WO2019024933A1 - Cellule car nk ciblant gpc3 - Google Patents

Cellule car nk ciblant gpc3 Download PDF

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WO2019024933A1
WO2019024933A1 PCT/CN2018/098623 CN2018098623W WO2019024933A1 WO 2019024933 A1 WO2019024933 A1 WO 2019024933A1 CN 2018098623 W CN2018098623 W CN 2018098623W WO 2019024933 A1 WO2019024933 A1 WO 2019024933A1
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cells
cell
cancer
gpc3
domain
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PCT/CN2018/098623
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Chinese (zh)
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李宗海
喻敏
高慧萍
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科济生物医药（上海）有限公司
上海市肿瘤研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464474—Proteoglycans, e.g. glypican, brevican or CSPG4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/53—Liver
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the invention belongs to the field of cellular immunotherapy, and in particular relates to NK cells or cell lines comprising GPC3-CAR.
  • Chimeric antigen receptor (CAR) technology combines single-chain antibodies recognizing tumor-associated antigens with activation motifs of immune cells, and can confer tumor-targeting and stronger killing activity on immune cells through gene transduction. And lasting vitality.
  • This technology was first implemented in T cells and has achieved great success in patients with CD19-positive B-lymphocytic leukemia.
  • CAR-T cells are administered by collecting T cells from patients and being modified, they are prepared in a single preparation, and the treatment cost is high, and a large amount of expansion of T cells in the body usually causes cytokine storms. Therefore there is a risk of security.
  • NK cells can also be targeted by CAR technology to identify and destroy tumor cells.
  • NK cells have multiple mature immortalized cell lines, of which NK-92 can be cultured and expanded in vitro for a long time, the gene phenotype is stable, and the results are reproducible.
  • Some researchers have tried to express CAR in NK-92 cells. (For example, CN201580032729.X), but because tumor tissue has a very complex microenvironment, whether CAR-NK-92 cells can kill tumor cells in vivo will be affected by many factors, which has great uncertainty.
  • CAR-NK research and development is mostly the direct imitation and transformation of CAR-T.
  • the present invention urgently requires an effective CAR-NK cell.
  • the NK cells may be primary NK cells or NK92- cells.
  • the invention provides an NK-92 cell or cell line that expresses a chimeric antigen receptor that specifically recognizes GPC3.
  • Another object of the invention is to provide the use of said NK-92 cells or cell lines for the prevention and/or treatment of cancer, preferably GPC3 expressing cancer.
  • a further object of the invention is to provide the use of the NK-92 cell or cell line as a targeted cell therapeutic and/or for immunotherapy of adoptive cancer.
  • a further object of the present invention is to provide a method for producing the NK-92 cell or cell line, a method for identifying the NK-92 cell or cell line, and an NK-92 cell or cell line obtained or identified by the method. And its use.
  • the present invention provides a genetically engineered NK cell, characterized in that said cell expresses a chimeric receptor that specifically recognizes GPC3, said chimeric receptor comprising an extracellular domain that recognizes GPC3, spans Membrane domains, and/or intracellular domains.
  • the NK cell is an NK-92 cell.
  • the extracellular domain has HCDR1, HCDR2, HCDR3 and/or as shown in SEQ ID NOs: 7, 8, 9 and LCDR1, LCDR2, LCDR3 as shown in SEQ ID NOs: 10, 11, and 12. .
  • the extracellular domain has HCDR1, HCDR2, HCDR3 as shown in SEQ ID NOs: 7, 8, and 9, and LCDR1, LCDR2, LCDR3 as shown in SEQ ID NOs: 10, 11, and 12.
  • the extracellular domain that specifically recognizes GPC3 comprises the heavy chain variable region set forth in SEQ ID NO: 13 and the light chain variable region set forth in SEQ ID NO: 14.
  • the extracellular domain that specifically recognizes GPC3 has the sequence set forth in SEQ ID NO:1.
  • the chimeric receptor has the amino acid sequence set forth in SEQ ID NO: 16, 17, 18, or 19.
  • the transmembrane domain is selected from the transmembrane domain of the following proteins: alpha, beta or ⁇ chain of a T cell receptor; CD28; CD3 epsilon; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33 CD37;CD64;CD80;CD86;CD134;CD137;CD154;KIRDS2;OX40;CD2;CD27;LFA-1 (CD11a;CD18);ICOS(CD278);4-1BB(CD137);GITR;CD40;BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 (KLRF1); CD160; CD19; IL2R ⁇ ; IL2R ⁇ ; IL7R ⁇ ; ITGA1; VLA1; CD49a; ITGA4; IA4; CD49D; ITGA6; VLA-6; CD49f; ITGAD; CD11d; ITGAE; CD103; ITGAL; CD11a; LFA-1
  • SLAMF6 NTB-A, Ly108
  • SLAM SLAMF1, CD150, IPO-3
  • BLAME SLAMF8
  • SELPLG CD162
  • LTBR TBR
  • PAG/Cbp PAG/Cbp
  • NKp44 NKp44
  • NKp30 NKp46
  • NKG2D NKG2D
  • NKG2C NKG2C
  • the intracellular domain comprises: a transcription factor binding domain; a primary signaling domain and/or a costimulatory signaling domain, wherein:
  • the primary signaling domain comprises a functional signal transduction selected from the group consisting of: CD3 ⁇ ; CD3 ⁇ ; CD3 ⁇ ; CD3 ⁇ ; common FcR ⁇ (FCER1G); FcR ⁇ (Fc ⁇ R1b); CD79a; CD79b; Fc ⁇ RIIa; DAP10; Domain, or a combination thereof; and/or
  • the costimulatory signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD27; CD28; 4-1BB (CD137); OX40; CD30; CD40; PD-1; ICOS; Related antigen-1 (LFA-1); CD2; CD7; LIGHT; NKG2C; B7-H3; ligand for specific binding to CD83; CDS; ICAM-1; GITR; BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 ( KLRF1);CD160;CD19;CD4;CD8 ⁇ ;CD8 ⁇ ;IL2R ⁇ ;IL2R ⁇ ;IL7R ⁇ ;ITGA4;VLA1;CD49a;ITGA4;IA4;CD49D;ITGA6;VLA-6;CD49f;ITGAD;CD11d;ITGAE;CD103;ITGAL;CD11a ;LFA-1;ITGAM;CD11b;ITGAX;CD11c;ITGB1;CD29;IT
  • the present invention provides the use of the cell of the first aspect of the invention for the preparation of a medicament for preventing and/or treating cancer, the cancer expressing GPC3, preferably from liver cancer, lung cancer, gastric cancer, breast cancer, Melanoma, ovarian cancer, yolk sac tumor, neuroblastoma.
