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WO2018058431A1 - Molécule de récepteur antigénique chimérique et son utilisation - Google Patents

Molécule de récepteur antigénique chimérique et son utilisation Download PDF

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WO2018058431A1
WO2018058431A1 PCT/CN2016/100796 CN2016100796W WO2018058431A1 WO 2018058431 A1 WO2018058431 A1 WO 2018058431A1 CN 2016100796 W CN2016100796 W CN 2016100796W WO 2018058431 A1 WO2018058431 A1 WO 2018058431A1
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cells
peptide
chimeric antigen
cell
antigen receptor
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李华顺
韩昆昆
薛亚男
王保垒
任宝永
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70517—CD8
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • 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
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • 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
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention relates to the field of molecular biology, in particular to a chimeric antigen receptor molecule and application thereof.
  • a malignant tumor is a disease that seriously threatens human life.
  • the pathogenesis of malignant tumors or cancers is diverse.
  • the common manifestation is that the mutant tumor cells are not cleared by the body's immune system, can propagate and spread without restriction, and destroy the normal cells and functions of surrounding tissues.
  • the medical profession has made a lot of efforts in trying to heal and control the course of tumor diseases, but the results have been minimal.
  • the clinical adjuvant treatment of mainstream medical circles is still radiation therapy, chemical drug therapy and antibody therapy.
  • the recognition and killing effect of immunocompetent cells on tumor cells depends on the expression of receptor molecules on the surface of tumor cell membranes. At least two factors lead to the inability of T lymphocytes in vivo to recognize cancer cells well: (1) Downregulation of antigens by cancer cells The expression of the presenting molecule, (2) the presented antigen has a weak affinity for the T cell receptor. Although there are highly specific T lymphocytes in cancer cells in cancer patients, the number is too small. Can not play a role in the treatment of cancer.
  • the chimeric antigen receptor is mainly composed of two parts, one end is located outside the cell and can specifically recognize an antigen on the surface of the cancer cell, and the other end is located in the cell containing a signal activation element (such as a Zeta chain of T cell receptor) to transmit a signal. Activate the role of T cells.
  • a signal activation element such as a Zeta chain of T cell receptor
  • CAR-T cell therapy developed with CD19 has achieved remarkable results in the clinical treatment of both primary and secondary hematological tumors, but the progress of CAR-T cell therapy in solid tumor research is slow. There is no significant breakthrough.
  • One of the reasons is that 95% of lymphocytic leukemia cells express the B cell antigen CD19, while the specific antigen expression of other solid tumor cells is between 40-70%; therefore, in the treatment of solid tumors, a single monoclonal antibody CAR - T cells are unlikely to kill cancer cells that express other tumor-specific antigens.
  • the second reason is the rate and number of immune killer cells infiltrating into the solid tumor through the blood/lymph circulation.
  • the third reason is the negative regulation of tumor tissue cells on immunocompetent cells.
  • T-regulated cells mainly controlled by Regulatory T cells (Treg).
  • T-regulated cells When the control of T-regulated cells is weakened, it may cause hyperimmune or autoimmune diseases. When the control is enhanced, the immune function will be low. , breeding tumors or other viral skin diseases.
  • CTL4 Cytotoxic T lymphocyte-associated antigen-4
  • programmed death protein 1 programmed yellow cell-associated antigen 1 (Programmed cell death protein) 1/Programmed cell death protein 1 ligand 1, PD-1/PD-L1) and other molecules.
  • Inhibitory T-regulated cells express CTLA4, which interacts with B7-type protein subunits on the surface of immune cells DC and T cells to inhibit immune cell function.
  • B7 protein family include B7-H1 (PD-L1), B7-H2 (PD-1L2) and the like.
  • B7-H1 (PD-L1) can also further inhibit the active function of target cells by binding to lymphocyte PD-1 molecules.
  • the inhibition of T cell function by PD-1 and PD-L1 interaction is a major obstacle to the treatment of tumors by immune cells. In most solid tumor tissues, cancer cells express an increased level of PD-L1/PD-1, which directly inhibits activated lymphocytes infiltrating into cancerous tissues.
