cell. 279. A method of increasing luciferase activity in a cell, the method comprising: contacting a cell-penetrating agent with the cell, wherein the cell-penetrating agent comprises (i) a luciferase enzyme and (ii) a CMIP covalently or non-covalently linked to the luciferase enzyme, thereby resulting in the internalization of the luciferase enzyme
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT into the cell, transfer of the luciferase enzyme to the cytosol, and an increase in luciferase activity in the cell. 280. The method of any one of claims 267 to 279, wherein the CMIP is covalently linked to the enzyme. 281. The method of any one of claims 267 to134, wherein the CMIP is covalently linked to the enzyme via a linker. 282. The method of claim 281, wherein the linker is a cleavable linker. 283. The method of claim 281, wherein the linker is a non-cleavable linker. 284. The method of any one of claims 281 to 283, wherein the linker is a polypeptide linker. 285. The method of claim 284, wherein the polypeptide linker comprises an amino acid sequence selected from Table 4. 286. The method of any one of claims 241 to 284, wherein the CMIP comprises an M- lycotoxin derivative. 287. The method of any one of claims 241 to 284, wherein the CMIP is a CMIP of any one of claims 1 to 159. 288. The method of any one of claims 241 to 284, wherein the CMIP comprises an amino acid sequence selected from the amino acid sequences of Table 3. 289. The method of any one of claims 241 to 288, wherein the cell is a mammalian cell. 290. The method of any one of claims 241 to 289, wherein the cell is in vitro. 291. The method of any one of claims 241 to 289, wherein the cell is in a subject. 292. A polypeptide comprising a CMIP and a light chain of an antibody, wherein the CMIP is a CMIP of any one of claims 1 to 159. 293. The polypeptide of claim 292, wherein the light chain of the antibody and the CMIP are connected via a polypeptide linker. 294. The polypeptide of claim 292 or 293, wherein the light chain is connected to the C- terminus of the CMIP. 295. The polypeptide of claim 292 or 293, wherein the light chain is connected to the N- terminus of the CMIP. 296. The polypeptide of any one of claims 292-295, comprising a CMIP and a heavy chain of an antibody.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 297. The polypeptide of claim 296, wherein the heavy chain of the antibody and the CMIP are connected via a polypeptide linker. 298. The polypeptide of claim 296 or 297, wherein the heavy chain is connected to the C- terminus of the CMIP. 299. The polypeptide of claim 296 or 297, wherein the heavy chain is connected to the N- terminus of the CMIP. 300. A polypeptide comprising a CMIP and a payload protein sequence, wherein the payload protein sequence is about 100 to about 1500 amino acids, wherein the CMIP is a CMIP of any one of claims 1 to 159. 301. A polypeptide comprising a CMIP and a payload protein sequence, wherein the payload protein sequence has a molecular weight of about 5 kD to about 1000 kD, wherein the CMIP is a CMIP of any one of claims 1 to 159. 302. A polypeptide comprising a CMIP and an antigen-binding domain, wherein the CMIP is a CMIP of any one of claims 1 to 159. 303. A polypeptide comprising a CMIP and an antibody, wherein the CMIP is a CMIP of any one of claims 1 to 159. 304. A polypeptide comprising a CMIP and a protein inhibitor of a kinase, wherein the CMIP is a CMIP of any one of claims 1 to 159. 305. A polypeptide comprising a CMIP and at least a portion of a protein binder of a target protein, wherein the CMIP is a CMIP of any one of claims 1 to 159. 306. The polypeptide of claim 305, wherein the protein binder is an antibody. 307. The polypeptide of claim 306, wherein at least a portion of the antibody is a heavy chain of an antibody. 308. The polypeptide of claim 306, wherein at least a portion of the antibody is a light chain of an antibody. 309. The polypeptide of any one of claims 305 to 308, wherein the target protein is selected from beta-actin, alpha tubulin, ERO1L, TOMM20, and Nesprin-1. 310. A polypeptide comprising a CMIP and an enzyme, wherein the CMIP is a CMIP of any one of claims 1 to 159. 311. A polypeptide comprising a CMIP and a kinase, wherein the CMIP is a CMIP of any one of claims 1 to 159.
Attorney Docket No.: 50887-0007WO1 // Client Ref. No.: 793-PCT 312. A polypeptide comprising a CMIP and a GSK3β enzyme, wherein the CMIP is a CMIP of any one of claims 1 to 159. 313. A polypeptide comprising a CMIP and a protease, wherein the CMIP is a CMIP of any one of claims 1 to 159. 314. A polypeptide comprising a CMIP and a calpain protease, wherein the CMIP is a CMIP of any one of claims 1 to 159. 315. A polypeptide comprising a CMIP and a reporter enzyme, wherein the CMIP is a CMIP of any one of claims 1 to 159. 316. A polypeptide comprising a CMIP and a luciferase enzyme, wherein the CMIP is a CMIP of any one of claims 1 to 159. 317. A polypeptide comprising a CMIP and a payload protein sequence, wherein the CMIP is linked to the C-terminus of the payload protein sequence, wherein the CMIP is a CMIP of any one of claims 1 to 159. 318. A polypeptide comprising a CMIP and a payload protein sequence, wherein the CMIP is linked to the N-terminus of the payload protein sequence, wherein the CMIP is a CMIP of any one of claims 1 to 159. 319. A composition comprising the polypeptide of any one of claims 292-318. 320. The composition of claim319, wherein the composition is a pharmaceutical composition. 321. A kit comprising the composition of claim 319 or the pharmaceutical composition of claim 320. 322. A method comprising contacting a mammalian cell with the cell-penetrating agent of any one of claims 166 to 222, the polypeptide of any one of claims 292 to 318, or the composition of any one of claims 223, 319, and 320.