WO2022068850A1 - Kras突变特异性t细胞受体筛选及抗肿瘤用途 - Google Patents

Kras突变特异性t细胞受体筛选及抗肿瘤用途 Download PDF

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WO2022068850A1
WO2022068850A1 PCT/CN2021/121576 CN2021121576W WO2022068850A1 WO 2022068850 A1 WO2022068850 A1 WO 2022068850A1 CN 2021121576 W CN2021121576 W CN 2021121576W WO 2022068850 A1 WO2022068850 A1 WO 2022068850A1
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tcr
kras
hla
seq
gvgk
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PCT/CN2021/121576
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高福
谭曙光
卢丹
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中国科学院微生物研究所
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Priority to AU2021351815A priority Critical patent/AU2021351815B2/en
Priority to US18/247,018 priority patent/US20230374101A1/en
Priority to EP21874505.7A priority patent/EP4223771A1/en
Priority to KR1020237014640A priority patent/KR20230079259A/ko
Priority to JP2023519189A priority patent/JP2023542417A/ja
Priority to CA3193963A priority patent/CA3193963A1/en
Publication of WO2022068850A1 publication Critical patent/WO2022068850A1/zh

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Definitions

  • the invention belongs to the field of medicine, in particular to a T cell receptor (TCR) or an antigen-binding fragment thereof capable of specifically recognizing the antigenic polypeptides mutated in tumor KRAS gene G12V and G12C.
  • TCR T cell receptor
  • TCR alpha beta T cell receptor
  • Boone, Rosenberg, Old and others respectively found that there are some tumor-specific antigens in different tumor patients, which can be recognized by T cells and specifically kill tumor cells, which makes the hope of tumor immunotherapy rekindled.
  • a great deal of research is devoted to the research and development of therapeutic vaccines for tumors.
  • the immune anti-cancer therapy was rated as the first of the 10 scientific and technological breakthroughs of the year by Science magazine.
  • Adoptive T cell therapy is a highly personalized cancer treatment method, which can achieve anti-tumor effects by rebuilding the missing or weak immune system in cancer patients.
  • ACT therapy refers to the isolation of immune active cells from tumor patients, expansion and functional identification in vitro, and then infusion to patients, so as to directly kill tumors or stimulate the body's immune response to kill tumor cells. Finding antigens that are only expressed on cancer tissues and not on normal essential tissues has been a limiting factor for ACT therapy.
  • ACT therapy can include T cell receptor engineered cell (TCR-T) therapy technology and chimeric antigen receptor engineered T cell (CAR-T) therapy technology.
  • TCR-T T cell receptor engineered cell
  • CAR-T chimeric antigen receptor engineered T cell
  • ACT has been shown to be effective against a variety of cancers, such as melanoma, cervical cancer, lymphoma, leukemia, cholangiocarcinoma, and neuroblastoma.
  • CAR-T has also achieved major breakthroughs in the treatment of acute/chronic myeloid leukemia, lymphoma and other diseases, greatly improving the survival rate and quality of life of patients.
  • the therapeutic prospects of CAR-T cells are not clear due to the limited specific targets.
  • TCR is a characteristic marker on the surface of all T cells, which binds to CD3 non-covalently to form a TCR-CD3 complex.
  • TCR is composed of two peptide chains, ⁇ and ⁇ , and belongs to the immunoglobulin superfamily. The antigen specificity exists in the V region (CDR1, CDR2, CDR3), and CDR3 directly determines the antigen binding specificity of TCR. In peripheral blood, 90%-95% of T cells express TCR.
  • the T cells of the genetically modified TCR can specifically recognize the antigen molecules on the surface of tumor cells, and then generate an immune response against the tumor cells.
  • TCR-T cell immunotherapy is a new cell therapy technology developed in recent years, and it is a typical "precision medicine" treatment technology. At present, this technology has shown positive therapeutic prospects in the treatment of myeloma, melanoma, esophageal cancer, liver cancer, etc.
  • TCR-T cell immunotherapy was first used in the treatment of HIV at the end of the 20th century. In recent years, studies have found that tumor antigen-specific TCR engineering based on MART-1, MAGE-A4, NY-ESO-1, WT-1, etc. Autologous immune cells have shown good development prospects in the treatment of melanoma, esophageal cancer, multiple myeloma, and synovial cell sarcoma.
  • the protein encoded by the KRAS gene (Kirsten rat sarcoma virus oncogene homolog) is a small GTPase, which belongs to the RAS superprotein family and is involved in intracellular signal transmission.
  • the KRAS protein has 188 amino acids and a molecular weight of 21.6KD. It is a guanosine-binding protein with GTPase activity.
  • the KRAS protein transitions between inactive and activated states, when KRAS binds to guanosine diphosphate (GDP), it is in an inactive state, and when it binds to guanosine triphosphate (GTP) When , it is in an active state and can activate downstream signaling pathways, including MAPK signaling pathway, PI3K signaling pathway and Ral-GEFs signaling pathway. These signaling pathways play important roles in promoting cell survival, proliferation, and cytokine release.
  • GDP guanosine diphosphate
  • GTP guanosine triphosphate
  • KRAS gene mutations are found in nearly 90% of pancreatic cancers, 30-40% of colon cancers, 17% of endometrial cancers, 15-20% of lung cancers including lobular lung cancers, as well as cholangiocarcinoma, cervical cancer, Bladder cancer, etc. KRAS mutations account for 86% of the total RAS mutations. Among KRAS mutations, 97% are the 12th or 13th amino acid residues mutated.
  • the 12th amino acid is changed to aspartic acid (G12D)
  • the 12th amino acid is changed to valine (G12V)
  • the 12th amino acid is changed to cysteine (G12C)
  • the 13th amino acid is changed.
  • KRAS has G12D, G12V, G13D mutations, it will keep KRAS bound to GTP by destroying GAP activity, locking KRAS in the active state of tyrosine kinase, and continuously activating downstream signaling pathways (such as PI3K, RAF-MEK-ERK (MAPK), RAL-GEF, etc.). When these downstream signaling pathways are opened, they stimulate cell proliferation, migration, and ultimately tumorigenesis.
  • downstream signaling pathways such as PI3K, RAF-MEK-ERK (MAPK), RAL-GEF, etc.
  • KRAS mutants covalent inhibitors have been developed, which target KRAS mutants through allosteric sites, so that the affinity of KRAS mutants with GTP is reduced to achieve the purpose of "locking" its activity.
  • Amgen's AMG510 is a KRAS-G12C inhibitor.
  • Mirati Therapeutics' KRAS-G12C inhibitor, MRTX1257 is still in preclinical development. There is currently no related therapeutic drug for other KRAS mutations, and there is also a lack of a tumor drug that can be detected and treated by utilizing the body's immune mechanism.
  • KRAS gene mutations are found in nearly 90% of pancreatic cancers, 30-40% of colon cancers, 17% of endometrial cancers, and 15-20% of lung cancers.
  • 97% are the 12th or 13th amino acid residues mutated.
  • the most important ones are G12D, G12V, G12C, and G13D mutations.
  • After KRAS mutation it can be presented to the cell surface by MHC molecules in cells and recognized by T cells to stimulate T cell immune response, and then remove tumor cells carrying KRAS mutation.
  • the body when used as an antigen, the body can produce a CD8 + CTL (cytotoxic lymphocyte, cytotoxic T lymphocyte) response. Mutations in some amino acid residues in KRAS polypeptides can be presented by HLA molecules and recognized by T cells.
  • CTL cytotoxic lymphocyte, cytotoxic T lymphocyte
  • One embodiment of the present invention includes screening for KRAS-G12V 8-16 (VVGA V GVGK) mutation (also referred to as G12V hereinafter) that specifically targets tumor KRAS gene through specific T cell receptor (TCR) single cell screening technology , G12V mutation, or KRAS gene G12V mutation), KRAS-G12C 8-16 (VVGA C GVGK) (hereinafter also referred to as G12C, G12C mutation, or KRAS gene G12C mutation) two TCRs.
  • KRAS-G12V 8-16 VVGA V GVGK mutation
  • TCR T cell receptor
  • One embodiment of the present invention includes providing specific T cell receptors and antigen-binding fragments thereof that target a G12V or G12C mutated epitope of the KRAS gene.
  • Another embodiment of the present invention includes the use of the above-mentioned T cell receptors and antigen-binding fragments thereof in the manufacture of a medicament for the treatment of tumors carrying G12V and G12C mutations of the KRAS gene.
  • the present invention is made based on the above-mentioned principle.
  • the KRAS mutant polypeptide-specific TCR or its antigen-binding fragment in the present invention is mutated by a complex molecule with KRAS G12V mutant polypeptide (VVGAVGVGK) and HLA-A11 and/or KRAS G12C mutation
  • the complex molecule of polypeptide (VVGACGVGK) and HLA-A11, or KRAS-G12V 8-16 (VVGA V GVGK) epitope and HLA-A03 complex molecule specifically bind to stimulate T cell activation and induce T cells to secrete IFN- ⁇ and other cytokines, and then kill KRAS mutant polypeptides, especially the G12V and or G12C mutation-positive tumor cells of the KRAS gene.
