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WO2018121602A1 - Système de changement de la fonction d'une protéine commandé par un médicament à petites molécules - Google Patents

Système de changement de la fonction d'une protéine commandé par un médicament à petites molécules Download PDF

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Publication number
WO2018121602A1
WO2018121602A1 PCT/CN2017/118970 CN2017118970W WO2018121602A1 WO 2018121602 A1 WO2018121602 A1 WO 2018121602A1 CN 2017118970 W CN2017118970 W CN 2017118970W WO 2018121602 A1 WO2018121602 A1 WO 2018121602A1
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protein
protease
switch system
protein function
target protein
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PCT/CN2017/118970
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Chinese (zh)
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邹庆剑
翟珍珍
高斌
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天津天锐生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
    • C12N9/506—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses derived from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the invention relates to the field of biotechnology, in particular to a protein function switching system controlled by a small molecule drug.
  • protein function regulation methods are mainly divided into three categories: 1 using small molecules or light to regulate the binding or dissociation of two heterodimers; 2 using small molecules to regulate protein structure changes; 3 using small molecules to regulate protein stability Sex.
  • the techniques involved in the regulation of each type of protein function are described below:
  • CID chemical inducers of dimerization
  • FK506 the immunosuppressive drug FK506.
  • FKBP12 FK506 binding protein
  • calcineurin FK506 binding protein
  • FK1012 FK506 binding protein
  • rapamycin a macrocyclic natural product that mediates the interaction between FKBP12 and the FRB domain of mTOR (Brown et al., 1994). If the two proteins of interest are fused to FKBP and FRB, respectively, they can be close to each other in the presence of rapamycin, and rapamycin can rapidly achieve FKBP-FRB binding at low doses. However, cellular endogenous FKBP and mTOR compete with FKBP and FRB fusion proteins for binding to rapamycin, resulting in ineffective interactions. Binding of rapamycin to mTOR also leads to cell cycle arrest (Haruki et al., 2008).
  • the optimized method to obtain Rimiducid-regulated FKBP-FRB dimerization can effectively reduce the CID off-target effect by site-directed mutagenesis of rapamycin derivatization and FKBP or FRB domain (Patent No.: US2015/065629) .
  • the dexamethasone-methotrexate (Dex-Mtx) complex induces an interaction between the glucocorticoid receptor (GR) and dihydrofolate reductase (DHFR).
  • GR glucocorticoid receptor
  • DHFR dihydrofolate reductase
  • Mtx is an inhibitor of DHFR, which limits its use.
  • trimethoprim-SLF (TMP-SLF) compound triggers the interaction between the DHFR-FKBP12 fusions (Czlapinski et al., 2008). This compound does not bind endogenous proteins, but these small molecules are expensive and require multiple steps of synthesis, which limits their widespread use.
  • Abscisic acid is one of the hormones that stimulate plant development. It inhibits protein phosphatase (PP2Cs) by binding to pyribactin resistance (PYR)/PYR1-like (PYL) (Cutler et al., 2010; patent number: US2012043121). After merging the PYL and PP2C domains with the DNA binding domain Gal4 and the transcriptional activation domain VP64, respectively, Gal4 transcriptional activity can be reconstituted.
  • Another phytohormone, gibberellin (GA3) can also be used as CID. GA3 binds to GID1 and promotes its interaction with GAI (Hirano et al., 2008; Miyamoto et al., 2012). Plant hormones are relatively economical, safe and responsive, making them useful.
  • the protein of interest is split into two inactive fragments, and small molecules of specific compounds can induce association of these fragments, thereby restoring the structure and function of the target protein. This process is also called fragment complementation.
  • Muir and colleagues developed an intein-based conditional protein splicing system (Mootz and Muir, 2002). The intein is divided into N- and C-terminal halves. When present alone, both inteins are inactive, but once they are close to each other, an active domain is formed, and the proteins at both ends are spliced together to form an activity. protein. For example, two former fusions are between FKBP and maltose binding protein (MBP), which is fused between the FRB and His tags.
  • MBP maltose binding protein
  • fusions dimerize under rapamycin induction and the intein domain is close, which restores intein splicing activity and leads to the formation of His-tagged MBP.
  • Intein splicing is an irreversible process, and the major disadvantage of intein-based fragment-complementing systems is the lack of reversibility.
  • conditional protein degradation is accomplished using the ubiquitin-proteasome system, so the regulation of protein stability can be achieved by using small molecules to regulate various aspects of protein degradation.
  • Degradants degrons
  • Most degradation-mediated degradation is accomplished by the ubiquitin-proteasome degradation pathway, or by autophagy.
  • a number of degrading sequences have been identified, for example: ddFKBP is an unstable domain (DD domain) that is rapidly degraded after expression.
  • DD domain unstable domain
  • the chemical small molecule shield-1 can bind this structure to make it stable without rapid degradation.
  • the target protein is bound to ddFKBP, and the stability and degradation process of the protein can be effectively regulated by shield-1 (Banaszynski et al., 2006).