  • the invention provides the use of a cell of the first aspect of the invention for the manufacture of a medicament for use as a targeted cell therapeutic and/or for immunotherapy of adoptive cancer.
  • the prophylactic and/or therapeutic cancer drug, target is described in the instructions for use in the prevention and/or treatment of cancer, a targeted cell therapeutic, or a drug for adoptive cancer immunotherapy.
  • the drug to the cell therapy agent or the immunotherapy for adoptive cancer is administered simultaneously with the chemotherapeutic drug or radiation therapy.
  • the medicament for preventing and/or treating cancer, a targeted cell therapeutic, or a medicament for adoptive cancer immunotherapy further comprises a chemotherapeutic drug.
  • the medicament for preventing and/or treating cancer, a targeted cell therapeutic, or a medicament for adoptive cancer immunotherapy is for treating liver cancer.
  • the chemotherapeutic agent is a chemotherapeutic drug for treating liver cancer; preferably a compound of formula I or formula II:
  • the instructions for the prevention and/or treatment of cancer, a targeted cell therapeutic, or a drug for adoptive cancer immunotherapy describe the individual to be administered prior to administration of the cell. Pretreatment is performed.
  • the pretreatment is to administer a chemotherapeutic drug or radiation therapy to control an increase in tumor burden.
  • the drug for preventing and/or treating cancer, a targeted cell therapeutic agent, or a drug for adoptive cancer immunotherapy further comprises a lymphocyte depleting agent as a pretreatment reagent to consume the Individual lymphocytes.
  • the lymphocyte depleting agent is fludarabine or cyclophosphamide.
  • the present invention provides a method for producing NK cells of the present invention, which comprises: preparing NK cells expressing a chimeric antigen receptor, and further treating the NK cells expressing the chimeric antigen receptor by irradiation; preferably NK cells expressing chimeric antigen receptors were pretreated with gamma irradiation.
  • the NK cell is an NK-92 cell.
  • the present invention provides a method of preventing and/or treating cancer in a subject, the cancer expressing GPC3, comprising administering a cell of the present invention to a subject in need of treatment for cancer.
  • the cancer is preferably selected from the group consisting of liver cancer, lung cancer, gastric cancer, breast cancer, melanoma, ovarian cancer, yolk sac tumor, neuroblastoma; preferably liver cancer.
  • the method of the invention for preventing and/or treating cancer in a subject comprises simultaneously administering to a subject in need of treatment of cancer a cell of the invention and a chemotherapeutic drug or radiation therapy.
  • the chemotherapeutic agent is a chemotherapeutic agent for treating liver cancer; preferably a compound of formula I or formula II:
  • the method of the invention for preventing and/or treating cancer in a subject comprises pretreating an individual to be administered prior to administration of the cells of the invention.
  • the pretreatment is to administer a chemotherapeutic drug or radiation therapy to control an increase in tumor burden.
  • the pretreatment is to consume lymphocytes of the individual using a lymphocyte depleting agent.
  • the lymphocyte depleting agent is fludarabine or cyclophosphamide.
  • Figure 1 shows the results of flow cytometry of NK-92/9.28.z cells
  • Figure 2A compares the in vitro killing activity of parental NK-92, Mock and NK-92/9.28.z cells;
  • Figure 2B shows the in vitro killing results of NK-92/9.28.z cells at inefficient target ratios;
  • Figure 2C shows the in vitro killing results of NK-92/9.28.z cells under hypoxic conditions;
  • Figure 2D shows the in vitro killing results of NK-92/9.28.z cells in the presence of TGF- ⁇ ;
  • Figure 2E shows serum The effect of horizontal GPC3 on the killing effect of NK-92/9.28.z cells in vitro;
  • Figure 3A shows the release of NK-92/9.28.z cell degranzyme
  • Figure 3B shows the secretion of IFN-gama
  • Figure 3C shows the secretion of NK-92/9.28.z IFN-gamma and the surface of tumor cells GPC3 expression value is proportional;
  • Figure 4A shows the anti-tumor effect of NK-92/9.28.z cells in SK-HEP-1 and SK-HEP-1/GPC3 xenograft models
  • Figure 4B shows NK-92/9.28.z cells infiltrating tumors locally. The results of anti-tumor effects
  • Figure 4C shows the results of local immunohistochemistry of NK-92/9.28.z cells infiltrating tumors;
  • Figure 5 shows the effect of NK-92/9.28.z cell therapy on important organ organs
  • Figure 6A shows the anti-tumor effect of NK-92/9.28.z on endogenous high-expression GPC3 Huh-7 subcutaneous xenografts;
  • Figure 6B shows the anti-tumor effect of NK-92/9.28.z on orthotopic tumors;
  • Figure 6D shows the changes in body weight of mice in the orthotopic transplantation model;
  • Figure 6E shows the NK-92/9.28.z in the orthotopic transplantation model. Infiltration;
  • Figure 7A shows tumor volume changes in the PLC/PRF/5 subcutaneous xenograft model
  • Figure 7B shows tumor weight changes in the PLC/PRF/5 subcutaneous xenograft model
  • Figure 7C shows PLC/PRF/5 subcutaneous xenografts Changes in mouse body weight in the model
  • Figure 8A shows the release of cytokines during killing
  • Figure 8B shows the results of release of IL-6 by monocytes after killing target cells by NK-92/9.28.z;
  • Figure 9A shows in vitro cell killing of PBNK/9.28.z
  • Figure 9B shows interferon release levels of PBNK/9.28.z
  • Figure 10 shows the killing of normal cells by PBNK/9.28.z
  • Figure 11 shows the cell killing of sorafenib in combination with NK-92/9.28.z cells.