  • KRAS mutant polypeptide-specific TCR or "murine KRAS mutant polypeptide-specific TCR” is a CTL epitope polypeptide (the sequence of which is VVGAVGVGK and/or VVGACGVGK) restricted to HLA-A11 in the KRAS mutant polypeptide ) of the murine TCR, referred to as 1-2C TCR or 1-2C, 3-2E TCR or 3-2E in a specific embodiment of the present invention.
  • the present application includes TCRs or derivatives that specifically bind to the complex molecules of VVGAVGVGK and/or VVGACGVGK polypeptides mutated at amino acid 12, derived from KRAS mutant polypeptides, and HLA-A11, and also include TCRs that exhibit substantially the same function as the original TCR.
  • Antigen-specific TCR fragments “Fragments of TCRs” or “antigen-binding fragments” refer to antigen-binding fragments and TCR analogs of TCRs, which typically include at least a portion of the antigen-binding or variable regions of a parent TCR, eg, one or more CDRs. Fragments of the TCR retain at least some of the binding specificity of the parent TCR.
  • Specific binding when referring to a ligand/receptor, antibody/antigen or other binding pair refers to determining the presence or absence of a protein such as VVGAVGVGK and/or VVGACGVGK in a heterogeneous population of proteins and/or other biological agents Binding reactions of polypeptides to HLA-A11 complex molecules.
  • a specific ligand/antigen binds to a specific receptor/antibody, and does not bind to other proteins present in the sample in significant amounts.
  • the present invention also provides pharmaceutical compositions containing one or both of the KRAS mutant polypeptide-specific TCRs of the present invention, or antigen-binding fragments thereof.
  • various desired dosage forms can be prepared by mixing a KRAS mutant polypeptide-specific TCR or an antigen-binding fragment thereof with a pharmaceutically acceptable carrier or excipient.
  • Examples of the dosage form of the pharmaceutical composition of the present invention include tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film coatings, pellets, Sublingual tablets, ointments, etc., as non-oral preparations, injections, suppositories, transdermal preparations, ointments, plasters, external liquid preparations, etc., can be listed, and those skilled in the art can select appropriate drugs according to the route of administration and the object of administration, etc. dosage form.
  • the dosage of the active ingredient of the pharmaceutical composition of the present invention varies depending on the administration object, target organ, symptoms, administration method, etc., and can be considered in consideration of the type of dosage form, administration method, age and weight of the patient, The patient's symptoms, etc., are determined by the doctor's judgment.
  • the pharmaceutical composition of the present invention may also contain other agents, including but not limited to cytotoxic agents, cytostatic agents, anti-angiogenic or anti-metabolite drugs, targeted tumor drugs, immunostimulatory or immunomodulatory agents or in combination with cytotoxic agents, cellular Growth inhibitor or other toxic drug conjugated TCR.
  • agents including but not limited to cytotoxic agents, cytostatic agents, anti-angiogenic or anti-metabolite drugs, targeted tumor drugs, immunostimulatory or immunomodulatory agents or in combination with cytotoxic agents, cellular Growth inhibitor or other toxic drug conjugated TCR.
  • the present invention provides the following solutions.
  • T cell receptor or antigen-binding fragment thereof capable of complexing with KRAS- G12V8-16 ( VVGAVGVGK ) epitope and HLA-A11, or KRAS- G12C8
  • the -16 (VVGA C GVGK) epitope binds to the HLA-A11 complex, or the KRAS-G12V 8-16 (VVGA V GVGK) epitope and the HLA-A03 complex
  • the TCR contains the alpha chain variable region and beta A chain variable region, characterized in that the TCR or its antigen-binding fragment comprises the following alpha chain complementarity determining regions (CDRs) and beta chain complementarity determining regions (CDRs):
  • TCR T cell receptor
  • the beta chain variable region shown in the sequence of SEQ ID NO:17.
  • TCR or antigen-binding fragment thereof according to item 1 or 2, wherein the TCR is a murine TCR, a human-mouse chimeric TCR or a humanized TCR.
  • Polynucleotide the TCR described in any one of its encoding items 1-3 or its antigen-binding fragment, which is selected from SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: one or more sequences from the group consisting of 16.
  • An expression vector comprising the polynucleotide of item 4, the expression vector is preferably a lentiviral vector, eg.
  • a host cell comprising the expression vector of item 5.
  • TCR or the antigen-binding fragment thereof described in any one of items 1-3 is recovered from the host cell or its culture medium.
  • a pharmaceutical composition comprising the TCR or antigen-binding fragment thereof of any one of items 1-3, and a pharmaceutically acceptable carrier.
  • TCR or its antigen-binding fragment according to any one of items 1-3 in the preparation of a drug for improving the level of cytokines secreted by T cells of IFN- ⁇ , wherein the drug is, for example, a protein drug, A drug in which an ADC drug or a TCR is combined with an antigen.
  • the TCR or its antigen-binding fragment according to any one of items 1 to 3 is used in the preparation of tumor cells expressing KRAS-G12V 8-16 (VVGA V GVGK) or KRAS-G12C 8-16 (VVGA C GVGK) mutation
  • KRAS-G12V 8-16 VVGA V GVGK
  • KRAS-G12C 8-16 VVGA C GVGK
  • described TCR or its antigen-binding fragment is combined with KRAS-G12V 8-16 (VVGA V GVGK)/HLA-A11 or KRAS- G12C 8-16 (VVGA C GVGK)/HLA-A11 specific binding, or
  • the mutant polypeptide specifically binds, wherein the HLA molecule is preferably HLA-A31, HLA-A33, HLA-A68, and HLA-A30.
  • the TCR according to any one of items 1 to 3 or an antigen-binding fragment thereof in the preparation of an antitumor drug for the treatment of a patient with a KRAS gene G12V and G12C mutated tumor, such as the pancreas Cancer, colorectal cancer, lung cancer, such as non-small cell lung cancer, preferably, the G12V and G12C mutations of the KRAS gene are KRAS-G12V 8-16 (VVGA V GVGK) mutations of the KRAS gene or KRAS-G12C 8 -16 (VVGA C GVGK) mutation.
  • T lymphocytes (TCR-T) expressing the TCR T lymphocytes (TCR-T) expressing the TCR
  • TCR-T T lymphocytes
  • the positive tumor cells with the G12V or G12C mutation of the KRAS gene can be effectively identified and killed. It is expected to further inhibit the growth of tumors, especially solid tumors, and achieve the effect of tumor therapy.
  • the two specific T cell receptors targeting the G12V or G12C mutated epitope of the KRAS gene of the present invention and the T cells expressing them have the characteristics of high infection efficiency and high binding characteristics, so they can be used for pre-drug research.
  • Figure 1 The results of molecular sieve chromatography and biotinylation level detection of different mutant polypeptides of KRAS and HLA-A11 complex protein.
  • FIG. 1 Single cell sorting of KRAS-G12V/HLA-A11 tetramer-specific T cells.
  • Panel A shows the ELISPOT detection of specific T cells in spleen cells of mice immunized with KRAS-G12V polypeptide. Among them, mock is a negative control without stimulus, KRAS-G12V 8-16 is a peptide stimulation test well, phorbol ester (PMA) is a positive control, and T1-T6 and TF1-TF6 are mouse numbers.
  • PMA phorbol ester
  • Panel B shows the sorting of epitope-specific T cells in spleen cells of mice immunized with polypeptides
  • WT is the negative control of unimmunized mice
  • the abscissa is KRAS-G12V 8-16 /HLA-A11 tetramer staining
  • the ordinate is CD8 positive staining
  • the KRAS- G12V8-16 /HLA-A11 tetramer-positive cells are the part circled in the figure.
  • FIG. 3 Validation of 3-2E and 1-2C TCR specific binding to KRAS-G12V 8-16 /HLA-A11.
  • A shows the flow cytometry results of the binding experiment of 3-2E or 1-2C TCR with specific binding KRAS-G12V 8-16 /HLA-A11 tetramer after transient transfection of 293T cells.
  • B shows the flow cytometry results of the binding experiment of 1-2C or 3-2E TCR with specific binding KRAS-G12V 7-16 /HLA-A11 tetramer after transient transfection of 293T cells.
  • FIG. 4 Cross-recognition of 1-2C and 3-2E TCRs with KRAS-G12V 8-16 /HLA-A3.
  • Panel A shows flow cytometry results of staining analysis of KRAS-G12V 8-16 /HLA-A11 with 293T cell tetramers expressing 1-2C or 3-2E TCR.