  • ecDHFR can also regulate its own stability by binding to trimethoprim (Iwamoto et al., 2010; patent number: 09487787).
  • the plant hormone binding domain (AID) IAA17 binds to TIR1 mediated by indoleacetic acid and is recognized by ubiquitin ligase and is degraded by ubiquitination in one step (Nishimura et al., 2009; :US20120115232).
  • the existing protein activity control technologies mainly include: 1. Using rapamycin to regulate the polymerization and dissociation of FKBP and FRB; 2. Using the interaction between abscisic acid regulation (PYR) and PYR1-like (PYL) 3. Using gibberellin to regulate the interaction between CID and GAI; 4. Using intein to achieve splicing of two-stage protein; 5. Using regulatable degradants to achieve regulation of fusion protein activity, such as shield-1 regulation of ddFKBP fusion The activity of the protein, TMP regulates the activity of the DHFR fusion protein, and the acetic acid regulates the activity of the AID fusion protein.
  • the technical problem to be solved by the present invention is how to regulate the target protein.
  • the present invention first provides a small molecule drug controlled protein function switching system.
  • the switch system provided by the invention consists of an operon original and a control sub-unit, the control is a single viral protease, or a protease-degradant formed by fusion of a viral protease and a protein degradation agent; the operon is located inside the target protein Or located between the target protein and the protease-degrading structure, and consists of a ligation peptide of the protease; the whole system regulates the target protein through the inhibitor compound of the small molecule of the protease as a switch.
  • the regulation of the protein of interest involves air conditioning control of the structure and/or function and/or location of the protein of interest.
  • the regulation of the structure and function includes regulating the primary structure, the tertiary structure, the expression of the protein, the stability, the functional activity, and the intracellular position of the target protein.
  • the switching system further comprises an inhibitor compound of a small molecule of a protease.
  • the operon element is a linker peptide recognizable and cleaved by the protease; the number of the operon elements is one or two or more.
  • the protease may be a viral protease.
  • the viral protease may be a hepatitis C virus protease (NS3pro/4A) or a protease having 70% or more homology with the hepatitis C virus protease NS3pro/4A. Mutant or isomerase.
  • the amino acid sequence of the NS3pro/4A protease is SEQ ID No. 7.
  • the invention mutates the NS3pro and 4A structural linking portions of the N34d protein to obtain the NS3pro/4A protease, and the NS3pro/4A protease has stable structure and loses the function of the degradation, but retains the complete protease function.
  • the N34d protein is an unstable and easily degradable protein formed by removing the NS3 RNA helicase region from the NS3/4A structure.
  • the viral protease can also be ligated into a degrading domain (degron).
  • the degrading domain may be an NS4A degrader which, after being fused to the corresponding protein, degrades in a very short period of time, thereby affecting protein function.
  • the protease-degrading protein is an N34d protein having an amino acid sequence of SEQ ID No. 6.
  • the degrading domain can also be a small molecule-regulated degrader, such as ddFKBP or exDHFR.
  • the two domains can be stabilized for a long time without being degradation. After removal of the ligand, the two structures are extremely unstable and, after expression, degrade in a very short time.
  • linker peptide is one of the following sequences:
  • 4B5A Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp;
  • 5A5B Glu Asp Val Val Cys Cys Ser Met Ser Tyr.
  • the inhibitor compound of the protease small molecule is one of the following compounds: Telaprevir (CAS Number: 402957-28-2); Boceprevir (CAS Number: 394730-60-0); Simeprevir (CAS Number: 923604- 59-5); Faldaprevir (CAS Number: 801283-95-4); Asunaprevir (CAS Number: 630420-16-5); Furaprevir (CAS Number: 1435923-88-8).
  • the degradation agent is an NS3/4A unstable region, a ddFKBP unstable region, or an ecDHFR unstable region.
  • the target protein is a single protein or a fusion protein.
  • Each protein may be a protein having one or several domains, or a fusion protein composed of a plurality of functional proteins.
  • the operon element may be located inside the target protein (such as between domains) or between the fusion moieties.
  • the target protein may be any one of a fluorescent protein, a drug resistance gene, an antibody, a cytokine, and an antigen recognition molecule.
  • the target protein is a single protein, the target protein, the control subunit, and the operon element constitute a fusion protein, the operon element is used to link the target protein and the control sub-assembly; the control The sub-origin is a protease-degrading agent;
  • the target protein is a fusion protein having several domains or several functional proteins
  • a viral protease recognition sequence can be inserted between each domain or a functional protein.
  • the target protein, the control subunit, and the operon element constitute a fusion protein, the operon element is used to link the target protein and the control subunit, or to link a domain of the target protein Or functional protein.
  • the protein of interest may be a fusion protein of two different fluorescent proteins, the linker peptide being located between two different fluorescent proteins, joining two different fluorescent proteins, and fluorescent protein fusion expression in the case where protease activity is inhibited It will co-localize in the cell. Under the protease active state, the fluorescent protein expressed by fusion will be cleaved into two proteins by protease and expressed separately. Or a chimeric antigen receptor (CAR) having multiple domains, the linker peptides being located between different domains, linking different domains, allowing the various domains of the chimeric antigen receptor to be cascaded, and the protease activity is inhibited.
