TW202515923A - Multi-targeting protein complex and methods of use thereof - Google Patents

Abstract

This disclosure relates to protein complexes targeting CD47, PD-L1, and/or TIGIT, and methods of use thereof. In one aspect, the protein complexes include a CD47-binding domain having all or a portion of the SIRPα extracellular region; a PD-L1-binding domains having all or a portion of the PD-1 extracellular domain; and a TIGIT ligand-binding domain having all or a portion of the TIGIT extracellular region.

Description

Multi-targeted protein complex and method of use thereof

The present disclosure relates to protein complexes targeting CD47, PD-L1 and TIGIT ligands and methods of using the same.

PD-1/PD-L1 blockade monotherapy has become a promising approach for cancer treatment due to its ability to enhance anti-tumor immune responses. However, the effectiveness of PD-1/PD-L1 blockade as a monotherapy is often limited by various factors. For example, many types of cancer are resistant to anti-PD-1/PD-L1 therapy. In addition, although the PD-1/PD-L1 axis plays a central role in regulating T cell function, there are many other co-inhibitory receptor-ligand interactions, including TIGIT, that may inhibit anti-tumor function. As an immunotherapy strategy for the treatment of cancer, TIGIT blockade therapy has shown encouraging results, however, the anti-tumor efficacy of anti-TIGIT antibodies alone is still not good enough. There is an urgent need to develop immunotherapy combinations that effectively target co-inhibitory molecules expressed by T cells to enhance anti-tumor efficacy.

The present disclosure relates to protein complexes targeting CD47, PD-L1 and TIGIT ligands (e.g., PVR or fibronectin-2) and methods of using the same.

In one aspect, the present disclosure relates to a protein complex comprising: (a) Fc; (b) a TIGIT (T cell immunoreceptor with immunoglobulin and ITIM domains) ligand binding domain; (c) a PD-L1 (programmed death ligand 1) binding domain; and (d) a CD47 binding domain.

In some embodiments, the TIGIT ligand binding domain can bind to cells (e.g., cancer cells) expressing a TIGIT ligand (e.g., PVR or adhesion protein-2) and/or can block the interaction between TIGIT and the TIGIT ligand. In some embodiments, the TIGIT ligand binding domain is or includes a TIGIT extracellular domain. In some embodiments, the TIGIT extracellular domain includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1.

In some embodiments, the PD-L1 binding domain can bind to cells expressing PD-L1 (e.g., cancer cells) and/or can block the interaction between PD-1 (programmed cell death protein 1) and PD-L1. In some embodiments, the PD-L1 binding domain is or includes a PD-1 extracellular domain. In some embodiments, the PD-1 extracellular domain includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2. In some embodiments, the PD-1 extracellular domain includes a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36. In some embodiments, the amino acid corresponding to S39 of SEQ ID NO: 36 is H. In some embodiments, the PD-1 extracellular domain further comprises a PD-L1 surface interaction sequence. In some embodiments, the PD-L1 surface interaction sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 37, 38, 39 or 40. In some embodiments, the PD-L1 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 17.

In some embodiments, the CD47 binding domain can bind to cells expressing CD47 (e.g., cancer cells) and/or can block the interaction between CD47 and signal regulatory protein α (SIRPα). In some embodiments, the CD47 binding domain is or includes a SIRPα extracellular domain. In some embodiments, the SIRPα extracellular domain includes a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3. In some embodiments, the SIRPα extracellular domain includes one or more amino acid mutations at positions corresponding to H24, I31, E54, G55, H56 and/or D73 of SEQ ID NO: 3. In some embodiments, the SIRPα extracellular domain comprises one or more of the following: (a) the amino acid corresponding to H24 of SEQ ID NO: 3 is R; (b) the amino acid corresponding to I31 of SEQ ID NO: 3 is T; (c) the amino acid corresponding to E54 of SEQ ID NO: 3 is A; (d) the amino acid corresponding to G55 of SEQ ID NO: 3 is K; (e) the amino acid corresponding to H56 of SEQ ID NO: 3 is Q; and (f) the amino acid corresponding to D73 of SEQ ID NO: 3 is I. In some embodiments, the CD47 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 18.

In some embodiments, the TIGIT ligand binding domain is linked to the N-terminus of the CH2 domain in the Fc, optionally through a hinge region. In some embodiments, the PD-L1 binding domain is linked to the N-terminus of the TIGIT ligand binding domain, optionally through a first linker peptide. In some embodiments, the CD47 binding domain is linked to the C-terminus of the CH3 domain in the Fc, optionally through a second linker peptide. In some embodiments, the CD47 binding domain is linked to the C-terminus of the CH3 domain in the Fc, optionally through a first linker peptide. In some embodiments, the PD-L1 binding domain is linked to the C-terminus of the CD47 binding domain, optionally through a second linker peptide. In some embodiments, the PD-L1 binding domain is linked to the N-terminus of the CH2 domain in the Fc, optionally through a hinge region. In some embodiments, the TIGIT ligand binding domain is linked to the N-terminus of the PD-L1 binding domain, optionally through a first linker peptide. In some embodiments, the CD47 binding domain is linked to the C-terminus of the CH3 domain in the Fc, optionally through a second linker peptide. In some embodiments, the CD47 binding domain is linked to the N-terminus of the CH2 domain in the Fc, optionally through a hinge region. In some embodiments, the PD-L1 binding domain is linked to the N-terminus of the CD47 binding domain, optionally through a first linker peptide. In some embodiments, the TIGIT ligand binding domain is linked to the C-terminus of the CH3 domain in the Fc, optionally via a second linker peptide.

In some embodiments, the Fc is human IgG1 Fc. In some embodiments, the Fc is human IgG4 Fc. In some embodiments, the hinge region is a human IgG4 hinge region, which optionally has an S228P mutation according to EU numbering.

In one aspect, the present disclosure relates to a protein complex comprising (a) a first polypeptide comprising, from N-terminus to C-terminus: a first PD-L1 binding domain, an optionally selected first linker peptide, a first TIGIT ligand binding domain, an optionally selected first hinge region, a first Fc region, an optionally selected second linker peptide, and a first CD47 binding domain; and (b) a second polypeptide comprising, from N-terminus to C-terminus: a second PD-L1 binding domain, an optionally selected third linker peptide, a second TIGIT ligand binding domain, an optionally selected second hinge region, a second Fc region, an optionally selected fourth linker peptide, and a second CD47 binding domain. In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 2 or 17. In some embodiments, the first TIGIT ligand binding domain and/or the second TIGIT ligand binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 1. In some embodiments, the first CD47 binding domain and/or the second CD47 binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 3 or 18. In some embodiments, the first hinge region and/or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO: 8. In some embodiments, the first Fc region and/or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the first hinge region and/or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO: 10. In some embodiments, the first Fc region and/or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO: 11. In some embodiments, the first linker peptide and/or the third linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 4. In some embodiments, the second linker peptide and/or the fourth linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 5. In some embodiments, the first polypeptide and/or the second polypeptide comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 12, 13, 19 or 20.

In one aspect, the present disclosure relates to a protein complex comprising (a) a first polypeptide comprising, from N-terminus to C-terminus: a first TIGIT ligand binding domain, an optionally selected first linker peptide, a first PD-L1 binding domain, an optionally selected first hinge region, a first Fc region, an optionally selected second linker peptide, and a first CD47 binding domain; and (b) a second polypeptide comprising, from N-terminus to C-terminus: a second TIGIT ligand binding domain, an optionally selected third linker peptide, a second PD-L1 binding domain, an optionally selected second hinge region, a second Fc region, an optionally selected fourth linker peptide, and a second CD47 binding domain. In some embodiments, the first TIGIT binding domain and/or the second TIGIT binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 1. In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 2 or 17. In some embodiments, the first CD47 binding domain and/or the second CD47 binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 3 or 18. In some embodiments, the first hinge region and/or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO: 8. In some embodiments, the first Fc region and/or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the first hinge region and/or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO: 10. In some embodiments, the first Fc region and/or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO: 11. In some embodiments, the first linker peptide and/or the third linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 4. In some embodiments, the second linker peptide and/or the fourth linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 5. In some embodiments, the first polypeptide and/or the second polypeptide comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 14 or 21.

In one aspect, the present disclosure relates to a protein complex comprising (a) a first polypeptide comprising, from N-terminus to C-terminus: a first TIGIT ligand binding domain, an optionally selected first hinge region, a first Fc region, an optionally selected first linker peptide, a first CD47 binding domain, an optionally selected second linker peptide, and a first PD-L1 binding domain; and (b) a second polypeptide comprising, from N-terminus to C-terminus: a second TIGIT ligand binding domain, an optionally selected second hinge region, a second Fc region, an optionally selected third linker peptide, a second CD47 binding domain, an optionally selected fourth linker peptide, and a second PD-L1 binding domain. In some embodiments, the first TIGIT binding domain and/or the second TIGIT binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 1. In some embodiments, the first CD47 binding domain and/or the second CD47 binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 3 or 18. In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 2 or 17. In some embodiments, the first hinge region and/or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO: 8. In some embodiments, the first Fc region and/or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the first hinge region and/or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO: 10. In some embodiments, the first Fc region and/or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO: 11. In some embodiments, the first linker peptide and/or the third linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 5. In some embodiments, the second linker peptide and/or the fourth linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 4. In some embodiments, the first polypeptide and/or the second polypeptide comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 15 or 22.

In one aspect, the present disclosure relates to a protein complex comprising (a) a first polypeptide comprising, from N-terminus to C-terminus: a first PD-L1 binding domain, an optionally selected first linker peptide, a first CD47 binding domain, an optionally selected first hinge region, a first Fc region, an optionally selected second linker peptide, and a first TIGIT ligand binding domain; and (b) a second polypeptide comprising, from N-terminus to C-terminus: a second PD-L1 binding domain, an optionally selected third linker peptide, a second CD47 binding domain, an optionally selected second hinge region, a second Fc region, an optionally selected fourth linker peptide, and a second TIGIT ligand binding domain. In some embodiments, the first TIGIT binding domain and/or the second TIGIT binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 1. In some embodiments, the first CD47 binding domain and/or the second CD47 binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 3 or 18. In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 2 or 17. In some embodiments, the first hinge region and/or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO: 8. In some embodiments, the first Fc region and/or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO: 9. In some embodiments, the first hinge region and/or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO: 10. In some embodiments, the first Fc region and/or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO: 11. In some embodiments, the first linker peptide and/or the third linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 4. In some embodiments, the second linker peptide and/or the fourth linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 5. In some embodiments, the first polypeptide and/or the second polypeptide comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 16.

In one aspect, the disclosure relates to a nucleic acid comprising a polynucleotide encoding a protein complex described herein. In some embodiments, the nucleic acid is a DNA (e.g., cDNA) or an RNA (e.g., mRNA). In one aspect, the disclosure relates to a vector comprising one or more of the nucleic acids described herein. In one aspect, the disclosure relates to a cell comprising a vector described herein. In some embodiments, the cell is a CHO cell. In one aspect, the disclosure relates to a cell comprising one or more of the nucleic acids described herein.

In one aspect, the disclosure relates to a method of producing a protein complex, the method comprising (a) culturing a cell described herein under conditions sufficient for the cell to produce the protein complex; and (b) collecting the protein complex produced by the cell.

In one aspect, the disclosure relates to a protein conjugate comprising a protein complex as described herein, the protein conjugate being covalently bound to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxic agent or a cytostatic agent.

In one aspect, the disclosure relates to a method of treating an individual having cancer, the method comprising administering to the individual a therapeutically effective amount of a composition comprising a protein complex or protein conjugate described herein. In some embodiments, the individual has cancer cells expressing PVR, nectin-2, CD47, and/or PD-L1. In some embodiments, the cancer is breast cancer, prostate cancer, non-small cell lung cancer, pancreatic cancer, diffuse large B-cell lymphoma, mesothelioma, lung cancer, ovarian cancer, colon cancer, pleural tumor, neuroglioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, stomach cancer, bile duct cancer, head and neck cancer, blood cancer, or a combination thereof.

In one aspect, the disclosure relates to a method of reducing the growth rate of a tumor, the method comprising contacting tumor cells with an effective amount of a composition comprising a protein complex or protein conjugate described herein.

In one aspect, the disclosure relates to a method of killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising a protein complex or protein conjugate described herein.

In one aspect, the present disclosure relates to a pharmaceutical composition comprising the protein complex described herein and a pharmaceutically acceptable carrier.

As used herein, the term "protein complex" or "protein construct" refers to a complex having one or more polypeptides. In some embodiments, the protein complex has two or more polypeptides, wherein the polypeptides can bind to each other to form dimers or multimers.

As used herein, the term "TIGIT ligand binding domain" refers to a protein domain that can bind to a TIGIT ligand (e.g., PVR or adhesion protein-2). In some embodiments, the TIGIT ligand binding domain may be an anti-PVR antibody or an anti-adhesion protein-2 antibody, an antigen-binding fragment thereof (e.g., scFv or VHH), or a PVR or adhesion protein-2 binding protein or a portion thereof. In some embodiments, the TIGIT ligand binding domain may have one or more self-stabilizing domains. In some embodiments, the TIGIT ligand binding domain includes or consists of a TIGIT extracellular domain. The TIGIT may be wild-type TIGIT, human TIGIT, a polypeptide derived from wild-type TIGIT (e.g., having a mutation), or a portion thereof (e.g., an extracellular region of TIGIT or an IgV domain of TIGIT). In some embodiments, the polypeptide derived from wild-type TIGIT may have one or more mutations. In some embodiments, the TIGIT extracellular domain includes or consists of substantially the entire extracellular region of TIGIT or a variant thereof. In some embodiments, the TIGIT extracellular domain includes or consists of the IgV domain of TIGIT or a variant thereof. In some embodiments, the IgV domain has one or more mutations. In some embodiments, the TIGIT extracellular domain includes or consists of amino acids 22-137 of the human TIGIT protein (NCBI deposit number: NP_776160.2; SEQ ID NO: 42). In some embodiments, the TIGIT extracellular domain has one or more mutations.

As used herein, the term "CD47 binding domain" refers to a protein domain that can bind to CD47. In some embodiments, the CD47 binding domain may be an anti-CD47 antibody, an antigen-binding fragment thereof (e.g., scFv or VHH), or a CD47 binding protein or a portion thereof. In some embodiments, the CD47 binding domain may have one or more self-stabilizing domains. In some embodiments, the CD47 binding domain includes or consists of the extracellular domain of SIRPα. The SIRPα may be wild-type SIRPα, human SIRPα, a polypeptide derived from wild-type SIRPα (e.g., having a mutation), or a portion thereof (e.g., an extracellular region of SIRPα or an IgV domain of SIRPα). In some embodiments, a polypeptide derived from wild-type SIRPα may have one or more mutations. In some embodiments, the SIRPα extracellular domain includes or consists of substantially the entire extracellular region of SIRPα or a variant thereof. In some embodiments, the SIRPα extracellular domain includes or consists of the IgV domain of SIRPα or a variant thereof. In some embodiments, the IgV domain has one or more mutations. In some embodiments, the SIRPα extracellular domain includes or consists of amino acids 31-148 of human SIRPα protein (NCBI deposit number: AAH26692.1; SEQ ID NO: 41). In some embodiments, the SIRPα extracellular domain has one or more mutations (e.g., mutations at positions corresponding to H24, I31, E54, G55, H56 and/or D73 of SEQ ID NO: 3).

As used herein, the term "PD-L1 binding domain" refers to a protein domain that can bind to PD-L1. In some embodiments, the PD-L1 binding domain may be an anti-PD-L1 antibody, an antigen-binding fragment thereof (e.g., scFv or VHH), or a PD-L1 binding protein or a portion thereof. In some embodiments, the PD-L1 binding domain may have one or more self-stabilizing domains. In some embodiments, the PD-L1 binding domain includes or consists of a PD-1 extracellular domain. PD-1 may be wild-type PD-1, human PD-1, a polypeptide derived from wild-type PD-1 (e.g., having a mutation), or a portion thereof (e.g., an extracellular region of PD-1 or an IgV domain of PD-1). In some embodiments, the polypeptide derived from wild-type PD-1 may have one or more mutations. In some embodiments, the PD-1 extracellular domain includes substantially the entire extracellular region of PD-1 or a variant thereof or consists thereof. In some embodiments, the PD-1 extracellular domain includes a portion of the extracellular region of PD-1 or a variant thereof or consists thereof. In some embodiments, the PD-1 extracellular domain includes or consists of the IgV domain of PD-1 or a variant thereof. In some embodiments, the IgV domain has one or more mutations. In some embodiments, the PD-1 extracellular domain includes or consists of amino acids 26-170 or 35-170 of human PD-1 protein (NP_005009.2; SEQ ID NO: 35). In some embodiments, the PD-1 extracellular domain has one or more mutations (e.g., a mutation at a position corresponding to S39 of SEQ ID NO: 36). In some embodiments, the PD-1 extracellular domain comprises one or more PD-L1 surface interaction sequences described herein, optionally fused to the N-terminus of the PD-1 extracellular domain (e.g., any PD-1 extracellular domain described herein).

As used herein, the term "cancer" refers to cells capable of autonomous growth. Examples of such cells include cells having an abnormal state or condition characterized by a rapid, explosive growth of the cells. The term is intended to include cancerous growths, such as tumors; oncogenic processes, metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of the type of tissue pathology or stage of invasiveness. Also included are malignancies of various organ systems, such as the respiratory, cardiovascular, renal, reproductive, hematologic, nervous, hepatic, gastrointestinal, and endocrine systems, as well as adenocarcinomas, which include malignancies such as most colon cancers, renal cell carcinomas, prostate cancers and/or testicular tumors, non-small cell lung cancer, and small intestinal cancer. "Naturally occurring" cancers include any cancer that is not experimentally induced by transplanting cancer cells into an individual, and include, for example, cancers that arise spontaneously, cancers that result from exposure of a patient to a carcinogen, cancers that result from the insertion of a transgenic oncogene or the knockout of a tumor suppressor gene, and cancers that result from infection (e.g., viral infection). The term "carcinoma" is art-recognized and refers to a malignancy of epithelial or endocrine tissue. The term also includes carcinosarcoma, which includes malignancies composed of cancerous tissue and sarcoma tissue. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or a carcinoma in which the neoplastic cells form a recognizable glandular structure. The term "sarcoma" is art-recognized and refers to a malignancy derived from mesenchymal cells. The term "hematopoietic neoplastic disorder" includes diseases involving proliferative/neoplastic cells of hematopoietic origin. Hematopoietic neoplastic disorders may arise from cells of the myeloid, lymphoid, or erythroid lineages or their precursors. Blood cancer is a cancer that begins in blood-forming tissues such as the bone marrow or in cells of the immune system. Examples of blood cancer include leukemia, lymphoma, and multiple myeloma.

As used herein, the terms "individual" and "patient" are used interchangeably throughout the specification and describe an animal, human, or non-human to whom treatment is provided according to the methods of the present invention. Veterinary applications and non-veterinary applications are contemplated in this disclosure. A human patient may be an adult human or an adolescent human (e.g., a human under the age of 18). In addition to humans, patients include, but are not limited to, mice, rats, hamsters, guinea pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkeys, chimpanzees, gorillas, etc.), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, porcine animals (e.g., pigs, miniature pigs), horses, dogs, cats, cattle and other livestock animals, farm animals and zoo animals.

As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length of at least two amino acids.

As used herein, the terms "polynucleotide," "nucleic acid molecule," and "nucleic acid sequence" are used interchangeably herein to refer to a polymer of nucleotides of any length of at least two nucleotides, and include but are not limited to DNA, RNA, DNA/RNA hybrids, and modifications thereof.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art may also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail.

Other features and advantages of the present invention will become apparent from the following detailed description and accompanying drawings as well as the scope of the claims.

CROSS-REFERENCE TO RELATED APPLICATIONS This disclosure claims priority to and the benefit of U.S. Provisional Patent Application Serial No. 63/509,693, filed on June 22, 2023, which is incorporated herein by reference in its entirety.

SEQUENCE LISTING This application contains a sequence listing that has been filed electronically as an XML file named "52246-0015WO1_SL_ST26.XML". The size of the XML file created on June 12, 2024 is 55,931 bytes. The material in the XML file is hereby incorporated by reference in its entirety.

Specific implementation methods Immunotherapy has become a promising method for treating various cancers in the human population due to the enhanced recognition and elimination of abnormal cells, including cancer cells. Although immunotherapy has shown impressive clinical success, a large proportion of patients do not respond or develop resistance over time. Limitations and challenges that need to be addressed include loss of antigen presentation, impaired T cell function, activation of alternative immune checkpoints, and tumor immune escape. Co-inhibitory molecules expressed on immune cells (e.g., T cells) play a key role in regulating immune responses and maintaining immune homeostasis. Targeting these co-inhibitory molecules can be a promising strategy for enhancing T cell function and releasing a powerful anti-tumor immune response.

Signal regulatory protein alpha (SIRPα) is a regulatory membrane glycoprotein from the SIRP family. It is mainly expressed in bone marrow cells and additional expression can be detected in stem cells or neurons. SIRPα acts as an inhibitory receptor that interacts with the ubiquitously expressed transmembrane protein CD47. This interaction negatively regulates effector functions of innate immune cells, such as host cell phagocytosis. SIRPα diffuses laterally on the macrophage membrane and accumulates at phagocytic synapses to bind to CD47, thereby inhibiting the cytoskeletal density process of phagocytosis by macrophages. CD47 provides a "do not eat" signal by binding to the N-terminus of signal regulatory protein alpha (SIRPα). CD47 has been found to be overexpressed in many different tumor cells. Targeting CD47 and/or SIRPα can be used for cancer immunotherapy. However, given that CD47 is also expressed on red blood cells (RBCs) and platelets, inhibition of CD47/SIRPα interaction may lead to phagocytosis of RBCs and platelets.

