WO2022171196A1 - 抗cd87抗体及其特异性嵌合抗原受体 - Google Patents

抗cd87抗体及其特异性嵌合抗原受体 Download PDF

Info

Publication number
WO2022171196A1
WO2022171196A1 PCT/CN2022/076087 CN2022076087W WO2022171196A1 WO 2022171196 A1 WO2022171196 A1 WO 2022171196A1 CN 2022076087 W CN2022076087 W CN 2022076087W WO 2022171196 A1 WO2022171196 A1 WO 2022171196A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
seq
car
antibody
cell
Prior art date
Application number
PCT/CN2022/076087
Other languages
English (en)
French (fr)
Inventor
焦作义
王�琦
秦龙
孙辉
王龙
王彦
张俊昶
文飞
蔡伟文
杨志良
Original Assignee
兰州大学第二医院
兰州华帜天成生物科技有限责任公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 兰州大学第二医院, 兰州华帜天成生物科技有限责任公司 filed Critical 兰州大学第二医院
Publication of WO2022171196A1 publication Critical patent/WO2022171196A1/zh

Links

Images

Classifications

    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • 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
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464429—Molecules with a "CD" designation not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • 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
    • 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
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors
    • 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
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • 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
    • A61K2239/51—Stomach
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

Definitions

  • the present invention relates to the fields of biology and medicine, in particular to a novel anti-CD87 antibody and its specific chimeric antigen receptor, CAR-T cells comprising the specific chimeric antigen receptor and its application in gastric cancer.
  • Gastric cancer is one of the most common malignant tumors in human beings. There are more than 600,000 new cases of gastric cancer in my country every year, ranking second in the incidence of malignant tumors, seriously endangering human life and health. Due to the high heterogeneity of gastric cancer tissue, traditional treatment methods such as surgery, chemotherapy and radiotherapy are not satisfactory, and the five-year survival rate of advanced gastric cancer is less than 30%. It showed that patients with diffuse gastric cancer according to Lauren's classification had the worst clinical prognosis, and the 5-year overall survival rate was about 8% lower than that of intestinal gastric cancer patients. Therefore, the traditional treatment methods for gastric cancer are not effective, and it is urgent to develop new treatment models to improve their efficacy.
  • CAR-T therapy is a new way to treat tumors. Mainly used in hematological tumors. In 2017, the US FDA has approved two CAR-T cell drugs targeting CD19 for the treatment of leukemia and lymphoma. The progress of CAR-T therapy in the field of solid tumors is slow. The main reason is that CAR-T cells will be inhibited by the inhibitory tumor microenvironment (Tumor Microenvironment TME) after infiltrating into solid tumor tissue.
  • Tuor Microenvironment TME Tumor Microenvironment
  • scientistss express chemotaxis in CAR-T cells. factor receptors to enhance the migration ability and anti-tumor activity of CAR-T cells to tumors, or directly target proteins related to the tumor microenvironment. Therefore, research on CAR-T therapy targeting solid tumors and their microenvironment is a difficulty to be broken through in this field in the future.
  • Li Zonghai's group developed a high-affinity humanized monoclonal antibody specific for Claudin18.2, and developed the second generation of CAR-T cells to study the antitumor activity in vitro and in vivo.
  • T cells showed good antitumor activity in the human-derived gastric cancer PDX mouse model, and gastric cancer has not been specifically involved.
  • CAR-T cell therapy for gastric cancer has good application prospects, however, no effective specific CAR-T cell therapy has been developed for gastric cancer with poor traditional therapy.
  • CD87 is a urokinase-type plasminogen activator (uPA) receptor that is anchored to cell membranes by GPIs and is involved in inflammation, tissue remodeling, and It is highly expressed in various cancers and has a poor prognosis.
  • uPA urokinase-type plasminogen activator
  • CD87 regulates the degradation of extracellular matrix proteins and activates multiple intracellular signaling pathways by cooperating with uPA, integrin and vitronectin, and plays an important role in tumor cell migration and proliferation. target.
  • CD87 is related to the tumor microenvironment. It not only participates in the degradation of extracellular matrix, but also promotes the infiltration of immune cells. It can be used as a target for CAR-T therapy in gastric cancer.
  • CD87 is highly expressed on the surface of gastric cancer cells and is lowly expressed in normal tissues.
  • Targeting CD87 can promote the degradation of extracellular matrix, improve the physical barrier of the tumor microenvironment, and promote immune cell infiltration.
  • CAR-T is used as a target. The therapy can overcome the physical barrier of the tumor microenvironment and achieve better tumor killing effect.
  • the invention provides an anti-CD87 antibody comprising at least 90% (eg at least 91%, 92%, 93%, 94%, 95%, 96%) of SEQ ID NO: 10 or 14 , 97%, 98%, 99% or 100%) sequence identity to VH sequences and have at least 90% (e.g. at least 91%, 92%, 93%, 94%, 95%, VL sequences of 96%, 97%, 98%, 99% or 100%) sequence identity.
  • the anti-CD87 antibody comprises the VH sequence of SEQ ID NO: 10 or 14 and the VL sequence of SEQ ID NO: 12 or 16.
  • the invention provides an anti-CD87 antibody comprising at least 90% (eg at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) heavy chain sequence identity and have at least 90% (e.g. at least 91%, 92%, 93%, 94%, 95%, 96%) with SEQ ID NO: 4 or 8 %, 97%, 98%, 99% or 100%) sequence identity of the light chain.
  • the anti-CD87 antibody comprises the heavy chain of SEQ ID NO: 2 or 6 and the light chain of SEQ ID NO: 4 or 8.
  • the present invention finds that anti-CD87 antibodies can be used to alter the tumor microenvironment. Accordingly, in one embodiment, the present invention provides the use of an anti-CD87 antibody for altering the tumor microenvironment. In a preferred embodiment, the anti-CD87 antibody is an anti-CD87 antibody as defined above.
  • the present invention provides the use of an anti-CD87 antibody as defined above for the treatment of gastric cancer in a patient, eg a human.
  • the present invention provides a CD87-specific chimeric antigen receptor (CAR) polypeptide comprising a CD87 antigen binding domain, a transmembrane domain and an intracellular signaling domain.
  • the CD87 antigen binding domain comprises a variable heavy chain (VH) and a variable light chain (VL), the VH comprising at least 90% (eg, at least 90% of SEQ ID NO: 10 or 14) and a variable light chain (VL) 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity
  • the VL comprises a sequence with at least SEQ ID NO: 12 or 16 Sequences of 90% (eg, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity.
  • VH comprises the sequence of SEQ ID NO: 10 or 14
  • VL comprises the sequence of SEQ ID NO: 12 or 16.
  • the CD87-specific chimeric antigen receptor (CAR) polypeptide further comprises a signal peptide, such as the signal peptide IL2-SP (eg, the sequence encoded by SEQ ID NO: 18 or at least 80% identical thereto). sequences of sequence identity).
  • the CD87-specific chimeric antigen receptor (CAR) polypeptide further comprises a linker polypeptide between the VH and VL of the CD87 antigen binding domain (eg, the sequence encoded by SEQ ID NO: 19 or sequences with at least 80% sequence identity).
  • the CD87-specific chimeric antigen receptor (CAR) polypeptide further comprises a hinge region between the CD87 antigen-binding domain and the transmembrane domain, such as the hCD8a hinge (e.g., represented by SEQ ID NO: 20 encoded sequences or sequences having at least 80% sequence identity therewith).
  • the transmembrane domain may be, for example, CD8tTM (eg, the sequence encoded by SEQ ID NO: 21 or a sequence having at least 80% sequence identity thereto).
  • the intracellular signaling domain comprises a costimulatory domain and a signaling domain
  • the costimulatory domain may be, for example, a 4-1BB costimulatory domain (e.g., represented by SEQ ID NO: 22 encoded sequence or a sequence having at least 80% sequence identity therewith)
  • the signaling domain may be, for example, a CD3zeta signaling domain (e.g., the sequence encoded by SEQ ID NO: 23 or a sequence having at least 80% sequence identity therewith) sexual sequence).
  • the present invention provides the use of a CD87-specific chimeric antigen receptor polypeptide as described above for altering the tumor microenvironment.
  • the present invention provides the use of a CD87-specific chimeric antigen receptor polypeptide as described above for the treatment of gastric cancer in a patient (eg, a human).
  • the present invention provides an isolated nucleic acid sequence encoding an antibody or chimeric antigen receptor polypeptide as described above.
  • the present invention provides a vector comprising the isolated nucleic acid sequence as described above.
  • the present invention provides a cell comprising the vector as described above.
  • the cell is selected from the group consisting of: ⁇ T cells, ⁇ T cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, innate lymphocytes (ILCs), cytokine-induced killing (CIK) cells, cytotoxic T lymphocytes (CTL), lymphokine-activated killer (LAK) cells, regulatory T cells, or any combination thereof.
  • the cell expresses a CD87-specific chimeric antigen receptor polypeptide as described above.
  • the present invention provides the use of a cell as described above for altering the tumor microenvironment.
  • the present invention provides the use of a cell as described above for the treatment of gastric cancer in a patient.
  • CD87 inhibitors alter the tumor microenvironment in the body by targeting CD87, thereby helping PD-1 inhibitors work better therapeutically.