  • CAR chimeric antigen receptor
  • the chimeric antigen receptor is expressed in T cells and has the function of recognizing a tumor antigen delivery signal and activating T cell function. In the protease active state, each domain of the chimeric antigen receptor is cleaved into two or more Independent structure that affects T cells to recognize tumor antigens and transmit signals.
  • the target protein can be expressed in fusion with viral proteases or separately. Fusion expression allows the viral protease to be located closer to the target protein and function faster. Separate expression reduces the effect on the structure of the target protein and more precisely regulates protein activity.
  • protein functional switching system is used in eukaryotic mammalian cells.
  • the present invention further provides a nucleic acid molecule, a plasmid, a cell line, and a kit comprising the above elements for expressing a protein function switching system.
  • the nucleic acid molecule is a DNA molecule represented by SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4 or SEQ ID No. 5;
  • An expression vector for a nucleic acid molecule which may specifically be a lentiviral vector Pltr vector;
  • the cell line is a cell line containing the plasmid, and the cell line may specifically be a 293T cell or a 3T3 cell.
  • the present invention also provides a novel use of the above protein functional switch system or nucleic acid molecule, plasmid, cell line or kit.
  • the invention provides the use of the above protein functional switch system or nucleic acid molecule, plasmid, cell line or kit for regulating the structure and/or function of a protein of interest.
  • the invention also provides the use of a protein functional switch system or nucleic acid molecule, plasmid, cell line or kit as described above for the preparation of a product that modulates the structure and/or function of a protein of interest.
  • the invention also provides the use of the above protein function switch system or nucleic acid molecule, plasmid, cell line or kit for regulating the activity of a target protein.
  • the invention also provides the use of the above protein functional switch system or nucleic acid molecule, plasmid, cell line or kit for the preparation of a product for regulating the activity of a target protein.
  • the invention also provides the use of the above protein function switch system or nucleic acid molecule, plasmid, cell line or kit for regulating the stability of a target protein.
  • the invention also provides the use of the above protein functional switch system or nucleic acid molecule, plasmid, cell line or kit for the preparation of a product for regulating the stability of a target protein.
  • the invention also provides the use of the above protein function switch system or nucleic acid molecule, plasmid, cell line or kit for regulating the spatiotemporal position of a target protein.
  • the invention also provides the use of the above protein functional switch system or nucleic acid molecule, plasmid, cell line or kit for the preparation of a product for regulating the spatiotemporal position of a target protein.
  • the present invention finally provides a method of regulating a target protein.
  • the method for regulating a target protein comprises the steps of: expressing a fusion protein comprising a promoter subgenus, an operon element and a target protein in a recipient cell, and then using an inhibitor compound of a protease small molecule to regulate the controller The activity of the original, thereby achieving the regulation of the target protein;
  • the control element is a single viral protease, or a protease-degradant fused by a viral protease and a protein degrader; the operon element is a linker peptide that the protease recognizes and cleaves.
  • the number of the operon originals is one or two or more.
  • the viral protease is a hepatitis C virus protease.
  • the hepatitis C virus protease is specifically a hepatitis C virus protease NS3pro/4A or a protease mutant or isomerase having 70% or more homology with the hepatitis C virus protease NS3pro/4A.
  • the linker peptide is one of the following sequences:
  • 4B5A Glu Cys Thr Thr Pro Cys Ser Gly Ser Trp;
  • 5A5B Glu Asp Val Val Cys Cys Ser Met Ser Tyr.
  • the inhibitor compound of the protease small molecule is one of the following compounds: Telaprevir (CAS Number: 402957-28-2); Boceprevir (CAS Number: 394730-60-0); Simeprevir (CAS Number: 923604) -59-5); Faldaprevir (CAS Number: 801283-95-4); Asunaprevir (CAS Number: 630420-16-5); Furaprevir (CAS Number: 1435923-88-8).
  • the proton is an NS3/4A unstable region, a ddFKBP unstable region, or an ecDHFR unstable region.
  • the protease-degrading agent is an N43d protein.
  • the target protein is a single protein or a fusion protein.
  • Each protein may be a protein having one or several domains, or a fusion protein composed of a plurality of functional proteins.
  • the operon element may be located inside the target protein (such as between domains) or between the fusion moieties.
  • the target protein may be any one of a fluorescent protein, a drug resistance gene, an antibody, a cytokine, and an antigen recognition molecule.
  • the method of expressing the fusion protein comprising the promoter element, the operon element and the target protein in the recipient cell, and then using the inhibitor compound of the protease small molecule to regulate the activity of the control element is a vector expressing the control element, the operon element, and the target protein is introduced into the recipient cell to obtain a recombinant cell; the recombinant cell is cultured in a culture system to obtain the fusion protein; Addition or removal of the inhibitor compound in the culture system can effect modulation of the activity of the control element.