Programmed cell death ligand 1 (PD-L1) is a transmembrane protein that is considered a co-inhibitory factor in immune responses. The ligand binds to programmed cell death protein 1 (PD-1) to reduce the proliferation of PD-1 positive cells, inhibit their interleukin secretion, and induce apoptosis. PD-L1 also plays an important role in various malignancies, where it can weaken the host immune response to tumor cells. Therefore, the PD-1/PD-L1 axis is responsible for cancer immune escape and has a huge impact on cancer therapy.

T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT) has emerged as an attractive immuno-oncology target. In naive cells, TIGIT expression is generally low. However, upon activation, both T cells and natural killer (NK) cells have been observed to upregulate TIGIT expression. Typically, TIGIT interacts with the following four ligands: CD155 (also known as PVR) and CD112 (also known as nectin-2 or PVRL2), CD113 (also known as nectin-3, PVRL3) and CD114 (nectin-4), which are expressed on antigen presenting cells and tumor cells. TIGIT functions as an immune checkpoint receptor that inhibits the activity of effector T cells and NK cells while promoting regulatory T cell (Treg) function. For example, TIGIT can inhibit CD8 ⁺ T cell or NK cell-mediated killing of tumor cells. TIGIT expression has been observed in various tumor-infiltrating immune cells, and its upregulation has been associated with immune escape and tumor progression. Therefore, targeting TIGIT by blocking therapy has become a promising approach for enhancing anti-tumor immune responses and overcoming immunosuppression.

The present disclosure provides protein complexes that bind to CD47, PD-L1, and TIGIT ligands (e.g., PVR or adhesion protein-2). These protein complexes can simultaneously target the CD47/SIRPα pathway, the PD-1/PD-L1 pathway, and the TIGIT/PVR pathway. The results show that the protein complex can effectively bind to cancer cells expressing CD47 and block the interaction between endogenous SIRPα and CD47, thereby inducing an innate immune response (e.g., phagocytosis of cancer cells by macrophages). On the other hand, the protein complex shows minimal binding to red blood cells or platelets, thereby inhibiting the clearance of host cells, as observed by the anti-CD47 antibody magrolimab. In addition, the results showed that the protein complex can selectively bind to cancer cells expressing PD-L1, block the interaction between endogenous PD-1 and PD-L1, and induce phagocytic activity against PD-L1 ⁺ tumor cells. In addition, by binding to TIGIT ligands (e.g., PVR), the protein complex can also compete with endogenous TIGIT, thereby exhibiting TIGIT-PVR blocking activity similar to the blocking activity observed by the anti-TIGIT antibody Tiragolumab. Therefore, the protein complex can bind to activated T cells and synergistically induce higher cytokine (e.g., IL-2 and IFN-γ) expression than the combination of individual functional domains.

Thus, the protein complexes described herein can be used in cancer treatment, wherein tumor immunogenicity and antigen presentation are enhanced by increasing phagocytosis by macrophages (e.g., by inactivation of CD47-mediated inhibition of phagocytosis); and T cell activation is enhanced by inhibition of PD-1/PD-L1 and TIGIT/PVR signaling pathways.

TIGIT extracellular domain TIGIT (T cell immunoglobulin and ITIM domain) is a type I transmembrane protein that is expressed on various immune cells, including T cells, regulatory T cells (Treg), natural killer (NK) cells, and dendritic cell subsets. It is also present in certain non-immune tissues. TIGIT belongs to the immunoglobulin superfamily and consists of an extracellular domain, a transmembrane domain, and a cytoplasmic tail. The extracellular region is responsible for ligand binding and is composed of an immunoglobulin variable (IgV) domain, followed by an Ig constant (IgC) domain. The IgV domain of TIGIT is responsible for binding to its ligand, mainly binding to CD155 (PVR) and CD112 (adhesion protein-2). The cytoplasmic region of TIGIT contains an inhibitory motif (ITIM) based on immunoreceptor tyrosine. After TIGIT activation, the ITIM motif recruits phosphatases, resulting in inhibition of downstream signaling pathways and blockage of immune responses. TIGIT also interacts with CD226 (DNAM-1), another protein of the immunoglobulin superfamily that acts as a co-stimulatory receptor. Binding of TIGIT to CD226 can inhibit CD226-mediated activation signals, thereby further regulating immune cell function. TIGIT expression has been observed in various tumor-infiltrating immune cells, and its upregulation is associated with immune escape and tumor progression. Therefore, targeting TIGIT by blocking therapy has become a promising method for enhancing anti-tumor immune responses and overcoming immunosuppression.

Detailed descriptions of TIGIT and its functions can be found in, for example, Manieri, N.A. et al. "TIGIT: a key inhibitor of the cancer immunity cycle". Trends in Immunology 38.1 (2017): 20-28; and Harjunpää, H. et al. "TIGIT as an emerging immune checkpoint". Clinical & Experimental Immunology 200.2 (2020): 108-119; each of which is incorporated herein by reference in its entirety.

According to the UniProt identifier Q495A1, the extracellular region of human TIGIT corresponds to amino acids 22-141 of SEQ ID NO: 42, the transmembrane region of human TIGIT corresponds to amino acids 142-162 of SEQ ID NO: 42, and the cytoplasmic region of human TIGIT corresponds to amino acids 163-244 of SEQ ID NO: 42. The TIGIT extracellular region also has an IgV domain, which corresponds to amino acids 22-124 of the human TIGIT protein (NP_776160.2; SEQ ID NO: 42). The signal peptide corresponds to amino acids 1-21 of SEQ ID NO: 42.

In some embodiments, the protein complex described herein includes one or more TIGIT ligand binding domains. In some embodiments, the TIGIT ligand binding domain is or includes a TIGIT extracellular domain. As used herein, "TIGIT extracellular domain" refers to all or a portion of the extracellular region of TIGIT or a variant thereof, wherein the portion of the extracellular region can bind to a TIGIT ligand. The TIGIT extracellular domain may have one or more protein domains that can fold independently and form a self-stabilizing structure. In some embodiments, the TIGIT extracellular domain includes or consists of an IgV domain.

In some embodiments, the amino acid sequence of the TIGIT extracellular domain described herein is provided. In some embodiments, the TIGIT extracellular domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to amino acids 22-137 of the human TIGIT protein (NCBI accession number: NP_776160.2; SEQ ID NO: 42). In some embodiments, the TIGIT extracellular domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1. In some embodiments, the TIGIT ligand binding domain or TIGIT extracellular domain described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1, and also comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) amino acid mutations.

In some embodiments, the TIGIT ligand binding domain or TIGIT extracellular domain described herein comprises the IgV domain of human TIGIT protein (wild type or mutated). In some embodiments, the TIGIT ligand binding domain or TIGIT extracellular domain described herein comprises the IgV domain of mouse TIGIT protein (wild type or mutated).

The extracellular domain of PD-1 PD-1 (programmed cell death protein 1) and its ligand PD-L1 (programmed cell death ligand 1) play a key role in immune regulation and have attracted great attention in the fields of immunology and cancer immunotherapy. PD-1 is a cell surface receptor expressed primarily on activated T cells, B cells, natural killer (NK) cells, and myeloid cells. On the other hand, PD-L1 is a ligand expressed on various immune cells as well as non-immune cells such as tumor cells. The interaction between PD-1 and PD-L1 plays a key role in maintaining immune homeostasis and preventing excessive immune activation. Under normal circumstances, the binding of PD-1 to PD-L1 provides a negative regulatory signal that attenuates immune responses and helps prevent autoimmunity and tissue damage. Therefore, the PD-1/PD-L1 pathway is critical for suppressing anti-tumor immune responses and promoting the induction and maintenance of immune tolerance within the tumor microenvironment.

However, tumor cells and certain immune cells can exploit the PD-1/PD-L1 pathway to escape immune surveillance and promote immune tolerance. In the tumor microenvironment, cancer cells often upregulate PD-L1 expression, enabling it to bind to PD-1 on immune cells. This binding effectively inhibits anti-tumor immune responses and promotes immune escape. The discovery of the role of the PD-1/PD-L1 pathway in immune escape led to the development of PD-1/PD-L1 blockade therapies, also known as immune checkpoint inhibitors. These therapies aim to block the interaction between PD-1 and PD-L1, thereby unleashing the immune system's ability to recognize and eliminate tumor cells. PD-1/PD-L1 blockade has shown remarkable clinical success, resulting in durable responses and improved outcomes in various types of cancer. By releasing the "brakes" on the immune system, these therapies restore anti-tumor immune responses, enhance T cell activation and infiltration into tumors, and promote tumor cell killing.

PD-1 (programmed cell death protein 1) (also known as CD279) is a 55-kDa transmembrane protein containing 288 amino acids and two tyrosine bases with an extracellular N-terminal domain (IgV-like), a membrane permeable domain, and a cytoplasmic tail located at the N-terminus and C-terminus, respectively. PD-1 is an inhibitor of both adaptive and innate immune responses and is expressed on activated T cells, natural killer (NK) cells and B lymphocytes, macrophages, dendritic cells (DCs), and monocytes.

PD-L1 (Programmed Death Ligand 1) (also known as CD279 and B7-H1) belongs to the B7 family and is a 33-kDa type 1 transmembrane glycoprotein containing 290 amino acids with an IgV domain and an IgC domain in its extracellular region. PD-L1 is normally expressed on macrophages, certain activated T cells, B cells, dendritic cells (DCs), and some epithelial cells, especially in the presence of inflammatory conditions. In addition, tumor cells express PD-L1 as an "adaptive immune mechanism" to escape anti-tumor responses. PD-L1 is often associated with an immune environment characterized by the presence of CD8 T cells, the production of Th1 interleukins, chemokines, interferons, and specific gene expression patterns. For example, lymphocyte-derived IFN-γ has been shown to induce PD-L1 upregulation and promote the progression of ovarian cancer. Inhibition of IFN-γ receptor 1 can reduce PD-L1 expression in mouse models of acute myeloid leukemia through the MEK/extracellular signal-regulated kinase (ERK) and MYD88/TRAF6 pathways. In addition, IFN-γ induces the expression of protein kinase D isoform 2 (PKD2), and inhibition of PKD2 activity inhibits PD-L1 expression, thereby enhancing the development of an effective anti-tumor immune response. NK cells secrete IFN-γ via the Janus kinase (JAK) 1, JAK2, and signal transducer and activator of transcription (STAT) 1 pathways, which subsequently upregulates the expression of PD-L1 on the surface of tumor cells. Studies on melanoma cells have also shown that T cells secrete IFN-γ via the JAK1/JAK2-STAT1/STAT2/STAT3-IRF1 pathway. IFN-γ secreted by T cells and NK cells has been found to induce the expression of PD-L1 on the surface of target cells, including tumor cells.

PD-L1 acts as a tumor promoter for cancer cells by engaging its receptors and activating proliferative and survival signaling pathways. This finding further supports the influence of PD-L1 in subsequent tumor progression. In addition, PD-L1 has been shown to exert non-immune proliferative effects on a variety of tumor cells. For example, PD-L1 has been observed to induce epithelial to mesenchymal transition (EMT) of kidney cancer cells and promote stem cell-like characteristics. This suggests that the intrinsic pathways of PD-L1 contribute to the progression of kidney cancer.

The use of PD-1 blockade to enhance anti-tumor immunity stems from observations in chronic infection models where prevention of PD-1 interactions reversed T cell exhaustion. Similarly, PD-1 blockade prevents T cell PD-1/tumor cell PD-L1 or T cell PD-1/tumor cell PD-L2 interactions, thereby restoring T cell-mediated anti-tumor immunity.

A detailed description of PD-L1 and its functions can be found in Han, Y. et al., “PD-1/PD-L1 pathway: current research in cancer”, Japanese Journal of Cancer Research 10.3 (2020): 727; and Liu, J. et al., “PD-1/PD-L1 checkpoint inhibitors in tumor immunotherapy”, Frontiers in Pharmacology 12 (2021), each of which is incorporated by reference in its entirety.

According to UniProt identifier Q15116, the extracellular region in human PD-1 corresponds to amino acids 24-170 of SEQ ID NO: 35, the transmembrane region of human PD-1 corresponds to amino acids 171-191 of SEQ ID NO: 35, and the cytoplasmic region of human PD-1 corresponds to amino acids 192-288 of SEQ ID NO: 35. The PD-1 extracellular region also has an IgV domain, which corresponds to amino acids 35-145 of the human PD-1 protein (NP_005009.2; SEQ ID NO: 35). The signal peptide corresponds to amino acids 1-23 of SEQ ID NO: 35.

In some embodiments, the protein complexes described herein include one or more PD-L1 binding domains. In some embodiments, the PD-L1 binding domain includes or consists of a PD-1 extracellular domain. As used herein, "PD-1 extracellular domain" refers to all or a portion of the extracellular region of PD-1 or a variant thereof, wherein the portion of the extracellular region can bind to PD-L1. The PD-1 extracellular domain may have one or more protein domains that can fold independently and form a self-stabilizing structure. In some embodiments, the PD-1 extracellular domain includes or consists of an IgV domain.

In some embodiments, the amino acid sequence of the PD-1 extracellular domain described herein is provided. In some embodiments, the PD-1 extracellular domain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to amino acids 26-170 or 35-170 of the human PD-1 protein (NP_005009.2; SEQ ID NO: 35). In some embodiments, the PD-L1 binding domain or PD-1 extracellular domain described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2 or 36. In some embodiments, the PD-L1 binding domain or PD-1 extracellular domain described herein comprises one or more mutations (e.g., a mutation at a position corresponding to S39 of SEQ ID NO: 36). In some embodiments, the PD-L1 binding domain or PD-1 extracellular domain described herein comprises histidine (H) at a position corresponding to S39 of SEQ ID NO: 36.

In some embodiments, the PD-L1 binding domain or PD-1 extracellular domain described herein comprises a PD-L1 surface interaction sequence (e.g., any one of the PD-L1 surface interaction sequences described herein). In some embodiments, the PD-L1 surface interaction sequence is fused at the N-terminus of the PD-L1 binding domain or PD-1 extracellular domain described herein (e.g., any one of SEQ ID NOs: 37, 38, 39, and 40). In some embodiments, the PD-L1 binding domain or PD-1 extracellular domain described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 17.

In some embodiments, the PD-L1 binding domain or the PD-1 extracellular domain comprises a PD-L1 surface interaction sequence comprising about or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, or 25 amino acids, wherein the PD-L1 surface interaction sequence comprises two or more histidine residues. In some embodiments, the PD-L1 surface interaction sequence comprises or consists of about or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, or 50 amino acids. In some embodiments, the PD-L1 surface interaction sequence comprises or consists of up to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, or 50 amino acids. In some embodiments, the PD-L1 surface interaction sequence comprises or consists of 5-15, 5-20, 5-30, 5-40, 10-15, 10-20, 10-30, 10-40, 15-20, 15-30, or 15-40 amino acids. In some embodiments, the PD-L1 surface interaction sequence comprises or consists of 5-15 amino acids.

In some embodiments, the PD-L1 surface interaction sequence comprises or consists of about or at least 2, 3, 4, 5 or 6 histidine residues. In some embodiments, the PD-L1 surface interaction sequence comprises or consists of up to 2, 3, 4, 5 or 6 histidine residues. In some embodiments, the PD-L1 surface interaction sequence comprises or consists of 2-3, 2-4, 2-5 or 2-6 histidine residues. In some embodiments, the PD-L1 surface interaction sequence comprises or consists of 2-4 histidine residues.

In some embodiments, the PD-L1 surface interaction sequence comprises about or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 positively charged amino acid residues (e.g., histidine, lysine or arginine) or consists thereof. In some embodiments, the PD-L1 surface interaction sequence comprises or consists of at most 2, 3, 4, 5, 6, 7, 8, 9 or 10 positively charged amino acid residues (e.g., histidine, lysine or arginine). In some embodiments, the PD-L1 surface interaction sequence comprises or consists of 2-3, 2-4, 2-5, 2-6, 2-10, 3-10 or 5-10 positively charged amino acid residues (e.g., histidine, lysine or arginine). In some embodiments, the PD-L1 surface interaction sequence comprises or consists of 2-4 positive amino acid residues (e.g., histidine, lysine, or arginine).

In some embodiments, the PD-L1 surface interaction sequence is selected from the group consisting of SHGHGGG (SEQ ID NO: 37), SHHGHGHGGGG (SEQ ID NO: 38), SHGHHGHGGGG (SEQ ID NO: 39), and SHGHGHHGGGG (SEQ ID NO: 40).

In some embodiments, the PD-L1 binding domain or PD-1 extracellular domain described herein comprises the IgV domain of human PD-1 protein (wild type or mutated). In some embodiments, the PD-L1 binding domain or PD-1 extracellular domain described herein comprises the IgV domain of mouse PD-1 protein (wild type or mutated).

SIRPα extracellular domain signal regulatory protein α (SIRPα, SIRPa, Sirpa or CD172A) is a transmembrane protein. The signal regulatory protein α has an extracellular region including three Ig-like domains and a cytoplasmic region containing an immunoreceptor tyrosine-based inhibitory motif that mediates the binding of protein tyrosine phosphatases SHP1 and SHP2. Tyrosine phosphorylation of SIRPα is regulated by various growth factors and interleukins, as well as integrin-mediated cell adhesion to extracellular matrix proteins. SIRPα is mainly present in bone marrow cells, which include macrophages and dendritic cells, and SIRPα is expressed at a higher level in these bone marrow cells. In contrast, SIRPα is expressed at a relatively low level in T cells, B cells, NK cells, and NKT cells.

The extracellular region of SIRPα can interact with its ligand CD47. The interaction between SIRPα on macrophages and CD47 on erythrocytes prevents macrophages from phagocytosing Ig-opsonized erythrocytes in vitro and in vivo. When CD47, CD47 expressed on neighboring cells, binds to SIRPα on phagocytic cells, it triggers phosphorylation of the cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM) contained in SIRPα. This phosphorylation event promotes the recruitment of SHP-1 and SHP-2 phosphatases to the SIRPα cytoplasmic domain. Therefore, one downstream effect of SIRPα expression in bone marrow cells is to prevent myosin-IIA accumulation at phagocytic synapses, thereby inhibiting phagocytosis. Thus, the CD47-SIRPα interaction functions as a negative immune checkpoint that sends a "don't eat me" signal to ensure that healthy self cells are not inappropriately engulfed.

However, overexpression of CD47 has been observed in a wide range of tumor types, including but not limited to acute myeloid leukemia, non-Hodgkin's lymphoma, bladder cancer, and breast cancer. Negative regulation of macrophages can be attenuated by inhibiting the binding of CD47 to SIRPα. Therefore, agents that block the interaction between CD47 and SIRPα can enhance antibody-dependent cellular phagocytosis (ADCP) and, in certain cases, can trigger antibody-dependent cellular cytotoxicity (ADCC), thereby leading to the recognition and elimination of cancer cells. The mechanism of blocking the binding between CD47 and SIRPα may be involved in the treatment of various types of tumors and cancers, such as solid tumors, hematological malignancies (e.g., relapsed or refractory hematological malignancies), acute myeloid leukemia, non-Hodgkin's lymphoma, breast cancer, bladder cancer, ovarian cancer, and small cell lung cancer tumors.

Additionally, SIRPα acts by inhibiting the clearance of CD47-expressing host cells such as red blood cells and platelets by macrophages in vivo. Furthermore, the CD47-SIRPα interaction also plays a key role in the successful engraftment of hematopoietic stem cells. Therefore, inhibiting the interaction between CD47 and SIRPα may inadvertently lead to the destruction of healthy red blood cells, which can lead to anemia and induce inflammation. Therefore, it is important to carefully regulate the interaction of SIRPα targeting agents with CD47 in order to limit or control the effects on red blood cells.

Detailed descriptions of SIRPα and its functions can be found in, for example, Yanagita et al., “Anti-SIRPα antibodies as a potential new tool for cancer immunotherapy.” JCI insight 2.1 (2017); Seiffert et al., “Signal-regulatory protein α (SIRPα) but not SIRPβ is involved in T ^- cell activation, binds to CD47 with high affinity, and is expressed on immature CD34 ⁺ CD38 ⁻ hematopoietic cells.” Blood 97.9 (2001 ⁾ : 2741-2749, which is incorporated herein by reference in its entirety.

Human SIRPα is a member of the signal regulatory protein (SIRP). Signal regulatory proteins are cell surface Ig superfamily proteins that mediate essential cell surface protein interactions and signal transduction. SIRPs all contain an N-terminal extracellular region, a single transmembrane domain, and a C-terminal intracellular region.

The extracellular region of human SIRPα (UniProt identifier: P78324) has an IgV domain, an Ig-like C1 type 1 domain, and an Ig-like C1 type 2 domain. The corresponding region of its specific amino acid range (NP_542970.1) includes amino acids 32-137, amino acids 148-247, and amino acids 254-348. The region of amino acids 1-30 is used for the signal peptide. Human SIRPα also has a long intracellular domain that includes two putative immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Activation of SIRPα ITIMs delivers inhibitory signals that negatively regulate cellular responses.

In some embodiments, the protein complex includes one or more CD47 binding domains. In some embodiments, the CD47 binding domain includes or consists of a SIRPα extracellular domain. As used herein, "SIRPα extracellular domain" refers to all or a portion of the extracellular region of SIRPα or a variant thereof, wherein the portion of the extracellular region can bind to CD47. The SIRPα extracellular domain may have one or more protein domains that can fold independently and form a self-stable structure. In some embodiments, the SIRPα extracellular domain includes or consists of one or more domains selected from the following: IgV domain, Ig-like C1 type 1 domain and Ig-like C1 type 2 domain. In some embodiments, the SIRPα extracellular domain includes or consists of an IgV domain. In some embodiments, the SIRPα extracellular domain includes or consists of an IgV domain and an Ig-like C1 type 1 domain. In some embodiments, the SIRPα extracellular domain includes or consists of an IgV domain, an Ig-like C1 type 1 domain, and an Ig-like C1 type 2 domain.