  • Panel B shows flow cytometry results of staining analysis of KRAS-G12V 8-16 /HLA-A03 with 293T cell tetramers expressing 1-2C or 3-2E TCR.
  • the first row represents the detection of tetramers from PBMCs of two volunteers D1 and D2, which were not infected with TCR lentivirus, as a negative control; the second row (horizontal) and the third row (horizontal) were the use of Flow cytometry using KRAS- G12V8-16 /HLA-A11 tetramers of TCR-T cells prepared from PBMCs or CD8 T cells of volunteers D1 (second row (lateral)) or D2 (third row (lateral)) As a result of cell staining, the value in the quadrant shows the positive rate of TCR expression.
  • FIG. 6 Response of 1-2C or 3-2E TCR-T cells to different mutant polypeptides of KRAS.
  • Panels A and B represent the images and histograms of ELISPOT of 1-2C or 3-2E TCR-T cells and KRAS-G12 wild-type and different mutant polypeptides prepared from PBMC cells, D1 or D2 for volunteers No.;
  • Panel C shows ELISA statistics of IFN- ⁇ levels produced by incubation of 1-2C or 3-2E TCR-T cells with KRAS-G12 wild-type and different mutant polypeptides, incubation of TCR-T cells with culture medium As a negative control (mock), PMA stimulation served as a positive control.
  • Panels D, E and F represent images of ELISPOT of 1-2C or 3-2E TCR-T cells with KRAS-G12 wild-type and different mutant polypeptides, respectively, Panel D is a histogram of the results, IFN- ELISA statistics of gamma levels.
  • Figure 7 In vitro renaturation and purification results of two TCR proteins 1-2C and 3-2E.
  • Figure A and Figure B show the purification results of 1-2C TCR molecules after ion column and molecular sieve chromatography, respectively.
  • Figures C and D represent the purification results of 3-2E TCR molecules after ion column and molecular sieve chromatography, respectively.
  • Panels are SDS-PAGE results of asterisk-marked peak proteins.
  • FIG. 8 Binding properties of different mutant polypeptides of 1-2C or 3-2E TCR and KRAS-G12 to HLA-A11.
  • A-D diagrams represent the affinity detection of 1-2C TCR with KRAS-G12 wild type and different mutant polypeptides and HLA-A11 complex protein;
  • E-H diagrams represent 3-2E TCR and KRAS-G12 wild type and different mutant polypeptides Affinity detection with HLA-A11 complex proteins.
  • Figure 9 Evaluation of tumor suppressor effect of 1-2C TCR-T cells in NCG immunodeficiency mouse tumor model.
  • Figure A shows the flow chart of the mouse tumor inhibition experiment. PANC-1 tumor cells were inoculated on day 0 (DO), TCR-T cells were injected intratumorally on day 7, and measurements were observed every 3-4 days thereafter.
  • Panel B represents a comparison of tumor weights isolated at the end of the experiment between different treatment groups.
  • Figure C shows the growth and comparison of tumor volume in different treatment groups, and each point represents the mean ⁇ standard deviation of tumor volume in each group of mice at that time point.
  • D-G graphs represent the growth of tumor volume in a single mouse in each group. Statistical differences between groups were calculated by T-test, where **: p ⁇ 0.01, ***: p ⁇ 0.001; ns, p>0.05.
  • HLA-A11-restricted epitope polypeptides predicted to have KRAS-G12 mutation were synthesized first, and these polypeptides were used to immunize mice, and the T cell response was screened by ELISPOT experiment, and the mutant polypeptides with immunogenicity were selected.
  • tetramers of these KRAS mutant polypeptides and HLA-A11 were prepared, and KRAS-G12 was obtained by selecting CD3 + CD8 + T cells from immunized mouse spleen cells by co-staining with CD3 and CD8 antibodies and sorting. Mutant polypeptide-specific T cells.
  • HLA-A11-restricted T cell epitope peptide prediction was performed on the KRAS-G12 mutant peptide.
  • the results showed that the KRAS-G12V epitope has a strong affinity with HLA-A11.
  • the entrusted company (Zhongke Yaguang Company) synthesized KRAS-G12 wild-type polypeptide 8-16 (sequence shown in SEQ ID NO: 34), G12V 8-16 (sequence shown in SEQ ID NO: 35), G12D 8 -16 (sequence shown in SEQ ID NO: 36), G12C 8-16 (sequence shown in SEQ ID NO: 37) mutant polypeptide, KRAS-G12 wild-type polypeptide 7-16 (sequence shown in SEQ ID NO: 38) shown), G12V 7-16 (sequence shown as SEQ ID NO:39), G12D 7-16 (sequence shown as SEQ ID NO:40), G12C 7-16 (sequence shown as SEQ ID NO:41) .
  • ⁇ 2m ⁇ 2-microglobulin, light chain gene of HLA-A11
  • HLA-A11 heavy chain gene IMGT /HLA Acc No: HLA00043
  • SEQ ID NO:48 The obtained ⁇ 2m nucleic acid sequence is as shown in SEQ ID NO:48
  • the encoded amino acid sequence is as shown in SEQ ID NO:47
  • the obtained HLA-A11 heavy chain gene is as shown in SEQ ID NO:44
  • the encoded amino acid sequence is as shown in SEQ ID NO:44.
  • the amino acid sequence is shown in SEQ ID NO:43.
  • Biotin-tag the amino acid sequence is shown in SEQ ID NO: 33
  • the entrusted company synthesized these DNA sequences respectively (Nanjing GenScript Company), and introduced the restriction sites Nde I and Xho I respectively, wherein the Nde I restriction site was located at the 5' end of the sequence, and the restriction restriction site Xho I was located in the sequence 3' end.
  • the restriction sites Nde I and Xho I the DNA sequences of the synthesized ⁇ 2 m and HLA-A11 heavy chain genes were cloned into the expression vector pET-21a (Invitrogen), respectively, and the ⁇ 2 m and HLA-A11 heavy chains were established. Prokaryotic recombinant expression of the protein plasmids ⁇ 2m-pET 21a and HLA-A11-pET 21a.
  • the two expression plasmids were transferred into E.coli.BL21 (DE3) competent cells (purchased from Tianenze Biotechnology) by heat excitation respectively, and IPTG was added to induce expression, and the E. coli was disrupted and homogenized to extract inclusion bodies to obtain the inclusion body state. Inclusion body proteins of ⁇ 2m and HLA-A11 heavy chains.
  • 1L of renaturation solution (20mM Tris-HCL, 400mM L-arginine, EDTA 2mM, GSH/GSSG 5mM/1mM)
  • the heavy chain inclusion bodies of -A11 were slowly added dropwise to the above-mentioned renaturation solution and renatured for more than 8 hours.
  • KRAS-G12 wild-type or mutant polypeptide/HLA-A11 complex purification After taking out the samples, centrifuge at 12,000rpm for 10min at 4°C, transfer the supernatant to an ultrafiltration tube and concentrate to about 0.5-1ml, and pass through superdex200 molecular sieves (purchased from GE Healthcare) for KRAS-G12 wild-type or mutant polypeptide/HLA-A11 complex purification.
  • the KRAS-G12 wild-type or mutant polypeptide//HLA-A11 complex protein peaks peak at about 15.8 mL
  • KRAS-G12 wild-type or mutant polypeptide//HLA-A11 complex protein samples purified by molecular sieves were collected in an ultrafiltration concentration tube, concentrated to about 500 ⁇ L, and then centrifuged at 4 °C to remove the precipitate to obtain KRAS-G12 wild-type or mutant polypeptide/HLA-A11 complex protein samples.
  • Biotinylation reaction system (purchased from AVIDITY): 500 ⁇ l in total
  • KRAS-G12 wild type and mutant polypeptide/HLA-A11 complex protein sample 1mg/ml 200 ⁇ l
  • Buffer A N-bis(hydroxyethyl)glycine buffer 50 ⁇ l
  • Buffer B (ATP, biotin) 50 ⁇ l
  • the above biotinylation reaction system sample was passed through Superdex200 molecular sieve, and the complex after biotinylation was purified to remove excess biotin. 0.1-0.2 mg, the peak of KRAS-G12 wild-type or mutant polypeptide//HLA-A11 complex protein peak is about 15.8 mL (shown in Figure 1).
  • the above biotinylated KRAS-G12/HLA-A11 complex was concentrated to about 500 ⁇ l, and a sample was taken for SDS-PAGE shift test to verify the biotinylation effect.
  • the KRAS-G12V 8-16 (VVGA V GVGK) mutant polypeptide was used to immunize HLA-A11 transgenic mice (entrusted by Beijing Biositu Co., Ltd.) to induce the production of KRAS-G12V 8-16 mutations in the mice.
  • polypeptide-specific T cells in order to further obtain the specific TCR of the KRAS-G12V 8-16 mutant polypeptide.
  • mice were sacrificed, the spleen was removed, and the mouse splenocytes were obtained by grinding.