  • the present invention prepares a high affinity chimeric antigen with higher specificity and lethality by modifying the PD-1 molecule of the extracellular domain of the chimeric antigen receptor molecule (CAR) and selecting a molecule having higher affinity than PD-1 and PDL1. Receptor molecule.
  • CAR chimeric antigen receptor molecule
  • an object of the present invention is to provide a chimeric antigen receptor molecule having a higher affinity for a PDL-1 molecule on a tumor surface and an application thereof.
  • a first aspect of the invention provides a chimeric antigen receptor molecule comprising a sequence of extracellular domain peptides, a transmembrane domain peptide and an intracellular domain peptide, wherein the extracellular domain peptide comprises a HAC peptide, Its sequence is shown in SEQ ID NO: 1. According to the article "Engineering high-affinity PD-1 variants for optimized immunotherapy and immuno-PET imaging", the mutant HAC of PD-1 has higher affinity with the tumor surface PDL-1 molecule.
  • the transmembrane region peptide is a region spanning a cell membrane in a protein sequence, usually an ⁇ -helical structure, about 20-25 amino acid residues, and the amino acid is mostly a hydrophobic amino acid; wherein
  • the transmembrane region peptide includes, but is not limited to, a CD8 transmembrane region peptide or a PD-1 transmembrane region peptide, and when the transmembrane region peptide is a CD8 transmembrane region peptide, the HAC peptide segment and the CD8 transmembrane region The peptides are also linked by the hinge region of the CD8.
  • the sequence of the CD8 transmembrane region peptide is shown in SEQ ID NO: 2
  • the sequence of the hinge region of CD8 is shown in SEQ ID NO: 3
  • the sequence of the PD-1 transmembrane region peptide is as SEQ ID NO: 4. Shown.
  • the intracellular domain peptide is a costimulatory signal molecule selected from one or more of the intracellular domain peptides of 4-1BB (also known as CD137), CD28, and CD3 ⁇ .
  • the intracellular domain peptide is selected from the group consisting of interconnected 4-1BB and CD3 sputum intracellular domain peptides, the sequences of which are set forth in SEQ ID NOS: 5 and 6, respectively.
  • the chimeric antigen receptor molecule further comprises a signal peptide.
  • the signal peptide can enhance the secretion of the fusion protein of the chimeric antigen receptor molecule, and the signal peptide and the other amino acid sequence of the fusion protein After being expressed, it is finally excised by proteases.
  • the protease has a certain recognition sequence, and the signal peptide is fused with the peptide after it to form a new amino acid sequence. Therefore, if the selected signal peptide is improper, the protease may be miscut and the protein may be inactivated.
  • the signal peptide may be selected from a signal peptide of an immunoglobulin light chain or a signal peptide secreted by a PD-1 protein, the sequences of which are shown in SEQ ID NOS: 7 and 8, respectively.
  • the chimeric antigen receptor molecule of the present invention comprises a signal peptide of an immunoglobulin light chain, a HAC peptide, a hinge region peptide of CD8, a transmembrane region peptide of CD8, and a 4-1BB intracellular domain peptide.
  • the segment and the CD3 sputum intracellular domain peptide are sequentially ligated, and the sequence thereof is shown in SEQ ID NO: 9.
  • a second aspect of the invention provides a nucleotide encoding a chimeric antigen receptor molecule provided by the first aspect of the invention.
  • a third aspect of the invention provides a recombinant vector comprising the nucleotide provided by the second aspect of the invention.
  • the vector is a lentiviral vector, which can efficiently integrate a foreign gene or an exogenous shRNA into a host chromosome, thereby achieving the effect of persistently expressing the sequence of interest. It can effectively infect various types of cells such as neuron cells, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, stem cells, etc. in terms of infection ability, thereby achieving a good gene therapy effect.