In some embodiments, the SIRPα extracellular domain described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to amino acids 31-148 of human SIRPα protein (NCBI accession number: AAH26692.1; SEQ ID NO: 41). In some embodiments, the CD47 binding domain or SIRPα extracellular domain described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3. In some embodiments, the CD47 binding domain or SIRPα extracellular domain described herein comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) amino acid mutations at positions corresponding to H24, I31, E54, G55, H56, and/or D73 of SEQ ID NO: 3. In some embodiments, the CD47 binding domain or SIRPα extracellular domain described herein comprises one or more of the following (e.g., 1, 2, 3, 4, 5, or 6): (a) the amino acid corresponding to H24 of SEQ ID NO: 3 is R; (b) the amino acid corresponding to I31 of SEQ ID NO: 3 is T; (c) the amino acid corresponding to E54 of SEQ ID NO: 3 is A; (d) the amino acid corresponding to G55 of SEQ ID NO: 3 is K; (e) the amino acid corresponding to H56 of SEQ ID NO: 3 is Q; and (f) the amino acid corresponding to D73 of SEQ ID NO: 3 is I. In some embodiments, the CD47 binding domain or SIRPα extracellular domain described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 18.

In some embodiments, the CD47 binding domain or SIRPα extracellular domain described herein comprises the IgV domain of human SIRPα protein (wild type or mutated). In some embodiments, the CD47 binding domain or SIRPα extracellular domain described herein comprises the IgV domain of mouse SIRPα protein (wild type or mutated).

Protein complexes targeting TIGIT ligands, PD-L1 and CD47 The present disclosure provides protein complexes that can specifically bind to TIGIT ligands (e.g., PVR or adhesion protein-2). In some embodiments, these protein complexes can block TIGIT/PVR and/or TIGIT/adhesion protein-2 signaling pathways. In some embodiments, these protein complexes can block the immunosuppressive signaling of TIGIT on NK cells, T cells and Treg cells. The present disclosure also provides protein complexes that can specifically bind to PD-L1. In some embodiments, these protein complexes can block the PD-1/PD-L1 signaling pathway, thereby increasing the immune response. In some embodiments, these protein complexes can induce T cell activation, proliferation and/or interleukin release. The disclosure also provides protein complexes that can specifically bind to CD47. In some embodiments, these protein complexes can block the SIRPα/CD47 signaling pathway, thereby increasing the immune response. In some embodiments, these protein complexes can induce phagocytosis.

In one aspect, the present disclosure provides a protein complex or protein construct comprising or consisting of Fc, one or more TIGIT ligand binding domains, one or more PD-L1 binding domains and/or one or more CD47 binding domains. As used herein, the term "Fc" refers to a fragment crystallizable region of an antibody (e.g., IgG, IgE, IgM, IgA, or IgD). The term "Fc region" or "Fc region sequence" refers to the heavy chain constant domains (e.g., CH2 and CH3) in a heavy chain peptide that form the Fc region. In some embodiments, the protein complex or protein construct comprises 1, 2, 3, 4, 5, or 6 TIGIT ligand binding domains. In some embodiments, the protein complex or protein construct comprises 1, 2, 3, 4, 5, or 6 PD-L1 binding domains. In some embodiments, the protein complex or protein construct comprises 1, 2, 3, 4, 5, or 6 CD47 binding domains.

In some embodiments, the protein complex or protein construct comprises or consists of Fc, a first domain that specifically binds to a TIGIT ligand (e.g., PVR or adhesion protein-2), a second domain that specifically binds to PD-L1, and a third domain that specifically binds to CD47.

In some embodiments, the first domain can bind to cells (e.g., cancer cells) expressing one or more TIGIT ligands (e.g., PVR and/or adhesion protein-2) and/or can block the interaction between TIGIT and the one or more TIGIT ligands. In some embodiments, the first domain includes all or part of the extracellular region of TIGIT. In some embodiments, the TIGIT is a human TIGIT extracellular domain, optionally with one or more mutations of the human TIGIT extracellular domain. In some embodiments, the first domain includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1.

In some embodiments, the second domain can bind to cells expressing PD-L1 (e.g., cancer cells) and/or can stimulate T cell activation and proliferation. In some embodiments, the second domain includes all or part of the extracellular region of PD-1. In some embodiments, PD-1 is a human PD-1 extracellular domain with one or more mutations (e.g., any PD-1 mutation described herein). In some embodiments, the second domain includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2, 17 or 36.

In some embodiments, the third domain can bind to cells expressing CD47 (e.g., cancer cells) and/or can block the interaction between CD47 and signal regulatory protein α (SIRPα). In some embodiments, the third domain includes all or a portion of the extracellular region of SIRPα. In some embodiments, SIRPα is a human SIRPα extracellular domain having one or more mutations (e.g., any SIRPα mutation described herein). In some embodiments, the first domain includes an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3 or 18.

In some embodiments, the Fc is a human IgG1 Fc. In some embodiments, the Fc is a human IgG4 Fc. In some embodiments, the first domain is linked to the N-terminus of the CH2 domain in the Fc, optionally via a hinge region (e.g., any hinge region described herein). In some embodiments, the second domain is linked to the N-terminus of the CH2 domain in the Fc, optionally via a hinge region (e.g., any hinge region described herein). In some embodiments, the third domain is linked to the N-terminus of the CH2 domain in the Fc, optionally via a hinge region (e.g., any hinge region described herein). In some embodiments, the hinge region is a human IgG4 hinge region, which optionally has an S228P mutation according to EU numbering. In some embodiments, the first domain is linked to the C-terminus of the CH3 domain in Fc, optionally through a linker peptide (e.g., any linker peptide described herein). In some embodiments, the second domain is linked to the C-terminus of the CH3 domain in Fc, optionally through a linker peptide (e.g., any linker peptide described herein). In some embodiments, the third domain is linked to the C-terminus of the CH3 domain in Fc, optionally through a linker peptide (e.g., any linker peptide described herein).

In some embodiments, the order of the first domain, the second domain, the third domain and Fc described herein from N-terminus to C-terminus can be any of the following: first domain-second domain-Fc-third domain; first domain-third domain-Fc-second domain; second domain-first domain-Fc-third domain; second domain-third domain-Fc-first domain; third domain-first domain-Fc-second domain; third domain-second domain-Fc-first domain; first domain-Fc-second domain-third domain; first domain-Fc-third domain-second domain; second domain-Fc-first domain-third domain; second domain-Fc-third domain-first domain; third domain-Fc-first domain-second domain; and third domain-Fc-second domain-first domain.

In some embodiments, the protein complex comprises two or more first domains. In some embodiments, the protein complex comprises two or more second domains. In some embodiments, the protein complex comprises two or more third domains.

In some embodiments, one or more TIGIT ligand binding domains, one or more CD47 binding domains, and one or more PD-L1 binding domains are linked to the Fc region via any of the linker peptides or hinge region sequences as described herein.

Some embodiments of protein complexes are shown in Figures 1A to 1E and 15A to 15D . They are described in detail below.

TgPS-C1_v1 and TgPS-C1_v2 In one aspect, the present disclosure relates to a protein complex comprising a first polypeptide and a second polypeptide. The first polypeptide preferably comprises from N-terminus to C-terminus: a first PD-L1 binding domain, an optionally selected first linker peptide, a first TIGIT ligand binding domain, an optionally selected first hinge region, a first Fc region, an optionally selected second linker peptide, and a first CD47 binding domain. The second polypeptide preferably comprises from N-terminus to C-terminus: a second PD-L1 binding domain, an optionally selected third linker peptide, a second TIGIT ligand binding domain, an optionally selected second hinge region, a second Fc region, an optionally selected fourth linker peptide, and a second CD47 binding domain. Schematic structures of exemplary protein complexes having the TgPS-C1_v1 form and the TgPS-C1_v2 form are shown in FIG. 1A and FIG. 15A , respectively.

In any protein complex described herein, the first TIGIT ligand binding domain and/or the second TIGIT ligand binding domain may comprise a TIGIT extracellular domain (e.g., any TIGIT extracellular domain described herein). In some embodiments, the TIGIT extracellular domain comprises amino acids 22-137 of the human TIGIT protein (SEQ ID NO: 42). In some embodiments, the first TIGIT ligand binding domain and the second TIGIT ligand binding domain are the same. In some embodiments, the first TIGIT ligand binding domain and the second TIGIT ligand binding domain are different. In some embodiments, the first TIGIT ligand binding domain and/or the second TIGIT ligand binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the TIGIT extracellular domain comprises the IgV domain of TIGIT (e.g., human TIGIT).

In any protein complex described herein, the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises a PD-1 extracellular domain (e.g., any PD-1 extracellular domain described herein). In some embodiments, the PD-1 extracellular domain comprises amino acids 26-170 or 35-170 of the human PD-1 protein (SEQ ID NO: 35). In some embodiments, the PD-1 extracellular domain comprises one or more PD-L1 surface interaction sequences fused to the N-terminus of the PD-1 extracellular domain described herein (e.g., any one of SEQ ID NO: 37-40). In some embodiments, the PD-1 extracellular domain comprises one or more mutations (e.g., a mutation at a position corresponding to S39 of SEQ ID NO: 36). In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain are the same. In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain are different. In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2, 17 or 36.

In any protein complex described herein, the first CD47 binding domain and/or the second CD47 binding domain may comprise a SIRPα extracellular domain (e.g., any SIRPα extracellular domain described herein). In some embodiments, the SIRPα extracellular domain comprises amino acids 31-148 of human SIRPα protein (SEQ ID NO: 41). In some embodiments, the SIRPα extracellular domain comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) mutations (e.g., mutations at positions corresponding to H24, I31, E54, G55, H56, and/or D73 of SEQ ID NO: 3). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are the same. In some embodiments, the first CD47 binding domain and the second CD47 binding domain are different. In some embodiments, the first CD47 binding domain and/or the second CD47 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3 or 18. In some embodiments, the SIRPα extracellular domain comprises an IgV domain of SIRPα (e.g., human SIRPα) having one or more mutations at positions corresponding to H24, I31, E54, G55, H56 and/or D73 of SEQ ID NO: 3.

In some embodiments, the first hinge region and/or the second hinge region may comprise a hinge region of an immunoglobulin, for example, all or a portion of a human IgG1 hinge region (SEQ ID NO: 8). In some embodiments, the first hinge region and/or the second hinge region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 8. In some embodiments, the first hinge region and the second hinge region are identical. In some embodiments, the first hinge region and the second hinge region are different.

In some embodiments, the first Fc region and/or the second Fc region may be identical and may form Fc homodimers. In some embodiments, the first Fc region and/or the second Fc region comprise an Fc region of an immunoglobulin, for example, all or a portion of a human IgG1 Fc region (SEQ ID NO: 9). In some embodiments, the first Fc region and/or the second Fc region comprise an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 9.

In some embodiments, the first linker peptide and/or the third linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 4. In some embodiments, the second linker peptide and/or the fourth linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 5. In some embodiments, the first linker peptide, the second linker peptide, the third linker peptide and/or the fourth linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to one or more (e.g., 1, 2, 3, 4, 5 or 6) repeats of GSG (SEQ ID NO: 31) or GGGGS (SEQ ID NO: 33).

In some embodiments, the first polypeptide and/or the second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 12 or 19.

TgPS-C2_v1 and TgPS-C2_v2 In one aspect, the present disclosure relates to a protein complex comprising a first polypeptide and a second polypeptide. The first polypeptide preferably comprises from N-terminus to C-terminus: a first PD-L1 binding domain, an optionally selected first linker peptide, a first TIGIT ligand binding domain, an optionally selected first hinge region, a first Fc region, an optionally selected second linker peptide, and a first CD47 binding domain. The second polypeptide preferably comprises from N-terminus to C-terminus: a second PD-L1 binding domain, an optionally selected third linker peptide, a second TIGIT ligand binding domain, an optionally selected second hinge region, a second Fc region, an optionally selected fourth linker peptide, and a second CD47 binding domain. Schematic structures of exemplary protein complexes having the TgPS-C2_v1 form and the TgPS-C2_v2 form are shown in FIG. 1B and FIG. 15B , respectively.

In any protein complex described herein, the first TIGIT ligand binding domain and/or the second TIGIT ligand binding domain may comprise a TIGIT extracellular domain (e.g., any TIGIT extracellular domain described herein). In some embodiments, the TIGIT extracellular domain comprises amino acids 22-137 of the human TIGIT protein (SEQ ID NO: 42). In some embodiments, the first TIGIT ligand binding domain and the second TIGIT ligand binding domain are the same. In some embodiments, the first TIGIT ligand binding domain and the second TIGIT ligand binding domain are different. In some embodiments, the first TIGIT ligand binding domain and/or the second TIGIT ligand binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the TIGIT extracellular domain comprises the IgV domain of TIGIT (e.g., human TIGIT).

In any protein complex described herein, the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises a PD-1 extracellular domain (e.g., any PD-1 extracellular domain described herein). In some embodiments, the PD-1 extracellular domain comprises amino acids 26-170 or 35-170 of the human PD-1 protein (SEQ ID NO: 35). In some embodiments, the PD-1 extracellular domain comprises one or more PD-L1 surface interaction sequences fused to the N-terminus of the PD-1 extracellular domain described herein (e.g., any one of SEQ ID NO: 37-40). In some embodiments, the PD-1 extracellular domain comprises one or more mutations (e.g., a mutation at a position corresponding to S39 of SEQ ID NO: 36). In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain are the same. In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain are different. In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2, 17 or 36.

In any protein complex described herein, the first CD47 binding domain and/or the second CD47 binding domain may comprise a SIRPα extracellular domain (e.g., any SIRPα extracellular domain described herein). In some embodiments, the SIRPα extracellular domain comprises amino acids 31-148 of human SIRPα protein (SEQ ID NO: 41). In some embodiments, the SIRPα extracellular domain comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) mutations (e.g., mutations at positions corresponding to H24, I31, E54, G55, H56, and/or D73 of SEQ ID NO: 3). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are the same. In some embodiments, the first CD47 binding domain and the second CD47 binding domain are different. In some embodiments, the first CD47 binding domain and/or the second CD47 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3 or 18. In some embodiments, the SIRPα extracellular domain comprises an IgV domain of SIRPα (e.g., human SIRPα) having one or more mutations at positions corresponding to H24, I31, E54, G55, H56 and/or D73 of SEQ ID NO: 3.

In some embodiments, the first hinge region and/or the second hinge region may comprise a hinge region of an immunoglobulin, for example, all or a portion of a human IgG4 hinge region (SEQ ID NO: 10). In some embodiments, the first hinge region and/or the second hinge region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10. In some embodiments, the first hinge region and the second hinge region are identical. In some embodiments, the first hinge region and the second hinge region are different.

In some embodiments, the first Fc region and/or the second Fc region may be identical and may form Fc homodimers. In some embodiments, the first Fc region and/or the second Fc region comprise an Fc region of an immunoglobulin, for example, all or a portion of a human IgG4 Fc region (SEQ ID NO: 11). In some embodiments, the first Fc region and/or the second Fc region comprise an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 11. In some embodiments, the first hinge region and/or the second hinge region comprise a proline at position 228 according to EU numbering.

In some embodiments, the first linker peptide and/or the third linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 4. In some embodiments, the second linker peptide and/or the fourth linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 5. In some embodiments, the first linker peptide, the second linker peptide, the third linker peptide and/or the fourth linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to one or more (e.g., 1, 2, 3, 4, 5 or 6) repeats of GSG (SEQ ID NO: 31) or GGGGS (SEQ ID NO: 33).

In some embodiments, the first polypeptide and/or the second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 13 or 20.

TgPS-D_v1 and TgPS-D_v2 In one aspect, the present disclosure relates to a protein complex comprising a first polypeptide and a second polypeptide. The first polypeptide preferably comprises from N-terminus to C-terminus: a first TIGIT ligand binding domain, an optionally selected first linker peptide, a first PD-L1 binding domain, an optionally selected first hinge region, a first Fc region, an optionally selected second linker peptide, and a first CD47 binding domain. The second polypeptide preferably comprises from N-terminus to C-terminus: a second TIGIT ligand binding domain, an optionally selected third linker peptide, a second PD-L1 binding domain, an optionally selected second hinge region, a second Fc region, an optionally selected fourth linker peptide, and a second CD47 binding domain. Schematic structures of exemplary protein complexes having the TgPS-D_v1 form and the TgPS-D_v2 form are shown in FIG. 1C and FIG. 15C , respectively.

In any protein complex described herein, the first TIGIT ligand binding domain and/or the second TIGIT ligand binding domain may comprise a TIGIT extracellular domain (e.g., any TIGIT extracellular domain described herein). In some embodiments, the TIGIT extracellular domain comprises amino acids 22-137 of the human TIGIT protein (SEQ ID NO: 42). In some embodiments, the first TIGIT ligand binding domain and the second TIGIT ligand binding domain are the same. In some embodiments, the first TIGIT ligand binding domain and the second TIGIT ligand binding domain are different. In some embodiments, the first TIGIT ligand binding domain and/or the second TIGIT ligand binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the TIGIT extracellular domain comprises the IgV domain of TIGIT (e.g., human TIGIT).

In any protein complex described herein, the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises a PD-1 extracellular domain (e.g., any PD-1 extracellular domain described herein). In some embodiments, the PD-1 extracellular domain comprises amino acids 26-170 or 35-170 of the human PD-1 protein (SEQ ID NO: 35). In some embodiments, the PD-1 extracellular domain comprises one or more PD-L1 surface interaction sequences fused to the N-terminus of the PD-1 extracellular domain described herein (e.g., any one of SEQ ID NO: 37-40). In some embodiments, the PD-1 extracellular domain comprises one or more mutations (e.g., a mutation at a position corresponding to S39 of SEQ ID NO: 36). In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain are the same. In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain are different. In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2, 17 or 36.

In any protein complex described herein, the first CD47 binding domain and/or the second CD47 binding domain may comprise a SIRPα extracellular domain (e.g., any SIRPα extracellular domain described herein). In some embodiments, the SIRPα extracellular domain comprises amino acids 31-148 of human SIRPα protein (SEQ ID NO: 41). In some embodiments, the SIRPα extracellular domain comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) mutations (e.g., mutations at positions corresponding to H24, I31, E54, G55, H56, and/or D73 of SEQ ID NO: 3). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are the same. In some embodiments, the first CD47 binding domain and the second CD47 binding domain are different. In some embodiments, the first CD47 binding domain and/or the second CD47 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3 or 18. In some embodiments, the SIRPα extracellular domain comprises an IgV domain of SIRPα (e.g., human SIRPα) having one or more mutations at positions corresponding to H24, I31, E54, G55, H56 and/or D73 of SEQ ID NO: 3.

In some embodiments, the first hinge region and/or the second hinge region may comprise a hinge region of an immunoglobulin, for example, all or a portion of a human IgG4 hinge region (SEQ ID NO: 10). In some embodiments, the first hinge region and/or the second hinge region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10. In some embodiments, the first hinge region and the second hinge region are identical. In some embodiments, the first hinge region and the second hinge region are different.

In some embodiments, the first Fc region and/or the second Fc region may be identical and may form Fc homodimers. In some embodiments, the first Fc region and/or the second Fc region comprise an Fc region of an immunoglobulin, for example, all or a portion of a human IgG4 Fc region (SEQ ID NO: 11). In some embodiments, the first Fc region and/or the second Fc region comprise an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 11. In some embodiments, the first hinge region and/or the second hinge region comprise a proline at position 228 according to EU numbering.

In some embodiments, the first linker peptide and/or the third linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 4. In some embodiments, the second linker peptide and/or the fourth linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 5. In some embodiments, the first linker peptide, the second linker peptide, the third linker peptide and/or the fourth linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to one or more (e.g., 1, 2, 3, 4, 5 or 6) repeats of GSG (SEQ ID NO: 31) or GGGGS (SEQ ID NO: 33).

In some embodiments, the first polypeptide and/or the second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 14 or 21.

TgPS-E_v1 and TgPS-E_v2 In one aspect, the present disclosure relates to a protein complex comprising a first polypeptide and a second polypeptide. The first polypeptide preferably comprises from N-terminus to C-terminus: a first TIGIT ligand binding domain, an optionally selected first hinge region, a first Fc region, an optionally selected first linker peptide, a first CD47 binding domain, an optionally selected second linker peptide, and a first PD-L1 binding domain. The second polypeptide preferably comprises from N-terminus to C-terminus: a second TIGIT ligand binding domain, an optionally selected second hinge region, a second Fc region, an optionally selected third linker peptide, a second CD47 binding domain, an optionally selected fourth linker peptide, and a second PD-L1 binding domain. Schematic structures of exemplary protein complexes having TgPS-E_v1 form and TgPS-E_v2 form are shown in FIG. 1D and FIG. 15D , respectively.

In any protein complex described herein, the first TIGIT ligand binding domain and/or the second TIGIT ligand binding domain may comprise a TIGIT extracellular domain (e.g., any TIGIT extracellular domain described herein). In some embodiments, the TIGIT extracellular domain comprises amino acids 22-137 of the human TIGIT protein (SEQ ID NO: 42). In some embodiments, the first TIGIT ligand binding domain and the second TIGIT ligand binding domain are the same. In some embodiments, the first TIGIT ligand binding domain and the second TIGIT ligand binding domain are different. In some embodiments, the first TIGIT ligand binding domain and/or the second TIGIT ligand binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the TIGIT extracellular domain comprises the IgV domain of TIGIT (e.g., human TIGIT).