  • KRAS-G12V 8-16 VVGA V GVGK
  • HLA-A11-PE tetramer-specific T cell sorting and single-cell TCR gene amplification and sequencing
  • the mouse splenocytes obtained after immunization with KRAS-G12V 8-16 mutant polypeptide in step 1 were about 1 x 10 7 , centrifuged at 200-250g for 10min; washed three times with PBS containing 0.5% BSA, 200 -250g centrifugation for 10min; KRAS-G12V 8-16 (VVGA V GVGK)/HLA-A11-PE tetramer (obtained from the above tetramer preparation), PerCP-Cy5-CD8 (purchased from BD company) and FITC -CD3 fluorescent antibody (purchased from BD) was incubated with mouse splenocytes at a molar ratio of 1:1:1 at 25°C for 20 minutes; washed three times with PBS containing 0.5% BSA, and centrifuged at 200-250g for 10 minutes; Cells were resuspended in PBS containing 0.5% BSA.
  • 5' RACE is divided into three steps: reverse transcription (RT-PCR), the first round of PCR amplification and the second round of PCR amplification.
  • RT-PCR reverse transcription
  • the following uses Takara's D315-FullRACE Kit and follows the instructions.
  • RT-PCR the downstream primer used is the TCR gene constant region specific primer GSP1 (purchased from Takara), and the upstream primer is the target switch primer with oligoguanine deoxyribonucleic acid (Oligo dG) at the 3' end (target-switching primer) (Takara Corporation).
  • the first round of PCR the cDNA of the TCR obtained in the above (1) is used as the template, the upstream primer is the outer layer adapter primer 1 (5'RACE outer Primer, Takara company), and the downstream primer is a section upstream of the constant region GSP1
  • the specific primers for the constant region of TCR purchased from Takara Company were used to obtain the first-round PCR product of the ⁇ chain or ⁇ chain of TCR.
  • the second round of PCR the first round PCR product of the ⁇ chain or ⁇ chain of the TCR obtained in the above (2) is used as the template, and the upstream primer is the inner linker primer 2 (5'RACE inner Primer, Takara company)) , the downstream primer is a TCR constant region-specific primer (purchased from Takara Company D315-FullRACE Kit) upstream of the constant region GSP2, to obtain the second round PCR product of TCR ⁇ chain or ⁇ chain, respectively.
  • the upstream primer is the inner linker primer 2 (5'RACE inner Primer, Takara company)
  • the downstream primer is a TCR constant region-specific primer (purchased from Takara Company D315-FullRACE Kit) upstream of the constant region GSP2, to obtain the second round PCR product of TCR ⁇ chain or ⁇ chain, respectively.
  • Agarose gel electrophoresis was performed on the amplified products of the second round of PCR amplification containing TCR ⁇ chain and ⁇ chain variable region genes, and the target genes of TCR ⁇ chain or ⁇ chain variable region were obtained at the 500 bp position, respectively.
  • the target band was recovered, and the target gene fragment was ligated into T vector (pMD18T, Takara) with T4 ligase. Afterwards, the ligation product was transformed into DH5 ⁇ cells (purchased from Tiangen Biotechnology), and monoclonal gene sequencing was performed (entrusted by Ruibo Xingke).
  • the 1-2C TCR has an alpha chain variable region as shown in the sequence of SEQ ID NO:2, and a beta chain variable region as shown in the sequence of SEQ ID NO:7.
  • the 3-2E TCR has an alpha chain variable region as shown in the sequence of SEQ ID NO:12, and a beta chain variable region as shown in the sequence of SEQ ID NO:17.
  • the inventors further confirmed that the screened 1-2C and 3-2E TCRs have specific recognition for KRAS-G12V 8-16 (VVGA V GVGK)/HLA-A11.
  • this example also found that 1-2C and 3-2E can bind to KRAS-G12V 8-16 (VVGA V GVGK) presented by HLA-A11, as well as KRAS-G12 presented by HLA-A03.
  • the ⁇ chain and ⁇ chain variable regions (V regions) of the 1-2C and 3-2E TCRs (1-2C: the nucleic acid sequence is shown in SEQ ID NO: 1, and the amino acid sequence is shown in SEQ ID NO: 6)
  • 3-2E nucleic acid sequence shown in SEQ ID NO: 7, amino acid sequence shown in SEQ ID NO: 11) gene and human TCR ⁇ chain and ⁇ chain constant region (C region) gene (Hongxun Biological Company synthesis) ligation to obtain 1-2C and 3-2E chimeric TCR alpha and beta chain sequences.
  • the specific sequence of the chimeric sequence is shown in Table 1 below.
  • the ⁇ chain and ⁇ chain of the 1-2C and 3-2E TCRs are connected with the T2A sequence (the amino acid sequence of the T2A sequence is shown in SEQ ID NO: 42), and the lentivirus expression plasmid pCDH (purchased from Invitrogen)
  • T2A sequence the amino acid sequence of the T2A sequence is shown in SEQ ID NO: 42
  • lentivirus expression plasmid pCDH purchased from Invitrogen
  • chimeric 1-2C and 3-2E TCR lentiviral expression vectors were constructed, namely lentiviral expression vector 1-2C-pCDH and lentiviral expression vector 3-2E-pCDH.
  • HEK-293T cells were co-transfected with 1-2C or 3-2E-pCDH lentiviral expression vector and CD3-CD8-pCDH plasmid expressing CD3 and CD8 (purchased from Nanjing GenScript) at a ratio of 1:1. (purchased from ATCC). 24 hours after co-transfection, the cells were centrifuged at 200-250g for 10min; washed three times with PBS containing 0.5% BSA, and centrifuged at 200-250g for 10min to obtain HEK-293T cells expressing 1-2C or 3-2E TCR.
  • KRAS-G12 wild-type polypeptide 8-16 VVGA G GVGK
  • Tetramers of G12V 8-16 VVGA V GVGK
  • G12D 8-16 VVGA D GVGK
  • G12C 8-16 VVGA C GVGK mutant polypeptides/HLA-A11 expressing 1-2C or 3-2E TCR 293T cells were stained to evaluate their binding specificity.
  • HEK-293T cells obtained by co-transfection were treated with KRAS-G12V 8-16 (VVGA V GVGK)/HLA-A11-PE tetramer prepared above, as well as PerCP-Cy5-CD8 and FITC-CD3 Antibodies (BD Company) were incubated at a molar ratio of 1:1:1 for 30 min; washed three times with PBS containing 0.5% BSA, centrifuged at 200-250 g for 10 min; resuspended cells in PBS containing 0.5% BSA for detection of KRAS-G12V 8-16 (VVGA V GVGK)/HLA-A11 positive T cell frequency. Analysis was performed using flow cytometry (shown in Figure 3A).
  • Rows 1 and 2 (horizontal) in Figure 3A are flow cytometry analysis of staining of co-transfected HEK-293T cells by KRAS-G12V 8-16 (VVGA V GVGK)/HLA-A11-PE tetramer .
  • the results showed that the proportion of CD8 positive cells in 293T cells transfected with 1-2C or 3-2E TCR was about 25.9%. %.
  • CD8 T cell epitope polypeptides is generally 9-10 amino acids, and since the polypeptides detected above are all 9-amino acid polypeptides (9 peptides), it cannot be ruled out whether 1-2C and 3-2E TCR can bind to Possibility of 10 amino acid polypeptides (10 peptides).
  • HLA-A11 binding polypeptide According to the motif of HLA-A11 binding polypeptide, it is found that its N-terminal forward one amino acid may still become a T cell epitope, namely KRAS-G12 wild-type polypeptide 7-16 (as shown in SEQ ID NO: 38), G12V 7- 16 (as shown in SEQ ID NO:39), G12D 7-16 (as shown in SEQ ID NO:40), G12C 7-16 (as shown in SEQ ID NO:41).
  • these polypeptides with a length of 10 were further prepared into tetramers using the same conditions and operations as in Example 1, and the cell binding experiments were carried out with 293T cells expressing 1-2C or 3-2E TCR. The operation and conditions were the same as the above-mentioned G12D 8-16 polypeptide, and the results of flow cytometry were shown in Fig. 3B.
  • the results showed that, according to the upper right quadrant of the flow chart, the tetramers prepared from the 10-peptide KRAS wild-type and mutant polypeptides were unable to bind to 293T cells expressing 1-2C or 3-2E TCR. Therefore, it is further demonstrated that the 1-2C or 3-2E TCR of the present invention is highly specific for the recognition of KRAS-G12V8-16 (VVGA V GVGK) and KRAS- G12C 8-16 ( VVGA C GVGK) polypeptide epitopes.
  • HLA-A11 belongs to the molecular members of the HLA-A3 superfamily.
  • the molecular members of the HLA-A3 superfamily also include HLA-A03, HLA-A31, HLA-A33 and HLA-A68.
  • the HLA-A3 superfamily molecules have similar antigens Presentation features.