  • Such a combined effect, more particularly a synergistic effect, of a CD87 inhibitor and a PD-1 inhibitor would not have been expected by those skilled in the art.
  • a CD87 inhibitor can be any agent that inhibits CD87.
  • the CD87 inhibitor is an anti-CD87 antibody as described above, a CD87-specific chimeric antigen receptor (CAR) polypeptide or a cell comprising a CD87-specific chimeric antigen receptor (CAR) polypeptide.
  • a PD-1 inhibitor can be any agent that inhibits PD-1.
  • the PD-1 inhibitor is an anti-PD-1 antibody.
  • the present invention provides an anti-CD87 antibody, a CD87-specific chimeric antigen receptor (CAR) polypeptide, or a cell comprising a CD87-specific chimeric antigen receptor (CAR) polypeptide for use in combination with a PD-1 inhibitor Use in the treatment of gastric cancer in a patient.
  • the anti-CD87 antibody, CD87-specific chimeric antigen receptor (CAR) polypeptide or cell comprising a CD87-specific chimeric antigen receptor (CAR) polypeptide alters the tumor microenvironment in the patient.
  • a patient can be any mammalian or non-mammalian, preferably a human.
  • the anti-CD87 antibody, CD87-specific chimeric antigen receptor (CAR) polypeptide or cell comprising a CD87-specific chimeric antigen receptor (CAR) polypeptide is an anti-CD87 antibody as described above , CD87-specific chimeric antigen receptor (CAR) polypeptide or cells comprising CD87-specific chimeric antigen receptor (CAR) polypeptide.
  • Figure 1 shows the expression of CD87 provided by the present invention in gastric cancer and normal group.
  • Figure 2 shows the expression level of CD87 in gastric cancer cell lines, A: qRT-PCR; B: flow cytometry.
  • FIG. 3 is a cell experiment: CD87 knockout inhibits tumor cell proliferation: A: SNU216; B: AGS. C, D: Knockout of PLAUR in SNU-216 cells and AGS cells, respectively, MTT results showed that knockdown of PLAUR inhibited tumor cell proliferation.
  • Figure 4 is an animal experiment.
  • A tumor volume change; B: tumor weight; C: small animal imaging.
  • B Mouse tumor tissue weight.
  • C Small animal imaging results.
  • FIG. 5 Expression of extracellular matrix proteins after AGS and SNU-216 knockout PLAUR.
  • Figure 6 shows the preparation of CD87 monoclonal antibody (also known as anti-UPAR antibody): A: cellular immunity; B: positive B cell screening and antibody flow verification; C: two screened high-affinity monoclonal antibodies.
  • A cellular immunity
  • B positive B cell screening and antibody flow verification
  • C two screened high-affinity monoclonal antibodies.
  • Figure 7 is a flow-through validation of CD87 monoclonal.
  • FIG. 8 Anti-uPAR antibody competes with uPA for uPAR binding:
  • A Binding affinity (KD) of anti-uPAR monoclonal antibody (mAb) to human uPAR was assessed by surface plasmon resonance.
  • B-C Immunofluorescence (IF) (B) and immunoelectron microscopy (C) analysis of anti-uPAR binding to uPAR-expressing HEK-293T cells.
  • D-E Detection of different forms of uPAR by anti-uPAR mAbs shown by flow cytometry (FCM) (D) and Western blotting (E).
  • F Competitive binding assay showing that anti-uPAR mAb detected by FCM inhibits the binding of uPA to uPAR.
  • FIG. 9 Anti-uPAR antibody inhibits uPAR-dependent signaling in gastric cancer cells
  • A Phosphorylated ERK (p-ERK) and total ERK (p-ERK) in SNU-216 cells pretreated with anti-uPAR or ctrl mAb after pro-uPA stimulation T-ERK) Western blot.
  • B-E Growth curves (B, C), transwell invasion assay (D) and cell adhesion assay (E) of AGS and SNU-216 cells treated with anti-uPAR or ctrl mAb. Data are presented as mean ⁇ SEM (*p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001).
  • Figure 10 is a cell experiment: CD87 monoclonal antibody blocking inhibits tumor cell proliferation: A: AGS; B: SNU216
  • Figure 11 is the data of CD87 monoclonal antibody killing CDX model.
  • Figure 12 shows the changes of tumor tissue weight and mouse weight in CDX model group killed by CD87 monoclonal antibody.
  • Figure 13 shows the imaging results of CD87 monoclonal antibody killing the CDX model of small animals.
  • FIG. 14 Anti-uPAR alone or in combination with anti-PD-1 improves survival in humanized mice bearing patient-derived xenografts.
  • A Diagram of the experimental procedure.
  • Figure 15 Anti-uPAR alone or in combination with anti-PD-1 enhances infiltration and activation of cytotoxic CD8+ T cells.
  • AB IF staining (A) and quantification (B) of CD8+ T cells in patient A-derived PDX at the end of the experiment.
  • CD CD8 IHC staining and data analysis of the same PDX as in (AB).
  • E mRNA expression levels of chemokines in PDX as in (AB) detected by qRT-PCR.
  • F Activated cytotoxic T cells (CD45 + CD8 + CD107a + ), regulatory T cells (Tregs, CD45 + CD4 + FOXP3 + ) and Frequency of M2 macrophages (CD45 + CD11b + CD68 + CD206 + ) as indicated by FCM. Data are presented as mean ⁇ SEM (ns not significant, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001).
  • Figure 16 is a schematic diagram of the construction of CD87-CAR.
  • Figure 17 is a schematic diagram of the structure of CD87-CAR of the present invention.
  • Fig. 18 is a graph showing the infection efficiency of T cells.
  • the initial infection efficiency was 27.8%, and the infection efficiency reached 99.9% after sorting and enrichment.
  • Figure 19 shows the establishment of two groups of CD87-CAR-T+AGS-luciferase and control-T cells+AGS-luciferase for co-culture, and at 1:1, 2:1, 4:1, 8:1 and The luciferase release of tumor cells in each group was observed under the ratio of 16:1, and the line graph was drawn. (Effective cells: target cell binding/killing screening).
  • Figure 20 shows the CDX model established in NSG mice with MKN-45 cells, which are divided into T cell control group, CAR-T cell killing group and CAR-T cell combined with PD-1 antibody treatment group.
  • A Changes in tumor volume;
  • B Animal survival analysis.
  • Figure 21 shows the expression levels of IFN- ⁇ and Granzyme B in the serum of each group of animals.
  • Figure 22 shows the imaging results of small animals.
  • Figure 23 shows the experimental results of CAR-T cells killing PDX model. They were divided into T cell control group, CAR-T cell killing group and CAR-T cell combined with PD-1 antibody treatment group.
  • A Changes in tumor tissue volume after treatment;
  • B Analysis of survival curve after treatment.
  • Figure 24 shows the expression levels of IFN- ⁇ and Granzyme B in the serum of animals in each group.
  • Figure 25 shows the infiltration of CD8+ T cells in the combination group of CAR-T cells and PD-1 antibody.
  • CAR-T target screening and validation A total of 605 up-regulated genes were identified in the cancer and adjacent tissues of 16 gastric cancer patients for gene expression profiling analysis. Fifteen pairs of fresh surgical samples of gastric cancer and adjacent tissues were collected and analyzed by label-free quantitative proteomics. A total of 186 proteins that were highly expressed in cancer and not expressed adjacent to cancer were identified. Using the TCGA gastric cancer data, the differential genes of diffuse gastric cancer and normal tissues were analyzed. The above two data obtained were intersected with immune-related genes, and two key proteins, FCGR3A and CD87, were finally obtained, which were analyzed in the TISIDB database with immune cell infiltration and immune cells. Checkpoint dependencies.
  • CD87 is preliminarily determined as a gastric cancer CAR -T therapeutic targets.
  • CD87 in gastric cancer cell lines HGC-27, MKN-45, AGS, SNU-216, HS746T and NCI-N87 were detected by qRT-PCR and flow cytometry, and the results showed that CD87 in AGS, SNU-216 and HS746T High expression, low expression in HGC-27, MKN-45 and NCI-N87,
  • gRNA1 TTCCACACGGCAATCCCCGT.
  • gRNA2 GGACCACGATCGTGCGCTTG.
  • Knockout of CD87 in AGS and SNU-216 showed that knockout of CD87 in AGS and SNU216 inhibited tumor cell proliferation, and antibody blocking achieved the same effect, using 2 ⁇ 10 6 MKN-45 cells, MKN -45-CD87 -/- cells and CD87-reverting MKN-45-CD87 -/- cells formed tumors in NSG mice, and tumor volumes in mice were measured every two days, and the tumor-bearing mice were observed with a small animal imager. Tumor changes.
  • CD87 can change the tumor microenvironment by detecting the changes of ECM-related adhesion proteins. It is found that the expression of adhesion-related proteins such as Smad2, vitronectin and vimetin is significantly down-regulated, which makes the intercellular adhesion become loose, which may be related to the changes in the tumor microenvironment.
  • adhesion-related proteins such as Smad2, vitronectin and vimetin
  • the constructed plasmids and packaging plasmids pDD and pVSVg were transfected with PEI at a ratio of 1:1:0.5 to 293T cells. After culturing for 36 hours, the supernatant was collected, centrifuged and concentrated the virus. After measuring the titer, 293T cells were infected with puromycin. The cells stably expressing the antigen CD87 were screened and successfully obtained.
  • Lentiviral vector plasmid Lenti-CMV-CD87-T2A-GFP; packaging plasmid: pDD, pVsVG; 293T cells; 0.25% trypsin; DMEM; FBS; Opti-MEM; lip2000; BSA.
  • Inoculation of 293T cells Digest 293T cells in good growth state with 0.25% trypsin, adjust the cell concentration to 6 ⁇ 10 5 cells/mL, and inoculate 10 mL in a 10 cm petri dish, incubate at 37°C and 5% carbon dioxide for 18 hours , so that the cell confluency reaches 60%-70%, and the culture medium is changed to Opti-MEM half an hour before transfection.
  • Virus packaging Add 7ug Lenti-CMV-CD87-T2A-GFP plasmid, 5ug pDD and 3.5ug pVsVg to 500uL OPTI-MEM and mix well; add 30uL lip2000 to 500uL OPTI-MEM and mix well, let stand at room temperature for 5 minutes, slowly add it to the plasmid mixture and mix well, let it stand at room temperature for 15 minutes, add it dropwise to the petri dish, and mix well. After 6 hours, it was replaced with fresh DMEM medium containing 10% FBS and 1% BSA.