  • NK cells are important effector cell types for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance anti-tumor activity, but experience with CAR-engineered NK cells is limited and lacks clinically developed data. . In addition to primary cells, NK cells have many mature cell lines, including NK-92, NKG, YT, NK-YS, HANK-1, YTS, and NKL. In a specific embodiment of the present invention, when the NK-92 cell line or primary NK cells are used, the NK cells modified with the chimeric antigen receptor of the present invention are most effective.
  • CARs chimeric antigen receptors
  • Cellular therapeutic agent particularly “targeted cell therapeutic agent” or “targeted allogeneic cell therapeutic agent” refers to an immune cell suitable for administration to adoptive immunotherapy for adoptive cancer, which is genetically modified to express specificity An antigen-restricting antigen receptor expressed on the surface of a target tumor cell is recognized.
  • “Adoptive, target cell-specific immunotherapy” or “adoptive cancer immunotherapy” or “adoptent cell therapy (ACT)” refers to a form of treatment in which immune cells are transferred to a host carrying a tumor. Immune cells have anti-tumor reactivity and can mediate direct or indirect anti-tumor effects.
  • GPC3 is Glypican-3 (also known as DGSX, GTR2-2, MXR7, OCI-5, SDYS, SGB, SGBS or SGBS1) and is a cell surface protein belonging to sulfuric acid. Heparan glycoprotein family.
  • the GPC3 gene encodes a 70-kDa precursor core protein that can be cleaved by furin to produce a soluble 40-kDa-capable amino-terminal (N-terminal) peptide and 30- A membrane-bound carboxy-terminal (C-terminal) peptide containing two heparan sulfate (HS) sugar chains around kDa.
  • the GPC3 protein is attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor.
  • GPI glycosylphosphatidylinositol
  • chimeric antigen receptor refers to a polypeptide which, when administered in an immune effector cell, provides the cell with a target cell (usually a cancer cell) Specificity, and has intracellular signal production.
  • CAR typically includes at least one extracellular antigen binding domain, a transmembrane domain (also known as a transmembrane or transmembrane domain), and an intracellular domain (also referred to herein as an "intracellular region” or “intracellular domain”).
  • the polypeptide groups are contiguous with each other.
  • a polypeptide group includes a dimerization switch that can couple the polypeptides to each other in the presence of a dimerization molecule, for example, an antigen binding domain can be coupled to an intracellular signaling domain.
  • the "chimeric antigen receptor" is capable of specifically recognizing GPC3.
  • the intracellular domain comprises a primary signaling domain, or a costimulatory signaling domain, or a primary signaling domain and a costimulatory signaling domain.
  • the primary signaling domain may be selected from the group consisting of: CD3 ⁇ ; CD3 ⁇ ; CD3 ⁇ ; CD3 ⁇ ; common FcR ⁇ (FCER1G); FcR ⁇ (Fc ⁇ R1b); CD79a; CD79b; Fc ⁇ RIIa; DAP10; Conduction domain.
  • human CD3 can be selected (amino acid sequence as set forth in SEQ ID NO: 6, nucleotide sequence as set forth in SEQ ID NO: 22).
  • the costimulatory signaling domain can be selected from the signaling domains of proteins: CD27; CD28; 4-1BB (CD137); OX40; CD30; CD40; PD-1; ICOS; Related antigen-1 (LFA-1); CD2; CD7; LIGHT; NKG2C; B7-H3; ligand for specific binding to CD83; CDS; ICAM-1; GITR; BAFFR; HVEM (LIGHTR); SLAMF7; NKp80 ( KLRF1);CD160;CD19;CD4;CD8 ⁇ ;CD8 ⁇ ;IL2R ⁇ ;IL2R ⁇ ;IL7R ⁇ ;ITGA4;VLA1;CD49a;ITGA4;IA4;CD49D;ITGA6;VLA-6;CD49f;ITGAD;CD11d;ITGAE;CD103;ITGAL;CD11a ;LFA-1;ITGAM;CD11b;ITGAX;CD11c;ITGB1;CD29;ITGB
  • signaling domain refers to a functional portion of a protein that functions by transmitting information within a cell for regulating cells via a defined signaling pathway by generating a second messenger or by acting as an effector in response to such a messenger. Activity.
  • the intracellular domain of human CD137 amino acid sequence as set forth in SEQ ID NO: 25, nucleotide sequence as set forth in SEQ ID NO: 26
  • human CD28 intracellular domain amino acid sequence, eg, SEQ ID NO: 5
  • the nucleotide sequence is set forth in SEQ ID NO: 21, or a combination of the intracellular domain of human CD137 and the human CD28 intracellular domain.