  • the use of lentiviral vectors can greatly improve the transduction efficiency of the target gene or the target shRNA, and the target gene or the target shRNA is integrated into the host cell. The probability of genome is greatly increased, and the long-term and stable expression of the target gene or the target shRNA can be realized conveniently and quickly.
  • the lentiviral vector used in the present invention should include, but is not limited to, a pRRSLIN lentiviral expression vector and a pLVX vector, preferably a pRRSLIN lentiviral expression vector.
  • a fourth aspect of the invention provides a recombinant cell comprising the recombinant vector provided by the third aspect of the invention.
  • the recombinant cell is preferably a T cell or an NK cell.
  • the gene encoding the chimeric antigen receptor can be transferred to T cells or NK cells by the aforementioned vector, used to modify T cells or NK cells to become CAR-T or CAR-NK cells; and the chimeric antigen receptor is modified by the chimeric antigen receptor T cells or NK cells can kill tumor cells by identifying tissue factors on the surface of tumor cells for tumor treatment.
  • co-stimulating molecule refers to some adhesion molecules on the surface of immune cells, such as CD28, CD134/OX40, CD137/4-1BB, CD40, etc., which activate immune cells by binding to their ligands.
  • the second signal enhances the proliferative capacity of immune cells and the secretory function of cytokines, prolonging the survival time of activated immune cells.
  • extracellular domain refers to a segment of the membrane protein that is located outside the cell.
  • domain refers to a region of a protein biomacromolecule having a specific structure and an independent function.
  • the number of amino acid residues in a common domain is between 100 and 400, and the smallest domain is only 40 to 50 amino acid residues. Large domains can exceed 400 amino acid residues.
  • PD-1 refers to programmed cell death protein 1 and the gene name PDCD1_HUMAN.
  • the corresponding protein sequence number is UniProtKB-Q15116, which is a T cell immunosuppressive molecule whose extracellular domain resembles an immunoglobulin.
  • the variable region (V-section) of the protein has the property of specifically binding to its ligands PD-L1 and PD-L2 (Programmed cell death protein 1 ligand 1/2).
  • PD-1 is normally expressed in activated T lymphocytes and is also expressed in a variety of malignant cells.
  • P-L1 and P-L2 refer to the currently discovered human programmed death factor 1 ligand 1/2. Its extracellular domain domain has immunoglobulin-like V and C1 regions, which are combined with the V region of PD-1 via the V region (4zqk Structure 232341-2348, 2015). It is usually expressed in a small amount in dendritic cells DC, T regulatory cells and Th cells, macrophages, Mast cells and bone marrow, and is also expressed in various malignant cells.
  • the present invention has at least the following advantages: the chimeric antigen receptor molecule of the present invention has a HAC molecule having higher affinity with the PDL-1 molecule on the tumor surface, and is assembled in the extracellular segment of the CAR-T cell. To identify tumor cells with higher affinity and killing activity.
  • Figure 1 is a schematic diagram showing the construction of a lentiviral expression vector
  • Figure 2 is a flow chart of pRRSLIN-HAC infection for 3 days
  • Figure 3 is a graph showing the killing results of CAR-HAC against different target cells
  • Figure 4 is a graph showing the results of CAR-HAC detection of in vitro proliferation of different target cells
  • Figure 5 is a graph showing the results of detection of cytokines by different kinds of target cells by CAR-HAC;
  • Figure 6 is a graph showing the results of detection of four cell lines of MCF7/MCF7-PDL1/HeLa/SMC7721 by CAR-HAC;
  • Figure 7 is a graph comparing the killing effects of different cell lines CAR-PD-1 and CAR-HAC.
  • the invention provides a preparation method of a lentiviral expression vector for expressing a chimeric antigen receptor molecule, comprising the following steps:
  • HAC gene fragment is synthesized, and the known human CD8 cross is searched from the GenBank database.
  • the above gene sequences are sequentially linked according to human HAC gene, CD8 membrane region gene, human 4-1BB intracellular region gene and CD3 sputum intracellular region gene, and different enzyme cleavage sites are introduced at each sequence junction to form a complete HAC-CD8-4-1BB-CD3 ⁇ gene sequence information,
  • the gene sequence of HAC-CD8-4-1BB-CD3 ⁇ is ligated into the pRRSLIN vector by restriction enzyme transformation, and the upstream of the gene is the EP-1 ⁇ promoter.