  • Virus concentration after culturing for 60 hours, collect the supernatant, centrifuge at 3000rpm at 4°C for 10 minutes, filter with a 0.45um filter membrane, and ultracentrifuge at 25000rpm at 4°C for 2 hours, discard the supernatant, and use 300uL containing 10% FBS and 1% BSA The pellet was resuspended in DMEM medium, overnight at 4°C, aliquoted in 50uL/tube, quick-frozen on dry ice and stored at -80°C.
  • Monoclonal antibodies targeting CD87 were prepared using the prepared 293T-CD87 cells.
  • 293T-CD87 cells were used for cellular immunization: 4 female Balb/c mice of SPF grade 6-8 weeks were taken, the orbital blood was collected one day before immunization, and the serum was separated as a negative control. Mice were injected subcutaneously with 1 ⁇ 10 7 293T cells stably expressing CD87. The second and third immunizations were performed on days 10 and 20 with 5 ⁇ 106 293T cells stably expressing CD87, respectively. On the 25th day, the orbital blood of mice was collected, and the titer of CD87 antibody was detected by ELISA. The mouse with the highest antibody titer was selected and injected with 2 ⁇ 10 6 cells for booster immunization.
  • the spleen cells of the immunized mice were prepared, the positive B cells that could produce CD87 antibody were sorted by flow cytometry (FCM), and the VH and VL sequences were obtained and verified by single-cell RT-PCR technology. High affinity monoclonal antibodies against CD87 (A7 and E2).
  • the monoclonal antibodies targeting CD87 prepared in Example 3 were characterized, and the prepared monoclonal antibodies A7 and E2 were verified by flow cytometry, WB and laser confocal respectively.
  • mice were subcutaneously injected with about 2 ⁇ 10 6 MKN-45 cells overexpressing CD87-T2A-luciferase to establish a CDX model in mice. They were divided into control group, CD87 monoclonal antibody treatment group, PD-1 monoclonal antibody treatment group and CD87 monoclonal antibody treatment group.
  • control group when the tumor volume reached about 90 mm 3 , 2 ⁇ 10 7 human PBMCs were injected into the tail vein, respectively, and 10 mg/kg CD87 monoclonal antibody, PD-1 monoclonal antibody, and CD87 monoclonal antibody were administered in the administration group, respectively.
  • +PD-1 antibody the control group was given the same dose of human IgG antibody twice a week. The tumor size of the mice was measured every two days, and the small animal in vivo imaging system was used to analyze and detect the changes of the tumor every 4 days to verify the therapeutic effect of CD87 monoclonal antibody.
  • Tumor tissues from patients with pathologically confirmed CD87+ gastric cancer were collected, cut into pieces and transplanted subcutaneously into NSG mice. After the tumors grew up, they were taken out. Part of the tumor tissues were cryopreserved, the other part was analyzed for gene expression, and the remaining part was inoculated into NSG mice. The third-generation tumors were not different and could be used for tumor killing experiments. Twenty model mice were prepared and divided into control group, CD87 monoclonal antibody treatment group, PD-1 monoclonal antibody treatment group and CD87 monoclonal antibody combined with PD-1 antibody group.
  • mice 2 ⁇ 10 7 human PBMCs were injected, the administration group was given 10 mg/kg CD87 monoclonal antibody, PD-1 monoclonal antibody and CD87 monoclonal antibody + PD-1 antibody respectively, and the control group was administered the same dose of human IgG antibody, twice a week.
  • the tumor size of the mice was measured every two days, and the small animal in vivo imaging system was used to analyze and detect the changes of the tumor every 4 days to verify the effect of CD87 monoclonal antibody treatment.
  • the CD87-CAR structure consists of EF1a promoter nucleic acid sequence, IL2 signal peptide, CD87 scFv sequence, hCD8a hinge region nucleic acid sequence, CD8TM transmembrane region nucleic acid sequence, 41-BB costimulatory signal domain nucleic acid sequence , CD3 ⁇ signaling domains formed in series, named EF1a-CD87 scFv-hCD8a hinge-CD8TM-41-BB-CD3 ⁇ .
  • the nucleic acid sequence of the CD87-CAR gene structure constructed in this example is shown in SEQ ID No. 24 in the sequence listing.
  • the lenti-EF1a-hCAR-Puro plasmid was digested with EcoRI and BamHI, respectively, and the digested products were recovered by gel.
  • CAR plasmid named CD87-CAR plasmid.
  • Lentiviral vector plasmid CD87-CAR packaging plasmid: pMD2.G, pSPAX2; 293T cells; 0.25% trypsin; DMEM; FBS; Opti-MEM; lip2000; BSA.
  • Inoculation of 293T cells Digest 293T cells in good growth state with 0.25% trypsin, adjust the cell concentration to 6 ⁇ 10 5 cells/mL, and inoculate 10 mL in a 10 cm petri dish, incubate at 37°C and 5% carbon dioxide for 18 hours , so that the cell confluency reaches 60%-70%, and the culture medium is changed to Opti-MEM half an hour before transfection.
  • Virus packaging Add 7ug CD87-CAR plasmid, 5ug psPAX2 and 3.5ug pMD2.G to 500uL OPTI-MEM and mix well; add 30uL lip2000 to 500uL OPTI-MEM and mix well, let stand for 5 minutes at room temperature, and add slowly Mix well into the plasmid mixture, let stand for 15 minutes at room temperature, add dropwise to the petri dish, and mix well. After 6 hours, it was replaced with fresh DMEM medium containing 10% FBS and 1% BSA.
  • Virus concentration after culturing for 60 hours, collect the supernatant, centrifuge at 3000rpm at 4°C for 10 minutes, filter with a 0.45um filter membrane, and ultracentrifuge at 25000rpm at 4°C for 2 hours, discard the supernatant, and use 300uL containing 10% FBS and 1% BSA The pellet was resuspended in DMEM medium, overnight at 4°C, aliquoted in 50uL/tube, quick-frozen on dry ice and stored at -80°C.
  • lymphocyte separation solution (Lymphoprep, Catalog #07801) into a centrifuge tube (SepMate-50, Catalog #15450). The upper end of the lymphocyte separation solution should submerge the filter screen.
  • T cell sorting add 0.5-2 mL of T cell isolation buffer to PBMC, and use EasySep TM Human T Cell Isolation Kit (StemCell, Catalog #17951) to sort T cells: add 2 mL of prepared PBMC to the flow tube, add 50uL /mL isolation Cocktail, mix well, room temperature for 5min, vortex RapidSpheres for 30s to mix, add 40uL/mL, make up to 2.5mL, pipette 3 times to mix.
  • T cell activation add T cells to a 24-well plate, 2 ⁇ 10 6 cells/well, add 50uL of washed Dynabeads Human T-Activator CD3/CD28 (4 ⁇ 10 7 beads/mL): magnetic beads and T cells The cell ratio was 1:1, and the cells were incubated at 37°C for 24 hours in a CO2 incubator.
  • T cell culture 8-10 hours after centrifugation, the medium was removed by centrifugation, and all infected wells were combined and cultured with complete medium 10% FBS+RMPI-1640+IL2+1%P/S. The medium was changed every 48 hours.
  • CD87-CAR-T cell sorting and determination After 6 days of culture, remove the magnetic beads (place on a magnetic rack for 1-2 min, transfer the supernatant to a new culture flask, continue to culture for 24 hours, repeat if necessary This step), GFP detection of CAR expression.
  • Cryopreservation of CD87-CAR-T cells Cryopreservation of CAR-T cells in 10% DMSO-FBS, 3 ⁇ 10 6 cells/tube.
  • the CD87-CAR-T cells prepared in Example 7 were used to detect the killing effect of the CD87-CAR-T cells on tumor cell lines.
  • CD87-CAR-T cells were co-cultured with luciferase-overexpressing gastric cancer cell lines AGS, AGS-CD87 -/- and MKN-45 in a certain proportion. After co-culture at 37°C for 4 hours, 50uL of supernatant and prepared luciferin were taken. The enzyme reagent (Promina#E2610) was added to the 96-well plate (Corning#3904), and the release of luciferase in the supernatant was detected by a microplate reader and the killing ability was calculated. To verify the binding ability and killing effect of CD87-CAR-T cells on gastric cancer target cells.
  • the CD87-CAR-T cells prepared in Example 7 were used to detect the killing effect of the CD87-CAR-T cells on the gastric cancer cell line CDX model.
  • NSG mice were subcutaneously injected with about 2 ⁇ 10 6 MKN-45 cells overexpressing CD87-T2A-luciferase to establish a CDX model in mice, and they were divided into T cell control group, CD87-CAR-T cell treatment group and CD87-CAR-T cell treatment group In the combination group of cells and PD-1 antibody, when the tumor volume reached about 90mm 3 , 1 ⁇ 10 6 T cells, CD87-CAR-T cells, CD87-CAR-T cells and PD-1 antibody were injected into the tail vein respectively. .
  • Small animal in vivo imaging system was used to analyze the changes of tumor, and ELISA was used to detect the changes of IFN- ⁇ and Granzyme B cytokines in peripheral blood to verify the ability of CD87-CAR-T cells to bind gastric cancer target cells in vivo and their killing effect.
  • the CD87-CAR-T cells prepared in Example 7 were used to detect the killing effect of the CD87-CAR-T cells on the PDX model derived from gastric cancer patients.
  • Tumor tissues from patients with pathologically confirmed CD87+ gastric cancer were collected, cut into pieces and transplanted subcutaneously into NSG mice. After the tumors grew up, they were taken out. Part of the tumor tissues were cryopreserved, the other part was analyzed for gene expression, and the remaining part was inoculated into NSG mice. The third-generation tumors were not different and could be used for tumor killing experiments. Twenty model mice were prepared and divided into T cell control group, CD87-CAR-T cell treatment group, and CD87 - CAR-T cell and PD-1 antibody combination group. 1 ⁇ 10 6 T cells, CD87-CAR-T cells, CD87-CAR-T cells and PD-1 antibody were injected intravenously, respectively.
  • mice The body weight and tumor size of the mice were monitored, and the levels of cytokines such as IFN- ⁇ and Granzyme B in peripheral blood were detected by ELISA to verify the ability of CD87-CAR-T cells to kill the gastric cancer PDX model in vivo.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Microbiology (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Cell Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Endocrinology (AREA)
  • Virology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