  • the transmembrane domain is selected from the transmembrane domain of the following proteins: alpha, beta or ⁇ chain of T cell receptor, CD28; CD3 epsilon; CD45; CD4; CD5; CD8; CD9; CD16; CD22; CD33; CD37; CD64;CD80;CD86;CD134;CD137;CD154;KIRDS2;OX40;CD2;CD27;LFA-1(CD11a;CD18);ICOS(CD278);4-1BB(CD137);GITR;CD40;BAFFR;HVEM(LIGHTR ;SLAMF7;NKp80(KLRF1);CD160;CD19;IL2R ⁇ ;IL2R ⁇ ;IL7R ⁇ ;ITGA1;VLA1;CD49a;ITGA4;IA4;CD49D;ITGA6;VLA-6;CD49f;ITGAD;CD11d;ITGAE;CD103;ITGAL;CD11a ; LFA-1; ITGAM;
  • transmembrane domain of CD28 amino acid sequence as shown in SEQ ID NO: 4, nucleotide sequence as shown in SEQ ID NO: 20
  • transmembrane domain of CD8 amino acid sequence such as SEQ ID NO: 23
  • nucleotide sequence is as shown in SEQ ID NO:24).
  • the chimeric antigen receptor further comprises a hinge region.
  • the hinge region of CD8 can be selected and the amino acid sequence is set forth in SEQ ID NO:3.
  • the chimeric receptor of the invention has the amino acid sequence set forth in SEQ ID NO: 16, 17, 18, or 19.
  • the GPC3-specific NK-92 cells or primary NK cells of the present invention comprise a lentiviral vector encoding a chimeric antigen receptor comprising a GPC3-specific scFv antibody fragment, a hinge region, and a transmembrane of CD28 And intracellular domains and intracellular domains of CD3 ⁇ .
  • the NK-92 cells or primary NK cells of the invention are characterized by: reduced natural cytotoxicity or no natural cytotoxicity, ie reduced cytotoxicity or absence of GPC3-negative cells compared to GPC3-positive cells Cytotoxicity.
  • the reduced natural cytotoxicity constitutes an important safety feature of the cells or cell lines of the invention, particularly in clinical applications.
  • the NK-92 cells or primary NK cells of the present invention are opposite to unmodified NK-92 cells or primary NK cells - lysing GPC3-expressing tumor cells with high efficiency, but with unmodified NK-92 cells or primary NK Cells are less likely to attack GPC3-negative non-target cells.
  • the GPC3-specific scFv antibody fragment comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.
  • the GPC3-specific CAR used in the present invention comprises:
  • transmembrane domain in a specific embodiment, the transmembrane domain of CD28 or the transmembrane domain of CD8 may be selected, and other transmembrane domains commonly used in the technique of preparing CAR may also be selected.
  • the intracellular domain has an intracellular domain of CD3 ⁇ , and/or a costimulatory signal domain, alternatively, the costimulatory signal domain may be a costimulatory signal domain of CD28 or The costimulatory signal domain of CD137.
  • the CAR-NK-92 cells of the present invention can prevent cell replication by gamma-irradiation as a potential safety measure for clinical applications, while in vitro and in vivo anti-tumor activity is maintained.
  • NK-92 may be tumorigenic.
  • pretreatment of the cells by irradiation, preferably gamma irradiation.
  • the illumination is a safety measure and can be carried out in a variety of conventional manners.
  • NK-92/9.28.z has no enhanced killing effect on GPC3-negative liver cancer cells; when overexpressing GPC3, these cells are more sensitive to NK-92/9.28.z, reflecting NK-92/9.28.z cells.
  • the role of targeted killing One obstacle to the treatment of solid tumors by cells is that too few effector cells enter the local microenvironment, failing to achieve an effective target-to-kill ratio. When the target ratio is greatly reduced, if the incubation time is extended, it can reach a very large Good specific killing; hypoxia and TGF- ⁇ in the microenvironment are factors that restrict the effect of cell therapy, and the present invention shows that NK-92/9.28.z still has a good specificity in the presence of both.
  • NK-92/9.28.z cells sexual killing effect; there is secretory GPC3 in the blood, but it does not affect the toxic effect of NK-92/9.28.z cells on tumor cells in the present invention.
  • Our data indicate that NK-92 cells encoding GPC3-CAR can tolerate a variety of adverse conditions and are clinically useful for the treatment of various GPC3-positive malignancies.
  • CD107a is a protein on lysosome. It can indicate the fusion of lysosome and cell membrane when releasing granzyme, which is on NK-92 cells.
  • An activation indicator We can reveal the targeting of CAR from the NK cell activation level.
  • CAR-NK mediates the lysis of target cells caused by a variety of endogenous cytotoxic receptors, and tumors with heterogeneous expression of antigen may be more advantageous than CAR-T.
  • Allogeneic infusion of NK cells does not cause graft-versus-host disease, and its KIR-MHC mismatch-based allogeneic model can also avoid or reduce KIR-mediated inhibitory signals, making it possible to commercialize universal and efficient "NK blood products" .
  • NK-92/9.28.z also has an antigen-dependent antitumor effect in vivo, which acts by inducing tumor cell apoptosis and inhibiting proliferation; and does not infiltrate important organ tissue-inducing cells. Toxic effects; does not cause side effects associated with weight loss in treatment.
  • NK-92 cells are a tumor-derived killer cell line, which has the risk of tumor formation in the body. It is often necessary to infuse after irradiation.
  • This animal experiment established a set of irradiation of NK-92/9.28.z cells. The results suggest that the effect is basically the same as that of the NK-92/9.28.z in the non-irradiated group, providing an animal experimental basis for its clinical safety application.
  • NK-92/9.28.z cells retain target cell specificity in vivo and are capable of penetrating tissue and homing to distant tumor sites.