  • the specific method can be as follows (1) or (2):
  • (1-1) co-transfecting a receptor-derived cell with a protease-degradant expressing a target protein, a protease and a proteosome, and a vector of the protease-recognizing and cleavable linker peptide together with the small molecule inhibitor of the protease Recombinant cells are obtained; the recombinant cells are cultured in a recombinant cell culture system, and a fusion protein is expressed, wherein the inhibitor inhibits the recognition and cleavage activity of the protease in the fusion protein, and the degradant converts the fusion protein Degrading, the target protein in the fusion protein is inactivated;
  • the fusion protein consists of the target protein, the protease-degradant, and the linker peptide for ligation;
  • the vector expressing the control subunit, the operon original, and the target protein is a coding gene containing the control subunit, a coding gene of the operon original, and the target protein.
  • a fragment of the coding gene is inserted into the multiple cloning site of the expression vector.
  • the coding gene containing the control subunit, the coding gene of the operon original, and the fragment of the coding gene of the target protein may specifically be SEQ ID No. 1 or SEQ ID No. 2 or SEQ ID No. 3. Or the DNA molecule shown in SEQ ID No. 4 or SEQ ID No. 5.
  • the expression vector may specifically be a lentiviral vector Pltr vector.
  • the recipient cell may specifically be a 293T cell or a 3T3 cell.
  • the vector expressing the target protein including: functional protein, fusion protein or genetic engineering protein
  • viral protease or engineered viral protease
  • viral protease recognition and cleavable linker peptide is co-transformed.
  • the activity of the protease can be regulated by the addition and removal of small molecule inhibitors of the corresponding proteases, and the expression of the protease affects the function of the target protein, thereby indirectly controlling the function of the target protein.
  • the viral protease of the present invention may be a natural protease, a protease that is genetically engineered to reduce the molecular weight of the protease, or a protease-degradant fused to a degrading domain (degron).
  • Viral protease inhibitors are primarily drugs that are commercially available for treatment or for clinical trials.
  • the invention prepares a switching system of a virus protease-dependent small molecule drug for regulating the function of a target protein, and proposes a protein function regulation method which is fast, efficient, reversible, controllable, simple, economical and has good application prospect.
  • FIG. 1 is a diagram showing the SwichOFF system-green fluorescent protein expression vector in Example 1 of the present invention.
  • Fig. 2 is a diagram showing the expression of green fluorescent protein by small molecule ASV in Example 1 of the present invention. Above: Green fluorescent protein is stably expressed when no ASV is added; the following figure: Green fluorescent protein is degraded and not expressed after adding ASV.
  • Figure 3 is a rapid switching of fluorescent protein expression in Example 1 of the present invention. Above: 24 hours after the addition of ASV, green fluorescence rapidly diminished. Bottom: After removal of ASV, fluorescence is rapidly and stably expressed.
  • FIG. 4 is a diagram showing the SwichON system-fusion fluorescent protein expression vector in Example 2 of the present invention.
  • Figure 5 is a diagram showing the localization of a fluorescent protein in a cell by the SwichON system in Example 2 of the present invention.
  • the first column is red fluorescence
  • the second column is green fluorescence
  • the third column is fluorescence overlay.
  • the first row no ARV, green fluorescence is expressed in the cytoplasm; the second column: adding ASV, green fluorescence is expressed in mitochondria.
  • Figure 6 is a diagram showing the localization of the fast-switching fusion fluorescent protein in the SwichON system of Example 2 of the present invention.
  • the first column is red fluorescence
  • the second column is green fluorescence
  • the third column is fluorescence overlay. 24 hours after the addition of ASV, green fluorescence rapidly localized from the cytoplasm to the mitochondria.
  • Figure 7 is a diagram showing the localization of the fast-switching fusion fluorescent protein in the SwichON system of Example 2 of the present invention.
  • the first column is red fluorescence
  • the second column is green fluorescence
  • the third column is fluorescence overlay. 24 hours after the removal of ASV, green fluorescence was rapidly released from the mitochondria to the cytoplasm.
  • Figure 8 is a map of the SwichOFF system CAR regulation carrier in the third embodiment of the present invention.
  • Fig. 9 is a schematic diagram showing the regulation of CAR expression by the SwichOFF system in the third embodiment of the present invention.
  • Figure 10 is a map of the SwichON system CAR regulatory vector in Example 4 of the present invention.
  • Fig. 11 is a schematic diagram showing the function of regulating the CAR of the SwichON system in the fourth embodiment of the present invention.
  • Figure 12 is a map of the SwichON system CAR regulatory vector in Example 5 of the present invention.
  • Figure 13 is a schematic diagram showing the function of the SwichON system for regulating CAR in the fifth embodiment of the present invention.
  • Figure 14 is a Western blot analysis of the Switch protein regulatory system in Example 6 of the present invention.
  • fCAR-V1 The first column: a section of 63kd Flag-CAR fragment is expressed without ASV. Second column: After the addition of ASV, the fusion protein rapidly degraded.
  • fCAR-V2 group First column: Flag-CAR1 protein of 45 kd in size was detected without ASV. Second column: After adding ASV, the anti-flag antibody detected 97kd of intact protein.