提供一种新的抗CD87抗体及其特异性嵌合抗原受体、包含其特异性嵌合抗原受体的CAR-T细胞及其在胃癌中的应用。

Description

抗CD87抗体及其特异性嵌合抗原受体 技术领域
本发明涉及生物与医药技术领域，特别涉及一种新的抗CD87抗体及其特异性嵌合抗原受体、包含其特异性嵌合抗原受体的CAR-T细胞及其在胃癌中的应用。
背景技术
胃癌是人类最常见的恶性肿瘤之一，我国每年胃癌新发病例超过60余万，高居恶性肿瘤发病率第二位，严重危害人类生命健康。由于胃癌组织的异质性较高，手术、化疗及放疗等传统治疗手段效果不够理想，进展期胃癌五年生存率不足30％，徐瑞华报道了中国748例D2胃癌根术后患者的疗效，结果显示Lauren分型为弥漫型的胃癌患者临床预后最差，比肠型胃癌患者5年总体生存率低8％左右。因而，胃癌传统治疗方法效果欠佳，亟待研发新的治疗模式来提高其疗效。
CAR-T疗法是目前治疗肿瘤的新方法。主要应用于血液肿瘤。2017年美国FDA已经被批准了两款针对CD19靶点治疗白血病与淋巴瘤的CAR-T细胞药物。CAR-T疗法在实体瘤领域进展较慢，主要原因CAR-T细胞在浸润至实体肿瘤组织后会被抑制性肿瘤微环境(Tumor Microenvironment TME)抑制，科学家们通过在CAR-T 细胞表达趋化因子受体来增强CAR-T细胞向肿瘤的迁移能力和抗肿瘤活性，或者直接靶向与肿瘤微环境相关的蛋白。因而，研究靶向实体瘤及其微环境的CAR-T疗法是未来该领域有待突破的难点。
针对胃癌的CAR-T疗法较多。陈凛组报道了以HER2为靶点的CAR-T细胞杀伤胃癌细胞作用研究，结果表明体内和体外均具较强的杀伤效果，但HER2在胃癌中的阳性率较低，有报道仅为5％左右。李鹏组报道了以间皮素(Mesothelin)为靶点的胃癌CAR-T研究，在构建的NSI小鼠中展现了很好的杀伤效果，关于胃癌尚未重点关注。李宗海组研制了特异针对Claudin18.2的高亲和力人源化单克隆抗体，并开发了第二代CAR-T细胞进行了体外和体内的抗肿瘤活性研究，结果表明：针对Claudin18.2的CAR-T细胞在人源胃癌PDX小鼠模型中展现出了较好的抗肿瘤活性，关于胃癌也未专门涉及。综上，胃癌CAR-T细胞疗法具有良好的应用前景，然而，针对传统疗法较差的胃癌尚未开发有效的特异性CAR-T细胞疗法。
发明内容
本发明人收集15对病理确诊为胃癌的癌组织和癌旁组织样本，采用非标记定量蛋白质组学法(label-free)筛选到了胃癌特异蛋白CD87。CD87即PLAUR或uPAR(其在本文互换使用)为尿激酶型血浆纤溶酶原激活物(urokinase-type plasminogen，uPA)受体，通过GPI锚定在细胞膜上，在炎症、组织重构和多种癌症中高表达且预后不良。CD87通过与uPA、整合素(integrin)和玻连蛋白(vitronectin)协同作用调控细胞外基质蛋白质降解和激活多条胞内信号通路，在肿瘤细胞迁移、增殖过程中起重要作用，是治疗癌症潜 在靶点。我们研究发现CD87与肿瘤微环境相关，不仅可以参与细胞外基质的降解，而且能够促进免疫细胞浸润，可以作为胃癌CAR-T治疗的靶点。
目前CAR-T治疗胃癌的临床前研究和临床试验层出不穷，但是针对不同靶点的不同CAR-T治疗策略相对于CAR-T在血液系统肿瘤治疗上取得的成效而言仍然不理想。而CD87在胃癌细胞表面高表达，在正常组织中低表达，靶向CD87可促进细胞外基质的降解，改善肿瘤微环境的物理屏障，可以促进免疫细胞浸润，以此为靶点采用CAR-T疗法可以攻克肿瘤微环境的物理屏障，取得较好的肿瘤杀伤效果。
因此，在一个方面，本发明提供了一种抗CD87抗体，其包含与SEQ ID NO：10或14具有至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％)序列同一性的VH序列和与SEQ ID NO：12或16具有至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％)序列同一性的VL序列。
在优选的实施方案中，抗CD87抗体包含SEQ ID NO：10或14的VH序列和SEQ ID NO：12或16的VL序列。
在另一个方面，本发明提供了一种抗CD87抗体，其包含与SEQ ID NO：2或6具有至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％)序列同一性的重链和与SEQ ID NO：4或8具有至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％)序列同一性的轻链。
在优选的实施方案中，抗CD87抗体包含SEQ ID NO：2或6的重链和SEQ ID NO：4或8的轻链。
在另一个方面，本发明发现抗CD87抗体可用于改变肿瘤微环境。因此，在一个实施方案中，本发明提供了抗CD87抗体可用于改变肿瘤微环境的用途。在优选的实施方案中，抗CD87抗体是如上文定义的抗CD87抗体。
在另一个方面，本发明提供了如上文定义的抗CD87抗体用于治疗患者(例如人)的胃癌的用途。
在另一个方面，本发明提供了一种CD87特异性嵌合抗原受体(CAR)多肽，其包含CD87抗原结合结构域、跨膜结构域和细胞内信号传导结构域。在具体的实施方案中，所述CD87抗原结合结构域包含可变重链(VH)和可变轻链(VL)，所述VH包含与SEQ ID NO：10或14具有至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％)序列同一性的序列，所述VL包含与SEQ ID NO：12或16具有至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％)序列同一性的序列。在优选的实施方案中，VH包含SEQ ID NO：10或14的序列，VL包含SEQ ID NO：12或16的序列。
关于嵌合抗原受体，可参考M.Sadelain,I.Rivière,R.Brentjens,Targeting tumours with genetically enhanced T lymphocytes.Nat.Rev.Cancer 3,35–45(2003)。
在具体的实施方案中，所述CD87特异性嵌合抗原受体(CAR)多肽还包含信号肽，例如信号肽IL2-SP(例如由SEQ ID NO：18编 码的序列或与其具有至少80％的序列同一性的序列)。在具体的实施方案中，所述CD87特异性嵌合抗原受体(CAR)多肽还包含在CD87抗原结合结构域的VH和VL之间的接头多肽(例如由SEQ ID NO：19编码的序列或与其具有至少80％的序列同一性的序列)。在具体的实施方案中，所述CD87特异性嵌合抗原受体(CAR)多肽还包含在CD87抗原结合结构域和跨膜结构域之间的铰链区，例如hCD8a铰链(例如由SEQ ID NO：20编码的序列或与其具有至少80％的序列同一性的序列)。在具体的实施方案中，所述跨膜结构域可以是例如CD8tTM(例如由SEQ ID NO：21编码的序列或与其具有至少80％的序列同一性的序列)。在具体的实施方案中，所述细胞内信号传导结构域包含共刺激结构域和信号传导结构域，所述共刺激结构域可以是例如4-1BB共刺激结构域(例如由SEQ ID NO：22编码的序列或与其具有至少80％的序列同一性的序列)，所述信号传导结构域可以是例如CD3ζ信号传导域(例如由SEQ ID NO：23编码的序列或与其具有至少80％的序列同一性的序列)。
在另一个方面，本发明提供了如上文所述的CD87特异性嵌合抗原受体多肽用于改变肿瘤微环境的用途。
在另一个方面，本发明提供了如上文所述的CD87特异性嵌合抗原受体多肽用于治疗患者(例如人)的胃癌的用途。
在另一个方面，本发明提供了一种分离的核酸序列，其编码如上文所述的抗体或嵌合抗原受体多肽。
在另一个方面，本发明提供了一种载体，其包含如上文所述的分离的核酸序列。
在另一个方面，本发明提供了一种细胞，其包含如上文所述的载体。在具体的实施方案中，该细胞选自：αβT细胞、γδT细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、B细胞、先天淋巴细胞(ILC)、细胞因子诱导杀伤(CIK)细胞、细胞毒性T淋巴细胞(CTL)、淋巴因子活化杀伤(LAK)细胞、调节性T细胞、或其任何组合。在具体的实施方案中，该细胞表达如上文所述的CD87特异性嵌合抗原受体多肽。
在另一个方面，本发明提供了如上文所述的细胞用于改变肿瘤微环境的用途。
在另一个方面，本发明提供了如上文所述的细胞用于治疗患者的胃癌的用途。
本发明人还令人惊讶地发现，通过靶向CD87，还可以显著改善PD-1抑制剂在胃癌治疗中的作用。不受理论限制，据信CD87抑制剂通过靶向CD87改变了机体内的肿瘤微环境，从而帮助PD-1抑制剂更好地发挥治疗作用。CD87抑制剂和PD-1抑制剂的这种联合效果，更特别地，协同效果，对于本领域技术人员而言是无法预料到的。
如本文所用，CD87抑制剂可以是抑制CD87的任何试剂。优选地，CD87抑制剂是如上文所述的抗CD87抗体、CD87特异性嵌合抗原受体(CAR)多肽或包含CD87特异性嵌合抗原受体(CAR)多肽的细胞。如本文所用，PD-1抑制剂可以是抑制PD-1的任何试剂。优选地，PD-1抑制剂是抗PD-1抗体。
因此，在一个方面，本发明提供了抗CD87抗体、CD87特异性嵌合抗原受体(CAR)多肽或包含CD87特异性嵌合抗原受体(CAR) 多肽的细胞与PD-1抑制剂联合用于治疗患者的胃癌的用途。在具体的实施方案中，所述抗CD87抗体、CD87特异性嵌合抗原受体(CAR)多肽或包含CD87特异性嵌合抗原受体(CAR)多肽的细胞改变患者中的肿瘤微环境。如本文所用，患者可以是任何哺乳动物或非哺乳动物，优选为人。
在优选的实施方案中，所述抗CD87抗体、CD87特异性嵌合抗原受体(CAR)多肽或包含CD87特异性嵌合抗原受体(CAR)多肽的细胞是如上文所述的抗CD87抗体、CD87特异性嵌合抗原受体(CAR)多肽或包含CD87特异性嵌合抗原受体(CAR)多肽的细胞。
附图说明