  • the in vivo anti-tumor activity of irradiated NK-92/9.28.z cells was identical to that of non-irradiated cells. This may be of interest for future clinical applications of NK-92/9.28.z, where irradiation of cells can be performed as in previous phase I clinical trials of unmodified NK-92 cells (Arai et al., 2008; Tonn Etc., 2013) included as a security measure.
  • Immune cells in tumor patients are often functionally impaired due to the immunosuppressive activity of cancer. Therefore, for adoptive cancer immunotherapy with NK cells, donor-derived allogeneic cells are preferred because they do not recognize tumor cells as "self", thereby circumventing inhibitory signals (Geller and Miller; 2011) . We have shown that this advantage can be extended to CAR-engineered NK-92 cells. Such cells are clinically useful for the treatment of various GPC3-positive malignancies.
  • the primary NK cells obtained from peripheral blood culture can specifically kill GPC3-positive hepatoma cell line secreting IFN- ⁇ after CAR modification, while Mock and untransfected NK cells have weaker killing effect. The expression of IFN- ⁇ was also lower. In vitro toxicity experiments indicated that the modified primary NK cells had no obvious toxic effects on normal tissue cells.
  • this example selects antibody AB1 (the sequence of the scFv as shown in SEQ ID NO: 1) that targets GPC3 as the extracellular domain of the chimeric antigen receptor.
  • the pre-prepared CAR has the sequence shown in SEQ ID NO: 17.
  • PRRLSIN-cPPT.EF-1 ⁇ a lentiviral plasmid expressing the second-generation chimeric antigen receptor of antibody AB1, PRRLSIN-cPPT.EF-1 ⁇ -AB1-28Z.
  • the AB1-28Z sequence consists of the CD8 ⁇ signal peptide (SEQ ID NO: 2), the scFv of AB1 (SEQ ID NO: 30), the CD8hinge (SEQ ID NO: 15), the CD28 transmembrane region (SEQ ID NO: 20), and the intracellular signal.
  • the conduction domain (SEQ ID NO: 21) and the intracellular domain CD3 (CD ID NO: 22) of CD3 consist.
  • the target gene plasmids PRRLSIN-cPPT.EF-1 ⁇ -EGFP (Mock) and PRRLSIN-cPPT.EF-1 ⁇ -AB1-28Z 5.4 ⁇ g and packaging plasmid pRsv-REV 6.2 ⁇ g, RRE-PMDLg 6.2 ⁇ g, Vsvg 2.4 ⁇ g was dissolved in 800 ⁇ L of blank DMEM medium and mixed for 5 min at room temperature; 60 ⁇ g of PEI (1 ⁇ g/ ⁇ l) was dissolved in 800 ⁇ l of serum-free DMEM medium, gently mixed (or vortexed at 1000 rpm for 5 seconds); The mixed solution was added to the PEI mixture, gently added after the addition, and incubated at room temperature for 20 min to obtain a transfection complex;
  • the virus titers after concentration were: lentivirus AB1-28Z: 2.4 ⁇ 10 8 U/ml, lentivirus Mock: 2 ⁇ 10 8 U/ml.
  • Retronectin coated 24 well plates 380 ⁇ l of 5 ⁇ g/ml retronectin solution (PBS) was added to each well, and after overnight incubation at 4 degrees, the retronectin solution (PBS) in a 24-well plate was discarded, and washed twice with PBS;
  • Amplification culture The infected cells were passaged every other day at a density of 5 ⁇ 10 5 /mL, and a recombinant human IL-2 having a final concentration of 500 U/mL was supplemented in the lymphocyte culture solution.
  • Mock NK-92 cells (designated Mock) expressing empty vector or NK-92 cells expressing CAR (designated NK-92/9.28.z) were obtained.
  • Control cells were incubated with 50 ⁇ l of PE-SA (1:200 dilution) antibody for 45 min on ice, washed twice with PBS (2% NBS), and resuspended as a control;
  • Test group cells 50 ⁇ l 1:50 diluted biotin-goat anti-human IgG, F(ab') 2 antibody, incubated on ice for 45 min; PBS (2% NBS) twice; add 50 ⁇ l PE-SA (1 :200 dilution) antibody incubation on ice for 45 min;
  • the cells received by the sorting receiving tube are placed in a 37-degree, 5% CO 2 incubator for cultivation;
  • NK-92/9.28.z The positive rate of NK-92/9.28.z was detected by flow cytometry. The results are shown in Figure 1.
  • the flow-sorted NK-92/9.28.z cells were compared with the Mock group and the Parental NK-92 group.
  • the surface has a significantly enhanced expression of CAR.
  • the Mock group was NK-92 cells transfected with Mock plasmid
  • Parental NK-92 was the original NK-92 cells.
  • Cytotoxicity was determined using a CytoTox 96 non-radioactive cytotoxicity assay kit (Promega). The specific method is described in the instruction manual of CytoTox 96 non-radioactive cytotoxicity test kit, and the detection time is 6h.
  • Target cells 50 ⁇ L of 2 ⁇ 10 5 /mL HepG2; Huh-7; Hep3B; PLC/PRF/5; SK-HEP-1; SK-HEP-1/GPC3; SMMC7721; SMMC7721/GPC3 cells in 96well plate (GPC3-positive liver cancer cell lines: HepG2, Huh-7, SK-HEP-1/GPC3, PLC/PRF/5, SMMC7721/GPC3, GPC3-negative liver cancer cell lines: SK-HEP-1, SMMC7721).
  • Effector cells Mock; NK-92 and NK-92/9.28.z cells as described in Example 1 were added at a target ratio of 3:1, 1.5:1 or 0.75:1.