  • fCAR-V3 group The first column: the Flag-CAR1 protein with a size of 33 kd was expressed without ASV. Second column: After adding ASV, the anti-flag antibody detected 97kd of intact protein. Since the hinge site of CAR itself may naturally break, a group of 33kd protein can be detected in all groups.
  • Figure 15 shows the results of the chimeric antigen receptor killing function test.
  • Example 1 Small molecule drug modulates the activity of green fluorescent protein (SwichOFF system)
  • the green fluorescent protein is expressed by fusion with the self-degrading HCV protease, and the expression of the green fluorescent protein is regulated by the HCV protease inhibitor ASV by inhibiting the protease activity.
  • the fusion expression gene represented by SEQ ID No. 1 was inserted into a lentiviral vector Pltr vector (addgen, 25870) to obtain a recombinant vector Pltr-sfGFP-N3N4, and positions 1-714 of SEQ ID No. 1 were sfGFP gene, 721-
  • the 753 is the coding gene of the HCV viral protease recognition sequence 4A4B (DEMEECSQHLP), and the 842-1635 is the N34d gene (with the NS3/4A sequence and a stretcher sequence).
  • the amino acid sequence of the N34d protein expressed by the N34d gene is SEQ ID No. 6.
  • the NS3/4A structure contains NS3pro (protease region), NS3 RNA helicase region and 4A region.
  • the N34d protein is an unstable and easily degradable protein formed by removing the NS3 RNA helicase region from the NS3/4A structure.
  • Fig. 1 The structure of the recombinant vector Pltr-sfGFP-N3N4 is shown in Fig. 1.
  • ASV 0uM group one well was added to the medium without ASV (DMEM (GBICO) containing 10% serum (BI));
  • ASV 1uM group One well was added to the medium containing 1 uM ASV.
  • the specific transfection steps are as follows: 1) Take two 1.5ml EP tubes and add 50uL respectively. Medium, then add 3.0uL Lipofectamine to one of the tubes 3000 reagent (Thermo Fisher), 2 ug DNA plasmid (recombinant vector Pltr-sfGFP-N3N4) was added to the other tube, and mixed separately. 2) The liquids in the two EP tubes were mixed into one tube, and a total of 100 uL of the mixture was obtained, and the mixture was gently blown and mixed for 15 times, and allowed to stand at room temperature for 5 minutes. 3) Add 50 uL of the mixture to the corresponding wells of the ASV 0uM group and the ASV 1uM group, respectively, and change the solution after 6 hours.
  • the cells were observed to express fluorescence.
  • the result is shown in Figure 2. It can be seen from the figure: the first row: the fusion protein sfGFP-4A4B-NS3/4A-Degron expressed without ASV (ASV 0uM group). The protease has a cleavage function and the fusion protein is cleaved at 4A4B. Degron then mediates rapid degradation of the NS3/4A protease without affecting the function of sfGFP.
  • ASV ASV 1uM group
  • was added and the 3T3 cells also expressed the fusion protein sfGFP-4A4B-NS3/4A-Degron.
  • ASV inhibited the recognition and cleavage of 4A4B by the NS3/4A protease, allowing Degron to bring the entire fusion protein into the ubiquitin-proteasome pathway for rapid degradation.
  • sfGFP is rapidly degraded, rendering cells unable to express green fluorescent protein. This indicates that ASV can regulate the expression of target proteins.
  • the cell culture medium of ASV 0uM group was aspirated, and fresh medium containing 1 uM of ASV was added;
  • the ASV 1uM cell culture medium was aspirated, washed twice with PBS, and then fresh ASV-free medium was added.
  • the cells were observed to express fluorescence after adding ASV and removing ASV for 24 h.
  • the result is shown in Figure 3. It can be seen from the figure: The first row: without the addition of ASV, the expression of green fluorescent protein is very high in the cells, and the expression of green fluorescent protein drops sharply at 24 h after the addition of ASV, to a very weak level. The second row: under the condition of adding ASV, the cells could not express or only a few cells expressed very weak green fluorescent protein. After 24 hours of ASV, the expression of green fluorescent protein in the cells recovered to a high level. This indicates that ASV can quickly switch the expression of the target protein.
  • the green fluorescent protein, the red fluorescent protein and the self-degrading HCV protease are fused and expressed, and the HCV protease inhibitor ASV inhibits the localization of the green fluorescent protein and the red fluorescent protein by inhibiting the protease activity.
  • the fusion protein gene represented by SEQ ID No. 2 was inserted into the lentiviral vector Pltr vector to obtain the recombinant vector Pltr-tomDsRed-N3N4-GFP V2, and the 1-10th position of SEQ ID No. 2 was the mitochondrial membrane localization signal of the Tom20 gene.
  • the coding gene, the 118th-789th position is the DsRed ex gene, the 808-840 is the coding gene of the HCV viral protease recognition sequence 4A4B (DEMEECSQHLP), the 880-1590 is the sfGFP gene, and the 1606-1629 bits are the flag tag sequence.