  • the vector was transformed into Stbl3 Escherichia coli strain, then transferred to a solid medium containing ampicillin for propagation, screened, positive clones were obtained, plasmids were extracted, and clones were identified by restriction enzyme digestion. The vector was confirmed to be successfully constructed by sequencing, and pRRSLIN-HAC was obtained slowly.
  • FIG. 1 A schematic diagram of the construction of a viral expression vector and a lentiviral expression vector is shown in FIG.
  • the present invention provides a method for producing a lentivirus by expressing a lentiviral expression vector in Example 1, comprising the following step:
  • 293T cells were seeded into a 15 cm culture dish at approximately 8 x 10 6 per dish. Ensure that the cells are confluent at 80% and evenly distributed in the culture dish during transfection.
  • Solution B A mixture of the following plasmids was separately added: 112.5 ⁇ g pRRLSIN-HAC (target plasmid); 39.5 ⁇ g pMD2.G (VSV-G envelop); 73 ⁇ g pCMVR8.74 (gag, pol, tat, rev); 625 ⁇ L 2M calcium ion Solution.
  • the present invention provides a method for preparing CAR-T cells by lentiviral infection of cells in Example 2, comprising the steps of:
  • S1 take 0.5mL blood for rapid detection of pathogenic microorganisms, exclude microbial infections such as HBV, HCV, HDV and HEV, HIV-1/2, Treponema pallidum and parasites; under sterile conditions, use blood collection of heparin bottle 50mL (heparin antibiotic) Condensed), immediately (4 ° C, within 24 hours) to the cell preparation laboratory to ensure that this process is free of pathogenic microorganisms. After obtaining the patient's blood, in the GMP preparation room, the surface of the heparin bottle is wiped with an alcohol cotton ball for disinfection and then placed in a biological safety cabinet.
  • heparin bottle 50mL (heparin antibiotic) Condensed immediately (4 ° C, within 24 hours
  • PBMC peripheral blood mononuclear cells
  • V-VIVO15 added autologous AB (FBS) concentration of 5%, interleukin-2 (IL-2) concentration of 40 ng / mL, and the isolated PBMC was diluted to 2 ⁇ with the culture medium 10 6 /mL, 50 ⁇ L flow detection of the purity of T cells in PBMC.
  • FBS autologous AB
  • IL-2 interleukin-2
  • Example 2 S6 and Day 2 adjusted the cell density to 3-5 ⁇ 10 6 /mL, and the pRRSLIN-HAC lentiviral expression vector prepared in Example 1 was added at a ratio of viral carrier to cell 1:5, and polyglycolamine was added ( Polybrene) 4 ⁇ g/mL and 40 ng/mL IL-2. After 4 h, fresh complete medium was added to adjust the cell density to 1 ⁇ 10 6 /mL to continue the culture. All cells were centrifuged, fresh medium was added, and the culture was continued.
  • the CAR-T cells prepared in Example 3 were subjected to flow analysis, and the specific steps were as follows:
  • Figures A and B are the control group, which are T cells that do not infect the virus; FITC-conjugated antibodies for detecting CAR molecules do not detect CAR molecule expression; C and D maps are T transfected with PRRSLIN-HAC lentivirus Cells, after flow detection, compared with A and B, cells were successfully transfected; after infection of T cells by virus, the infection efficiency reached 53.26% after 3 days of flow detection, indicating successful preparation of HAC-CAR -T cells.
  • LDH release assay was used to detect the killing effect of HAC-CAR-T cells on engineered cell lines MCF-1/PDL1 and PDL1 high expressing cells. LDH release was detected by ELISA, including the following steps:
  • the target cells were adjusted to 5 ⁇ 10 4 /mL with RPMI-1640 medium containing 5% calf serum.