  • NK-92/9.28.z For GPC3-positive liver cancer cells, the in vitro toxicity of NK-92/9.28.z was significantly stronger than that of the Mock group and the Parental NK-92 group.
  • NK-92 /9.28.z has no enhanced killing effect; when overexpressing GPC3, these cells are more sensitive to NK-92/9.28.z, reflecting the targeted killing effect of NK-92/9.28.z cells.
  • NK-92/9.28.z To further confirm the effect of tumor microenvironment on NK-92/9.28.z, the cytotoxic effect of NK-92/9.28.z on hepatoma cell lines under different conditions and conditions was also examined.
  • the hypoxic incubator is used to simulate the hypoxic environment, with Huh-7 as the target cell.
  • the in vitro toxicity of different effective target ratios of NK-92/9.28.z was measured under different oxygen conditions (1% oxygen concentration in low oxygen environment and 20% oxygen concentration in normal oxygen environment). The results are shown in Fig. 2C. As shown, the killing function of NK-92/9.28.z was not significantly affected in a low oxygen environment.
  • TGF- ⁇ is another important factor that restricts the effect of cell therapy.
  • Huh-7 as the target cell
  • the results shown in Figure 2D TGF- ⁇ can significantly inhibit the killing function of CAR-T/9.28.z, while the NK-92/9.28.z cell surface for TGF- ⁇ It exhibited a considerable degree of resistance and its killing function was not significantly affected by TGF- ⁇ .
  • CAR-T/9.28.z is a T cell expressing the CAR of SEQ ID NO: 17, which is derived from peripheral blood, and which is infected with lentivirus AB1-28Z by T cells in a molecular biology routine in the art.
  • CAR-T/9.28.z is a T cell expressing the CAR of SEQ ID NO: 17, which is derived from peripheral blood, and which is infected with lentivirus AB1-28Z by T cells in a molecular biology routine in the art.
  • CN104140974A, CN106397593A and the like refer to CN104140974A, CN106397593A and the like.
  • GPC3 is a cell surface marker, but the N-terminal peptide of GPC3 protein can be cleaved into blood (GPC3N), and can also be cleaved by the noumase at the GPI site to form a soluble GPC3 protein (GPC3 ⁇ GPI). ) released into the blood. Serum levels of GPC3 may compete with GPC3 on the surface of liver cancer cells, affecting the killing effect of NK-92/9.28.z.
  • We expressed GPC3N and GPC3 ⁇ GPI proteins by protein recombination method, and added different concentrations of the above soluble GPC3 protein in the killing system, using Huh-7 as the target cell, and incubating for 6 hours under the condition of 3:1 effective target ratio. The results are shown in Figure 2E, and the two did not significantly affect the killing function of NK-92/9.28.z cells at each concentration.
  • NK cells have a rapid toxic killing effect depending on lysosome-releasing granzyme
  • a protein on CD107a lysosome can indicate the fusion of lysosome and cell membrane when releasing granzyme, so we detected NK- Degranulation of 92/9.28.z and Parental NK-92 cells co-incubated with tumors.
  • NK-92/9.28.z cells and control group NK-92 cells were co-cultured with SK-HEP-1, SK-HEP-1/GPC3 and K562 cells in different proportions by flow cytometry.
  • CD107a expression The medium was used as a negative control and PMA + IONO stimulation was used as a positive control.
  • NK-92/9.28.z cells were seeded into 24-well plates, and 2 ⁇ 10 5 target cells were inoculated at a final volume of 400 ul. No exogenous IL-2 was added, and culture was carried out. The suspension cells were collected at 6 hours in the culture of the box; the expression of CD107a was detected by flow cytometry, and the results are shown in Fig.
  • IFN- ⁇ is one of the most important cytokines secreted by NK cells.
  • NK-92/9.28.z we also compared the secretion of NK-92/9.28.z and NK-92 cells in the common hepatocellular carcinoma cell line at a target-to-target ratio of 1.5:1.
  • IFN- ⁇ levels The details are as follows: 1) 5 ⁇ 10 4 NK-92/9.28.z cells were seeded into a 24-well plate, and 5 ⁇ 10 4 target cells were inoculated at a final volume of 400 ul without exogenous IL-2. The supernatant was collected in an incubator for 24 hours; 2) the expression of IFN-gama was detected by ELISA;
  • Example 4 Therapeutic effect of NK-92/9.28.z cells on SK-HEP-1 and SK-HEP-1/GPC3 xenografts
  • mice were inoculated with SK-HEP-1 and SK-HEP-1/GPC3 xenografts
  • the above cells were inoculated subcutaneously, 2 ⁇ 10 6 /piece, and the tumor volume reached 30-50 mm 3 after about 15 days;
  • mice On day d17, the transplanted tumor volume of Nod-Scid mice was measured and grouped. The mice were divided into 3 groups, including: saline control group, Mock control group, NK-92/9.28.z group, 6-8 mice in each group;
  • Nod-Scid mouse cells were subjected to adoptive immunotherapy. 200ul normal saline or 200ul NK-92, Mock and NK-92/9.28.z cells were injected through the tail vein once every 5 days;
  • the volume of SK-HEP-1 and SK-HEP-1/GPC3 xenografts was measured every 3-4 days, the changes in tumor volume of each group of mice were recorded, and the growth curve of tumor volume with time was plotted.
  • NK-92/9.28.z cell infiltration was detected by flow cytometry.
  • NK-92/9.28.z infiltration and detection of tumor tissue proliferation were detected by immunohistochemistry.
  • Apoptosis index; important organs of SK-HEP-1/GPC3 transplanted tumor mice were taken for HE and immunohistochemical staining.