  • the antigen presentation of HLA-A3 superfamily molecules is characterized in that the C-terminus of the presented polypeptide is generally lysine (K) or arginine (R), and the two amino acids starting from the N-terminus of the polypeptide and the amino acid at the C-terminus are inserted.
  • HLA-A3 superfamily molecules such as HLA-A03, HLA-A31, HLA-A33 and HLA-A68 are more than 70% conserved in the ⁇ 1 and ⁇ 2 helices that can interact with TCR.
  • the TCR screened in Example 1 can also recognize HLA-A3 superfamily molecules such as HLA-A03, HLA-A31, HLA-A33 and HLA-A68 and KRAS-G12V 8-16 (VVGAVGVGK) or other mutant polypeptides the ability of the complexes formed.
  • HLA-A03 and KRAS-G12V 8-16 (VVGAVGVGK) were used to prepare tetramers, and the binding ability of HLA-A03 to the 1-2C and 3-2E TCRs screened above was tested by flow cytometry. To determine its specific binding ability to KRAS-G12 mutant polypeptides presented by other HLA-A3 superfamily molecules.
  • the HLA-A03 heavy chain gene used therein is shown in SEQ ID NO: 46, and the encoded amino acid sequence is shown in SEQ ID NO: 45. The rest of the materials and procedures used were the same as those used in HLA-A11.
  • the tetrameric protein of HLA-A03 (IMGT/HLA Acc No: HLA00037) and KRAS-G12V 8-16 (VVGAVGVGK) polypeptide was also prepared by the method shown in the above Example 1, and the tetrameric protein of HLA-G12V 8-16 (VVGAVGVGK) polypeptide was prepared with KRAS-G12V 8- 16 (VVGAVGVGK)/HLA-A11 tetramers were used for staining analysis of 293T cells expressing 1-2C or 3-2E TCR, respectively (Figure 4).
  • 1-2C or 3-2E TCR gene was introduced into peripheral blood mononuclear cells (PBMC) or isolated CD8 T cells isolated from healthy volunteers with HLA-A11 genetic background as TCR-T effector cells.
  • KRAS-G12 wild-type polypeptide 8-16 VVGA G GVGK
  • G12V 8-16 VVGA V GVGK
  • G12D 8-16 VVGA D GVGK
  • G12C 8-16 VVGA C GVGK mutant polypeptides were added Co-cultured in the above-mentioned TCR-T effector cell system, the levels of IFN- ⁇ secreted by effector cells and target cells presenting KRAS wild-type and mutant polypeptides were detected.
  • the effect of wild-type and mutant polypeptides/HLA-A11 on target cells was evaluated. The specific operations are as follows.
  • the 1-2C and 3-2E TCR lentiviral expression plasmids (1-2C-pCDH and 3-2E-pCDH) in Example 2 were combined with the lentiviral packaging plasmids PLP1, PLP2 and VSVG (purchased from Addgene) according to PLP1:
  • PEI polyetherimide
  • DMEM fetal calf serum
  • the peripheral blood lymphocytes of two healthy volunteers were collected to obtain PBMCs, and a portion of the PBMCs were taken to separate CD8 T cells by negative selection with magnetic beads (Biolegend).
  • Anti-CD3/anti-CD28-coated microspheres (ThermoFisher) were added to PBMC or CD8 T cells at a ratio of 1:1 for activation and culture overnight, and then 1-2C or 3-2E TCR lentivirus was added according to 1:1: Add 1 volume ratio to PBMC or CD8T cells, mix well, set a virus-free well as a control, and culture in a 37°C, 5% CO2 incubator. After 24 hours, the culture medium was changed to complete medium and the culture was continued until day 10.
  • the anti-CD3/anti-CD28 microspheres were removed under a magnetic field, and washed twice with the same medium as the cell culture to obtain the 1-2C or 3-2E TCR-T effector cells of this example.
  • 1-2C or 3-2E TCR-T cells (cultured to 10 days) prepared by using PBMC or CD8T cells were cultured, and KRAS-G12V 8-16 (VVGA V GVGK)/HLA-A11 tetrameric Body staining was performed by flow cytometry in the same manner as in Example 2, and the expression of 1-2C or 3-2E TCR was confirmed (Fig. 5, upper right quadrant).
  • 1-2C, 3-2E from volunteer D1 and D1, 3-2E-D2TCR-T cells from volunteer D2 prepared by PBMC or CD8+ T cells could detect 0.26%-2.38 % of tetramer-positive T cells with different positive rates, among which, in PBMC, 1-2C: 0.26%, 0.77%, 3-2E: 1.03%, 0.55%, respectively, and in CD8+ T cells, respectively 1-2C: 0.68%, 0.91%, 3-2E: 2.38%, 2.37%.
  • TCR-T effector cells of the present invention can specifically bind to KRAS-G12V 8-16 (VVGA V GVGK)/HLA-A11.
  • T cell detection methods IFN- ⁇ -ELISPOT and IFN- ⁇ -ELISA were used to act on 1-2C or 3-2E TCR-T cells and target cells presenting KRAS wild-type and mutant polypeptides, respectively. IFN- ⁇ levels were detected.
  • Antigen-presenting cells were added to the ELISPOT plate pre-coated with anti-IFN- ⁇ antibody according to the volume of 100 ⁇ l of 1 ⁇ 10 5 cells/well, and KRAS wild-type and mutant polypeptides (100 ⁇ l volume, 10 ⁇ g/ml) were added at the same time.
  • the 1-2C or 3-2E TCR-T cells prepared from the PBMCs or CD8 T cells of the two volunteers D1 and D2 were mixed with the PBMCs of the two volunteers D1 and D2 in a ratio of 1:1 to serve as a antigen presenting cells.
  • the prepared cell suspension was plated into a 96-well plate, 100 ⁇ l of cell suspension per well (2 ⁇ 10 5 cells), and three replicates were performed in each well, in which the PMA/ionomycin (ION) group was performed according to each well.
  • 1 ⁇ l of PMA/Ionomycin mixture (250 ⁇ ) was added to 250 ⁇ l of cell culture medium and diluted in advance to form a working solution, which was used as a positive stimulation control.
  • T cells (D1mock and D2mock) not infected with 1-2C or 3-2E TCR lentiviruses were added in parallel as negative controls. After culturing at 37°C for 20 h, the supernatant from the culture wells of the 96-well plate was taken and centrifuged at 500 g for 5 min to remove the remaining cells. The supernatant was added to the ELISA detection plate ( BD Company), the level of IFN- ⁇ in the supernatant was detected, and the results are shown in Figure 6C and F.
  • 1-2C and 3-2E TCR-T cells can specifically recognize target cells presenting KRAS- G12V 8-16 (VVGA V GVGK )/HLA-A11
  • the target cells of GVGK have certain cross-reactivity and can specifically secrete the cytokine IFN- ⁇ .
  • the 1-2C and 3-2E TCR-T cells of the present invention have potential target cell killing activity and tumor therapeutic value.
  • the inventors further used surface plasmon resonance (SPR) to detect the affinity at the protein level. Since the functional domain of the 1-2C or 3-2E TCR is the extracellular domain, and the extracellular domain without the transmembrane domain is a soluble protein, the extracellular domain of the 1-2C or 3-2E TCR was synthesized. The specific operations are as follows.
  • the extracellular region genes of 1-2C or 3-2E TCR ⁇ chain and ⁇ chain were optimized according to prokaryotic codons, and the DNA sequences of the extracellular regions of 1-2C and 3-2E TCR chimeric ⁇ chain and ⁇ chain were synthesized ( 1-2C: ⁇ chain, SEQ ID NO: 29; ⁇ chain, SEQ ID NO: 30; 3-2E: ⁇ chain, SEQ ID NO: 31; ⁇ chain SEQ ID NO: 32), wherein, 1-2C TCR Has an alpha chain variable region shown in the sequence of SEQ ID NO:2, and a beta chain variable region shown in the sequence of SEQ ID NO:7.
  • the 3-2E TCR has an alpha chain variable region as shown in the sequence of SEQ ID NO:12, and a beta chain variable region as shown in the sequence of SEQ ID NO:17.
  • the restriction sites Nde I and Xho I were introduced respectively, wherein the Nde I restriction site was located at the 5' end of the sequence, and the restriction restriction site Xho I was at the 3' end of the sequence.
  • the DNA sequences of the extracellular regions of the synthesized 1-2C or 3-2E TCR ⁇ chain and ⁇ chain were cloned into the expression vector pET21a (Invitrogen Company) using the enzyme cleavage sites Nde I and Xho I, respectively, to establish 1-2C or 3 -2E Prokaryotic recombinant expression plasmid for the extracellular domain proteins of TCR ⁇ chain and ⁇ chain.