  • the 1660-2352 position is the NS3pro/4A protease gene.
  • the amino acid sequence of the NS3pro/4A protease encoded by the NS3pro/4A protease gene is SEQ ID No. 7.
  • the NS3pro/4A protease is a protease obtained by mutating the NS3pro and 4A structural linking portions of the N34d protein.
  • the NS3pro/4A protease is structurally stable and loses its proteolytic function, but retains intact protease function.
  • the mitochondrial membrane localization signal sequence of the Tom20 gene is as follows: Met Val Gly Arg Asn Ser Ala Ile Ala Ala Gly Val Cys Gly Ala Leu Phe Ile Gly Tyr Cys Ile Tyr Phe Asp Arg Lys Arg Arg Ser Asp Pro Asn Phe Lys.
  • the flag tag sequence is as follows: Asp Tyr Lys Asp Asp Asp Asp Lys.
  • ASV 0uM group one well was added to the medium without ASV;
  • ASV 1uM group One well was added to the medium containing 1 uM ASV.
  • the specific transfection steps are as follows: 1) Take two 1.5ml EP tubes and add 50uL respectively. Medium, then add 3.0uL Lipofectamine to one of the tubes 3000 reagent (Thermo Fisher), 2 ug DNA plasmid (recombinant vector Pltr-sfGFP-N3N4) was added to the other tube, and mixed separately. 2) The liquids in the two EP tubes were mixed into one tube, and a total of 100 uL of the mixture was obtained, and the mixture was gently blown and mixed for 15 times, and allowed to stand at room temperature for 5 minutes. 3) Add 50 uL of the mixture to the corresponding wells of the ASV 0uM group and the ASV 1uM group, respectively, and change the solution after 6 hours.
  • the cells were observed to express fluorescence.
  • the result is shown in Figure 5. It can be seen from the figure that the first row: the fusion protein MTS-DsRed-4A4B-sfGFP-NS3/4A was expressed without ASV (ASV 0uM group).
  • the protease is active and can cleave the fusion protein at 4A4B to form two proteins, MTS-DsRed and sfGFP-NS3/4A. Due to the mitochondrial localization signal, MTS-DsRed expression was localized on the mitochondrial outer membrane, while the sfGFP-NS3/4A fusion protein was widely distributed in the cytoplasm.
  • the cell culture medium of ASV 0uM group was aspirated, and fresh medium containing 1 uM of ASV was added;
  • the ASV 1uM cell culture medium was aspirated, washed twice with PBS, and then fresh ASV-free medium was added.
  • the cells expressed fluorescence after 24 hours of ASV addition The result is shown in Figure 6. As can be seen from the figure: The first row: green fluorescent protein and red fluorescent protein are expressed separately without ASV. Second row: After 24 hours of ASV addition, the localization of green fluorescent protein in the cells was gradually transferred to the mitochondrial surface.
  • the chimeric antigen receptor (CAR) is expressed by fusion with a self-degrading HCV protease, and the chimeric antigen receptor activity is regulated by the HCV protease inhibitor ASV by inhibiting protease activity.
  • the fusion protein gene represented by SEQ ID No. 3 was inserted into the lentiviral vector Pltr vector to obtain the recombinant vector Pltr-CAR19-N34d, the 1st to 27th of SEQ ID No. 3 was the Flag tag sequence, and the 28th to 1491th were CAR Genes, positions 1498-1530 are the genes encoding the HCV viral protease recognition sequence 4A4B (DEMEECSQHLP), and positions 1588-2388 are the N34d gene (with the NS3/4A sequence and a stretcher sequence).
  • the structure of the recombinant vector Pltr-CAR19-N34d is shown in Fig. 8.
  • the antigen recognized by the chimeric antigen receptor is human CD19, and the CAR is expressed by fusion with the self-degrading HCV protease, and the protease recognition site 4A4B is located between the two proteins.
  • the HCV protease inhibitor ASV regulates CAR expression by inhibiting protease activity.
  • Figure 9 Left: In the absence of ASV, NS3/4A cleaves the 4A4B sequence, CAR is separated from protease, protease is degraded by degron into the ubiquitin-proteasome system, and CAR can express and recognize antigen And the function of activating T cells internal signals. At this point, the switch is in the on state.
  • CAR chimeric antigen receptor
  • the fusion protein gene represented by SEQ ID No. 4 was inserted into the lentiviral vector Pltr vector to obtain the recombinant vector Pltr-CAR19-N3V4V2, the 1st to 27th of SEQ ID No. 4 was the Flag tag sequence, and the positions 28 to 1530 were CAR.
  • the structure of the recombinant vector Pltr-CAR19-N34d is shown in FIG.
  • the chimeric antigen receptor recognizes an antigen that is human CD19, and fuses CAR and HCV protease, and the protease recognition site 4A4B is located between the 4-1BB and CD3zata domains of the CAR.
  • the structure and function of CAR is controlled by HCV protease by ASV.