  • NK-92/9.28.z had no significant effect with the control group; whereas for SK-HEP-1/GPC3, NK-92/9.28.
  • the tumor in the treatment group was significantly smaller than the control group, suggesting that NK-92/9.28.z also has an antigen-dependent antitumor effect in vivo.
  • Infiltration was detected by flow cytometry in the animal model SK-HEP-1/GPC3, and the results were detected in the SK-HEP-1/GPC3 tumor model treated with NK-92/9.28.z as shown in Fig. 4B.
  • SK-HEP-1/GPC3 transplanted tumor mice were taken for HE staining. The results are shown in Fig. 5. There was no significant difference between the groups and no pathological changes were observed. This cell is safe to treat and does not infiltrate important organ tissues to induce cytotoxicity.
  • Example 5 Therapeutic effect of NK-92/9.28.z on endogenous high GPC3 Huh-7 subcutaneous and orthotopic transplantation tumors
  • Huh-7 subcutaneous and orthotopic transplantation tumors were inoculated with NOD/SCID mice.
  • the subcutaneous xenograft model was established: subcutaneously inoculated Huh-7 cells, 2 ⁇ 10 6 /piece, and the tumor volume reached 30-50 mm 3 after about 14 days, and a mouse subcutaneous xenograft model was obtained.
  • In situ animal models were more able to simulate the real situation in patients, and an animal model of in situ Huh-7 liver cancer was established by inoculation, and four mice in each group were randomly assigned.
  • Adoptive NK-92/9.28.z and Parental NK-92 cells were used for adoptive immunotherapy of subcutaneous and orthotopic xenografts by subcutaneous and orthotopic transplantation of intraperitoneal cyclophosphamide at 12 and 13 days, respectively. 100mg/kg).
  • Nod-Scid mouse xenograft volume was measured and grouped on day D15.
  • Subcutaneous xenograft mice were divided into three groups, including: saline control group, NK-92 group, NK-92/9.28.z group, each group of mice 6-8; in situ tumor group was divided into two groups Including: NK-92 group and NK-92/9.28.z group, 4 mice per group. D15 and 17 days, adoptive immunotherapy for grouped NOD/SCID mouse cells. 200ul normal saline or 200ul NK-92, Mock and NK-92/9.28.z cells were injected through the tail vein once every 5 days;
  • the volume of Huh-7 subcutaneous xenografts was measured every 3-4 days, the changes in tumor volume of each group of mice were recorded, and the growth curve of tumor volume with time was plotted. The results are shown in Fig. 6A. The growth of Huh-7 subcutaneous xenografts was shown. The tumor volume and tumor weight of the NK-92/9.28.z treatment group were significantly lower than the other two groups.
  • Fig. 6D The growth of liver tumors in situ was measured by Luciferase every other week, and the results are shown in Fig. 6D.
  • the liver was taken for immunohistochemical detection, and the results are shown in Fig. 6E.
  • Figure 6D shows an increase in the body weight of each group of mice, suggesting that NK-92/9.28.z does not cause treatment-related weight loss side effects; while residual tissue is detected, the results are shown in Figure 6E, found in tumor tissue
  • the infiltrating NK-92/9.28.z was significantly higher than the control group, but its expression was not detected in the liver, suggesting that NK-92/9.28.z does not cause liver infiltration, indicating NK-92/9.28.z safety. it is good.
  • Example 6 Therapeutic effect of NK-92/9.28.z cells on endogenous low expression of GPC3PLC/PRF/5 subcutaneous xenografts
  • NOD/SCID mice were inoculated with PLC/PRF/5 subcutaneous xenografts, and the above cells were inoculated subcutaneously, 2 ⁇ 10 6 /piece, and the tumor volume reached 30-50 mm 3 after about 15 days;
  • Subcutaneous xenografts were injected intraperitoneal cyclophosphamide (100 mg/kg) on days 12 and 13 respectively.
  • mice On day d17, the transplanted tumor volume of Nod-Scid mice was measured and grouped. The mice were divided into 3 groups, including: saline control group, Mock control group, NK-92/9.28.z group and NK-92/9.28.z irradiation group, 6-8 mice in each group;
  • Fig. 7 The experimental results are shown in Fig. 7, and Figs. 7A and 7B suggest that they also have a good antitumor effect in vivo.
  • Figure 7C suggests that each mouse is well-balanced, suggesting that NK-92/9.28.z treatment does not cause weight loss side effects; NK-92 cells are a tumor-derived killer cell line that has a risk of tumor formation in the body and is often clinically needed. After irradiation, the infusion was performed. In this animal experiment, a group of NK-92/9.28.z cells irradiation group (Irradiated NK-92/9.28.z) was established. The results showed that the effect was the same as that of the non-irradiated group. 92/9.28.z is basically consistent, providing an animal test basis for its clinical safety applications.
  • CRS is a common complication of CAR-T cell therapy and may even be life-threatening. There may be multiple mechanisms of cross-reactivity and activation of CRS. It is reported that IL6, IL-1 ⁇ and IL-2 are also involved in causing or expanding cells. An important cytokine of factor release syndrome.
  • NK-92/9.28.z NK-92 cells during the killing of SK-HEP-1 and SK-HEP-1/GPC3 at a target-to-target ratio of 1.5:1.
  • the secretion of IL6, IL-1 ⁇ and IL-2 by NK-92/9.28.z was detected.
  • NK-92/9.28.z cells did not release IL-1 ⁇ , IL during the killing process. -2 and IL-6.
  • NK-92/9.28.z can induce IL-6 release from monocytes after killing target cells.