  • the expression plasmid was transferred into E.coli.BL21(DE3) competent cells by heat shock method, and IPTG was added to induce expression to obtain the extracellular domain proteins of 1-2C or 3-2E TCR ⁇ chain and ⁇ chain in inclusion body state.
  • Inclusion bodies of the extracellular regions of 1-2C or 3-2E TCR ⁇ and ⁇ chains were dropped in 1L of prepared renaturation solution (5M urea, 20mM Tris-HCL, 400mM L-arginine, EDTA 2mM, GSH/GSSG 5mM/1mM) according to the mass ratio of 2:1 , added dropwise twice, 3 mL each time, with a minimum interval of 8h between the two dropwise additions, and then concentrated with a concentrating cup (Millipore Company).
  • the target protein was identified by SDS-PAGE.
  • the target protein was concentrated with a concentration cup (Millipore Company), and the buffer was exchanged with 20 mM Tris-HCl, 150 mM NaCl, pH 8.0, and purified with Superdex 200 pg molecular sieve (GE Healthcare) after concentration to obtain 1-2C or 3 About 2-3 mg of -2E TCR protein, reducing (containing dithiothreitol (DTT)) and non-reducing (without dithiothreitol (DTT)) SDS-PAGE detected the target protein (Figure 7).
  • KRAS wild-type and different mutant 9 peptide/HLA-A11 complex proteins were diluted to 20 ⁇ g/ml and immobilized on SA chips (GE Health), followed by gradients (0 ⁇ M, 6.25 ⁇ M, 12.5 ⁇ M, 25 ⁇ M, 50 ⁇ M, 100 ⁇ M)
  • the diluted 1-2C and 3-2E TCR proteins flowed through each channel of the SA chip respectively, and the binding kinetic parameters were analyzed by BIA evaluation software, and the affinity constant was calculated.
  • Affinities of 1-2C and 3-2E TCRs to wild-type and KRAS wild-type and different mutant 9-peptide/HLA-A11 complex proteins were tested (Figure 8).
  • the 3-2E TCR was unable to bind KRAS wild-type KRAS-G12 8-16 (VVGA G GVGK)/HLA-A11 and KRAS-G12D 8-16 (VVGA D GVGK)/HLA-A11.
  • 1-2C and 3-2E TCRs have good binding properties and affinity, and it can be speculated that when 1-2C and 3-2E TCRs are used in anti-tumor therapy, they can bind to tumor cells carrying G12V and G12C mutations of the gene KRAS. Produce IFN- ⁇ , and then kill tumor cells to achieve the effect of treating tumors.
  • Example 5 Tumor suppressive activity of 1-2C TCR-T cells in tumor mouse model
  • NCG immunodeficient mouse PANC-1 tumor model was used to evaluate the tumor suppressive effect of 1-2C TCR-T cells.
  • the experimental steps of tumor suppression in NCG mice by TCR-T cells include:
  • NCG mice were obtained from Nanjing University-Nanjing Institute of Biomedicine. Each NCG mouse was inoculated subcutaneously with PANC-1 tumor cells (Peking Union Cell Resource Center) carrying the KRAS-G12V mutant gene, and a human-derived tumor cell was established in NCG mice. Source immune system of mice:
  • Inoculation site subcutaneous on the back
  • mice with relatively uniform tumor formation were selected for grouping, and then intratumoral injection of 1-2C TCR-T cells was performed.
  • the injection group of T cells (1 ⁇ 10 7 ) not transferred into TCR was used as the negative control, with 6 mice in each group, including 3 mice in each of the TCR-T cell treatment groups prepared from D1 and D2.
  • the information and processing of each group are shown in the following table:
  • the tumor size was detected every three to four days after tumor formation, and the experiment ended when the maximum tumor volume of the mice was 4000 mm 3 .
  • the mice were sacrificed and the tumors were isolated and weighed.

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Abstract

本发明提供一种靶向KRAS基因的G12V或G12C突变的表位的两种特异性T细胞受体及抗肿瘤用途，该两种T细胞受体分别由α、β两条肽链组成，还提供其抗原结合片段，以及编码它的核酸，包含该核酸的载体，包含该载体的宿主细胞；还提供一种制备KRAS的G12V突变特异性T细胞受体或其抗原结合片段的方法。该特异性T细胞受体及其抗原结合片段能够作为免疫效应活化物刺激机体的免疫反应，从而产生抗肿瘤等疾病的作用效果。

Description

KRAS突变特异性T细胞受体筛选及抗肿瘤用途 技术领域
本发明属于医药领域，具体涉及一种能够特异性识别肿瘤KRAS基因G12V和G12C突变的抗原多肽的T细胞受体(TCR)或其抗原结合片段。
背景技术
2011年，癌症超过心脏病，成为全球第一大死亡原因。WHO在2013年12月公布，全球每年新增癌症患者数已经超过1400万名，这与2008年的统计结果1270万人相比，人数大幅增加。截至2020，癌症每年造成960万人死亡，全球每年约1万亿美金投入癌症诊疗。