In any protein complex described herein, the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises a PD-1 extracellular domain (e.g., any PD-1 extracellular domain described herein). In some embodiments, the PD-1 extracellular domain comprises amino acids 26-170 or 35-170 of the human PD-1 protein (SEQ ID NO: 35). In some embodiments, the PD-1 extracellular domain comprises one or more PD-L1 surface interaction sequences fused to the N-terminus of the PD-1 extracellular domain described herein (e.g., any one of SEQ ID NO: 37-40). In some embodiments, the PD-1 extracellular domain comprises one or more mutations (e.g., a mutation at a position corresponding to S39 of SEQ ID NO: 36). In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain are the same. In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain are different. In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2, 17 or 36.

In any protein complex described herein, the first CD47 binding domain and/or the second CD47 binding domain may comprise a SIRPα extracellular domain (e.g., any SIRPα extracellular domain described herein). In some embodiments, the SIRPα extracellular domain comprises amino acids 31-148 of human SIRPα protein (SEQ ID NO: 41). In some embodiments, the SIRPα extracellular domain comprises one or more (e.g., 1, 2, 3, 4, 5, or 6) mutations (e.g., mutations at positions corresponding to H24, I31, E54, G55, H56, and/or D73 of SEQ ID NO: 3). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are the same. In some embodiments, the first CD47 binding domain and the second CD47 binding domain are different. In some embodiments, the first CD47 binding domain and/or the second CD47 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3 or 18. In some embodiments, the SIRPα extracellular domain comprises an IgV domain of SIRPα (e.g., human SIRPα) having one or more mutations at positions corresponding to H24, I31, E54, G55, H56 and/or D73 of SEQ ID NO: 3.

In some embodiments, the first hinge region and/or the second hinge region may comprise a hinge region of an immunoglobulin, for example, all or a portion of a human IgG4 hinge region (SEQ ID NO: 10). In some embodiments, the first hinge region and/or the second hinge region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10. In some embodiments, the first hinge region and the second hinge region are identical. In some embodiments, the first hinge region and the second hinge region are different.

In some embodiments, the first Fc region and/or the second Fc region may be identical and may form Fc homodimers. In some embodiments, the first Fc region and/or the second Fc region comprise an Fc region of an immunoglobulin, for example, all or a portion of a human IgG4 Fc region (SEQ ID NO: 11). In some embodiments, the first Fc region and/or the second Fc region comprise an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 11. In some embodiments, the first hinge region and/or the second hinge region comprise a proline at position 228 according to EU numbering.

In some embodiments, the first linker peptide and/or the third linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 5. In some embodiments, the second linker peptide and/or the fourth linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 4. In some embodiments, the first linker peptide, the second linker peptide, the third linker peptide and/or the fourth linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to one or more (e.g., 1, 2, 3, 4, 5 or 6) repeats of GSG (SEQ ID NO: 31) or GGGGS (SEQ ID NO: 33).

In some embodiments, the first polypeptide and/or the second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 15 or 22.

TgPS-F_v1 In one aspect, the present disclosure relates to a protein complex comprising a first polypeptide and a second polypeptide. The first polypeptide preferably comprises from N-terminus to C-terminus: a first PD-L1 binding domain, an optionally selected first linker peptide, a first CD47 binding domain, an optionally selected first hinge region, a first Fc region, an optionally selected second linker peptide, and a first TIGIT ligand binding domain. The second polypeptide preferably comprises from N-terminus to C-terminus: a second PD-L1 binding domain, an optionally selected third linker peptide, a second CD47 binding domain, an optionally selected second hinge region, a second Fc region, an optionally selected fourth linker peptide, and a second TIGIT ligand binding domain. A schematic structure of an exemplary protein complex having the form of TgPS-F_v1 is shown in FIG . 1E .

In any protein complex described herein, the first TIGIT ligand binding domain and/or the second TIGIT ligand binding domain may comprise a TIGIT extracellular domain (e.g., any TIGIT extracellular domain described herein). In some embodiments, the TIGIT extracellular domain comprises amino acids 22-137 of the human TIGIT protein (SEQ ID NO: 42). In some embodiments, the first TIGIT ligand binding domain and the second TIGIT ligand binding domain are the same. In some embodiments, the first TIGIT ligand binding domain and the second TIGIT ligand binding domain are different. In some embodiments, the first TIGIT ligand binding domain and/or the second TIGIT ligand binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 1. In some embodiments, the TIGIT extracellular domain comprises the IgV domain of TIGIT (e.g., human TIGIT).

In any of the protein complexes described herein, the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises a PD-1 extracellular domain (e.g., any PD-1 extracellular domain described herein). In some embodiments, the PD-1 extracellular domain comprises amino acids 26-170 or 26-170 of the human PD-1 protein (SEQ ID NO: 35). In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain are the same. In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain are different. In some embodiments, the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2.

In any protein complex described herein, the first CD47 binding domain and/or the second CD47 binding domain may comprise a SIRPα extracellular domain (e.g., any SIRPα extracellular domain described herein). In some embodiments, the SIRPα extracellular domain comprises amino acids 31-148 of human SIRPα protein (SEQ ID NO: 41). In some embodiments, the first CD47 binding domain and the second CD47 binding domain are identical. In some embodiments, the first CD47 binding domain and the second CD47 binding domain are different. In some embodiments, the first CD47 binding domain and/or the second CD47 binding domain comprise an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3. In some embodiments, the SIRPα extracellular domain comprises the IgV domain of SIRPα (e.g., human SIRPα).

In some embodiments, the first hinge region and/or the second hinge region may comprise a hinge region of an immunoglobulin, for example, all or a portion of a human IgG4 hinge region (SEQ ID NO: 10). In some embodiments, the first hinge region and/or the second hinge region comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 10. In some embodiments, the first hinge region and the second hinge region are identical. In some embodiments, the first hinge region and the second hinge region are different.

In some embodiments, the first Fc region and/or the second Fc region may be identical and may form Fc homodimers. In some embodiments, the first Fc region and/or the second Fc region comprise an Fc region of an immunoglobulin, for example, all or a portion of a human IgG4 Fc region (SEQ ID NO: 11). In some embodiments, the first Fc region and/or the second Fc region comprise an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 11. In some embodiments, the first hinge region and/or the second hinge region comprise a proline at position 228 according to EU numbering.

In some embodiments, the first linker peptide and/or the third linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 4. In some embodiments, the second linker peptide and/or the fourth linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to SEQ ID NO: 5. In some embodiments, the first linker peptide, the second linker peptide, the third linker peptide and/or the fourth linker peptide described herein comprises an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to one or more (e.g., 1, 2, 3, 4, 5 or 6) repeats of GSG (SEQ ID NO: 31) or GGGGS (SEQ ID NO: 33).

In some embodiments, the first polypeptide and/or the second polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 16.

Properties of Protein Complexes In some embodiments, a protein complex may include any TIGIT ligand binding domain, PD-L1 binding domain, and/or CD47 binding domain as described herein. The disclosure also provides a nucleic acid comprising a polynucleotide encoding a polypeptide described herein.

In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison (e.g., a gap can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal comparison, and non-homologous sequences can be ignored for the purpose of comparison). The length of the reference sequence aligned for the purpose of comparison is at least 80% of the length of the reference sequence, and in some embodiments at least 90%, 95% or 100%. The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For example, comparison of sequences and determination of the percent identity between two sequences can be accomplished using the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

The protein complexes described herein may comprise the Fc region of an antibody. Such antibodies may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) class or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE1, IgE2). In some embodiments, the Fc region is derived from human IgG (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the Fc region is an IgG4 Fc region (e.g., a human IgG4 Fc region).

In some embodiments, the protein complexes described herein are linked to the Fc region via an antibody hinge region (e.g., IgG, IgE hinge region). In addition, the Fc region can be modified to provide desired effector functions or serum half-life.

The protein complexes described herein can block the binding of endogenous TIGIT derived from immune cells to TIGIT ligands (e.g., PVR and adhesion protein-2). In some embodiments, by binding to TIGIT ligands, the protein complexes described herein can inhibit the interaction between one or more TIGIT ligands (e.g., TIGIT ligands expressed on tumor cells) and endogenous TIGIT expressed on immune cells (e.g., bone marrow cells, macrophages and/or dendritic cells). This therefore leads to interruption of TIGIT/TIGIT ligand signaling pathways (e.g., TIGIT/PVR and TIGIT/adhesion protein-2 signaling pathways), thereby enhancing T cell activation and upregulating immune responses.

The protein complexes described herein can block the binding between PD-L1 and endogenous PD-1 expressed on immune cells. In some embodiments, by binding to PD-L1, the protein complexes described herein can inhibit the binding of PD-L1 (e.g., expressed on tumor cells) to endogenous PD-1 derived from immune cells (e.g., T cells), thereby blocking the PD-1/PD-L1 pathway, upregulating immune responses, promoting T cell proliferation and cytokine release.

The protein complexes described herein can block the binding between CD47 and endogenous SIRPα expressed on immune cells. In some embodiments, by binding to CD47, the protein complexes described herein can inhibit the binding of CD47 (e.g., expressed on tumor cells) to endogenous SIRPα expressed on immune cells (e.g., bone marrow cells, macrophages, and dendritic cells), thereby blocking the CD47/SIRPα pathway and further enhancing immune response and phagocytosis.

In some embodiments, the protein complexes described herein can increase the immune response, activity, or number of immune cells (e.g., bone marrow cells, macrophages, dendritic cells, antigen presenting cells) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, or 20-fold.

In some embodiments, the protein complexes described herein can bind to CD47 (e.g., human CD47, monkey CD47, or mouse CD47), PD-L1 (e.g., human PD-L1, monkey PD-L1, or mouse PD-L1), or TIGIT ligand (e.g., human TIGIT ligand, monkey TIGIT ligand, or mouse TIGIT ligand) with an off rate (koff) of less than 0.1 s ^-1 , less than 0.01 s ^-1 , less than 0.001 s ^-1 , less than 0.0001 s ^-1 , or less than 0.00001 s ^-1 . In some embodiments, the dissociation rate (koff) is greater than 0.01 s ^-1 , greater than 0.001 s ^-1 , greater than 0.0001 s ^-1 , greater than 0.00001 s ^-1 , or greater than 0.000001 s ^-1 . In some embodiments, the kinetic association rate (kon) is greater than 1 × 10 ² /Ms, greater than 1 × 10 ³ /Ms, greater than 1 × 10 ⁴ /Ms, greater than 1 × 10 ⁵ /Ms, or greater than 1 × 10 ⁶ /Ms. In some embodiments, the kinetic association rate (kon) is less than 1 × 10 ⁵ /Ms, less than 1 × 10 ⁶ /Ms, or less than 1 × 10 ⁷ /Ms. Affinity can be derived from the quotient of the kinetic rate constants (KD = koff/kon). In some embodiments, KD is less than 1 × ^10-6 M, less than 1 × ^10-7 M, less than 1 × ^10-8 M, less than 1 × ^10-9 M, or less than 1 × ^10-10 M. In some embodiments, the KD is less than 300 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 90 pM, 80 pM, 70 pM, 60 pM, 50 pM, 40 pM, 30 pM, 20 pM, or 10 pM. In some embodiments, KD is greater than 1× ^10-7 M, greater than 1× ^10-8 M, greater than 1× ^10-9 M, greater than 1× ^10-10 M, greater than 1× ^10-11 M, or greater than 1× ^10-12 M.

Common techniques for measuring affinity include, for example, ELISA, RIA, and surface plasmon resonance (SPR). In some embodiments, the protein complexes described herein can bind to monkey CD47 and/or mouse CD47. In some embodiments, the protein complexes described herein cannot bind to monkey CD47 and/or mouse CD47. In some embodiments, the protein complexes described herein can bind to monkey PD-L1 and/or mouse PD-L1. In some embodiments, the protein complexes described herein cannot bind to monkey PD-L1 and/or mouse PD-L1. In some embodiments, the protein complexes described herein can bind to monkey TIGIT ligands and/or mouse TIGIT ligands. In some embodiments, the protein complexes described herein cannot bind to monkey TIGIT ligands and/or mouse TIGIT ligands.

In some embodiments, thermal stability is determined. The Tm of the protein complexes described herein may be greater than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C. In some embodiments, Tm is less than 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C or 95°C.

In some embodiments, the protein complexes described herein have a tumor growth inhibition percentage (TGI%) greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200%. In some embodiments, the protein complexes described herein have a tumor growth inhibition percentage less than 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200%. TGI% can be determined, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days after the start of treatment, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the start of treatment. As used herein, the tumor growth inhibition percentage (TGI%) is calculated using the following formula: TGI (%) = [1-(Ti-T0)/(Vi-V0)] × 100 Ti is the average tumor volume of the treatment group on day i. T0 is the average tumor volume of the treatment group on day zero. Vi is the average tumor volume of the control group on day i. V0 is the average tumor volume of the control group on day zero.

In some embodiments, the tumor inhibitory effect of the protein complexes described herein is comparable to the tumor inhibitory effect of an anti-CD47 reference antibody (e.g., mololimab (Hu5F9-G4)) and/or an anti-TIGIT reference antibody (e.g., tisleliumab). Mololimab and tisleliumab are described, for example, in Sikic et al., "First-in-human, first-in-class phase I trial of the anti-CD47 antibody Hu5F9-G4 in patients with advanced cancers." Journal of Clinical Oncology 37.12 (2019): 946; and Rousseau, A. et al., "Anti-TIGIT therapies for solid tumors: a systematic review." ESMO Open 8.2 (2023): 101184; each of which is incorporated herein by reference in its entirety. In some embodiments, the tumor inhibition effect of the protein complex described herein is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 2-fold or 5-fold that of the anti-CD47 reference antibody or anti-TIGIT reference antibody described herein.

In some embodiments, the tumor inhibition of the protein complexes described herein is comparable to that of an anti-PD-L1 reference antibody (e.g., atezolizumab (MPDL3280A)) or an anti-PD-1 antibody (e.g., pembrolizumab). MPDL3280A is described, for example, in Powles, T. et al. "MPDL3280A (anti-PD-L1) treatment leads to clinical activity in metastatic bladder cancer". Nature 515.7528 (2014): 558-562, which is incorporated herein by reference in its entirety.

In some embodiments, the protein complexes described herein have a functional Fc. In some embodiments, the Fc is from human IgG1, human IgG2, human IgG3, or human IgG4. In some embodiments, the effector function of the functional Fc is antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the effector function of the functional Fc region is phagocytosis. In some embodiments, the effector functions of the functional Fc are ADCC and phagocytosis. In some embodiments, the protein constructs as described herein have an Fc region without effector functions. In some embodiments, the Fc is human IgG4 Fc. In some embodiments, the Fc does not have a functional Fc region. For example, the Fc region has a LALA mutation (L234A and L235A mutations in EU numbering) or a LALA-PG mutation (L234A, L235A, P329G mutations in EU numbering).

Some other modifications may be made to the Fc region. For example, cysteine residues may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The resulting homodimeric fusion protein may have any increased half-life in vitro and/or in vivo.

In some embodiments, the IgG4 has an S228P mutation (EU numbering). The S228P mutation prevents IgG4 Fab arm exchange in vivo and in vitro.

In some embodiments, the Fc region is provided with a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an Fc region composition may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. For example, the amount of fucose is determined by calculating the average amount of fucose at Asn297 within the sugar chain relative to the sum of all sugar structures (e.g., complex, hybrid, and high mannose structures) attached to Asn 297 as measured by MALDI-TOF mass spectrometry as described in WO 2008/077546. Asn297 refers to the asparagine residue at approximately position 297 (Eu numbering of Fc region residues; or position 314 in Kabat numbering) in the Fc region; however, due to minor sequence variations in the Fc region sequence, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants may have improved ADCC function. In some embodiments, in order to reduce glycan heterogeneity, the Fc region may be further engineered to replace the asparagine at position 297 with alanine (N297A).

In some embodiments, the main peak of HPLC-SEC accounts for at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% of the protein complex after purification by Protein A-based affinity chromatography and/or size exclusion chromatography described herein.

In some embodiments, the protein complexes described herein can bind to a TIGIT ligand (e.g., PVR-ECD/Fc, PVR-ECD/His or nectin-2-ECD/Fc) with at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140% or at least 150% of the affinity of a reference protein (e.g., TIGIT/G4Fc or TIGIT/G1Fc). In some embodiments, the protein complexes described herein can bind to PD-L1 (e.g., PD-L1-ECD/Fc or PD-L1-ECD/His) with an affinity of at least 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 400%, or at least 500% of the affinity of a reference protein (e.g., PD-1/G4Fc or PD-1-mt13/G4Fc). In some embodiments, the protein complexes described herein can bind to CD47 (e.g., CD47-ECD/His) with at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the affinity of a reference protein (e.g., SIRPα/G4Fc, SIRPα-mt15/G4Fc, or SIRPα-mt15/G1Fc).

In some embodiments, the protein complexes described herein can bind to tumor cells expressing human PD-L1 (e.g., human PD-L1 tf CHO-S cells) with an affinity of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% of the affinity of a reference protein (e.g., PD-1/G4Fc, PD-1-mt13/G4Fc, or PD-1-mt13/G1Fc). In some embodiments, the protein complexes described herein can bind to tumor cells expressing human CD47 (e.g., human CD47 tf CHO-S cells) with at least 5%, at least 10%, at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% of the affinity of a reference protein (e.g., SIRPα/G4Fc, SIRPα-mt15/G4Fc, or SIRPα-mt15/G1Fc) or a reference anti-CD47 antibody (e.g., a mololimab analog). In some embodiments, the protein complexes described herein can bind to tumor cells expressing a TIGIT ligand (e.g., human PVR tf CHO-S cells) with at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, or at least 150% of the affinity of a reference protein (e.g., TIGIT/G4Fc or TIGIT/G1Fc).

In some embodiments, the protein complexes described herein can bind to tumor cells expressing human PD-L1 (e.g., PD-L1 tf OE19) with at least 10%, at least 20%, 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250%, or at least 300% of the affinity of a reference protein (e.g., PD-1/G4Fc). In some embodiments, the protein complexes described herein can selectively bind to cells expressing PD-L1 (e.g., PD-L1 tf OE19), for example, in a cell mixture of cells expressing PD-L1 and non-transfected cells. In some embodiments, the selectivity of the protein complex is equal to or greater than that of a reference protein (e.g., PD-1/G4Fc).

In some embodiments, the protein complexes described herein can bind to RBC cells or platelets (e.g., from human donors) with an affinity of less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, less than 3% or less than 1% of the affinity of an anti-CD47 reference antibody (e.g., a mololimab analog) or a reference protein (e.g., SIRPα/G4Fc, SIRPα/G1Fc, SIRPα-mt15/G4Fc or SIRPα-mt15/G1Fc).

In some embodiments, the protein complexes described herein can bind to activated T cells with at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110% or at least 120%, at least 130%, at least 140%, at least 150%, at least 170%, at least 180%, at least 190%, at least 200%, at least 250% or at least 300% of the affinity of a reference protein (e.g., TIGIT/G4Fc, TIGIT/G1Fc, PD-1/G4Fc, PD-1/G1Fc, SIRPα/G4Fc or SIRPα/G1Fc).

In some embodiments, the protein complexes described herein can block the interaction between a TIGIT ligand (e.g., human PVR or a fragment thereof) and TIGIT (e.g., human TIGIT or a fragment thereof). In some embodiments, the protein complexes described herein can block the interaction between cells expressing a human TIGIT ligand (e.g., PVR tf CHO-S cells) and human TIGIT. In some embodiments, the blocking ability of the protein complex described herein is at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140% or at least 150% of the blocking ability of a reference anti-TIGIT antibody (e.g., a tisleliumab analog) or a reference protein (e.g., TIGIT/G4Fc or TIGIT/G1Fc).

In some embodiments, the protein complexes described herein can block the interaction between PD-L1 (e.g., human PD-L1 or a fragment thereof) and PD-1 (e.g., human PD-1 or a fragment thereof). In some embodiments, the protein complexes described herein can block the interaction between cells expressing human PD-L1 (e.g., PD-L1 tf CHO-S cells) and human PD-1. In some embodiments, the blocking ability of the protein complex described herein is at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140% or at least 150% of the blocking ability of a reference protein (e.g., PD-1/G1Fc, PD-1-mt13/G4Fc or PD-1-mt13/G1Fc).

In some embodiments, the protein complex described herein can block the interaction between PD-1 and PD-L1, thereby increasing the signal of the NFAT-Luc signaling blockade assay by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% compared to the signal of a reference protein (e.g., PD-1-mt13/G4Fc or PD-1-mt13/G1Fc).

In some embodiments, the protein complexes described herein can block the interaction between CD47 (e.g., human CD47 or a fragment thereof) and SIRPα (e.g., human SIRPα or a fragment thereof). In some embodiments, the protein complexes described herein can block the interaction between cells expressing human CD47 (e.g., CD47 tf CHO-S cells) and human SIRPα. In some embodiments, the blocking ability of the protein complex described herein is at least 20, at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140% or at least 150% of the blocking ability of an anti-CD47 reference antibody (e.g., a mololimab analog) or a reference protein (e.g., SIRPα/G1Fc, SIRPα-mt15/G4Fc or SIRPα-mt15/G1Fc).

In some embodiments, the protein complexes described herein do not induce hemagglutination. In some embodiments, the protein complexes described herein can induce hemagglutination at a minimum concentration greater than 500 times, 2000 times, 5000 times, 20000 times, or 50000 times the minimum concentration of an anti-CD47 reference antibody (e.g., a mololimab analog).

In some embodiments, the protein complexes described herein can induce macrophages (e.g., mouse RAW264.7 cells) to phagocytose tumor cells. In some embodiments, the ability of the protein complexes described herein to induce macrophages to phagocytose tumor cells (e.g., FaDu cells or tumor cells expressing PD-L1 (e.g., PD-L1 tf OE19)) is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the ability of a reference anti-CD47 antibody (e.g., mololimab analog) or a reference protein (e.g., SIRPα/G1Fc, SIRPα-mt15/G4Fc, or SIRPα-mt15/G1Fc). In some embodiments, the ability of a protein complex described herein to induce macrophages to phagocytose RBC cells or platelets (e.g., mouse RAW264.7 cells) is weaker (e.g., less than 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%) than the ability of an anti-CD47 reference antibody (e.g., a mololimab analog) or a reference protein (e.g., SIRPα/G1Fc, SIRPα-mt15/G4Fc or SIRPα-mt15/G1Fc) to induce macrophages to phagocytose RBC cells or platelets.