图1为本发明提供的CD87在胃癌和正常组中的表达情况。A：利用qRT-PCR验证胃癌患者PLAUR基因表达水平，结果表明PLAUR在胃癌组织中高表达，在癌旁组织中低表达(P<0.05，n＝12)。B、C：利用Western Blot验证胃癌患者PLAUR蛋白表达水平，结果表明PLAUR在胃癌组织中显著高表达，在癌旁组织中低表达(P<0.01，n＝8)。
图2为CD87在胃癌细胞系中的表达水平，A：qRT-PCR；B：流式细胞。A：利用qRT-PCR验证PLAUR在胃癌细胞系(MKN-45、AGS、SNU-216、N87、HGC-27和GES)中的表达水平，结果表明PLAUR在AGS和SNU-216细胞中高表达。B：利用流式细胞仪检测PLAUR在胃癌细胞系(MKN-45、AGS、SNU-216、N87、HGC-27和GES)中的表达水平，结果同样表明PLAUR在AGS和SNU-216 细胞中高表达。
图3为细胞实验：CD87敲除后抑制肿瘤细胞增殖：A：SNU216；B：AGS。C，D：分别在SNU-216细胞和AGS细胞中敲除PLAUR，MTT结果表明，敲除PLAUR抑制了肿瘤细胞增殖。
图4为动物实验。A：肿瘤体积变化；B：肿瘤重量；C：小动物成像。A：NSG小鼠皮下分别接种MKN-45细胞、MKN-45-PLAUR -/-和MKN-45-PLAUR OE细胞，结果表明，在MKN-45细胞中敲除PLAUR抑制了肿瘤细胞增殖(p<0.05)，回复PLAUR使得功能恢复。B：小鼠肿瘤组织重量。C：小动物成像结果。
图5：AGS和SNU-216敲除PLAUR之后细胞外基质蛋白表达情况。
图6为CD87单克隆抗体(也称为抗UPAR抗体)制备：A：细胞免疫；B：阳性B细胞筛选及抗体流式验证；C：筛选到的两种高亲和力单克隆抗体。
图7为CD87单克隆流式验证。
图8：抗uPAR抗体与uPA竞争uPAR结合：(A)通过表面等离子共振评估抗uPAR单克隆抗体(mAb)与人uPAR的结合亲和力(KD)。(B-C)免疫荧光(IF)(B)和免疫电子显微镜(C)分析抗uPAR与表达uPAR的HEK-293T细胞的结合。(D-E)通过流式细胞术(FCM)(D)和蛋白质印迹法(E)显示的抗uPAR mAb检测不同形式的uPAR。(F)竞争性结合测定显示通过FCM检测到的抗uPAR mAb抑制uPA与uPAR的结合。
图9：抗uPAR抗体抑制胃癌细胞中uPAR依赖性信号传导 (A)在pro-uPA刺激后用抗uPAR或ctrl mAb预处理的SNU-216细胞中磷酸化ERK(p-ERK)和总ERK(T-ERK)的蛋白质印迹。(B-E)用抗uPAR或ctrl mAb处理的AGS和SNU-216细胞的生长曲线(B、C)、transwell侵袭试验(D)和细胞粘附试验(E)。数据表示为平均值±SEM(*p<0.05,**p<0.01,***p<0.001)。
图10为细胞实验：CD87单抗封闭抑制肿瘤细胞增殖：A：AGS；B：SNU216
图11为CD87单克隆抗体杀伤CDX模型数据。
图12为CD87单克隆抗体杀伤CDX模型组肿瘤组织重量及小鼠重量变化。
图13为CD87单克隆抗体杀伤CDX模型小动物成像结果。
图14：单独使用抗uPAR或与抗PD-1联合使用可提高携带患者来源异种移植物的人源化小鼠的存活率。(A)实验程序图。(B-C)抗体治疗携带来自患者的PDX的小鼠的肿瘤生长(B)和存活曲线(C)(n＝5/组，10mg/kg/抗体，I.P.，从8dpi开始每5天)；表示为平均值±SEM(ns不显著，*p<0.05，**p<0.01，***p<0.001)。
图15：单独使用抗uPAR或与抗PD-1一起使用可增强细胞毒性CD8+T细胞的浸润和活化。
(A-B)在实验结束时源自患者A的PDX中的CD8+T细胞的IF染色(A)和量化(B)。(C-D)与(A-B)中相同的PDX的CD8 IHC染色和数据分析。(E)qRT-PCR检测到的如(A-B)中的PDX中趋化因子的mRNA表达水平。(F)肿瘤接种后第21天用 不同抗体治疗的患者A衍生的PDX中的活化细胞毒性T细胞(CD45 +CD8 +CD107a +)、调节性T细胞(Tregs,CD45 +CD4 +FOXP3 +)和M2巨噬细胞(CD45 +CD11b +CD68 +CD206 +)的频率，如FCM所示。数据表示为平均值±SEM(ns不显著，*p<0.05，**p<0.01，***p<0.001)。
图16为CD87-CAR的构建的示意图。
图17为本发明的CD87-CAR结构示意图。
图18为对T细胞进行感染的感染效率图，初始感染效率为27.8％，分选富集后感染效率达到99.9％。
图19为设立CD87-CAR-T+AGS-荧光素酶和对照-T细胞+AGS-荧光素酶两组进行共同培养，并且在1:1、2:1、4:1、8:1和16:1的比例下观察各组肿瘤细胞的荧光素酶释放情况绘制成的折线图。(效应细胞：靶细胞结合力/杀伤力筛选)。
图20为以MKN-45细胞在NSG小鼠中造CDX模型，分为T细胞对照组，CAR-T细胞杀伤组和CAR-T细胞联合PD-1抗体治疗组。A：肿瘤体积变化；B：动物生存分析。
图21为各组动物血清中IFN-γ和Granzyme B表达水平。
图22为小动物成像结果。
图23为CAR-T细胞杀伤PDX模型实验结果。分为T细胞对照组，CAR-T细胞杀伤组和CAR-T细胞联合PD-1抗体治疗组。A：治疗后肿瘤组织体积变化；B：治疗后生存曲线分析。
图24为各组动物血清中IFN-γ和Granzyme B表达水平。
图25为CAR-T细胞和PD-1抗体联用组CD8+T细胞浸润图。
实施例
下面结合实施例对本发明实施方案进行详细描述，但是本领域技术人员将会理解，下面实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中的未注明具体条件者，按照常规条件或制作商建议的条件进行。所用试剂或仪器未注明生产商者，均为可以通过市购获得的常规产品。
实施例1
CAR-T靶点筛选及验证：收集16名胃癌患者癌和癌旁组织进行了基因表达谱分析，共鉴定了605个表达上调基因。收集15对胃癌和癌旁组织的新鲜手术样本，采用非标记定量蛋白质组学法(label-free)分析，共鉴定了186个在癌中高表达，癌旁不表达的蛋白。利用TCGA胃癌数据做了弥漫性胃癌与正常组织的差异基因，将得到的上述2个数据与免疫相关基因求交集，最终得到2个关键蛋白FCGR3A和CD87，在TISIDB数据库分析与免疫细胞浸润和免疫检查点的相关性。按照CAR-T膜蛋白须在人体正常组织器官表达水平较低，癌组织表达较高且与其生物学行为有关的基本原则，结合CD87在人体正常多组织器官表达分布情况，初步确定CD87为胃癌CAR-T治疗靶点。
实施例2
通过qRT-PCR和流式细胞仪检测胃癌细胞株HGC-27、MKN-45、AGS、SNU-216、HS746T和NCI-N87中CD87的表达水平，结果表明在AGS、SNU-216和HS746T中CD87高表达，在HGC-27、MKN-45和NCI-N87中低表达，
在此基础上设计了待敲除基因CD87的gRNA序列分别为：gRNA1:TTCCACACGGCAATCCCCGT。gRNA2：GGACCACGATCGTGCGCTTG。对AGS、SNU-216中的CD87进行敲除，结果表明在AGS和SNU216中敲除CD87抑制了肿瘤细胞增殖，抗体封闭取得了同样的效果，分别用2×10 6个MKN-45细胞、MKN-45-CD87 -/-细胞和回复CD87的MKN-45-CD87 -/-细胞在NSG小鼠中成瘤，每隔两天测量小鼠肿瘤体积，用小动物成像仪观察荷瘤小鼠的肿瘤变化情况。
CD87可通过改变肿瘤微环境：检测ECM相关黏附蛋白的变化发现Smad2、vitronectin和vimetin等黏附相关蛋白表达明显下调，使得细胞间黏附变得松散，这可能与肿瘤微环境的改变相关。
实施例3
CD87单克隆抗体制备
制备CD87单克隆抗体构建慢病毒质粒Lenti-CMV-CD87-T2A-GFP：
将构建好的质粒和包装质粒pDD和pVSVg按1:1:0.5的比例利用PEI转染293T细胞，培养36小时后收上清，离心并浓缩病毒，测定滴度后感染293T细胞，嘌呤霉素筛选并成功获得了稳定表达抗原CD87的细胞。
实验步骤：
1.1利用Gibson连接体系设计引物分别扩增CD87序列和GFP序列，PCR产物胶回收。
1.2将Lenti-CMV-Puro质粒BamHI酶切，胶回收酶切产物。
1.3利用Gibson连接体系连接、转化、提取质粒测序并表达验证。
1.4鉴定正确者，命名为Lenti-CMV-CD87-T2A-GFP质粒。
构建稳定表达CD87的293T细胞系：293T-CD87：
实验材料
慢病毒载体质粒Lenti-CMV-CD87-T2A-GFP；包装质粒：pDD、pVsVG；293T细胞；0.25％胰酶；DMEM；FBS；Opti-MEM；lip2000；BSA。
实验步骤
1)293T细胞接种：将生长状态良好的293T细胞用0.25％胰酶消化，调整细胞浓度为6×10 5个/mL，取10mL接种于10cm培养皿中，37℃，5％二氧化碳培养18小时，使细胞融合度达到60％-70％，转染前半小时将培养液换为Opti-MEM。
2)病毒包装：将7ug Lenti-CMV-CD87-T2A-GFP质粒、5ug pDD和3.5ug pVsVg加入到500uL OPTI-MEM中混匀；取30uL lip2000加入500uL OPTI-MEM中混匀，室温静置5分钟，缓慢加入到质粒混合液中混匀，室温静置15分钟，逐滴加入到培养皿中，充分混匀。6小时后更换为含10％FBS 1％BSA的DMEM新鲜培养液。
3)病毒浓缩：培养60小时之后，收集上清，4℃3000rpm离心10分钟，0.45um滤膜滤过，4℃25000rpm超高速离心2小时，弃上清，用300uL含10％FBS 1％BSA的DMEM培养液重悬沉淀，4℃过夜，50uL/管分装，干冰速冻后储存在-80℃。
4)利用慢病毒感染293T细胞构建稳定表达CD87的293T细胞 系，命名为293T-CD87。
利用制备的293T-CD87细胞制备靶向CD87的单克隆抗体。