在20世纪80年代早期，Allison及其他研究者确定了在T细胞表面负责识别抗原的αβT细胞受体(TCR)的基因结构。80年代后期，Boone、Rosenberg、Old等人分别研究发现，不同肿瘤病人体内均存在一些肿瘤特异性抗原，能够被T细胞所识别并特异性杀伤肿瘤细胞，使得肿瘤免疫治疗的希望重新燃起，大量研究致力于肿瘤治疗性疫苗的研究和开发。2013年免疫抗癌疗法被Science杂志评为年度10大科技突破之首。
近年来，随着干细胞生物学、免疫学、分子技术、组织工程技术等的快速发展，细胞免疫治疗作为一种安全而有效的治疗手段，在肿瘤等治疗中的作用越来越突出。当前，新型细胞治疗技术的研究和开发已经成为解决肿瘤等相关疾病的重要研究领域。
过继性T细胞疗法(Adoptive cell therapy，ACT)是一种高度个性化的癌症治疗方法，它可以通过重建癌症患者体内缺失或较弱的免疫系统，以达到抗肿瘤的效果。ACT疗法是指从肿瘤患者体内分离免疫活性细胞，在体外进行扩增和功能鉴定，然后向患者回输，从而达到直接杀伤肿瘤或激发机体的免疫应答杀伤肿瘤细胞的目的。寻找仅表达在癌症组织上而非正常必需组织上的抗原成为ACT疗法的限制因素。
目前，ACT疗法可以包括T细胞受体工程化细胞(TCR-T)治疗技术和嵌合抗原受体工程化T细胞(CAR-T)治疗技术来实现。通过这些方法，ACT对多种癌症，例如黑色素瘤、宫颈癌、淋巴瘤、白血病、胆总管癌和成神经细胞瘤都有很好的疗效。
当前，CAR-T在急/慢性粒细胞性白血病、淋巴瘤等疾病的治疗中也实现了重大突破，大大提高了患者生存率及生存质量。然而，在实体瘤治疗研究中，由于有限的特异性靶点使得CAR-T细胞的治疗前景并不明朗。
与CAR-T细胞利用抗体靶向胞外抗原不同，TCR是所有T细胞表面的特征性标志，以非共价键与CD3结合，形成TCR-CD3复合物。TCR是由α、β两条肽链组成，属于免疫球蛋白超家族，抗原特异性存在于V区(CDR1、CDR2、CDR3)，CDR3直接决定了TCR的抗原结合特异性。外周血中，90％-95％的T细胞表达TCR。经过基因改造的TCR的T细胞，可以对肿瘤细胞表面上的抗原分子进行特异性识别，进而针对肿瘤细胞产生免疫反应。
TCR-T细胞免疫治疗是近年发展起来的细胞治疗新技术，是典型的“精准医疗”治疗技术。目前该技术已在骨髓瘤、黑色素瘤、食管癌、肝癌等治疗中表现出积极的治疗前景。TCR-T细胞免疫治疗最早在20世纪末应用于HIV的治疗中，近年来的研究发现基于MART-1、MAGE-A4、NY-ESO-1、WT-1等肿瘤抗原特异性TCR工程化改造的自体免疫细胞治疗黑色素瘤、食管癌、多发性骨髓瘤、滑膜细胞肉瘤等表现出了良好的开发前景。
特别是2015年报道的NY-ESO-1特异性TCR工程化细胞免疫治疗20例多发性骨髓瘤的临床I/II期报道，80％的病例在接受了TCR-T治疗后表现出积极的临床治疗效果。当前，TCR-T细胞免疫治疗技术已经成为国际肿瘤及传染病治疗研究的热点领域，一些技术和产品已经进入临床前或临床研究阶段。
KRAS基因(Kirsten大鼠肉瘤病毒癌基因同源物，Kirsten rat sarcoma virus oncogene homolog)编码的蛋白是一种小GTP酶(small GTPase)， 它属于RAS超蛋白家族，参与细胞内的信号传递。KRAS蛋白质有188个氨基酸，分子量是21.6KD，拥有GTPase酶活性的鸟嘌呤核苷结合蛋白。在细胞内，KRAS蛋白在失活和激活状态之间转变，当KRAS与鸟嘌呤核苷二磷酸(GDP)结合时，它处于失活状态，当它与鸟嘌呤核苷三磷酸(GTP)结合时，它处于激活状态，并且可以激活下游信号通路，其中包括MAPK信号通路，PI3K信号通路和Ral-GEFs信号通路。这些信号通路在促进细胞生存、增殖和细胞因子释放方面具有重要作用。
在人类癌症中，其为肿瘤学领域最著名的致癌基因之一，曾被认为是“不可成药”的靶点。KRAS基因突变出现在接近90％的胰腺癌中，30-40％的结肠癌中，17％的子宫内膜癌中，15-20％的肺癌包括小叶性肺癌中，以及胆管癌、宫颈癌、膀胱癌等。KRAS的基因突变占RAS基因突变总数的86％。在KRAS的基因突变中，有97％是第12号或者第13号氨基酸残基发生了突变。其中最主要的是第12号氨基酸变为天冬氨酸(G12D)、第12号氨基酸变为缬氨酸(G12V)、第12号氨基酸变为半胱氨酸(G12C)、第13号氨基酸变为天冬氨酸(G13D)这几种突变。
结构学研究表明，这些基因突变大多干扰KRAS水解GTP的能力。当KRAS发生G12D、G12V、G13D这几种突变后，会通过破坏GAP活性而使KRAS一直保持与GTP结合，将KRAS锁定在有酪氨酸激酶活跃状态，并不断激活下游信号通路(如PI3K，RAF-MEK-ERK(MAPK)，RAL-GEF等)。这些下游的信号通路打开之后，就会刺激细胞增殖、迁移，最终促成肿瘤发生。
近年来，针对KRAS突变体，研究开发了共价抑制剂，其通过异构位点(allosteric)靶向KRAS突变体，使得KRAS突变体与GTP的亲和力降低而达到“锁死”其活性的目的。例如安进公司的为KRAS-G12C抑制剂的AMG510。MiratiTherapeutics公司的仍然处于临床前开发阶段的KRAS-G12C抑制剂MRTX1257。针对其他KRAS突变目前尚无相关治疗性药物，也缺少一种通过利用机体的免疫机制进行检测以及治疗的肿瘤药物。
发明内容
在人类癌症中，KRAS基因突变出现在接近90％的胰腺癌中，30-40％的结肠癌中，17％的子宫内膜癌中，15-20％的肺癌中。在KRAS的基因突变中，97％是第12号或者第13号氨基酸残基发生了突变。其中最主要的是G12D、G12V、G12C、G13D这几种突变。KRAS突变后能够被细胞中的MHC分子呈递到细胞表面，并被T细胞所识别，以激发T细胞免疫反应，进而清除携带KRAS突变的肿瘤细胞。
具体而言，KRAS突变多肽作为抗原时可以使机体产生CD8 +CTL(cytotoxic lymphocyte，细胞毒性T淋巴细胞)反应。KRAS多肽中的一些氨基酸残基发生的突变可以被HLA分子呈递并被T细胞识别。
本发明的一个实施方式包括通过特异性T细胞受体(TCR)单细胞筛选技术，筛选出特异性靶向于肿瘤KRAS基因的KRAS-G12V 8-16(VVGA VGVGK)突变(以下也简称G12V，G12V突变，或KRAS基因的G12V突变)、KRAS-G12C 8-16(VVGA CGVGK)(以下也简称G12C，G12C突变，或KRAS基因的G12C突变)的两种TCR。
本发明的一个实施方式包括提供了靶向KRAS基因的G12V或G12C突变的表位的特异性T细胞受体及其抗原结合片段。本发明的另一个实施方式包括上述T细胞受体及其抗原结合片段在制备用于治疗携带KRAS基因的G12V和G12C突变的肿瘤的药物中的用途。
本发明是基于上述的原理作出的，本发明中的KRAS突变多肽特异性TCR或其抗原结合片段通过与KRAS的G12V突变多肽(VVGAVGVGK)和HLA-A11的复合物分子和/或KRAS的G12C突变多肽(VVGACGVGK)和HLA-A11的复合物分子，或KRAS-G12V 8-16(VVGA VGVGK)表位和HLA-A03复合物分子特异性结合，从而刺激T细胞活化，诱导T细胞分泌IFN-γ等细胞因子，进而杀伤表达KRAS突变多肽，特别是KRAS基因的G12V和或G12C突变阳性的肿瘤细胞。
在本发明中，表述“KRAS突变多肽特异性TCR”或“鼠源KRAS突变多肽特异性TCR”为针对KRAS突变多肽中HLA-A11限制性的CTL 表位多肽(其序列为VVGAVGVGK和/或VVGACGVGK)的鼠源TCR，在本发明具体的实施方式中称为1-2C TCR或1-2C，3-2E TCR或3-2E。
本申请包括与KRAS突变多肽来源的第12位氨基酸突变的VVGAVGVGK和/或VVGACGVGK多肽与HLA-A11的复合物分子特异性结合的TCR或衍生物，也包括与原来的TCR显示实质上功能相同的抗原特异性的TCR片段。“TCR的片段”或“抗原结合片段”是指TCR的抗原结合片段及TCR类似物，其通常包括至少部分母体TCR的抗原结合区或可变区，例如一个或多个CDR。TCR的片段保留母体TCR的至少某些结合特异性。
当提及配体/受体、抗体/抗原或其它结合对时，“特异性”结合是指在蛋白和/或其它生物试剂的异质群体中确定是否存在所述蛋白例如VVGAVGVGK和/或VVGACGVGK多肽与HLA-A11复合物分子的结合反应。因此，在所指定的条件下，特定的配体/抗原与特定的受体/抗体结合，并且并不以显著量与样品中存在的其它蛋白结合。
本发明还提供含有本发明KRAS突变多肽特异性TCR中的一种或两种，或其抗原结合片段的药物组合物。为了制备药物组合物，可以通过使KRAS突变多肽特异性TCR或其抗原结合片段与药用载体或赋形剂混合，制备成各种所需的剂型。作为本发明的药物组合物的剂型的种类，例如可以列举作为口服剂的片剂、粉末剂、丸剂、散剂、颗粒剂、细粒剂、软/硬胶囊剂、薄膜包衣剂、小丸剂、舌下片、膏剂等，作为非口服剂，可以列举注射剂、栓剂、经皮剂、软膏剂、硬膏剂、外用液剂等，本领域的技术人员能够根据给药途径和给药对象等选择适当的剂型。
本发明的药物组合物的有效成分的给药量，根据给药对象、对象脏器、症状、给药方法等不同而存在差异，可以考虑剂型的种类、给药方法、患者的年龄和体重、患者的症状等，根据医生的判断来确定。
本发明药物组合物还可以含有其它药剂，包括但不限于细胞毒剂、细胞生长抑制剂、抗血管形成药物或抗代谢药物、靶向肿瘤药物、免疫刺激剂或免疫调节剂或与细胞毒剂、细胞生长抑制剂或其它毒性药物结合的TCR。
具体地，本发明提供以下方案。