In some embodiments, the protein complexes described herein can induce phagocytosis of tumor cells expressing PD-L1 by mouse macrophages (e.g., RAW264.7 cells). In some embodiments, the protein complexes described herein can induce phagocytosis of tumor cells expressing PD-L1 by human macrophages (e.g., MDM cells). In some embodiments, the protein complexes described herein can induce phagocytosis of tumor cells expressing CD47 by mouse macrophages (e.g., RAW264.7 cells). In some embodiments, the protein complexes described herein can induce phagocytosis of tumor cells expressing CD47 by human macrophages (e.g., MDM cells).

Endogenous expression of CD47 on a variety of cell types, including red blood cells, creates a strong "antigen sink" that may limit the efficacy of CD47-targeted therapies. Therefore, the reduced ability of the protein complexes described herein to induce phagocytosis of RBC cells and/or platelets may increase the in vivo efficacy of the protein complexes. Additionally, the protein complexes may be administered at lower dose levels and/or less frequent dosing schedules with similar efficacy compared to anti-CD47 reference antibodies (e.g., mololimab analogs).

In some embodiments, the protein complexes described herein can enhance T cell responses (e.g., in MLR assays). The principle of mixed lymphocyte reaction (MLR) is that T cells from one donor will proliferate in the presence of APCs from different donors. This is due to the recognition of HLA mismatches between two unrelated donors, which stimulates an immune response from T cells. MLR is typically used as a means of inducing general stimulation/activation of T cells in culture. In some embodiments, the protein complex can increase T cell proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the control molecule or combination thereof used herein. In some embodiments, the protein complex described herein can increase the production of cytokines (e.g., IFN-γ and/or IL-2) by at least 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1000-fold, 2000-fold, or 10000-fold compared to a control molecule or a combination thereof used herein. In some embodiments, the protein complex described herein can increase the production of cytokines (e.g., IFN-γ and/or IL-2) by at least 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold compared to a combination of individual functional domains (TIGIT/Fc + PD-1/Fc + SIRPα/Fc).

In some embodiments, the protein complexes described herein do not induce interleukin storm in the human body. In some embodiments, the protein complexes described herein are not super agonists. Details of interleukin storm and super agonists can be found, for example, in Shimabukuro-Vornhagen, A. et al. "Cytokine release syndrome". Journal for ImmunoTherapy of Cancer 6.1 (2018): 1-14, which is incorporated herein by reference in its entirety.

In some embodiments, the protein complexes described herein can inhibit tumor growth. In some embodiments, the protein complexes (e.g., any of the protein complexes described herein) can significantly inhibit tumor growth compared to a vehicle control. In some embodiments, the protein complexes described herein can inhibit tumor growth at a TGI value that is at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 2.5 times, at least 3 times, at least 3.5 times, at least 4 times, at least 4.5 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or at least 10 times the TGI value of a reference antibody or reference protein in a mouse xenograft model. In some embodiments, tumor cells are subcutaneously inoculated into immunocompromised or immunodeficient mice to generate a xenograft model. For example, the immunodeficient mice can be NPG™ mice (NOD.Cg- PrkdcscidIl2rgtm1Vst /Vst mice), which are NOD/SCID mice in which the interleukin-2 gamma chain receptor is genetically knocked out. In some embodiments, the tumor volume of the mice can be analyzed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days after inoculation. In some embodiments, the TGI value of mice treated with the protein complexes described herein may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, or at least 90%.

Methods for preparing protein complexes Variants of the protein complexes described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding the polypeptide or a portion thereof or by peptide synthesis. Such variants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence.

Screening can be performed to improve the binding affinity of the CD47 binding domain, PD-L1 binding domain, and/or TIGIT ligand binding domain. Any combination of deletions, insertions, and/or combinations can be made to obtain variants with improved binding affinity for the target. Amino acid changes introduced into the variants can also alter the polypeptide or introduce new post-translational modifications into the polypeptide, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence so that a different sugar is attached by an enzyme present in the cell), or introducing a new glycosylation site.

The CD47 binding domain, PD-L1 binding domain and/or TIGIT ligand binding domain can be derived from any animal species, including mammals. Non-limiting examples of binding domain variants include sequences derived from humans, primates (e.g., monkeys and apes), cows, pigs, horses, sheep, camelids (e.g., camels and camels), chickens, goats, and rodents (e.g., rats, mice, hamsters, and rabbits).

The present disclosure also provides recombinant vectors (e.g., expression vectors) comprising an isolated polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein), host cells into which the recombinant vectors are introduced (i.e., such that the host cell contains the polynucleotide and/or the vector comprising the polynucleotide), and the production of the recombinant polypeptide or fragment thereof by recombinant techniques.

As used herein, a "vector" is any construct capable of delivering one or more polynucleotides of interest to a host cell when the vector is introduced into the host cell. An "expression vector" is capable of delivering and expressing one or more polynucleotides of interest as encoded polypeptides in a host cell into which the expression vector has been introduced. Thus, in an expression vector, the polynucleotide of interest is positioned for expression in the vector by being operably linked to regulatory elements such as a promoter, enhancer, and/or poly-A tail within the vector or in the genome of the host cell at, near, or flanking the site of integration of the polynucleotide of interest, such that the polynucleotide of interest will be translated in the host cell into which the expression vector has been introduced.

The vector can be introduced into the host cell by methods known in the art, such as electroporation, chemical transfection (e.g., DEAE-dextran), transformation, transfection, and infection and/or transduction (e.g., using recombinant viruses). Thus, non-limiting examples of vectors include viral vectors (which can be used to produce recombinant viruses), naked DNA or RNA, plasmids, cosmids, phage vectors, and DNA or RNA expression vectors conjugated to a cationic condensing agent.

In some embodiments, a polynucleotide disclosed herein (e.g., a polynucleotide encoding a polypeptide disclosed herein) is introduced using a viral expression system (e.g., vaccinia or other poxvirus, retrovirus, or adenovirus), which may involve the use of a non-pathogenic (defective) replication-competent virus, or a replication-defective virus may be used. Techniques for incorporating DNA into such expression systems are well known to those of ordinary skill in the art. The DNA may also be "naked." Uptake of naked DNA may be increased by coating the DNA onto biodegradable beads, which are efficiently transported into cells.

For expression, a DNA insert comprising a polynucleotide encoding a polypeptide disclosed herein may be operably linked to an appropriate promoter (e.g., a heterologous promoter), such as the bacteriophage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters, and the promoters of the retroviral LTR, to name a few. Other suitable promoters are known to the skilled artisan. In some embodiments, the promoter is a cytomegalovirus (CMV) promoter. The expression construct may further contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct may include a transcription initiator at the start and a stop codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vector may contain at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culture in E. coli and other bacteria. Representative examples of suitable hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces , and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells, such as nymph S2 and Spodoptera Sf9 cells; animal cells, such as CHO, COS, Bowes melanoma, and HEK293 cells; and plant cells. Appropriate culture media and conditions for the host cells described herein are known in the art.

Non-limiting vectors for use in bacteria include pQE70, pQE60, and pQE-9, which are available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, which are available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5, which are available from Pharmacia. Non-limiting eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1, and pSG, which are available from Strater Genomics, and pSVK3, pBPV, pMSG, and pSVL, which are available from Pharmacia. Other suitable vectors will be apparent to the skilled artisan.

Non-limiting bacterial promoters suitable for use include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters, and the trp promoter. Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, promoters from retrovirus LTRs, such as the promoter from the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-I promoter.

In the yeast Saccharomyces cerevisiae , a number of vectors containing constitutive or inducible promoters such as α-factor, alcohol oxidase, and PGH are available.

Introduction of the construct into the host cell may be effected by calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986), which is incorporated herein by reference in its entirety.

Transcription of the DNA encoding the polypeptide disclosed in the code book by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. An enhancer is a cis-acting element of DNA, usually about 10 bp to 300 bp, that acts to increase the transcriptional activity of a promoter in a given host cell type. Examples of enhancers include the SV40 enhancer located on the back side of the replication origin at base pairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the back side of the replication origin, and the adenovirus enhancer.

In order to secrete the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, an appropriate secretion signal may be incorporated into the expressed polypeptide. The signal may be endogenous to the polypeptide, or it may also be a heterologous signal.

The polypeptide may be expressed in a modified form such as a fusion protein (e.g., GST fusion) or with a histidine tag and may include not only a secretion signal but also additional heterologous functional regions. For example, additional amino acids, particularly a region of charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell during purification or during subsequent transport and storage. Similarly, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. Adding peptide moieties to polypeptides to cause secretion or excretion, improve stability, and facilitate purification, etc., is well known and within the skill of the art.

Therapeutic Methods The protein constructs or polypeptides disclosed herein can be used for a variety of therapeutic purposes.

In one aspect, the present disclosure provides methods for treating cancer in an individual, reducing the rate at which a tumor in an individual increases in size over time, reducing the risk of developing metastasis, or reducing the risk of additional metastasis in an individual. In some embodiments, treatment can stop, slow, delay, or inhibit the progression of cancer. In some embodiments, treatment can reduce the number, severity, and/or duration of one or more symptoms of cancer in an individual.

In one aspect, the disclosure is characterized by a method comprising administering a therapeutically effective amount of a protein construct or polypeptide disclosed herein to a subject in need thereof (e.g., a subject having, or identified or diagnosed as having, cancer), such as breast cancer (e.g., triple-negative breast cancer), carcinoid, cervical cancer, endometrial cancer, neuroglioma, head and neck cancer, liver cancer, lung cancer, small cell lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, prostate cancer, kidney cancer, colorectal cancer, gastric cancer, testicular cancer, thyroid cancer, bladder cancer, urethral cancer, or hematological malignancy. In some embodiments, the cancer is unresectable melanoma or metastatic melanoma, non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), bladder cancer, or metastatic hormone-refractory prostate cancer. In some embodiments, the individual has a solid tumor. In some embodiments, the cancer is squamous cell carcinoma of the head and neck (SCCHN), renal cell carcinoma (RCC), triple negative breast cancer (TNBC), or colorectal cancer. In some embodiments, the cancer is melanoma, pancreatic cancer, mesothelioma, a hematological malignancy, particularly non-Hodgkin's lymphoma, lymphoma, chronic lymphocytic leukemia, or an advanced solid tumor.

In some embodiments, the compositions and methods disclosed herein can be used to treat patients at risk of developing cancer. Patients with cancer can be identified using various methods known in the art.

As used herein, "effective amount" means an amount or dosage sufficient to produce beneficial or desired results, including stopping, slowing, delaying or inhibiting the progression of a disease (e.g., cancer). The effective amount will vary depending on, for example, the age and weight of the individual to whom the protein construct or polypeptide, vector comprising a polynucleotide encoding the protein construct or polypeptide, and/or a composition thereof is to be administered, the severity of the symptoms, and the route of administration, and thus administration can be determined on an individual basis.

An effective amount can be administered in one or more administrations. For example, an effective amount of a protein construct or polypeptide is an amount sufficient to improve, prevent, stabilize, reverse, inhibit, slow down and/or delay the progression of a patient's cancer, or an amount sufficient to improve, stop, stabilize, reverse, slow down and/or delay the proliferation of cells (e.g., biopsy cells, any of the cancer cells described herein, or cell lines (e.g., cancer cell lines)) in vitro. As is understood in the art, an effective amount can vary, particularly depending on the patient's medical history and other factors, such as the type (and/or dose) of protein construct or polypeptide used.

The effective amount and schedule for administering the protein constructs or polypeptides, polynucleotides encoding the protein constructs or polypeptides, and/or compositions disclosed herein can be determined empirically, and making such determinations is within the skill of the art. Those skilled in the art will understand that the dosage that must be administered will vary depending on, for example, the mammal to which the protein constructs or polypeptides, polynucleotides, and/or compositions disclosed herein will be administered, the route of administration, the specific type of polynucleotide and/or composition disclosed herein used, and other drugs administered to the mammal.

Typical daily dosages of effective amounts of protein constructs and/or polypeptides are 0.1 mg/kg to 100 mg/kg (mg per kg of patient body weight). In some embodiments, the dosage may be less than 100 mg/kg, 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.1 mg/kg. In some embodiments, the dosage may be greater than 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, 1 mg/kg, 0.5 mg/kg, or 0.1 mg/kg. In some embodiments, the dosage is about 10 mg/kg, 9 mg/kg, 8 mg/kg, 7 mg/kg, 6 mg/kg, 5 mg/kg, 4 mg/kg, 3 mg/kg, 2 mg/kg, or 1 mg/kg. In some embodiments, the dosage is about 1 mg/kg to 10 mg/kg, about 1 mg/kg to 5 mg/kg, or about 2 mg/kg to 5 mg/kg.

In any of the methods described herein, the protein construct or polypeptide can be administered to a subject at least once a week (e.g., once a week, twice a week, three times a week, four times a week, once a day, twice a day, or three times a day).

In some embodiments, one or more additional therapeutic agents can be administered to a subject before or after administration of a protein construct or polypeptide. In some embodiments, the one or more additional therapeutic agents are administered to a subject such that the one or more additional therapeutic agents overlap with the biological activity time period of the protein construct or polypeptide in the subject.

In some embodiments, one or more additional therapeutic agents may be administered to a subject. The additional therapeutic agent may include one or more inhibitors selected from the group consisting of: B-Raf inhibitors, EGFR inhibitors, MEK inhibitors, ERK inhibitors, K-Ras inhibitors, c-Met inhibitors, anaplastic lymphoma kinase (ALK) inhibitors, phosphatidylinositol 3-kinase (PI3K) inhibitors, Akt inhibitors, mTOR inhibitors, dual PI3K/mTOR inhibitors, Bruton's tyrosine kinase (BTK) inhibitors, and isocitrate dehydrogenase 1 (IDH1) and/or isocitrate dehydrogenase 2 (IDH2) inhibitors. In some embodiments, the additional therapeutic agent is an inhibitor of indoleamine 2,3-dioxygenase-1 (IDO1) (e.g., epacadostat).

In some embodiments, the additional therapeutic agent may include one or more inhibitors selected from the group consisting of: HER3 inhibitors, LSD1 inhibitors, MDM2 inhibitors, BCL2 inhibitors, CHK1 inhibitors, activated hedgehog signaling pathway inhibitors, and agents that selectively degrade estrogen receptors.

In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of Trabectedin, nab-paclitaxel, Trebananib, Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine, IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent, temsirolimus, axitinib, everolimus, sorafenib, enib), Votrient, Pazopanib, IMA-901, AGS-003, cabozantinib, Vinflunine, Hsp90 inhibitors, Ad-GM-CSF, Temozolomide, IL-2, IFNa, vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide, azacytidine, lenalidomide, bortezomid, amrubicine, carfilzomib, pralatrexate, and enzastaurin.

In some embodiments, the additional therapeutic agent may include one or more therapeutic agents selected from the group consisting of: adjuvants, TLR agonists, tumor necrosis factor (TNF) α, IL-1, HMGB1, IL-10 antagonists, IL-4 antagonists, IL-13 antagonists, IL-17 antagonists, HVEM antagonists, ICOS agonists, therapeutic targeting CX3CL1, therapeutic targeting CXCL9, therapeutic targeting CXCL10, therapeutic targeting CCL5, LFA-1 agonists, ICAM1 agonists, and selectin agonists.

In some embodiments, the subject is administered carboplatin, albumin-bound paclitaxel, paclitaxel, cisplatin, pemetrexed, gemcitabine, FOLFOX, or FOLFIRI.

In some embodiments, the additional therapeutic agent is an anti-OX40 antibody, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-SIRPα antibody, an anti-CD47 antibody, an anti-LAG-3 antibody, an anti-TIGIT antibody, an anti-BTLA antibody, an anti-CTLA-4 antibody, or an anti-GITR antibody. In some embodiments, the additional therapeutic agent is an anti-CD20 antibody (e.g., rituximab) or an anti-EGF receptor antibody (e.g., cetuximab).

Pharmaceutical compositions and routes of administration Also provided herein are pharmaceutical compositions containing the protein constructs or polypeptides described herein. The pharmaceutical compositions can be formulated in any manner known in the art.

The pharmaceutical composition is formulated to be compatible with its intended route of administration (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal). The composition may include a sterile diluent (e.g., sterile water or saline), a non-volatile oil, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents, an antibacterial or antifungal agent such as benzyl alcohol or methyl paraben, chlorobutanol, phenol, ascorbic acid, thimerosal and the like; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate or phosphate; and an isotonic agent such as a sugar (e.g., dextrose), a polyol (e.g., mannitol or sorbitol) or a salt (e.g., sodium chloride), or any combination thereof. Liposome suspensions may also be used as pharmaceutically acceptable carriers. Formulations of the composition may be formulated and packaged in ampoules, disposable syringes, or multidose vials. Where necessary (e.g., in injectable formulations), appropriate fluidity may be maintained, for example, by the use of coatings such as lecithin or surfactants. Absorption of the agent may be prolonged by including agents that delay absorption (e.g., aluminum monostearate and gelatin). Alternatively, controlled release may be achieved by implants and microencapsulated delivery systems that may include biodegradable, biocompatible polymers (e.g., ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid).

Compositions containing the protein constructs or polypeptides described herein may be formulated for parenteral (e.g., intravenous, intraarterial, intramuscular, intradermal, subcutaneous, or intraperitoneal) administration in dosage unit form (i.e., physically discrete units containing a predetermined amount of active compound for ease of administration and uniformity of dosage).

Pharmaceutical compositions for parenteral administration are preferably sterile and substantially isotonic and prepared under good manufacturing practice (GMP) conditions. Pharmaceutical compositions can be provided in unit dosage form (i.e., a dose for a single administration). Pharmaceutical compositions can be formulated using one or more physiologically acceptable carriers, diluents, excipients or adjuvants. The formulation depends on the selected route of administration. For injection, the medicament can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, to reduce discomfort at the injection site. The solution may contain formulating agents, such as suspending agents, stabilizers and/or dispersing agents. Alternatively, the protein construct or polypeptide may be in lyophilized form for constitution with a suitable vehicle (e.g., sterile, pyrogen-free water) prior to use.

The toxicity and therapeutic efficacy of the composition can be determined by standard pharmaceutical procedures in cell culture or experimental animals (e.g., monkeys). For example, the LD50 (the dose that is lethal to 50% of the population) and the ED50 (the dose that is therapeutically effective in 50% of the population) can be determined: the therapeutic index is the ratio of LD50:ED50. Agents that exhibit high therapeutic indices are preferred. In the event that an agent exhibits undesirable side effects, care should be taken to minimize potential harm (i.e., reduce undesirable side effects). Toxicity and therapeutic efficacy can be determined by other standard pharmaceutical procedures.

Exemplary dosages include milligram or microgram amounts of any protein construct or polypeptide described herein per kilogram of body weight of an individual (e.g., about 1 μg/kg to about 500 mg/kg; about 100 μg/kg to about 500 mg/kg; about 100 μg/kg to about 50 mg/kg; about 10 μg/kg to about 5 mg/kg; about 10 μg/kg to about 0.5 mg/kg; about 1 μg/kg/ to about 50 μg/kg; about 1 mg/kg to about 10 mg/kg; or about 1 mg/kg to about 5 mg/kg). Although these dosages cover a broad range, one of ordinary skill in the art will appreciate that the potency of a therapeutic agent can vary and that an effective amount can be determined by methods known in the art. Typically, a relatively low dose is administered first, and the attending health care professional or veterinary professional (in the case of therapeutic applications) or the researcher (when still working in the development phase) can then and gradually increase the dose until an appropriate response is obtained. In addition, it should be understood that the specific dose level for any particular individual will depend on a variety of factors, including the activity of the specific compound employed, the individual's age, weight, general health, sex and diet, time of administration, route of administration, rate of excretion and half-life in vivo.

The pharmaceutical composition can be contained in a container, package or dispenser together with instructions for administration. The present disclosure also provides methods for preparing protein constructs or polypeptides for various uses described herein.

EXAMPLES The present invention is further described in the following examples, which do not limit the scope of the present invention described in the patent application.

Example 1 : Design of Designer Fc -Based Biologics (FBDB™) with TIGIT × PD-1 × SIRPα Domains The following example demonstrates the application and efficacy of Designer Fc-Based Biologics (FBDB™) with TIGIT × PD-1 × SIRPα Domains in enhancing immune responses. These triple-targeted formats of FBDB™ were developed and optimized to enhance the synergistic ability between innate immunity (e.g., targeting SIRPα/CD47 and TIGIT/PVR pathways) and adaptive immunity (e.g., targeting PD-1/PD-L1 and TIGIT/PVR pathways).

Each format contains at least three different types of immune modules directly or indirectly linked to the Fc region of IgG (e.g., human IgG4 or human IgG1). The three immune modules are: (1) one or more PD-1 extracellular domains (ECDs) acting as PD-1 baits that can block the PD-1/PD-L1 pathway to enhance T cell function and direct TIGIT × PD-1 × SIRPα molecules to tumors expressing PD-L1; (2) one or more SIRPα extracellular domains acting as SIRPα baits that can stimulate antigen presentation by inducing phagocytosis; and (3) one or more TIGIT extracellular domains acting as TIGIT baits that can interact with all natural TIGIT ligands and block TIGIT’s immunosuppressive signaling to NK cells, T cells, and Treg cells. For example, one or more PD-1 ectodomains can disrupt the interaction between PD-1 expressed on T cells and PD-L1 present on tumor cells, thereby releasing the inhibitory brakes on effector T cells and promoting T cell function; one or more SIRPα ectodomains can disrupt the interaction between CD47 expressed on tumor cells and SIRPα present on macrophages, thereby enabling macrophages to overcome the "don't eat me" inhibitory brake; and a TIGIT ectodomain can block inhibitory signals on effector T cells and NK cells by blocking the interaction between TIGIT and PVR, while inhibiting activation signals on regulatory T cells.