293T-CD87的细胞进行细胞免疫：取6～8周龄SPF级Balb/c雌鼠4只，免疫前一天眼眶采血，分离血清用作阴性对照。将1×10 7个稳定表达CD87的293T细胞皮下多点注射小鼠。第10天和20天分别用5×106个稳定表达CD87的293T细胞进行第二次和第三次免疫。第25天小鼠眼眶采血，应用ELISA检测CD87抗体效价。选取抗体效价最高的小鼠注射2×10 6个细胞加强免疫。第30天制备免疫小鼠脾细胞，流式细胞仪(FCM)对可产生CD87抗体的阳性B细胞进行分选，利用单细胞RT-PCR技术获取VH和VL序列并验证，最终筛选出2种针对CD87的高亲和力单克隆抗体(A7和E2)。
实施例4
实施例3制备的靶向CD87的单克隆抗体表征，分别利用流式细胞术，WB和激光共聚焦验证制备的单克隆抗体A7和E2。
实施例5
实施例3制备的靶向CD87的单克隆抗体对胃癌细胞系CDX模型的治疗效果及联合PD-1治疗效果。
NSG小鼠皮下注射约2×10 6个过表达CD87-T2A-luciferase的MKN-45细胞造小鼠CDX模型，分为对照组、CD87单抗治疗组、PD-1单抗治疗组和CD87单抗联用PD-1抗体组，待瘤体体积约达到90mm 3时，尾静脉分别注射2×10 7个人PBMC、给药组分别给予10mg/kgCD87单抗、PD-1单抗和CD87单抗+PD-1抗体，对照组给予相同剂量人IgG抗体，每周给药两次。没两天测量小鼠肿瘤大小， 每隔4天用小动物活体成像系统分析检测瘤体变化情况，验证CD87单抗治疗效果。
实施例6
实施例3制备的靶向CD87的单克隆抗体对胃癌患者来源的PDX模型的治疗效果及联合PD-1治疗效果。
取病理确诊CD87+胃癌患者的肿瘤组织，组织切块后皮下移植至NSG小鼠，待瘤体长大后取出，一部分肿瘤组织冻存，另一部分进行基因表达分析，剩余部分继续接种NSG小鼠，第三代肿瘤无差异可用于肿瘤杀伤实验。制备该模型小鼠20只，分为对照组、CD87单抗治疗组、PD-1单抗治疗组和CD87单抗联用PD-1抗体组，待瘤体体积约达到90mm3时，尾静脉分别注射2×10 7个人PBMC、给药组分别给予10mg/kgCD87单抗、PD-1单抗和CD87单抗+PD-1抗体，对照组给予相同剂量人IgG抗体，每周给药两次。没两天测量小鼠肿瘤大小，每隔4天用小动物活体成像系统分析检测瘤体变化情况，验证CD87单抗治疗效果。
实施例7
制备CD87-CAR
制备CD87-CAR质粒，所述CD87-CAR结构由EF1a启动子核酸序列、IL2的信号肽、CD87 scFv序列、hCD8a铰链区核酸序列、CD8TM跨膜区域核酸序列、41-BB协同刺激信号域核酸序列、CD3ζ信号传导结构域串联而成，命名为EF1a-CD87 scFv-hCD8a hinge-CD8TM-41-BB-CD3ζ。本实施例构建的CD87-CAR基因结构的核酸序列如序列表中的SEQ ID No.24所示。
实验步骤：
1.利用Gibson连接体系设计引物扩增CD87 scFv序列，PCR产物胶回收。
2.将lenti-EF1a-hCAR-Puro质粒分别用EcoRI和BamHI双酶切，胶回收酶切产物。
3.利用Gibson连接体系连接、转化、提取质粒测序并表达验证。
4.鉴定正确者为CAR质粒，命名为CD87-CAR质粒。
利用构建的CD87-CAR质粒包装及浓缩慢病毒
实验材料
慢病毒载体质粒CD87-CAR；包装质粒：pMD2.G、pSPAX2；293T细胞；0.25％胰酶；DMEM；FBS；Opti-MEM；lip2000；BSA。
实验步骤
1)293T细胞接种：将生长状态良好的293T细胞用0.25％胰酶消化，调整细胞浓度为6×10 5个/mL，取10mL接种于10cm培养皿中，37℃，5％二氧化碳培养18小时，使细胞融合度达到60％-70％，转染前半小时将培养液换为Opti-MEM。
2)病毒包装：将7ug CD87-CAR质粒、5ug psPAX2和3.5ug pMD2.G加入到500uL OPTI-MEM中混匀；取30uL lip2000加入500uL OPTI-MEM中混匀，室温静置5分钟，缓慢加入到质粒混合液中混匀，室温静置15分钟，逐滴加入到培养皿中，充分混匀。6小时后更换为含10％FBS 1％BSA的DMEM新鲜培养液。
3)病毒浓缩：培养60小时之后，收集上清，4℃3000rpm离心10分钟，0.45um滤膜滤过，4℃25000rpm超高速离心2小时，弃 上清，用300uL含10％FBS 1％BSA的DMEM培养液重悬沉淀，4℃过夜，50uL/管分装，干冰速冻后储存在-80℃。
利用提供的慢病毒感染人外周血T细胞
实验材料
EasySep TM Human T Cell Isolation Kit(StemCell,Catalog#17951)；Dynabeads Human T-Activator CD3/CD28(Life Technologies,Catalog number：11131D)；PBS；FBS；RMPI-1640；human IL-2；Lymphoprep(Catalog#07801)；SepMate-50(Catalog#15450)。
实验步骤
1)人PBMC细胞准备：移液管量取15mL淋巴细胞分离液(Lymphoprep，Catalog#07801)加入离心管中(SepMate-50，Catalog#15450)，淋巴细胞分离液上端应淹没过滤网。取新鲜血浆10-15mL，用等体积2％FBS-PBS稀释，轻轻吹吸混匀，保持离心管垂直，沿管壁加入混匀后的样品(滤网上部离心液与样本混合)，室温1200g离心10分钟，倒出上层液体至新的离心管中，300g室温离心8分钟(如有必要2％FBS-PBS洗一次)。获得的PBMC用于T细胞分选。
2)T细胞分选：PBMC加入0.5-2mL T细胞分离buffer，用EasySep TM Human T Cell Isolation Kit(StemCell,Catalog#17951)分选T细胞：流式管中加入2mL准备好的PBMC，加入50uL/mL isolation Cocktail混匀，常温5min，将RapidSpheres涡旋30s使混匀，加入40uL/mL，补充至2.5mL，吹吸3次使混匀。至于磁力架 上5min，小心吸出上清至一新的5mL流式管中，再次置于磁力架上5min，小心吸出上清置于离心管中离心，将T细胞用完全培养基10％FBS+RMPI-1640+IL2+1％P/S培养。
3)T细胞活化：将T cell加入24孔板中，2×10 6cell/孔，加入50uL清洗好的Dynabeads Human T-Activator CD3/CD28(4×10 7beads/mL)：磁珠和T细胞比率为1：1，CO2培养箱37℃培养24小时。
4)T细胞感染：激活的T细胞活化24-48小时，加入浓缩的病毒和4.4ug/mL Polybrene，32℃离心90min(1000×g)感染。(MOI＝5-10)
5)T细胞培养：离心感染后8-10小时离心移去培养基，合并所有感染孔用完全培养基10％FBS+RMPI-1640+IL2+1％P/S培养。每48小时更换培养基。
6)CD87-CAR-T细胞分选和测定：培养6天后，移去磁珠(在磁力架上放置1-2min，上清转移至新的培养瓶中，继续培养24小时，有必要时重复此步骤)，GFP检测CAR表达情况。
7)CD87-CAR-T细胞冻存：10％DMSO-FBS冻存CAR-T细胞,3×10 6cell/管。
8)CD87-CAR基因结构表达的测序验证：取1×10 6个CAR-T细胞，TRIzol提取细胞总RNA并逆转录成cDNA，PCR扩增含CD87-CAR基因结构片段，其中上游引物为：5’-GTGTCGTGATCTAGAGCTAGCGGCCACCATGTACAGGATGCAACTCCTG-3’；下游引物为：5’ -GACTTCCTCTGCCCTCAGCGGCCGCCCGAGGCGGCAGGGCCTGCATGTG-3’。扩增结果送测序鉴定。
实施例8
利用实施例7制备的CD87-CAR-T细胞，检测此CD87-CAR-T细胞对肿瘤细胞系的杀伤效果。
将CD87-CAR-T细胞与过表达luciferase的胃癌细胞系AGS、AGS-CD87 -/-、MKN-45按一定比例共培养，37℃共培养4小时后，取50uL上清和准备好的荧光素酶试剂(Promina#E2610)加入96孔板中(Corning#3904)，酶标仪检测上清中luciferase释放情况并计算杀伤能力。验证CD87-CAR-T细胞对胃癌靶细胞的结合能力及杀伤效果。
实施例9
利用实施例7制备的CD87-CAR-T细胞，检测此CD87-CAR-T细胞对胃癌细胞系CDX模型的杀伤效果。
NSG小鼠皮下注射约2×10 6个过表达CD87-T2A-luciferase的MKN-45细胞造小鼠CDX模型，分为T细胞对照组、CD87-CAR-T细胞治疗组和CD87-CAR-T细胞和PD-1抗体联用组，待瘤体体积约达到90mm 3时，尾静脉分别注射1×10 6个T细胞、CD87-CAR-T细胞，CD87-CAR-T细胞和PD-1抗体。小动物活体成像系统分析检测瘤体变化情况、应用ELISA检测外周血中IFN-γ和Granzyme B细胞因子水平变化，验证CD87-CAR-T细胞体内结合胃癌靶细胞的能力及杀伤效果。
实施例10
利用实施例7制备的CD87-CAR-T细胞，检测此CD87-CAR-T细胞对胃癌患者来源PDX模型的杀伤效果。
取病理确诊CD87+胃癌患者的肿瘤组织，组织切块后皮下移植至NSG小鼠，待瘤体长大后取出，一部分肿瘤组织冻存，另一部分进行基因表达分析，剩余部分继续接种NSG小鼠，第三代肿瘤无差异可用于肿瘤杀伤实验。制备该模型小鼠20只，分为T细胞对照组、CD87-CAR-T细胞治疗组和CD87-CAR-T细胞和PD-1抗体联用组，待瘤体体积约达到90mm 3时，尾静脉分别注射1×10 6个T细胞、CD87-CAR-T细胞，CD87-CAR-T细胞和PD-1抗体。监测小鼠体重、瘤体大小、应用ELISA检测外周血中IFN-γ和Granzyme B等细胞因子水平变化，验证CD87-CAR-T细胞体内杀伤胃癌PDX模型的能力。