  • target cells were added to a 96-well cell culture plate, and 100 ⁇ L per well was added. Three wells were used as effector cells (HAC-CAR-T cells) to naturally release control wells, and no target cells were added, and only 100 ⁇ L of the culture solution was added.
  • HAC-CAR-T cells effector cells
  • effector cells 100 ⁇ L were added to each well, and the ratio of effector cells to target cells was 10:1; 5:1; 1:1.
  • the natural release well was added with only 100 ⁇ L of the culture medium without effector cells, and the effector cells were incubated with the target cells for 6 hours, and three replicate wells were placed for each experiment.
  • the optical density (OD value) of each well was measured on an enzyme-linked detector, and the detection wavelength was 490 nm or 492 nm, and the measurement was completed within 1 hour.
  • Killing rate experimental group LDH (OD) / maximum LDH release group (OD).
  • the cytokine secretion was measured by a CBA kit, and the proliferation of each group of CAR-T cells was calculated, and the ratio of CD8-positive T cells in the proliferating T cells was confirmed by staining with CD3 and CD8 antibodies.
  • HAC CAR-T can significantly kill SMCCC7721 tumor cells, and the killing effect on MCF-7 cells with high expression of PDL-1 is better than that of MCF-7 common tumor cells.
  • the abscissa indicates CAR-T cells. Different from tumor cells, the ordinate indicates the killing efficiency, and different types of histograms indicate different tumor cells.
  • the abscissa indicates the ratio of T cells to tumor or CAR-T cells to tumors
  • the ordinate indicates the number of cells
  • T represents T cells
  • HAC represents CAR-T cells
  • the abscissa indicates the different target ratio of CAR-T cells to tumor cells
  • the ordinate indicates the cytokine content
  • the cytokines in the culture supernatant were detected
  • the CAR-T killing experimental group IL-2 was found.
  • the secretion of Figure 5A) and TNF-[alpha] (Figure 5B) was significantly elevated.
  • the CD3/CD8 flow antibody was used to detect the ratio of CAR-T to CD8T cells after killing MCF7/MCF7-PDL1/HeLa/SMC7721 cell lines
  • the ordinate PE signal showed specific detection of CD3 molecule expression.
  • the abscissa FITC signal indicates that the expression of CD8 molecules is detected, and the specific proliferation of CD8 T cells is mainly detected by flow cytometry after activation of HAC CAR-T cells.
  • CAR-PD-1 indicates that the extracellular domain of the CAR molecule is a normal PD-1 molecule
  • CAR-PD-1-HAC indicates the HAC-CAR chimeric antigen receptor molecule constructed by the present invention
  • HAC-CART cells can specifically activate and proliferate after exposure to tumor cells, release cytokines and kill tumor cells, and CD8-positive T cells play a major role.
  • the killing effect of PD-1 CAR-T and HAC CAR-T on tumors was compared.
  • the results showed that HAC-CAR-T was significantly better than PD-1 CAR-T.

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Abstract

L'invention concerne une molécule du récepteur antigénique chimérique et son utilisation. La molécule du récepteur antigénique chimérique comprend un fragment de peptide dans une région extracellulaire, un fragment de peptide dans une région transmembranaire et un fragment de peptide dans un domaine intracellulaire en tandem successive. Le fragment peptidique dans la région extracellulaire est un fragment de peptide HAC, et sa séquence est telle que représentée dans SEQ ID NO : 1. L'invention concerne également un nucléotide, un vecteur recombinant et une cellule recombinante utilisant la molécule du récepteur antigénique chimérique.
PCT/CN2016/100796 2016-09-28 2016-09-29 Molécule de récepteur antigénique chimérique et son utilisation Ceased WO2018058431A1 (fr)

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CN107082813A (zh) * 2017-05-17 2017-08-22 董克海 靶向pd‑1的嵌合抗原受体分子及其应用
CN108977453A (zh) * 2017-06-02 2018-12-11 阿思科力(苏州)生物科技有限公司 一种以robo1为靶点的嵌合抗原受体细胞及其制备和应用
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