TgPS is a triple-targeted FBDB™ fusion protein platform designed to have wild-type or variant domains of TIGIT × PD-1 × SIRPα, which can be arranged in different configurations to produce a wide range of fusion proteins. For example, a fusion protein group named "TgPS_v1" was developed, which includes five forms, namely TgPS-C1_v1, TgPS-C2_v1, TgPS-D_v1, TgPS-E_v1 and TgPS-F_v1 as shown in Figures 1A to 1E , respectively. Each form includes the wild-type TIGIT extracellular domain (TIGIT-ECD-WT; SEQ ID NO: 1), the wild-type PD-1 extracellular domain (PD-1-ECD-WT; SEQ ID NO: 2) and the SIRPα extracellular domain (SIRPα-ECD-WT; SEQ ID NO: 3). Mutations can also be introduced into the wild-type PD-1 extracellular domain and the wild-type SIRPα extracellular domain to generate PD-1-ECD-mt13 (SEQ ID NO: 17) and SIRPα-ECD-mt15 (SEQ ID NO: 18), respectively. Therefore, another fusion protein group named "TgPS_v2" was developed, which includes four forms, namely TgPS-C1_v2, TgPS-C2_v2, TgPS-D_v2 and TgPS-E_v2 as shown in Figures 15A to 15D , respectively. Each form includes TIGIT-ECD-WT, PD-1-ECD-mt13 and SIRPα-ECD-mt15.

TgPS-C1_v1 (schematic structure shown in FIG1A ) contains two identical polypeptide chains, and each polypeptide chain has the amino acid sequence shown in SEQ ID NO: 12. Specifically, each polypeptide chain comprises PD-1-ECD-WT ( SEQ ID NO: 2), TIGIT-ECD-WT (SEQ ID NO: 1), a human IgG1 Fc region containing a hinge region (SEQ ID NO: 6), and SIRPα-ECD-WT (SEQ ID NO: 3) from the N-terminus to the C-terminus. PD-1-ECD-WT is linked to the N-terminus of TIGIT-ECD-WT via a (GSG) ₅ linker peptide (SEQ ID NO: 4). SIRPα-ECD-WT is linked to the C-terminus of human IgG1 Fc via an A3S2(G4S) ₂ linker peptide (SEQ ID NO: 5).

TgPS-C2_v1 (schematic structure shown in FIG . 1B ) contains two identical polypeptide chains, and each polypeptide chain has the amino acid sequence shown in SEQ ID NO: 13. Specifically, each polypeptide chain comprises PD-1-ECD-WT (SEQ ID NO: 2), TIGIT-ECD-WT (SEQ ID NO: 1), a human IgG4 (S228P) Fc region containing a hinge region (SEQ ID NO: 7), and SIRPα-ECD-WT (SEQ ID NO: 3) from the N-terminus to the C-terminus. PD-1-ECD-WT is linked to the N-terminus of TIGIT-ECD-WT via a (GSG) ₅ linker peptide (SEQ ID NO: 4). SIRPα-ECD-WT is linked to the C-terminus of human IgG4 (S228P) Fc via an A3S2(G4S) ₂ linker peptide (SEQ ID NO: 5).

TgPS-D_v1 (schematic structure shown in FIG1C ) contains two identical polypeptide chains, and each polypeptide chain has the amino acid sequence shown in SEQ ID NO: 14. Specifically, each polypeptide chain comprises TIGIT-ECD-WT (SEQ ID NO: 1), PD-1-ECD-WT (SEQ ID NO: 2), a human IgG4 (S228P) Fc region containing a hinge region (SEQ ID NO: 7), and SIRPα-ECD-WT (SEQ ID NO: 3) from the N-terminus to the C-terminus. TIGIT-ECD-WT is linked to the N-terminus of PD-1-ECD-WT via a (GSG) ₅ linker peptide (SEQ ID NO: 4). SIRPα-ECD-WT is linked to the C-terminus of human IgG4 (S228P) Fc via an A3S2(G4S) ₂ linker peptide (SEQ ID NO: 5).

TgPS-E_v1 (schematic structure shown in FIG . 1D ) contains two identical polypeptide chains, and each polypeptide chain has the amino acid sequence shown in SEQ ID NO: 15. Specifically, each polypeptide chain comprises TIGIT-ECD-WT (SEQ ID NO: 1), a human IgG4 (S228P) Fc region containing a hinge region (SEQ ID NO: 7), SIRPα-ECD-WT (SEQ ID NO: 3), and PD-1-ECD-WT (SEQ ID NO: 2) from the N-terminus to the C-terminus. SIRPα-ECD-WT is linked to the C-terminus of human IgG4 (S228P) Fc via an A3S2(G4S) ₂ linker peptide (SEQ ID NO: 5). PD-1-ECD-WT is linked to the C-terminus of SIRPα-ECD-WT via a (GSG) ₅ linker peptide (SEQ ID NO: 4).

TgPS-F_v1 (schematic structure shown in FIG. 1E ) contains two identical polypeptide chains, and each polypeptide chain has the amino acid sequence shown in SEQ ID NO: 16. Specifically, each polypeptide chain comprises PD-1-ECD-WT (SEQ ID NO: 2), SIRPα-ECD-WT (SEQ ID NO: 3), a human IgG4 (S228P) Fc region containing a hinge region (SEQ ID NO: 7), and TIGIT-ECD-WT (SEQ ID NO: 1) from the N-terminus to the C-terminus. PD-1-ECD-WT is linked to the N-terminus of SIRPα-ECD-WT via a (GSG) ₅ linker peptide (SEQ ID NO: 4). TIGIT-ECD-WT is linked to the C-terminus of human IgG4 (S228P) Fc via an A3S2(G4S) ₂ linker peptide (SEQ ID NO: 5).

TgPS-C1_v2 (schematic structure shown in FIG. 15A ) contains two identical polypeptide chains, and each polypeptide chain has the amino acid sequence shown in SEQ ID NO: 19. Specifically, each polypeptide chain comprises PD-1-ECD-mt13 (SEQ ID NO: 17), TIGIT-ECD-WT (SEQ ID NO: 1), a human IgG1 Fc region containing a hinge region (SEQ ID NO: 6), and SIRPα-ECD-mt15 (SEQ ID NO: 18) from the N-terminus to the C-terminus. PD-1-ECD-mt13 is linked to the N-terminus of TIGIT-ECD-WT via a (GSG) ₅ linker peptide (SEQ ID NO: 4). SIRPα-ECD-mt15 is linked to the C-terminus of human IgG1 Fc via an A3S2(G4S) ₂ linker peptide (SEQ ID NO: 5).

TgPS-C2_v2 (schematic structure shown in FIG. 15B ) contains two identical polypeptide chains, and each polypeptide chain has the amino acid sequence shown in SEQ ID NO: 20. Specifically, each polypeptide chain comprises PD-1-ECD-mt13 (SEQ ID NO: 17), TIGIT-ECD-WT (SEQ ID NO: 1), human IgG4 (S228P) Fc region containing hinge region (SEQ ID NO: 7), and SIRPα-ECD-mt15 (SEQ ID NO: 18) from N-terminus to C-terminus. PD-1-ECD-mt13 is linked to the N-terminus of TIGIT-ECD-WT via a (GSG) ₅ linker peptide (SEQ ID NO: 4). SIRPα-ECD-mt15 was linked to the C-terminus of human IgG4 (S228P) Fc via an A3S2(G4S) ₂ linker peptide (SEQ ID NO: 5).

TgPS-D_v2 (schematic structure shown in FIG. 15C ) contains two identical polypeptide chains, and each polypeptide chain has the amino acid sequence shown in SEQ ID NO: 21. Specifically, each polypeptide chain comprises TIGIT-ECD-WT (SEQ ID NO: 1), PD-1-ECD-mt13 (SEQ ID NO: 17), a human IgG4 (S228P) Fc region containing a hinge region (SEQ ID NO: 7), and SIRPα-ECD-mt15 (SEQ ID NO: 18) from the N-terminus to the C-terminus. TIGIT-ECD-WT is linked to the N-terminus of PD-1-ECD-mt13 via a (GSG) ₅ linker peptide (SEQ ID NO: 4). SIRPα-ECD-mt15 was linked to the C-terminus of human IgG4 (S228P) Fc via an A3S2(G4S) ₂ linker peptide (SEQ ID NO: 5).

TgPS-E_v2 (schematic structure shown in FIG. 15D ) contains two identical polypeptide chains, and each polypeptide chain has the amino acid sequence shown in SEQ ID NO: 22. Specifically, each polypeptide chain comprises TIGIT-ECD-WT (SEQ ID NO: 1), a human IgG4 (S228P) Fc region containing a hinge region (SEQ ID NO: 7), SIRPα-ECD-mt15 (SEQ ID NO: 18), and PD-1-ECD-mt13 (SEQ ID NO: 17) from the N-terminus to the C-terminus. SIRPα-ECD-mt15 is linked to the C-terminus of human IgG4 (S228P) Fc via an A3S2(G4S) ₂ linker peptide (SEQ ID NO: 5). PD-1-ECD-mt13 is linked to the C-terminus of SIRPα-ECD-mt15 via a (GSG) ₅ linker peptide (SEQ ID NO: 4).

The expressed protein was purified by a protein A column and then by HPLC-SEC (high performance liquid chromatography related to size exclusion chromatography; Agilent Technologies, Inc.). Specifically, TgPS protein was expressed in CHO-S cells. The culture supernatant was collected and subjected to protein A purification. Next, the protein A column was equilibrated with 10× column volumes of equilibration buffer (25 mM Tris, 150 mM NaCl, pH 8.0), and the culture supernatant was then loaded onto the equilibrated protein A column. The column was then washed with 6× column volumes of equilibration buffer (25 mM Tris, 150 mM NaCl, pH 8.0). The protein samples were eluted with 6× column volumes of elution buffer (100 mM acetate, 20 mM NaCl, pH 3.0) and the pH was adjusted to 7.0-7.2 with a buffer containing 1 M HEPES, pH 8.0. The results showed that all TgPS proteins could be expressed and collected at high purity.

In addition, the amino acid sequences of SEQ ID NOs: 12-16 and 19-22 were analyzed using a deimmunization tool (Immune Epitope Database and Analysis Resource; Dhanda et al. "Development of a strategy and computational application to select candidate protein analogues with reduced HLA binding and immunogenicity". Immunology 153.1 (2018): 118-132) to identify immunogenic regions. No immunogenicity was identified.

Example 2 : Binding ability of TgPS protein to TIGIT ligand, PD-L1 and CD47 detected by ELISA ELISA assay was performed to test the binding ability of TgPS_v1 protein to TIGIT ligand, for example, a fusion protein (recombinant PVR-ECD/Fc fusion protein; SEQ ID NO: 48) fused to Fc comprising the recombinant human PVR extracellular domain (SEQ ID NO: 47), as shown in FIG2A . Specifically, 1 μg/mL of TgPS_v1 protein and TIGIT/G4Fc were used to coat the wells of a 96-well EIA microplate at 4°C overnight. TIGIT/G4Fc was used as a positive control, which comprises a TIGIT extracellular domain having an amino acid sequence shown in SEQ ID NO: 23 fused to IgG4 Fc. After blocking with 5% skim milk diluted in phosphate buffered saline (PBS), biotin-PVR-ECD/Fc diluted serially in PBS was added and incubated at 25°C for 1 hour. Unbound biotin-PVR-ECD/Fc was removed by washing 3 times with PBST (1× PBS supplemented with 0.05% ^Tween® 20). HRP-conjugated avidin secondary antibody (BioLegend, catalog number: 405103) was added to each well and incubated at 25°C for 1 hour. After washing 3 times with PBST (containing 0.1% Tween® 20) to remove excess secondary antibody, TMB substrate was then added to each well and incubated. Then, the reaction was stopped and the HRP activity was measured as optical density (OD) at 450 nm using a spectrophotometer (Varioskan LUK™, Thermo Scientific, Model 3020).

A similar experiment was performed to test the binding ability of TgPS_v1 protein to another TIGIT ligand, for example, a fusion protein comprising the extracellular domain of recombinant human nectin-2 fused to Fc (recombinant human nectin-2-ECD/Fc fusion protein), as shown in FIG2B . Specifically, 1 μg/mL human nectin-2-ECD/Fc (Sino Biological, catalog number: 10005-H02H) was used to coat the wells of a 96-well EIA microplate. Biotin-TgPS_v1 protein or biotin-TIGIT/G4Fc in PBS was added to each well of the microplate for ligand binding. The other materials used and the general ELISA steps performed were the same as those described above.

As shown in Figures 2A and 2B , TgPS-C2/D/E/F_v1 proteins can bind to TIGIT ligands (i.e., both PVR and fibronectin-2), while TgPS-C1_v1 can only bind to PVR. TgPS-D_v1 and TgPS-E_v1 showed stronger TIGIT ligand binding activity than TgPS-C1_v1, TgPS-C2_v1, TgPS-F_v1, and the positive control protein TIGIT/G4Fc.

ELISA assays were also performed to test the binding ability of TgPS_v1 protein to PD-1 ligand PD-L1 ( FIG. 2C ) and SIRPα ligand CD47 ( FIG. 2D ). Similar experiments were performed as above. In FIG. 2C , PD-1/G4Fc was used as a positive control, and the positive control included a PD-1 extracellular domain having an amino acid sequence shown in SEQ ID NO: 24 fused to IgG4 Fc. In FIG . 2D , SIRPα/G4Fc was used as a positive control, and the positive control included a SIRPα extracellular domain having an amino acid sequence shown in SEQ ID NO: 25 fused to IgG4 Fc.

As shown in Figures 2C to 2D , all five TgPS_v1 proteins showed suitable and comparable binding abilities to PD-L1 and CD47, respectively, as detected by ELISA. Specifically, all TgPS_v1 proteins bound to PD-L1 more effectively than the positive control PD-1/G4Fc. In addition, all TgPS_v1 proteins showed significant binding abilities to CD47 compared to the negative control, but did not reach the level of efficacy exhibited by the positive control SIRPα/G4Fc.

ELISA assays were also performed to test the binding ability of TgPS_v2 protein to recombinant human PVR-ECD/His (ACRO Biosystems; Catalog No.: CD5-H5223), PD-L1-ECD/His (Yixuan Shenzhou; Catalog No.: 10084-H08H), and CD47-ECD/His (Yixuan Shenzhou; Catalog No.: 12283-H08H) proteins, which contain His tags at the C-terminus of the PVR extracellular domain, PD-L1 extracellular domain, and CD47 extracellular domain, respectively. Specifically, 1 µg/mL PVR-ECD/His ( FIG. 16A ), PD-L1-ECD/His ( FIG. 16B ), or CD47-ECD/His ( FIG. 16C ) was used to coat the wells of a 96-well EIA microplate. TgPS_v2 protein and corresponding control protein in PBS were added to each well for ligand binding. HRP-conjugated anti-human IgG Fcγ fragment specific secondary antibody (Jackson ImmunoResearch, catalog number: 109-035-008) and TMB substrate were used for signal measurement. Other materials used and general ELISA steps performed were the same as those described above. In FIG . 16A , TIGIT/G1Fc and TIGIT/G4Fc were used as positive controls. TIGIT/G1Fc comprises a TIGIT extracellular domain having an amino acid sequence shown in SEQ ID NO: 26 fused to IgG1 Fc. In FIG . 16B , PD-1-mt13/G4Fc was used as a positive control, which comprises PD-1-ECD-mt13 having an amino acid sequence shown in SEQ ID NO: 27 fused to IgG4 Fc. In FIG . 16C , SIRPα-mt15/G4Fc and SIRPα-mt15/G1Fc were used as positive controls. SIRPα-mt15/G4Fc comprises SIRPα-ECD-mt15 having an amino acid sequence shown in SEQ ID NO: 28 fused to IgG4 Fc. SIRPα-mt15/G1Fc comprises SIRPα-ECD-mt15 having the amino acid sequence shown in SEQ ID NO: 29 fused to IgG1 Fc.

As shown in Figure 16A , all four TgPS_v2 proteins showed significant binding ability to PVR, among which TgPS-D_v2 showed the strongest binding. The PVR binding activity of all four TgPS_v2 proteins and positive control proteins can be ranked as follows: TgPS-D_v2 > TgPS-C2_v2, TgPS-C1_v2 > TgPS-E_v2, TIGIT/G1Fc > TIGIT/G4Fc. Except for TgPS-E_v2, other TgPS_v2 proteins showed stronger PVR binding activity than the PVR binding activity of positive controls (TIGIT/G4Fc and TIGIT/G1Fc).

As shown in Figure 16B , all four TgPS_v2 proteins showed significant binding ability to PD-L1, among which TgPS-C2_v2 showed the strongest binding ability. The PD-L1 binding activity of all four TgPS_v2 proteins and positive control proteins can be ranked as follows: TgPS-C2_v2 > TgPS-C1_v2, TgPS-D_v2, PD-1-mt13/G4Fc > TgPS-E_v2. Specifically, TgPS-C1_v2, TgPS-C2_v2 and TgPS-D_v2 proteins showed stronger PD-L1 binding activity than PD-1-mt13/G4Fc.

As shown in FIG . 16C , all four TgPS_v2 proteins showed significant binding ability to CD47, but did not reach the level of efficacy exhibited by the positive control proteins SIRPα-mt15/G4Fc and SIRPα-mt15/G1Fc.

These results collectively suggest that variations in the form of TgPS fusion proteins may affect their ligand-specific binding ability detected by ELISA.

Example 3 : Simultaneous binding of TgPS_2 protein to PVR , PD-L1 and CD47 detected by biolayer interferometry (BLI) A three-way binding assay was performed using an Octet ^® RED96 instrument with an anti-human IgG Fc (AHC) biosensor tip. The Octet ® assay was run at 30°C and 1000 RPM using a 96-well black sample plate (200 µL/well). TgPS_v2 protein, recombinant human PVR, PD-L1 and CD47 proteins were prepared in assay buffer (PBS, pH 7.4, 0.05% Tween® 20). The three-way binding assay was performed as follows: (1) a baseline was set by running with assay buffer for 60 seconds; (2) 10 µg/mL of each TgPS_v2 protein was loaded onto the AHC biosensor tip for 600 seconds; (3) a baseline was set by running with assay buffer for an additional 60 seconds; (4) serially diluted human CD47 protein (100 nM) was allowed to associate for 180 seconds; (5) dissociation was allowed for 5 seconds in assay buffer; (6) steps (1)-(5) were repeated, however, the associated protein was replaced by human PD-L1 (1000 nM); and (7) steps (1)-(5) were repeated, however, the associated protein was replaced by human PVR (1500 nM). The biosensor tip was regenerated with 10 mM glycine at pH 1.5 to repeat the measurement. Details of the three-way binding assay are summarized in the table below. Table 1: Three-way binding of TgPS_v2 protein Three-way binding of TgPS molecules Ligand Analytes TgPS-C1_v2 TgPS-C2_v2 TgPS-D_v2 TgPS-E_v2 CD47-ECD/His PVR-ECD/His PD-L1-ECD/His Layout 1 2 3 4 5 6 7 8 9 10 11 12 A PBST+ pH7.4 10 μg 10 μg 10 μg 10 μg PBST+ pH7.4 0 nM 0 nM 0 nM Regeneration buffer PBST+ pH7.4 B 10 μg 10 μg 10 μg 10 μg 50 nM 50 nM 500 nM C 10 μg 10 μg 10 μg 10 μg 100 nM 1500nM 1000nM D E F G H

As shown in Figures 17A to 17D , the three-way binding data showed that the TgPS_v2 protein was able to interact with PVR, PD-L1 and CD47 simultaneously. The three-arm form of the TgPS_v2 protein did not affect the ligand binding activity of each arm. The results suggest that the TgPS_v2 protein may have the potential to target PVR ⁺ PD-L1 ⁺ CD47 ⁺ tumor cells.

Example 4 : Whole cell binding ability of TgPS proteins to PVR tf CHO-S cells, PD-L1 tf CHO-S cells and CD47 tf CHO-S cells The whole cell binding ability of TgPS_v1 and TgPS_v2 proteins was tested by the following method: 3 × 10 ⁴ cells from each type, i.e., cells transfected to express PVR (PVR tf cells), cells transfected to express PD-L1 (PD-L1 tf cells) or cells transfected to express CD47 (CD47 tf cells) were separately incubated with serially diluted TgPS proteins or corresponding control proteins in FACS buffer (supplemented with 4% After washing the cells with FACS buffer, total cell binding was detected by incubating the cells with R-phycoerythrin AffiniPure goat anti-human IgG Fcγ fragment specific antibody (Jackson ImmunoResearch Laboratories, catalog number: 109-115-098) for an additional 30 minutes at 4°C. The cells were then subjected to flow cytometric analysis using a CytoFLEX flow cytometer (Beckman Coulter Inc.).

As shown in Figures 3A to 3B , all five TgPS_v1 proteins were able to bind to PD-L1 tf CHO-S cells and CD47 tf CHO-S cells, respectively. Specifically, in Figure 3A , TgPS-C2_v1 showed the strongest PD-L1 binding activity among TgPS_v1 proteins and PD-1/G4Fc. The binding activity to PD-L1 tf CHO-S cells can be ranked as follows: TgPS-C2_v1 > TgPS-D_v1 > TgPS-E_v1, TgPS-C1_v1, PD-1/G4Fc > TgPS-F_v1. In Figure 3B , all TgPS_v1 proteins showed significant CD47 binding activity, but did not reach the level shown by SIRPα/G4Fc.