Claims (11)

  1. 一种抗CD87抗体，其包含与SEQ ID NO：10或14具有至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％)序列同一性的VH序列和与SEQ ID NO：12或16具有至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％)序列同一性的VL序列，
    优选地，其包含SEQ ID NO：10或14的VH序列和SEQ ID NO：12或16的VL序列，
    优选地，其包含与SEQ ID NO：2或6具有至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％)序列同一性的重链和与SEQ ID NO：4或8具有至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％)序列同一性的轻链，
    优选地，其包含SEQ ID NO：2或6的重链和SEQ ID NO：4或8的轻链。
  2. 权利要求1的抗CD87抗体用于改变肿瘤微环境的用途。
  3. 权利要求1的抗CD87抗体用于治疗患者的胃癌的用途。
  4. 一种嵌合抗原受体(CAR)多肽，其包含CD87抗原结合结构域、跨膜结构域和细胞内信号传导结构域，
    优选地，其中所述CD87抗原结合结构域包含可变重链(VH)和可变轻链(VL)，所述VH包含与SEQ ID NO：10或14具有至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％)序列同一性的序列，所述VL包含与SEQ ID NO：12或16具有至少90％(例如至少91％、92％、93％、94％、95％、96％、97％、98％、99％或100％)序列同一性的序列，
    优选地，VH包含SEQ ID NO：10或14的序列，VL包含SEQ ID NO：12或16的序列。
  5. 权利要求4的嵌合抗原受体多肽用于改变肿瘤微环境的用途。
  6. 权利要求4的嵌合抗原受体多肽用于治疗患者的胃癌的用途。
  7. 一种分离的核酸序列，其编码如权利要求1至6中任一项所述的抗体或嵌合抗原受体多肽。
  8. 一种载体，其包含权利要求7的分离的核酸序列。
  9. 一种细胞，其包含如权利要求8所述的载体。
    优选地，其中该细胞选自：αβT细胞、γδT细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、B细胞、先天淋巴细胞(ILC)、细胞因子诱导杀伤(CIK)细胞、细胞毒性T淋巴细胞(CTL)、淋巴因子活化杀伤(LAK)细胞、调节性T细胞、或其任何组合。
  10. 权利要求10的细胞用于改变肿瘤微环境的用途。
  11. 权利要求10的细胞用于治疗患者的胃癌的用途。
PCT/CN2022/076087 2021-02-11 2022-02-11 抗cd87抗体及其特异性嵌合抗原受体 WO2022171196A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110185661 2021-02-11
CN202110185661.6 2021-02-11