  • Figure 11 Left panel: In the absence of ASV, NS3/4A cleaves the 4A4B sequence, and the 4-1BB in the CAR protein is separated from the CD3zeta domain and cannot transmit cell proliferation signals. Even if CAR-T cells recognize tumor antigens, they cannot effectively kill the corresponding tumor cells because they cannot proliferate. At this point, the switch is in the off state.
  • NS3/4A is inhibited and can not cleave 4A4B sequence, CAR, protease fusion expression, CAR has a complete structure, and functions to recognize antigen and activate T cell internal signals. At this point, the switch is in the on state.
  • the fusion of the chimeric antigen receptor (CAR) with the HCV protease is different from that of the second embodiment in that the protease recognition region is increased within the CAR protein by three, which are located between the anti-CD19 scFv of the CAR and the transmembrane region of the CD8, Between the 4-1BB and CD3zata domains, between the CD8 transmembrane region and the 4-1BB domain.
  • the structure and function of CAR is controlled by HCV protease by ASV.
  • the fusion protein gene represented by SEQ ID No. 5 was inserted into the lentiviral vector Pltr vector to obtain the recombinant vector Pltr-CAR19-N3V4V3, the 1st to 28th of SEQ ID No. 5 was the Flag tag sequence, and the 28th to the 1296th was CAR
  • the gene encoding the HCV viral protease recognition sequence 4A4B is located between the scFv and CD8 transmembrane regions, the CD8 transmembrane region and the 4-1BB domain, and the HCV viral protease recognition sequence 5A5B is located between the 4-1BB and CD3zata domains, 1648.
  • the -2334 position is the NS3pro/4A protease gene.
  • the structure of the recombinant vector Pltr-CAR19-N34d is shown in FIG.
  • the chimeric antigen receptor recognizes the antigen as human CD19, and fuses the expression of CAR and HCV protease.
  • the protease recognition sites are located between the anti-CD19scFv of CAR and the transmembrane region of CD8, respectively.
  • Figure 13 Left: In the absence of ASV, NS3/4A cleaves the 4A4B sequence, and the CAR protein is cleaved into four separate domains, causing the corresponding T cells to fail to recognize the antigen and also fail to deliver cell activation and Proliferation signals cannot kill the corresponding tumor cells.
  • the switch is in the off state.
  • NS3/4A is inhibited and can not cleave 4A4B sequence, CAR and protease fusion expression, CAR has a complete structure, and functions to recognize antigen and activate T cell internal signal.
  • the switch is in the on state.
  • 293T cells (Clontech: 632180) were resuscitated and cultured, and fCAR-V1, fCAR-V2 and fCAR-V3 were transfected into 293T cells, respectively.
  • fCAR-V1, fCAR-V2 and fCAR-V3 were transfected into 293T cells, respectively.
  • One day before transfection 5 x 10 5 cells were added to each well in a 12-well plate.
  • the liquid exchange before transfection was divided into the following two groups according to whether or not the small molecule inhibitor Asunaprevir (ASV) was added:
  • ASV 0uM group one well was added to the medium without ASV;
  • ASV 1uM group One well was added to the medium containing 1 uM ASV.
  • the specific transfection steps are as follows: 1) Take two 1.5ml EP tubes and add 100uL respectively. Medium, then add 6.0uL Lipofectamine to one of the tubes For the 3000 reagent, add 4ug DNA plasmid (fCAR-V1, fCAR-V2 or fCAR-V3) to the other tube and mix separately. 2) The liquids in the two EP tubes were mixed into one tube, and a total of 200 uL of the mixture was obtained, and the mixture was gently blown and mixed for 15 times, and allowed to stand at room temperature for 5 minutes. 3) Add 100 uL of the mixture to each of the two wells, and change the solution after 6 hours.
  • fCAR-V1 group the first column: the fusion protein Flag-CAR-4A4B-NS3/4A-Degron expressed without ASV.
  • the protease has a cleavage function and the fusion protein is cleaved at 4A4B.
  • Degron mediates the rapid degradation of NS3/4A protease to form a 63kd size Flag-CAR fragment.
  • Second column After adding ASV, 293 cells also expressed the fusion protein Flag-CAR-4A4B-NS3/4A-Degron.
  • fCAR-V2 group The first column: the fusion protein Flag-CAR1-4A4B-CAR2-NS3/4A was expressed without ASV. The protease is active and can cleave the fusion protein at 4A4B to form a 45 kd Flag-CAR1 and a 52 kd CAR2-NS3/4A protein. 45 kd Flag-CAR1 was detected by anti-flag antibody.
  • Second column After addition of ASV, 293 cells expressed the fusion protein Flag-CAR-4A4B-CAR2-NS3/4A. Since ASV inhibits the recognition and cleavage of 4A4B by NS3/4A protease, the entire fusion protein can be stably present, and the anti-flag antibody detects a protein size of 97 kd.
  • fCAR-V3 group First column: The fusion protein Flag-CAR1-4A4B-CAR2-5A5B-CAR3-4A4B-CAR4-NS3/4A was expressed without ASV.