As shown in Figures 18A to 18C , all TgPS_v2 proteins can bind to PVR tf cells, PD-L1 tf cells and CD47 tf cells, respectively. Specifically, in Figure 18A , TgPS-D_v2 showed the strongest PVR binding activity. The PVR binding activity among all four TgPS_v2 proteins and positive control proteins can be ranked as follows: TgPS-D_v2 > TgPS-E_v2 > TgPS-C2_v2 > TgPS-C1_v2, TIGIT/G1Fc, TIGIT/G4Fc. In Figure 18B , TgPS-C2_v2 showed the strongest PD-L1 binding ability. The PD-L1 binding activity among all four TgPS_v2 proteins and the positive control proteins can be ranked as follows: TgPS-C2_v2 > TgPS-C1_v2, TgPS-D_v2, PD-1-mt13/G1Fc > PD-1-mt13/G4Fc > TgPS-E_v2. PD-1-mt13/G1Fc was used as a positive control, which contained PD-1-ECD-mt13 having the amino acid sequence shown in SEQ ID NO: 30 fused to IgG4 Fc. In Figure 18C , all four TgPS_v2 proteins showed considerable binding ability to CD47 tf cells, but did not reach the efficacy level shown by the positive control proteins, which included mololimab analogs (anti-CD47 antibody analogs; heavy chain sequence: SEQ ID NO: 43 and light chain sequence: SEQ ID NO: 44), SIRPα-mt15/G1Fc and SIRPα-mt15/G4Fc.

Taken together, these results suggest that variations in the form of TgPS fusion proteins may affect their ligand-specific binding capacity detected by whole-cell binding assays.

Example 5 : Binding ability of TgPS proteins to RBC and platelets The binding ability of TgPS_v1 and TgPS_v2 proteins to red blood cells (RBC) and platelets was tested by incubating 3 × 10 ⁵ RBCs ( FIG. 4A ) or 5 × 10 ⁵ platelets ( FIG. 4B ) freshly obtained from healthy human donors with serially diluted TgPS proteins or corresponding control proteins in modified FACS buffer (1× PBS supplemented with 4% FBS) at 4° C. for 30 minutes. After washing the cells with FACS buffer, binding to RBCs or platelets was detected by incubating them with R-phycoerythrin AffiniPure goat anti-human IgG Fcγ fragment specific antibody (Jackson ImmunoResearch Laboratories, catalog number: 109-115-098) for another 30 minutes at 4° C. After incubation, RBCs or platelets were subjected to flow cytometric analysis using a CytoFLEX flow cytometer (Beckman Coulter).

As shown in Figure 4A , none of the TgPS_v1 proteins bound to RBCs at any concentration tested, while the positive control (i.e., mololimab analog) showed significant RBC binding. Figure 4B shows that no platelet binding was detected for the TgPS-C1/C2/D/E_v1 proteins at any concentration tested. TgPS-F_v1 showed detectable levels of binding to platelets at its highest concentration tested (1000 nM). These results indicate that the TgPS_v1 proteins are less likely to induce binding to RBCs or platelets than the positive control (i.e., mololimab analog). SIRPα/G1Fc was used as a positive control, which included the SIRPα extracellular domain having the amino acid sequence shown in SEQ ID NO: 32 fused to IgG1 Fc.

As shown in Figures 19A to 19B , all TgPS_v2 proteins showed detectable RBC and platelet binding activity, but the binding activity was significantly weaker than that of positive control proteins (including mololimab analogs, SIRPα-mt15/G1Fc and SIRPα-mt15/G4Fc). These results indicate that the potential risk of TgPS_v2 proteins inducing RBC or platelet binding is low.

Example 6 : Selective Binding Ability of TgPS_v1 Protein to PD-L1 Overexpressing Tumor Cells The selective binding ability of TgPS_v1 protein to PD-L1 overexpressing tumor cells was tested by the following method: 2 × 10 ⁴ CellTrace ^™ -CFSE (Thermo Fisher Scientific, catalog number: C34554) labeled OE19 (PVR ⁺ CD47 ⁺ PD-L1 ^low ) cells and 2 × 10 ⁴ CellTrace ^™ -Violet (Thermo Fisher Scientific, catalog number: C34557) labeled PD-L1 tf OE19 cells (PVR ⁺ CD47 ⁺ PD-L1 ⁺ ) were incubated with serially diluted TgPS protein or corresponding control protein in FACS buffer (PBS supplemented with 4% FBS) at 4°C for 30 minutes. After washing the cells with FACS buffer, binding was detected by incubation with R-phycoerythrin AffiniPure goat anti-human IgG Fcγ fragment specific antibody (Jackson ImmunoResearch Laboratories, catalog number: 109-115-098) for another 30 minutes at 4°C. After incubation, the cells were subjected to flow cytometric analysis using a CytoFLEX flow cytometer (Beckman Coulter). Cell populations were gated based on PE signal, and the percentages of different cell types were calculated using Kaluza analysis software (Beckman Coulter).

As shown in FIG5A , little or no binding signal was detected between any of the tested proteins and OE19 cells. In contrast, in FIG5B , all TgPS_v1 proteins preferentially bound to PD-L1 tf OE19 cells in a mixture of OE19 cells and PD-L1 tf OE19 cells (ratio 1:1). The intensity of this selective binding observed for all TgPS_v1 proteins to PD-L1 tf OE19 cells was even stronger than that observed for the positive control PD-1/G4Fc to PD-L1 tf OE19 cells.

Example 7 : Binding ability of TgPS_v1 protein to activated T cells The binding ability of TgPS_v1 protein to activated T cells was tested as follows: 3 × 10 ⁴ Dynabeads™ human T activator CD3/CD28 (Thermo Fisher Scientific, catalog number: 11131D) activated T cells (bead-to-cell ratio = 1:1) were incubated with serially diluted TgPS_v1 protein and corresponding control protein in FACS buffer (PBS supplemented with 4% FBS) at 4°C for 30 minutes. The cells were washed with FACS buffer and binding was detected by incubating the cells with R-phycoerythrin AffiniPure goat anti-human IgG Fcγ fragment specific antibody (Jackson ImmunoResearch Laboratories, Cat. No. 109-115-098) for another 30 min at 4° C. The cells were then subjected to flow cytometric analysis using a CytoFLEX flow cytometer (Beckman Coulter).

As shown in Figure 6 , all TgPS_v1 proteins were able to bind to activated T cells expressing PVR (low), PD-L1 (low), and CD47 (high). Specifically, among the tested TgPS_v1 proteins and control proteins, TgPS-C1_v1 exhibited the strongest binding efficacy.

Example 8 : Inhibitory effect of TgPS protein on the interaction between TIGIT and PVR The inhibitory effect of TgPS_v1 and TgPS_v2 proteins on the interaction between TIGIT and PVR was determined as follows: 3 × 10 ⁴ PVR tf CHO-S cells were incubated with serially diluted TgPS protein or corresponding control protein and 250 nM biotin-TIGIT/G4Fc protein in FACS buffer (PBS supplemented with 4% FBS) at 4°C for 30 minutes. After washing, 0.3 µg of streptoavidin-PE (or "SA-PE"; eBioscience, catalog number: 12-4317-87) was added to each well, and the PE signal from the cells was analyzed using a CytoFLEX flow cytometer (Beckman Coulter).

The blocking activity in Figure 7B was calculated as follows:

[1 - (mean PE value of each tested protein at 1000 nM)/(the difference in mean PE value between the SA-PE group and the biotin-TIGIT/G4Fc group)] × 100%.

The blocking activity in Figure 20B was calculated as follows:

[1 - (mean PE value of each tested protein at 125 nM)/(the difference in mean PE value between the SA-PE group and the biotin-TIGIT/G4Fc group)] × 100%.

The calculated blocking activity value increased as TIGIT/PVR binding decreased ( Fig. 7A and Fig. 20A ) ( Fig. 7B and Fig. 20B ). The SA-PE group was used as a staining background control.

As shown in Figures 7A to 7B , all TgPS_v1 proteins were able to block the interaction between TIGIT and PVR, among which TgPS-D_v1 and TgPS-E_v1 showed blocking efficacy similar to that of tisleliumab analog (anti-TIGIT antibody analog; heavy chain sequence: SEQ ID NO: 45 and light chain sequence: SEQ ID NO: 46) and TIGIT/G4Fc. The blocking activity can be ranked as follows: TgPS-E_v1, TIGIT/G4Fc > tisleliumab analog (anti-TIGIT), TgPS-D_v1 > TgPS-C2_v1, TgPS-C1_v1 > TgPS-F_v1. It is noteworthy that the blocking activity of TgPS_v1 protein on TIGIT/PVR binding is consistent with its PVR binding ability.

As shown in Figures 20A to 20B , all TgPS_v2 proteins were able to block the interaction between TIGIT and PVR. In this analysis, TgPS-C1/C2/D/E_v1 was used together with all four TgPS_v2 proteins, and the blocking activity was ranked as follows: TgPS-E_v1, TgPS-E_v2, tisleliumab analog (anti-TIGIT), TgPS-D_v2 > TgPS-D_v1 > TgPS-C2_v1, TgPS-C2_v2, TIGIT/G1Fc, TIGIT/G4Fc > TgPS-C1_v1, TgPS-C1_v2. The similar trends and ordering observed between the different forms of TgPS_v1 and TgPS_v2 proteins may be attributed to the presence of the same TIGIT arm in all TgPS fusion proteins.

Example 9 : Inhibitory effect of TgPS protein on the interaction between PD-1 and PD-L1 The inhibitory effect of TgPS_v1 and TgPS_v2 proteins on the interaction between PD-1 and PD-L1 was determined as follows. 3 × 10 ⁴ cells PD-L1 tf CHO-S cells were incubated with serially diluted TgPS protein or corresponding control protein and 2 µg/mL biotin-PD-1/G4Fc in FACS buffer (PBS supplemented with 4% FBS) at 4°C for 30 minutes. After washing, 0.3 µg of streptozotocin-PE (or "SA-PE"; eBioscience, catalog number: 12-4317-87) was added to each well, and the PE signal from the cells was analyzed using a CytoFLEX flow cytometer (Beckman Coulter). The blocking activity in Figure 8B and Figure 21B was calculated as follows:

[1 - (mean PE value of each tested molecule at 125 nM)/(the difference in mean PE value between the SA-PE group and the biotin-PD-1/G4Fc group)] × 100%.

The calculated value of blocking activity increased as PD-1/PD-L1 binding decreased ( Figure 8A and Figure 21A ). PD-1/G1Fc was used as a positive control, which contained the PD-1 extracellular domain having the amino acid sequence shown in SEQ ID NO: 34 fused to IgG1 Fc. The SA-PE group was used as a staining background control.

The signaling blocking effect of TgPS_v2 protein on the interaction between PD-1 and PD-L1 was also confirmed by NFAT-Luc signaling blocking assay ( Figure 23 ). Briefly, 4 × 10 ⁴ PD-L1 tf aAPC/CHO-K1 cells and 5 × 10 ⁴ PD-1 tf NFAT-Luc Jurkat cells were co-cultured with serially diluted TgPS_v2 protein or corresponding control protein at 37°C for 4 hours. Then, steadylite™ plus reporter gene assay system reagent (PerkinElmer, catalog number 6066759) was added to each reaction. After 15 minutes of incubation, the reaction mixture was transferred to a 96-well plain white plate (Greiner Bio-One, catalog number: 655098), and the luciferase activity was measured by a spectrophotometer (Varioskan LUK™, Thermo Scientific, model 3020).

As shown in Figures 8A to 8B , all TgPS_v1 proteins were able to block the interaction between PD-1 and PD-L1, with TgPS-C1_v1 and TgPS-C2_v1 showing similar blocking efficacy to the positive control PD-1/G1Fc. The blocking activity can be ranked as follows: TgPS-C2_v1, TgPS-C1_v1, PD-1/G1Fc > TgPS-F_v1, TgPS-D_v1, TgPS-E_v1.

As shown in Figures 21A to 21B , all TgPS_v2 proteins were able to block the interaction between PD-1 and PD-L1. In this analysis, TgPS-C1/C2/D/E_v1 was used together with all four TgPS_v2 proteins, and the blocking activity could be ranked as follows: TgPS-C2_v1, TgPS-C2_v2, PD-1-mt13/G4Fc, PD-1-mt13/G1Fc > TgPS-C1_v2 > TgPS-D_v2 > TgPS-E_v2 > TgPS-C1_v1 > TgPS-D_v1 > TgPS-E_v1. Specifically, among all the proteins tested, TgPS-C2_v1 and TgPS-C2_v2 showed stronger blocking activity against PD-1/PD-L1 binding than other TgPS_v1 and TgPS_v2 proteins, and the blocking activity was similar to that of PD-1-mt13/G1Fc and PD-1-mt13/G4Fc. Overall, the blocking effect of TgPS_v2 protein was stronger than that of TgPS_v1 protein, and this result may be attributed to the presence of PD-1-ECD-mt13 in TgPS_v2 protein.

The results shown in FIG. 23 reinforce these findings, at least partially demonstrating in FIG. 21A to FIG . 21B that all TgPS_v2 proteins are capable of blocking the interaction between PD-1 and PD-L1, and TgPS-C2_v2 shows stronger blocking activity against PD-1/PD-L1 binding than other TgPS_v1 and TgPS_v2 proteins, as well as PD-1-mt13/G1Fc and PD-1-mt13/G4Fc. The blocking activity can be ranked as follows: TgPS-C2_v2 > TgPS-C1_v2 > TgPS-D_v2 > PD-1-mt13/G1Fc > PD-1-mt13/G4Fc, TgPS-E_v2. The results also showed that TgPS-E_v2 did not block the negative signaling of PD-1-PD-L1 interaction.

Example 10 : Inhibitory effect of TgPS protein on the interaction between SIRPα and CD47 The inhibitory effect of TgPS_v1 and TgPS_v2 proteins on the interaction between SIRPα and CD47 was determined as follows. 3 × 10 ⁴ cells of CD47 tf CHO-S cells were incubated with serially diluted TgPS molecules or corresponding control proteins and 1 µg/mL biotin-SIRPα/Fc in FACS buffer (PBS supplemented with 4% FBS) at 4°C for 30 minutes. After washing, 0.3 µg of streptoavidin-PE (or "SA-PE"; eBioscience, catalog number: 12-4317-87) was added to each well, and the PE signal from the cells was analyzed using a CytoFLEX flow cytometer (Beckman Coulter).

The blocking activity in Figure 9B was calculated as follows:

[1 - (mean PE value of each tested molecule at 125 nM)/(the difference in mean PE value between the SA-PE group and the biotin-SIRPα/G4Fc group)] × 100%.

The blocking activity in Figure 22B was calculated as follows:

[1 - (mean PE value of each tested molecule at 15.6 nM)/(the difference in mean PE value between the SA-PE group and the biotin-SIRPα/G4Fc group)] × 100%.

The calculated value of blocking activity increased as SIRPα/CD47 binding decreased ( Fig. 9A and Fig. 22A ) ( Fig. 9B and Fig. 22B ). The SA-PE group was used as a staining background control.

As shown in Figures 9A to 9B , all five TgPS_v1 proteins were able to block the interaction between SIRPα and CD47, but did not reach the blocking efficacy level shown by the positive control proteins (including mololimab analogs and SIRPα/G1Fc). Specifically, the blocking activity can be ranked as follows: mololimab analogs, SIRPα/G1Fc > TgPS-C1_v1, TgPS-C2_v1, TgPS-D_v1, TgPS-F_v1 > TgPS-E_v1.

As shown in Figures 22A to 22B , all TgPS_v2 molecules were able to block the interaction between SIRPα and CD47. In this analysis, TgPS-C1/C2/D/E_v1 was used together with all four TgPS_v2 proteins, and the blocking activity could be ranked as follows: SIRPα-mt15/G4Fc, SIRPα-mt15/G1Fc, mololimab analog (anti-CD47) > TgPS-C1_v2 > TgPS-C2_v2, TgPS-D_v2, TgPS-E_v2 > TgPS-C1_v1, TgPS-C2_v1, TgPS-D_v1 > TgPS-E_v1. Although the blocking efficacy level did not reach that of the positive control protein, TgPS_v2 protein showed stronger blocking activity than TgPS_v1 protein overall. This result may be attributed to the presence of SIRPα-ECD-mt15 in TgPS_v2 protein.

Example 11 : Hemagglutination (HA) activity induced by TgPS_v1 protein To determine the HA activity induced by TgPS_v1 protein, a 10% RBC solution was prepared from whole blood of healthy donors. RBCs were washed twice with 0.9% NaCl buffer and then diluted to 10% (by volume) in 0.9% NaCl buffer. The 10% RBC solution was then incubated with the serially diluted TgPS_v1 protein and the corresponding control protein in a round-bottom 96-well plate at room temperature overnight. The image of the plate was captured the next day. As a result, the agglutinated RBCs evenly coated the wells, while the non-agglutinated cells formed obvious red spots at the bottom of each well.

Figure 10 is an image captured the next day after overnight incubation of a 96-well plate. The image shows that only the mololimab analog induced significant HA activity, while none of the five TgPS_v1 proteins induced HA activity within the indicated concentration range.

Example 12 : TgPS proteins induce macrophage-mediated phagocytosis on tumor cells (FaDu) and PD-L1 tf tumor cells (PD-L1 tf OE19) To determine the macrophage-mediated phagocytosis induced by TgPS_v1 and TgPS_v2 proteins on tumor cells and RBCs, phagocytosis assays were performed as follows. FaDu cells ( FIG. 11A ) and PD-L1 overexpressing tumor cells (PD-L1 tf OE19) ( FIG . 11B ) were labeled with 5 μM or 10 μM CellTrace ^™ -CFSE (Thermo Fisher Scientific, catalog number: C34554) at room temperature for 10 minutes and then washed with complete medium. Then, 1 × 10 ⁵ - 5 × 10 ⁵ cells/well of CFSE-labeled FaDu cells or CFSE-labeled PD-L1 tf OE19 (target cells) were added to each well and incubated with serially diluted TgPS protein and corresponding control protein at 37°C for 30 minutes. Afterwards, 5 × 10 ⁴ mouse macrophage RAW264.7 cells were added to each well and incubated at 37°C for 2 hours. RAW264.7 cells were stained with PE-Cyanine7-conjugated F4/80 antibody (Invitrogen, catalog number: 25-4801-82). The phagocytic activity of the tested proteins was assessed by calculating the percentage of CFSE ⁺ F4/80 ⁺ macrophages (indicating CFSE-labeled target cells engulfed by macrophages) to the total F4/80 signal (indicating total macrophages) using a CytoFLEX flow cytometer (Beckman Coulter).

As shown in FIG11A , TgPS-C1_v1 was able to induce significant phagocytic activity against FaDu cells (PVR ^med PD-L1 ^med CD47 ^med ). TgPS-C2_v1 and TgPS-F_v1 induced slight phagocytic activity at higher concentrations. In FIG11B , TgPS-C1_v1, TgPS - C2_v1, and TgPS-D_v1 showed significant phagocytic activity against PD-L1 tf OE19 cells (PVR ^med PD-L1 ^high CD47 ^low ) at an efficacy level similar to that of the positive control protein SIRPα/G1Fc. These results suggest that some TgPS_v1 proteins can promote macrophage-mediated phagocytosis on PD-L1 overexpressing tumor cells.

As shown in FIG24A , among all the tested TgPS_v1 and TgPS_v2 proteins, the phagocytic activity induced by TgPS-C1_v2 and TgPS-C2_v2 on FaDu cells was stronger than that induced by other TgPS_v1 and TgPS_v2 proteins on FaDu cells. In general, TgPS_v2 proteins can promote macrophage-mediated phagocytosis more effectively than TgPS_v1 proteins. This result may be attributed to the presence of SIRPα-ECD-mt15 in TgPS_v2 proteins. In Figure 24B , TgPS-C1/C2/D/E_v1 and all four TgPS_v2 proteins showed significant efficacy in promoting phagocytosis on PD-L1 tf OE19 cells, with TgPS-C1_v2 and TgPS-C1_v1 showing the strongest ability. The phagocytosis-inducing activity was ranked as follows: SIRPα-mt15/G1Fc, mololimab analog (anti-CD47) > TgPS-C1_v2, TgPS-C1_v1 > TgPS-C2_v2, TgPS-E_v2, SIRPα-mt15/G4Fc > TgPS-C2_v1, TgPS-D_v1, TgPS-E_v1 > TgPS-D_v2.

Example 13 : TgPS protein induces macrophage-mediated phagocytosis on RBC and / or platelets Phagocytosis assays using RBC and platelets were performed according to the steps and materials described in Example 12. Specifically, for TgPS_v1 protein, phagocytosis assays were performed using only RBC, and the results are shown in Figure 11C . For TgPS_v2 protein, phagocytosis on both RBC and platelets was tested.

As shown in FIG11C , the positive control proteins (i.e., mololimab analogs (anti-CD47) and SIRPα/G1Fc) induced significant phagocytic activity on RBCs. In contrast, the TgPS_v1 protein induced minimal or no phagocytic activity on RBCs. Specifically, only TgPS-C1_v1 showed minimal effect in promoting phagocytosis at higher concentrations. These data suggest that the TgPS_v1 protein is unlikely to induce adverse events of phagocytosis on RBCs.

As shown in Figures 25A to 25B , the positive control proteins (i.e., mololimab analogs and SIRPα-mt15/G1Fc) induced significant phagocytic activity on RBCs and platelets. All TgPS_v1 and TgPS_v2 proteins tested induced only minimal or no phagocytic activity on RBCs and platelets at higher concentrations. The data reinforce and supplement the findings described in Figure 11C , which indicates that TgPS_v1 and TgPS_v2 proteins are unlikely to induce adverse events of phagocytosis on RBCs and platelets.