  • NK-92/9.28.z and CAR-T/9.28.z were incubated with tumor cells Huh-7 and monocytes for 36 h, and then detected the secretion of IL-6 in the culture system by ELISA. Specifically, 5 ⁇ 10 3 monocytes were seeded into 96-well plates, and 3 ⁇ 10 4 NK-92/9.28.z or CAR-T/9.28.z cells were inoculated simultaneously, and 2 ⁇ 10 4 target cells Huh were added.
  • NK-92/9.28.z did not stimulate the release of IL-6 by monocytes in the process of killing tumor cells compared with CAR-T cells, and is a very safe effector cell.
  • Example 8 CAR modification enhances anti-tumor effect of primary NK cells
  • PBMCs were co-cultured with K562-mbIL21 cells (exposed with membrane bound interleukin-21 and IL-2 on K562 cells, and NK cells were expanded in vitro, magnetic beads ( Sorted from Meitian), the primary NK cells were obtained and named PBNK cells.
  • PBNK cells were infected with a lentivirus of AB1-28Z to obtain a primary NK cell expressing CAR, designated PBNK/9.28.z.
  • PBNK cells were infected with an empty plasmid to obtain Mock' cells.
  • Fig. 9A The secretion level of IFN- ⁇ was examined, and the results are shown in Fig. 9B.
  • the normal PBMC was used as the target cell to detect the cell killing ability of Mock' cells and PBNK/9.28.z. As shown in Fig. 10, it was shown that the modified primary NK cells had no obvious toxic effects on normal tissue cells.
  • Example 9 Targeting GPC3 chimeric antigen receptor-modified NK cells in combination with sorafenib for hepatocellular carcinoma
  • Hepatoma Huh-7 cells (target cells) were inoculated. After 24 h, different concentrations of sorafenib (2 ⁇ M, 5 ⁇ M and 10 ⁇ M, respectively) or DMSO were added, with or without different target ratios of NK-92/9.28.z cells. (1:1, 1:5, and 1:10), after incubation for 24 h, cytotoxicity was determined using a CytoTox 96 non-radioactive cytotoxicity assay kit. The results of the experiment are shown in Table 1. NK-92/9.28.z cells have significant killing effect on Huh-7 and are effective in target-dependent ratio. The greater the target ratio, the more killing effect of NK-92/9.28.z cells.
  • the cell killing toxicity was 69.2% ⁇ 3.5%, 36.8% ⁇ 6.5%, 22.2% ⁇ 4.6% for the strong and effective target ratios of 1:1, 1:5, and 1:10; the sorafenib concentration was 10 ⁇ M, 5 ⁇ M.
  • the cell killing toxicity was 63.3.3 ⁇ 7.9%, 45.6 ⁇ 5.6% and 25.3 ⁇ 4.6%, respectively; the cytotoxicity of the combination group was 88.1% ⁇ 8.9%, 56.2% ⁇ 5.8% and 38.3%, respectively.
  • SK-HEP-1 and SK-HEP-1/GPC3 were mixed 50% each to prepare SK-HEP-1/GPC3 (50%) heterogeneous mixed cells.
  • the cytotoxicity of sorafenib, NK-92/9.28.z cells, or a combination thereof was determined using a CytoTox 96 non-radioactive cytotoxicity assay kit. The results of the experiment are shown in Figure 11.
  • the solanin 5 ⁇ M for 48h has approximately the same killing effect on SK-HEP-1/GPC3 and SK-HEP-1/GPC3 (50%) heterogeneous mixed cells, and the killing rate is respectively It was 54.5% ⁇ 6.7% and 56.8% ⁇ 5.6%.
  • NK-92/9.28.z cells alone had significant killing effect on SK-HEP-1/GPC3, and the effective target ratio was 33.2% killing rate of 73.2% ⁇ 9.2%.
  • NK-92/9.28.z cells were used alone, and the killing rate was 39.8% ⁇ at a target-to-target ratio of 3:1. 5.6%.
  • the inhibitory effect of sorafenib combined with NK-92/9.28.z cells on liver cancer was significantly enhanced, up to 93.6% ⁇ 10.2%, which was 1.28 times that of NK-92/9.28.z group.
  • the Fini single-agent group was 1.72 times; the latter heterogeneous mixed cells, the combined toxicity of the two was up to 88.9% ⁇ 7.8%, which was 2.23 times that of the NK-92/9.28.z group.
  • the Rafini single drug group was 1.56 times. This result shows that the combination of sorafenib and NK-92/9.28.z cells can increase the killing rate of heterogeneous cell populations to a greater extent.

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Abstract

L'invention concerne une cellule NK à récepteur d'antigène chimérique exprimant une reconnaissance spécifique pour GPC3 et sa méthode de préparation, ainsi qu'une utilisation de la cellule dans la prévention et/ou le traitement de cancers, en particulier un cancer exprimant GPC3. L'invention concerne également une utilisation de la cellule NK en tant qu'agent thérapeutique cellulaire de ciblage et/ou en immunothérapie adoptive de cancers.
PCT/CN2018/098623 2017-08-04 2018-08-03 Cellule car nk ciblant gpc3 WO2019024933A1 (fr)

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WO2022028623A1 (fr) 2020-08-07 2022-02-10 佧珐药业有限公司 Cellules modifiées et procédé de modification de cellules
WO2022214089A1 (fr) 2021-04-08 2022-10-13 克莱格医学有限公司 Utilisation d'immunothérapie cellulaire
WO2023274303A1 (fr) 2021-06-29 2023-01-05 科济生物医药（上海）有限公司 Polypeptide chimérique pour la régulation de l'activité physiologique cellulaire
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WO2023274303A1 (fr) 2021-06-29 2023-01-05 科济生物医药（上海）有限公司 Polypeptide chimérique pour la régulation de l'activité physiologique cellulaire
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