  • the protease is active and can cleave the fusion protein at 4A4B and 5A5B to form four proteins of Flag-CAR1, 33kd, CAR2, 5kd CAR3 and 52kd CAR4-NS3/4A.
  • the 33 kd Flag-CAR1 was detected by an anti-flag antibody.
  • Second column After addition of ASV, 293 cells expressed the fusion protein Flag-CAR1-4A4B-CAR2-5A5B-CAR3-4A4B-CAR4-NS3/4A.
  • the entire fusion protein can be stably present, and the anti-flag antibody detects a protein size of 97 kd. Since the hinge site of CAR itself may naturally break, a group of 33kd protein can be detected in all groups.
  • the fusion protein gene represented by SEQ ID No. 3 was inserted into the lentiviral vector Pltr vector to obtain a lentiviral vector Pltr-CAR19-N34d expressing the chimeric antigen receptor (CAR) and self-degradation of the Pltr-CAR19-N34d vector. Fusion protein of HCV protease.
  • the CAR gene shown in positions 28-1491 of SEQ ID No. 3 was inserted into the lentiviral vector Pltr vector, and the resulting lentiviral vector PLTR-CAR19 was obtained.
  • the lentiviral vector PLTR-CAR19 expresses a chimeric antigen receptor (CAR).
  • Lentiviral PLTR-CAR19-N34d and lentiviral vector PLTR-CAR19 were co-transfected into 293T cells with viral packaging plasmids to obtain viral particles. Two days later, the cell culture supernatant was collected and PEG-precipitated for virus concentration. The virus solution CAR19-N34d and the virus solution CAR19 were respectively obtained and stored frozen at -80 ° C for use.
  • T cells are cultured with X-vivo15 containing 0.5% HSA and 300U IL2).
  • Lonza cultured cells obtained three days after PBMC cells (Tianjin blood center), and regulatable CAR-T cells and common CAR-T cells were obtained, respectively.
  • the CAR-T cells expressing the chimeric antigen receptor are co-cultured with the raji target cells in a culture system containing the viral protease inhibitor ASV or DPV (Aladdin), and the virus protease inhibitor is not included as a control (Control) ).
  • Elisport was used to detect the expression of IFN-gamma in CAR-T cells. Specific steps are as follows:
  • Fig. 15 The results are shown in Fig. 15. As can be seen from the figure: the upper row: normal CAR-T cells release a large amount of IFN-gamma (black spots in the figure) after binding to raji cells, and the addition of the viral protease inhibitors ASV or DPV does not Inhibition of release of IFN-gamma by CAR-T cells. Lower row: Regulatory CAR-T cells also release large amounts of IFN-gamma after binding to raji cells, but the expression of IFN-gamma is greatly reduced by the addition of the viral protease inhibitors ASV or DPV to the culture system.
  • the invention discloses a small molecule drug controlled protein function switching system.
  • the system comprises a control element original and a operon original, the control element is a protease or a protease-degradant fused by a protease and a proton; the operon element is a linker peptide recognizable and cleaved by the protease;
  • the system also includes a small molecule inhibitor of the protease; the entire system modulates the activity of the protease or protease-degradant by a small molecule inhibitor of the protease, thereby regulating the activity of the protein of interest.
  • the small molecule drug can be used to precisely regulate the biological activity of the target protein.
  • the viral protease of the present invention may be a natural protease, a protease that is genetically engineered to reduce the molecular weight of the protease, or a protease-degradant fused to a degrading domain (degron).
  • Viral protease inhibitors are primarily drugs that are commercially available for treatment or for clinical trials.
  • the invention prepares a switching system of a virus protease-dependent small molecule drug for regulating the function of a target protein, and provides a protein function regulation method which is fast, efficient, reversible, controllable, simple, economical and has good application prospect.

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Abstract

L'invention concerne un système de changement de la fonction d'une protéine commandé par un médicament à petites molécules, constitué d'une protéine cible d'expression, d'une protéase ou d'un complexe protéase-dégron, d'un support d'un peptide de liaison pouvant être reconnu et clivé par la protéase et d'un inhibiteur à petites molécules de la protéase. L'activité biologique de la protéine cible peut être régulée avec précision à l'aide du médicament à petites molécules. La protéase virale peut être une protéase naturelle, et peut également être une protéase génétiquement modifiée, la protéase présentant un poids moléculaire réduit, ou il peut s'agir d'un complexe protéase-dégron formé par fusion avec un domaine dégron. L'inhibiteur de protéase virale est principalement un médicament destiné à être utilisé dans un traitement ou lors d'une expérience clinique sur le marché. Le système de changement préparé est un système utilisant un médicament à petites molécules dépendant d'une protéase virale pour réguler la fonction d'une protéine cible. L'invention concerne un procédé rapide, efficace, réversible, contrôlable, simple et économique de régulation de la fonction d'une protéine présentant de bonnes perspectives d'application.
PCT/CN2017/118970 2016-12-27 2017-12-27 Système de changement de la fonction d'une protéine commandé par un médicament à petites molécules WO2018121602A1 (fr)

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