Example 14 : Evaluation of TgPS protein-induced T cell responses by MLR assay A mixed lymphocyte reaction (MLR) assay was performed to determine the enhancement of T cell responses by TgPS_v1 and TgPS_v2 proteins. Specifically, 1 × 10 ⁵ total T cells and 1 × 10 ⁴ dendritic cells (DCs) were co-cultured with 100 nM, 10 nM, or 1 nM of TgPS protein or 100 nM of the corresponding control protein. After a five-day incubation period, the culture supernatants were collected to measure IL-2 and IFN-γ expression using Human IL-2 ELISA MAX™ Deluxe Kit (Bio-Pro, Catalog No. 431805) and Human IFN-γ ELISA MAX™ Deluxe Kit (Bio-Pro, Catalog No. 430105).

As shown in FIG12A , TgPS-C1_v1, TgPS-C2_v1, and TgPS-D_v1 induced IL-2 expression, and TgPS-C1_v1 induced the highest IL-2 expression. Specifically, the IL-2 expression shown by cells treated with TIGIT/Fc + PD-1/Fc + SIRPα/Fc was lower than that shown by cells treated with TgPS-C1_v1. As shown in FIG12B , TgPS-C1_v1 induced more IFN-γ expression than other TgPS_v1 proteins and the combination of TIGIT/Fc + PD-1/Fc + SIRPα/Fc.

As shown in Figure 26A , TgPS-C1_v2, TgPS-C2_v2, and TgPS-D_v2 induced IL-2 expression. Specifically, TgPS-C1_v2 and TgPS-C2_v2 induced the highest IL-2 expression. In addition, cells treated with the combination of TIGIT/Fc + PD-1-mt13/Fc + SIRPα-mt15/Fc did not induce IL-2 expression. As shown in Figure 26B , TgPS-C2_v2 induced more IFN-γ expression than TgPS-C1_v2, TgPS-D_v2, and TIGIT/Fc + PD-1-mt13/Fc + SIRPα-mt15/Fc combination.

These results suggest that certain forms of the TgPS_v1 and TgPS_v2 proteins are able to promote T cell activation and response.

Other embodiments It should be understood that although the present invention has been described in conjunction with its embodiments, the foregoing description is intended to illustrate and not limit the scope of the present invention, which is defined by the scope of the attached patent application. Other aspects, advantages and modifications are within the scope of the attached patent application.

without

Figures 1A to 1E show schematic structures of TgPS_v1 forms, which respectively include TgPS-C1_v1, TgPS-C2_v1, TgPS-D_v1, TgPS-E_v1 and TgPS-F_v1. Figure 2A shows the binding ability of TgPS_v1 protein to TIGIT ligand PVR detected by ELISA. Figure 2B shows the binding ability of TgPS_v1 protein to TIGIT ligand adhesion protein-2 detected by ELISA. Figure 2C shows the binding ability of TgPS_v1 protein to PD-L1 detected by ELISA. Figure 2D shows the binding ability of TgPS_v1 protein to CD47 detected by ELISA. Figure 3A shows the whole cell binding ability of TgPS_v1 protein to PD-L1 tf CHO-S cells. Figure 3B shows the whole cell binding ability of TgPS_v1 protein to CD47 tf CHO-S cells. Figure 4A shows the RBC binding curve of TgPS_v1 protein. Figure 4B shows the platelet binding curve of TgPS_v1 protein. Figure 5A shows the whole cell binding results of TgPS_v1 protein to CellTrace-CFSE ⁺ OE19 cells in a mixture of OE19 cells and PD-L1 tf OE19 cells. Figure 5B shows the whole cell binding results of TgPS_v1 protein and CellTrace-Violet ⁺ PD-L1 tf OE19 cells in a mixture of OE19 cells and PD-L1 tf OE19 cells. Figure 6 shows the whole cell binding results of TgPS_v1 protein and activated T cells. Figures 7A to 7B show the blocking effect of TgPS_v1 protein using PVR tf CHO-S cells on the interaction between TIGIT and PVR. Figures 8A to 8B show the blocking effect of TgPS_v1 protein using PD-L1 tf CHO-S cells on the interaction between PD-1 and PD-L1. Figures 9A to 9B show the blocking effect of TgPS_v1 protein on the interaction between SIRPα and CD47 using CD47 tf CHO-S cells. Figure 10 is the RBC hemagglutination activity induced by TgPS_v1 protein. Figure 11A shows the macrophage-mediated phagocytosis of FaDu cells induced by TgPS_v1 protein. Figure 11B shows the macrophage-mediated phagocytosis of PD-L1 tf OE19 induced by TgPS_v1 protein. Figure 11C shows the macrophage-mediated phagocytosis of RBC induced by TgPS_v1 protein. Figure 12A shows the IL-2 expression induced by TgPS_v1 protein in MLR assay. "TIGIT + PD-1 + SIRPα" represents the combination of TIGIT/G4Fc, PD-1/G1Fc and SIRPα/G4Fc. Figure 12B shows the expression of IFN-γ induced by TgPS_v1 protein in the MLR assay. "TIGIT + PD-1 + SIRPα" represents the combination of TIGIT/G4Fc, PD-1/G1Fc and SIRPα/G4Fc. Figure 13 is a table showing an overview of the in vitro studies specifically described in Figures 2A to 10 (I). Figure 14 is a table showing an overview of the in vitro studies specifically described in Figures 11A to 12B (II). Figures 15A to 15D show schematic structures of TgPS_v2 forms, which respectively include TgPS-C1_v2, TgPS-C2_v2, TgPS-D_v2 and TgPS-E_v2. Figure 16A shows the binding ability of TgPS_v2 protein to TIGIT ligand PVR detected by ELISA. Figure 16B shows the binding ability of TgPS_v2 protein to PD-L1 detected by ELISA. Figure 16C shows the binding ability of TgPS_v2 protein to CD47 detected by ELISA. Figures 17A to 17D show the simultaneous binding ability of TgPS_2 protein to PVR, PD-L1 and CD47 detected by biolayer interferometry (BLI). Figure 18A shows the whole cell binding ability of TgPS_v2 protein to PVR tf CHO-S cells. Figure 18B shows the whole cell binding ability of TgPS_v2 protein to PD-L1 tf CHO-S cells. Figure 18C shows the whole cell binding ability of TgPS_v2 protein to CD47 tf CHO-S cells. Figure 19A shows the RBC binding curve of TgPS_v2 protein. Figure 19B shows the platelet binding curve of TgPS_v2 protein. Figures 20A to 20B show the blocking effect of TgPS_v1 protein and TgPS_v2 protein on the interaction between TIGIT and PVR using PVR tf CHO-S cells. Figures 21A to 21B show the blocking effect of TgPS_v1 protein and TgPS_v2 protein on the interaction between PD1 and PD-L1 using PD-L1 tf CHO-S cells. Figures 22A to 22B show the blocking effect of TgPS_v1 protein and TgPS_v2 protein on the interaction between SIRPα and CD47 using CD47 tf CHO-S cells. Figure 23 shows the blocking effect of TgPS_v2 protein on the interaction between PD-1 and PD-L1 measured using NFAT-Luc signaling blocking assay. Figure 24A shows macrophage-mediated phagocytosis of FaDu cells induced by TgPS_v1 protein and TgPS_v2 protein. Figure 24B shows macrophage-mediated phagocytosis of PD-L1 tf OE19 induced by TgPS_v1 protein and TgPS_v2 protein. Figure 25A shows macrophage-mediated phagocytosis of RBC induced by TgPS_v1 protein and TgPS_v2 protein. Figure 25B shows macrophage-mediated phagocytosis of platelets induced by TgPS_v1 protein and TgPS_v2 protein. Figure 26A shows IL-2 expression induced by TgPS_v2 protein in MLR assay. "3 combinations (G1Fc)" means the combination of TIGIT/G1Fc, PD-1-mt13/G1Fc and SIRPα-mt15/G1Fc. "3 combinations (G4Fc)" means the combination of TIGIT/G4Fc, PD-1-mt13/G4Fc and SIRPα-mt15/G4Fc. Figure 26B shows the expression of IFN-γ induced by TgPS_v2 protein in the MLR assay. "3 combinations (G1Fc)" means the combination of TIGIT/G1Fc, PD-1-mt13/G1Fc and SIRPα-mt15/G1Fc. "3 combinations (G4Fc)" means the combination of TIGIT/G4Fc, PD-1-mt13/G4Fc and SIRPα-mt15/G4Fc. Figure 27 is a table showing an overview of the in vitro studies specifically described in Figures 16A to 23 (I). Figure 28 is a table showing an overview of the in vitro studies described in detail in Figures 24A to 26B (II). Figure 29 lists the amino acid sequences of proteins used in the present disclosure.

TW202515923A_113123161_SEQL.xml

Claims (90)

A protein complex comprising: (a) Fc; (b) TIGIT (T cell immunoreceptor with immunoglobulin and ITIM domains) ligand binding domain; (c) PD-L1 (programmed death ligand 1) binding domain; and (d) CD47 binding domain. The protein complex of claim 1, wherein the TIGIT ligand binding domain is capable of binding to cells (e.g., cancer cells) expressing a TIGIT ligand (e.g., PVR or adhesion protein-2) and/or is capable of blocking the interaction between TIGIT and the TIGIT ligand. The protein complex of claim 1 to 2, wherein the TIGIT ligand binding domain is or includes the TIGIT extracellular domain. The protein complex of claim 3, wherein the TIGIT extracellular domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 1. The protein complex of any one of claims 1 to 4, wherein the PD-L1 binding domain is capable of binding to cells expressing PD-L1 (e.g., cancer cells) and/or is capable of blocking the interaction between PD-1 (programmed cell death protein 1) and PD-L1. The protein complex of any one of claims 1 to 5, wherein the PD-L1 binding domain is or includes a PD-1 extracellular domain. The protein complex of claim 6, wherein the PD-1 extracellular domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 2. The protein complex of claim 6, wherein the PD-1 extracellular domain comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 36. The protein complex of claim 8, wherein the amino acid corresponding to S39 of SEQ ID NO: 36 is H. The protein complex of claim 8 or 9, wherein the PD-1 extracellular domain further comprises a PD-L1 surface interaction sequence, wherein the PD-L1 surface interaction sequence comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 37, 38, 39 or 40. The protein complex of any one of claims 1 to 6 and 8 to 10, wherein the PD-L1 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 17. The protein complex of claim 1 to 11, wherein the CD47 binding domain is capable of binding to cells expressing CD47 (e.g., cancer cells) and/or is capable of blocking the interaction between CD47 and signal regulatory protein α (SIRPα). The protein complex of any one of claims 1 to 12, wherein the CD47 binding domain is or includes the SIRPα extracellular domain. The protein complex of claim 13, wherein the SIRPα extracellular domain comprises a sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 3. The protein complex of claim 14, wherein the SIRPα extracellular domain comprises one or more amino acid mutations at positions corresponding to H24, I31, E54, G55, H56 and/or D73 of SEQ ID NO: 3. The protein complex of claim 14 or 15, wherein the SIRPα extracellular domain comprises one or more of the following: (a)   The amino acid corresponding to H24 of SEQ ID NO: 3 is R; (b)   The amino acid corresponding to I31 of SEQ ID NO: 3 is T; (c)   The amino acid corresponding to E54 of SEQ ID NO: 3 is A; (d)   The amino acid corresponding to G55 of SEQ ID NO: 3 is K; (e)   The amino acid corresponding to H56 of SEQ ID NO: 3 is Q; and (f)   The amino acid corresponding to D73 of SEQ ID NO: 3 is I. The protein complex of any one of claims 1 to 16, wherein the CD47 binding domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 18. The protein complex of any one of claims 1 to 17, wherein the TIGIT ligand binding domain is linked to the N-terminus of the CH2 domain in the Fc, optionally via a hinge region. A protein complex as claimed in claim 18, wherein the PD-L1 binding domain is linked to the N-terminus of the TIGIT ligand binding domain, optionally via a first linker peptide. The protein complex of claim 19, wherein the CD47 binding domain is linked to the C-terminus of the CH3 domain in the Fc, optionally via a second linker peptide. The protein complex of claim 18, wherein the CD47 binding domain is linked to the C-terminus of the CH3 domain in the Fc, optionally via a first linker peptide. The protein complex of claim 21, wherein the PD-L1 binding domain is linked to the C-terminus of the CD47 binding domain, optionally via a second linker peptide. The protein complex of any one of claims 1 to 17, wherein the PD-L1 binding domain is linked to the N-terminus of the CH2 domain in the Fc, optionally via a hinge region. A protein complex as claimed in claim 23, wherein the TIGIT ligand binding domain is linked to the N-terminus of the PD-L1 binding domain, optionally via a first linker peptide. The protein complex of claim 24, wherein the CD47 binding domain is linked to the C-terminus of the CH3 domain in the Fc, optionally via a second linker peptide. The protein complex of any one of claims 1 to 17, wherein the CD47 binding domain is linked to the N-terminus of the CH2 domain in the Fc, optionally via a hinge region. The protein complex of claim 26, wherein the PD-L1 binding domain is linked to the N-terminus of the CD47 binding domain, optionally via a first linker peptide. The protein complex of claim 27, wherein the TIGIT ligand binding domain is linked to the C-terminus of the CH3 domain in the Fc, optionally via a second linker peptide. The protein complex of any one of claims 1 to 28, wherein the Fc is human IgG1 Fc. The protein complex of any one of claims 1 to 28, wherein the Fc is human IgG4 Fc. A protein complex as claimed in any one of claims 18 to 28 and 30, wherein the hinge region is a human IgG4 hinge region, which optionally has an S228P mutation according to EU numbering. A protein complex comprising (a) a first polypeptide, which comprises from N-terminus to C-terminus: a first PD-L1 binding domain, an optionally selected first linker peptide, a first TIGIT ligand binding domain, an optionally selected first hinge region, a first Fc region, an optionally selected second linker peptide, and a first CD47 binding domain; and (b) a second polypeptide, which comprises from N-terminus to C-terminus: a second PD-L1 binding domain, an optionally selected third linker peptide, a second TIGIT ligand binding domain, an optionally selected second hinge region, a second Fc region, an optionally selected fourth linker peptide, and a second CD47 binding domain. The protein complex of claim 32, wherein the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 2 or 17. The protein complex of claim 32 or 33, wherein the first TIGIT ligand binding domain and/or the second TIGIT ligand binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 1. The protein complex of claim 32 to 34, wherein the first CD47 binding domain and/or the second CD47 binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 3 or 18. The protein complex of any one of claims 32 to 35, wherein the first hinge region and/or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO: 8. The protein complex of any one of claims 32 to 36, wherein the first Fc region and/or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO: 9. The protein complex of any one of claims 32 to 35, wherein the first hinge region and/or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO: 10. The protein complex of any one of claims 32 to 35 and 38, wherein the first Fc region and/or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO: 11. The protein complex of any one of claims 32 to 39, wherein the first linker peptide and/or the third linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 4. The protein complex of any one of claims 32 to 40, wherein the second linker peptide and/or the fourth linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 5. The protein complex of any one of claims 32 to 41, wherein the first polypeptide and/or the second polypeptide comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 12, 13, 19 or 20. A protein complex comprising (a) a first polypeptide, which comprises from N-terminus to C-terminus: a first TIGIT ligand binding domain, an optionally selected first linker peptide, a first PD-L1 binding domain, an optionally selected first hinge region, a first Fc region, an optionally selected second linker peptide, and a first CD47 binding domain; and (b) a second polypeptide, which comprises from N-terminus to C-terminus: a second TIGIT ligand binding domain, an optionally selected third linker peptide, a second PD-L1 binding domain, an optionally selected second hinge region, a second Fc region, an optionally selected fourth linker peptide, and a second CD47 binding domain. The protein complex of claim 43, wherein the first TIGIT binding domain and/or the second TIGIT binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 1. The protein complex of claim 43 or 44, wherein the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 2 or 17. The protein complex of any one of claims 43 to 45, wherein the first CD47 binding domain and/or the second CD47 binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 3 or 18. The protein complex of any one of claims 43 to 46, wherein the first hinge region and/or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO: 8. The protein complex of any one of claims 43 to 47, wherein the first Fc region and/or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO: 9. The protein complex of any one of claims 43 to 46, wherein the first hinge region and/or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO: 10. The protein complex of any one of claims 43 to 46 and 49, wherein the first Fc region and/or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO: 11. The protein complex of any one of claims 43 to 50, wherein the first linker peptide and/or the third linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 4. The protein complex of any one of claims 43 to 51, wherein the second linker peptide and/or the fourth linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 5. The protein complex of any one of claims 43 to 52, wherein the first polypeptide and/or the second polypeptide comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 14 or 21. A protein complex comprising (a) a first polypeptide, which comprises from N-terminus to C-terminus: a first TIGIT ligand binding domain, an optionally selected first hinge region, a first Fc region, an optionally selected first linker peptide, a first CD47 binding domain, an optionally selected second linker peptide, and a first PD-L1 binding domain; and (b) a second polypeptide, which comprises from N-terminus to C-terminus: a second TIGIT ligand binding domain, an optionally selected second hinge region, a second Fc region, an optionally selected third linker peptide, a second CD47 binding domain, an optionally selected fourth linker peptide, and a second PD-L1 binding domain. The protein complex of claim 54, wherein the first TIGIT binding domain and/or the second TIGIT binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 1. The protein complex of claim 54 or 55, wherein the first CD47 binding domain and/or the second CD47 binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 3 or 18. The protein complex of any one of claims 54 to 56, wherein the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 2 or 17. The protein complex of any one of claims 54 to 57, wherein the first hinge region and/or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO: 8. The protein complex of any one of claims 54 to 58, wherein the first Fc region and/or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO: 9. The protein complex of any one of claims 54 to 57, wherein the first hinge region and/or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO: 10. The protein complex of any one of claims 54 to 57 and 60, wherein the first Fc region and/or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO: 11. The protein complex of any one of claims 54 to 61, wherein the first linker peptide and/or the third linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 5. The protein complex of any one of claims 54 to 62, wherein the second linker peptide and/or the fourth linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 4. The protein complex of any one of claims 54 to 63, wherein the first polypeptide and/or the second polypeptide comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 15 or 22. A protein complex comprising (a) a first polypeptide, which comprises from N-terminus to C-terminus: a first PD-L1 binding domain, an optionally selected first linker peptide, a first CD47 binding domain, an optionally selected first hinge region, a first Fc region, an optionally selected second linker peptide, and a first TIGIT ligand binding domain; and (b) a second polypeptide, which comprises from N-terminus to C-terminus: a second PD-L1 binding domain, an optionally selected third linker peptide, a second CD47 binding domain, an optionally selected second hinge region, a second Fc region, an optionally selected fourth linker peptide, and a second TIGIT ligand binding domain. The protein complex of claim 65, wherein the first TIGIT binding domain and/or the second TIGIT binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 1. The protein complex of claim 65 or 66, wherein the first CD47 binding domain and/or the second CD47 binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 3 or 18. The protein complex of any one of claims 65 to 67, wherein the first PD-L1 binding domain and/or the second PD-L1 binding domain comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 2 or 17. The protein complex of any one of claims 65 to 68, wherein the first hinge region and/or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO: 8. The protein complex of any one of claims 65 to 69, wherein the first Fc region and/or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO: 9. The protein complex of any one of claims 65 to 68, wherein the first hinge region and/or the second hinge region comprises a sequence that is at least 80% identical to SEQ ID NO: 10. The protein complex of any one of claims 65 to 68 and 71, wherein the first Fc region and/or the second Fc region comprises a sequence that is at least 80% identical to SEQ ID NO: 11. The protein complex of any one of claims 65 to 72, wherein the first linker peptide and/or the third linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 4. The protein complex of any one of claims 65 to 73, wherein the second linker peptide and/or the fourth linker peptide comprises a sequence that is at least 80% identical to SEQ ID NO: 5. The protein complex of any one of claims 65 to 74, wherein the first polypeptide and/or the second polypeptide comprises a sequence that is at least 80%, 90%, 95% or 100% identical to SEQ ID NO: 16. A nucleic acid comprising a polynucleotide encoding the protein complex of any one of claims 1 to 75. A nucleic acid as in claim 76, wherein the nucleic acid is DNA (e.g., cDNA) or RNA (e.g., mRNA). A vector comprising one or more nucleic acids of claim 76 or 77. A cell comprising the vector of claim 78. The cell of claim 79, wherein the cell is a CHO cell. A cell comprising one or more nucleic acids of claim 76 or 77. A method for producing a protein complex, the method comprising: (a) culturing the cell under conditions sufficient for the cell to produce the protein complex; and (b) collecting the protein complex produced by the cell. A protein conjugate comprising the protein complex of any one of claims 1 to 75, wherein the protein conjugate is covalently bound to a therapeutic agent. The protein conjugate of claim 83, wherein the therapeutic agent is a cytotoxic agent or a cell growth inhibitory agent. A method for treating an individual suffering from cancer, the method comprising administering to the individual a therapeutically effective amount of a composition comprising the protein complex of any one of claims 1 to 75 or the protein binding of claim 83 or 84. The method of claim 85, wherein the individual has cancer cells expressing PVR, adhesion protein-2, CD47 and/or PD-L1. The method of claim 85 or 86, wherein the cancer is breast cancer, prostate cancer, non-small cell lung cancer, pancreatic cancer, diffuse large B-cell lymphoma, mesothelioma, lung cancer, ovarian cancer, colon cancer, pleural tumor, neuroglioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, stomach cancer, bile duct cancer, head and neck cancer, blood cancer, or a combination thereof. A method for reducing tumor growth rate, the method comprising contacting tumor cells with an effective amount of a composition comprising a protein complex as in any one of claims 1 to 75 or a protein conjugate as in claim 83 or 84. A method for killing tumor cells, the method comprising contacting the tumor cells with an effective amount of a composition comprising a protein complex as described in any one of claims 1 to 75 or a protein conjugate as described in claim 83 or 84. A pharmaceutical composition comprising the protein complex of any one of claims 1 to 75 and a pharmaceutically acceptable carrier.