Publications (1)

Publication Number Publication Date
WO2022171196A1 true WO2022171196A1 (zh) 2022-08-18

Family

ID=82804384

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/CN2022/076086 WO2022171195A1 (zh) 2021-02-11 2022-02-11 抗cd87抗体与抗pd1抗体联合治疗胃癌
PCT/CN2022/076087 WO2022171196A1 (zh) 2021-02-11 2022-02-11 抗cd87抗体及其特异性嵌合抗原受体

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/076086 WO2022171195A1 (zh) 2021-02-11 2022-02-11 抗cd87抗体与抗pd1抗体联合治疗胃癌

Country Status (2)

Country Link
CN (2) CN114920841B (zh)
WO (2) WO2022171195A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115925985A (zh) * 2022-08-26 2023-04-07 卡瑞济(北京)生命科技有限公司 Car-t细胞及其在非小细胞肺癌治疗中的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105368859A (zh) * 2015-11-25 2016-03-02 王任直 一种嵌合抗原受体hCD87-CAR及载有hCD87-CAR基因结构的慢病毒及质粒及其应用
CN105612183A (zh) * 2013-07-15 2016-05-25 诺和诺德股份有限公司 结合尿激酶纤溶酶原激活物的抗体
WO2020160518A1 (en) * 2019-02-01 2020-08-06 Memorial Sloan Kettering Cancer Center Senolytic car-t cells targeting upar, a cell surface and secreted senescence biomarker

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW200526684A (en) * 2003-11-21 2005-08-16 Schering Corp Anti-IGFR1 antibody therapeutic combinations
TW200813091A (en) * 2006-04-10 2008-03-16 Amgen Fremont Inc Targeted binding agents directed to uPAR and uses thereof
CN1919874B (zh) * 2006-09-18 2010-09-08 中国人民解放军军事医学科学院生物工程研究所 一种抗尿激酶型纤溶酶激活剂受体的抗体样分子ATF-Fc融合蛋白及其用途
CN101704892B (zh) * 2009-12-09 2014-02-12 中国人民解放军军事医学科学院生物工程研究所 一种抗uPAR人源化抗体及其编码基因与应用
US20140193436A1 (en) * 2011-06-24 2014-07-10 Centrose, Llc Extracellular targeted drug conjugates
US9255155B2 (en) * 2013-01-31 2016-02-09 The Regents Of The University Of California Antibodies specific for urokinase-type plasminogen activator and methods of treating cancer
CA2913052A1 (en) * 2013-05-24 2014-11-27 Board Of Regents, The University Of Texas System Chimeric antigen receptor-targeting monoclonal antibodies
JP2017522884A (ja) * 2014-07-29 2017-08-17 ファイザー・インク がん免疫療法のためのＥＧＦＲｖＩＩＩ特異的キメラ抗原受容体
PT3294764T (pt) * 2015-05-15 2021-02-15 Hope City Composições de recetores de antigénios quiméricos
CN109923123B (zh) * 2016-10-14 2024-01-23 爱默蕾大学 具有结合或阻断pd-l1的分子的纳米颗粒及其在治疗癌症中的用途
CN118562016A (zh) * 2016-10-21 2024-08-30 艾尔特生物科技公司 基于多聚体il-15的分子
CN107118267B (zh) * 2017-04-18 2020-07-24 浙江工商大学 一种用于缓解对虾原肌球蛋白致敏反应的改良蛋白mMet e 1及其制备方法和应用
EP3820522A4 (en) * 2018-07-12 2022-05-18 Corvus Pharmaceuticals, Inc. METHODS FOR DETECTING AND TREATING CANCERS HAVING ACTIVATION OF THE ADENOSINE PATHWAY
CN110343665B (zh) * 2019-05-05 2023-07-14 吉林大学 一种car-t细胞及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105612183A (zh) * 2013-07-15 2016-05-25 诺和诺德股份有限公司 结合尿激酶纤溶酶原激活物的抗体
CN105368859A (zh) * 2015-11-25 2016-03-02 王任直 一种嵌合抗原受体hCD87-CAR及载有hCD87-CAR基因结构的慢病毒及质粒及其应用
WO2020160518A1 (en) * 2019-02-01 2020-08-06 Memorial Sloan Kettering Cancer Center Senolytic car-t cells targeting upar, a cell surface and secreted senescence biomarker

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AMOR CORINA; FEUCHT JUDITH; LEIBOLD JOSEF; HO YU-JUI; ZHU CHANGYU; ALONSO-CURBELO DIRENA; MANSILLA-SOTO JORGE; BOYER JACOB A.; LI : "Senolytic CAR T cells reverse senescence-associated pathologies", NATURE, vol. 583, no. 7814, 17 June 2020 (2020-06-17), London, pages 127 - 132, XP037181070, ISSN: 0028-0836, DOI: 10.1038/s41586-020-2403-9 *
LI, Y. PARRY, G. CHEN, L. CALLAHAN, J.A. SHAW, D.E. MEEHAN, E.J. MAZAR, A.P. HUANG, M.: "An Anti-urokinase Plasminogen Activator Receptor (uPAR) Antibody: Crystal Structure and Binding Epitope", JOURNAL OF MOLECULAR BIOLOGY, vol. 365, no. 4, 30 December 2006 (2006-12-30), United Kingdom , pages 1117 - 1129, XP005733680, ISSN: 0022-2836, DOI: 10.1016/j.jmb.2006.10.059 *
MAZZI MARIANA TORRES, HAJDU KARINA LÔBO, RIBEIRO PRISCILA RAFAELA, BONAMINO MARTÍN HERNÁN: "CAR-T cells leave the comfort zone: current and future applications beyond cancer", IMMUNOTHERAPY ADVANCES, vol. 1, no. 1, 1 January 2021 (2021-01-01), pages 1 - 15, XP055959486, DOI: 10.1093/immadv/ltaa006 *
XU, GUANJUN ET AL.: "Research Progress of uPA System and Digestive System Neoplasms", THE JOURNAL OF PRACTICAL MEDICINE, vol. 27, no. 12, 31 December 2011 (2011-12-31), pages 2287 - 2289, XP055959476 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115925985A (zh) * 2022-08-26 2023-04-07 卡瑞济(北京)生命科技有限公司 Car-t细胞及其在非小细胞肺癌治疗中的应用
CN115925985B (zh) * 2022-08-26 2023-10-31 卡瑞济(北京)生命科技有限公司 Car-t细胞及其在非小细胞肺癌治疗中的应用

Also Published As

Publication number Publication date
CN114920841B (zh) 2023-06-09
CN114920841A (zh) 2022-08-19
CN114917329B (zh) 2023-07-21
WO2022171195A1 (zh) 2022-08-18
CN114917329A (zh) 2022-08-19

Similar Documents

Publication Publication Date Title
CN110872577B (zh) 修饰的免疫细胞及其应用
JP2020012000A (ja) 新規に単離された細胞の治療組成物の操作および送達
CN108409840B (zh) 抗cd123单链抗体及其组合的嵌合抗原受体和应用
CN111629734A (zh) 用于共刺激的新型平台、新型car设计以及过继性细胞疗法的其他增强
CN117305250A (zh) 工程化天然杀伤细胞及其用途
US20180369285A1 (en) Specific Chimeric Antigen Receptor T Cells Targeting To NKG2DL, Its Preparation Method and Application Thereof
TW201928052A (zh) 靶向ny-eso-1的基因修飾免疫細胞及其用途
WO2020108645A1 (en) Cd19-and bcma-based combined car-t immunotherapy
Maryamchik et al. New directions in chimeric antigen receptor T cell [CAR‐T] therapy and related flow cytometry
CN111944054B (zh) 抗bcma的car及其表达载体和应用
CN113214408B (zh) 一种嵌合抗原受体巨噬细胞及其制备方法和用途
WO2022135578A1 (zh) Claudin18.2嵌合抗原受体以及其用途
CN113416260B (zh) 靶向Claudin18.2的特异性嵌合抗原受体细胞及其制备方法和应用
WO2021031113A1 (zh) 抗bcma抗体及其在car-t领域中的应用
JP2022513689A (ja) 養子細胞療法におけるｂ細胞悪性腫瘍の投与および処置のための方法
US11014974B2 (en) Non-antibody binding proteins binding to PD-1 receptors and uses thereof
WO2022222846A1 (zh) 靶向cd19的嵌合抗原受体、制备方法及其应用
CN114478803A (zh) 一种新型双特异性嵌合抗原受体的构建及其应用
CN111944053B (zh) 抗bcma的car及其表达载体和应用
US11359012B1 (en) Specific chimeric antigen receptor cells targeting human CLDN18A2, preparation method and application thereof
WO2022171196A1 (zh) 抗cd87抗体及其特异性嵌合抗原受体
CN116143943B (zh) 一种靶向baffr嵌合抗原受体、car-t细胞及应用
CN110669138A (zh) 一种双嵌合抗原受体、t细胞及其构建方法与应用
CN113549157B (zh) 双靶向嵌合抗原受体及其应用
CN111533810A (zh) 含有双功能免疫开关分子的双嵌合抗原受体t细胞及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22752356

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22752356

Country of ref document: EP

Kind code of ref document: A1