1.T细胞受体(TCR)或其抗原结合片段，所述TCR或其抗原结合片段能够与KRAS-G12V 8-16(VVGA VGVGK)表位和HLA-A11复合物，或KRAS-G12C 8-16(VVGA CGVGK)表位和HLA-A11复合物，或KRAS-G12V 8-16(VVGA VGVGK)表位和HLA-A03复合物结合，并且所述TCR包含α链可变区和β链可变区，其特征在于，所述TCR或其抗原结合片段包含以下的α链互补决定区(CDR)和β链互补决定区(CDR)：
如SEQ ID NO:3所示的α链互补决定区CDR1；
如SEQ ID NO:4所示的α链互补决定区CDR2；
如SEQ ID NO:5所示的α链互补决定区CDR3；
如SEQ ID NO:8所示的β链互补决定区CDR1；
如SEQ ID NO:9所示的β链互补决定区CDR2；和
如SEQ ID NO:10所示的β链互补决定区CDR3，
或
如SEQ ID NO:13所示的α链互补决定区CDR1；
如SEQ ID NO:14所示的α链互补决定区CDR2；
如SEQ ID NO:15所示的α链互补决定区CDR3；
如SEQ ID NO:18所示的β链互补决定区CDR1；
如SEQ ID NO:19所示的β链互补决定区CDR2；和
如SEQ ID NO:20所示的β链互补决定区CDR3。
2.如项1所述的T细胞受体(TCR)或其抗原结合片段，其包含：
如SEQ ID NO:2的序列所示的α链可变区，和
如SEQ ID NO:7的序列所示的β链可变区；
或
如SEQ ID NO:12的序列所示的α链可变区，和
如SEQ ID NO:17的序列所示的β链可变区。
3.根据项1或2所述的TCR或其抗原结合片段，其中所述TCR为鼠源TCR、人鼠嵌合TCR或人源化TCR。
4.多核苷酸，其编码项1-3中任一项所述的TCR或其抗原结合片段，其为选自由SEQ ID NO:1，SEQ ID NO:6，SEQ ID NO:11，SEQ ID NO:16组成的组中的一个或多个序列。
5.表达载体，其包含项4所述的多核苷酸，所述表达载体优选为慢病毒载体，例如。
6.宿主细胞，其包含项5所述的表达载体。
7.制备项1-3中任一项所述的TCR或其抗原结合片段的方法，所述方法包括：
1)培养项6所述的宿主细胞；
2)从所述宿主细胞或其培养基中回收项1-3中任一项所述的TCR或其抗原结合片段。
8.药物组合物，其包含项1-3中任一项所述的TCR或其抗原结合片段，和药学上可接受的载体。
9.项1-3中任一项所述的TCR或其抗原结合片段在制备用于提高T细胞分泌IFN-γ的细胞因子水平的药物中的用途，其中所述药物例如为蛋白类药物、ADC药物或TCR与抗原组合的药物。
10.项1-3中任一项所述的TCR或其抗原结合片段在制备表达KRAS-G12V 8-16(VVGA VGVGK)或KRAS-G12C 8-16(VVGA CGVGK)突变的肿瘤细胞的检测试剂中的用途，或在制备检测或诊断肿瘤的试剂中的用途，优选地，所述的TCR或其抗原结合片段与KRAS-G12V 8-16 (VVGA VGVGK)/HLA-A11或KRAS-G12C 8-16(VVGA CGVGK)/HLA-A11特异性结合，或
所述TCR或其抗原结合片段与具有与HLA-A11或HLA-A03类似的抗原结合特性的HLA分子，与KRAS-G12V 8-16(VVGA VGVGK)或KRAS-G12C 8-16(VVGA CGVGK)突变多肽特异性结合，其中，所述HLA分子优选为HLA-A31,HLA-A33,HLA-A68，HLA-A30。
11.项1-3中任一项所述的TCR或其抗原结合片段在制备用于治疗携带KRAS基因的G12V和G12C突变的肿瘤的患者的抗肿瘤药物中的用途，所述肿瘤例如为胰腺癌、结直肠癌、肺癌，所述肺癌例如非小细胞肺癌，优选地，所述KRAS基因的G12V和G12C突变为KRAS基因的KRAS-G12V 8-16(VVGA VGVGK)突变或KRAS-G12C 8-16(VVGA CGVGK)突变。
本发明的优点
通过利用本发明的KRAS基因的G12V或G12C突变特异性TCR，制备表达该TCR的T淋巴细胞(TCR-T)，可以有效的识别并杀死KRAS基因的G12V或G12C突变的阳性肿瘤细胞，可以期待其进而抑制肿瘤特别是实体瘤的生长，达到肿瘤治疗的效果。
本发明的靶向KRAS基因的G12V或G12C突变的表位的两种特异性T细胞受体及表达其的T细胞具有感染效率高，结合特性高的特性，因此可以利用其进行药物前期研究，在试验模型等中开发靶向与肿瘤生长和进展相关突变的药物。由此，能够用于对于表达KRAS基因突变的各种肿瘤在各种时期的诊断，治疗的药物的制备，尤其是治疗突变频率较高的实体瘤的药物。
附图说明
图1.KRAS的不同突变体多肽与HLA-A11复合物蛋白的分子筛层析及生物素化水平检测结果。
图2.KRAS-G12V/HLA-A11四聚体特异性T细胞单细胞分选。A图表示为免疫KRAS-G12V多肽的小鼠脾细胞中特异性T细胞的ELISPOT检测图。其中，mock为不加刺激物的阴性对照，KRAS-G12V 8-16为多肽刺激检测孔，佛波酯(PMA)为阳性对照，T1-T6、TF1-TF6为小鼠编号。B图表示为多肽免疫的小鼠脾细胞中表位特异性T细胞的分选，WT为未免疫小鼠的阴性对照，横坐标为KRAS-G12V 8-16/HLA-A11四聚体染色，纵坐标为CD8阳性染色，KRAS-G12V 8-16/HLA-A11四聚体阳性细胞为图中用方框圈出的部分。
图3.3-2E和1-2C TCR特异性结合KRAS-G12V 8-16/HLA-A11验证。A表示为将3-2E或1-2C TCR瞬时转染293T细胞后，与特异性结合KRAS-G12V 8-16/HLA-A11四聚体结合实验的流式细胞结果。B表示为将1-2C或3-2E TCR瞬时转染293T细胞后与特异性结合KRAS-G12V 7-16/HLA-A11四聚体结合实验的流式细胞结果。
图4.1-2C和3-2E TCR与KRAS-G12V 8-16/HLA-A3的交叉识别。A图表示为KRAS-G12V 8-16/HLA-A11与表达1-2C或3-2E TCR的293T细胞四聚体的染色分析的流式细胞结果。B图表示为KRAS-G12V 8-16/HLA-A03与表达1-2C或3-2E TCR的293T细胞四聚体的染色分析的流式细胞结果。
图5.1-2C或3-2E TCR-T细胞的感染效率检测。第一行(横向)表示为未感染TCR慢病毒的来源于D1和D2两名志愿者PBMC的四聚体检测，作为阴性对照；第二行(横向)和第三行(横向)分别为利用D1(第二行(横向))或D2(第三行(横向))志愿者的PBMC或CD8T细胞制备的TCR-T细胞，利用KRAS-G12V 8-16/HLA-A11四聚体进行流式细胞染色的结果，象限中标的数值示出其TCR的表达阳性率。
图6.1-2C或3-2E TCR-T细胞与KRAS的不同突变体多肽的反应。A图和B图表示为利用PBMC细胞制备的1-2C或3-2E TCR-T细胞与KRAS-G12野生型及不同突变体多肽的ELISPOT的图像和直方统计图，D1或D2为志愿者的编号；图C表示为1-2C或3-2E TCR-T细胞与KRAS-G12野生型及不同突变体多肽孵育后产生的IFN-γ水平的ELISA 统计图，TCR-T细胞与培养基的孵育作为阴性对照(mock)，PMA刺激作为阳性对照。图D、E和F分别表示为1-2C或3-2E TCR-T细胞与KRAS-G12野生型及不同突变体多肽的ELISPOT的图像，图D结果的直方统计图，这些细胞中的IFN-γ水平的ELISA统计图。
图7.1-2C与3-2E两种TCR蛋白的体外复性及纯化结果。A图与B图分别表示1-2C TCR分子经过离子柱和分子筛层析后的纯化结果。C图与D图分别表示3-2E TCR分子经过离子柱和分子筛层析后的纯化结果。小图为星号标记的峰值蛋白的SDS-PAGE结果。
图8.1-2C或3-2E TCR与KRAS-G12不同突变体多肽与HLA-A11的结合特性。A-D图表示为1-2C TCR与KRAS-G12野生型及不同突变体多肽与HLA-A11复合物蛋白的亲合力检测；E-H图表示为3-2E TCR与KRAS-G12野生型及不同突变体多肽与HLA-A11复合物蛋白的亲合力检测。
图9.1-2C TCR-T细胞在NCG免疫缺陷鼠肿瘤模型中的抑瘤效果评价。A图表示为小鼠抑瘤实验的流程图。在第0天(D0)接种PANC-1肿瘤细胞，在第7天瘤内注射TCR-T细胞，之后每3-4天观察测量。B图表示为实验结束时分离的肿瘤重量在不同治疗组间的比较。C图表示为不同治疗组肿瘤体积生长情况和比较,每个点代表该时间点每组小鼠肿瘤体积的平均值±标准差。D-G图表示为每组内单只小鼠肿瘤体积生长情况。组间统计差异由T检验计算得出，其中**：p<0.01,***:p<0.001；ns,p>0.05.
具体实施方式
本发明通过具体实施方式和附图进一步阐述本发明的技术方案，但是本领域普通技术人员可以理解的是：以下具体实施方式以及实施例旨在阐述本发明，而不应理解为以任何方式限制本发明。本领域技术人员公知，在不背离本发明精神的情况下，可以对本发明做出许多修改，这样的修改也落入本发明的范围。
下述实验方法如无特殊说明，均为本领域常规的实验方法，所使用的实验材料如无特别说明，均属于可以容易的从商业公司获取的实验材料。
实施例1.KRAS-G12突变多肽特异性T细胞分选和TCR基因克隆
本实施例首先合成了预测具有KRAS-G12突变的HLA-A11限制性表位多肽，并利用这些多肽免疫小鼠，通过ELISPOT实验进行T细胞反应筛选，选出其中具有免疫原性的突变多肽。同时制备了这些KRAS突变多肽与HLA-A11的四聚体，通过利用CD3、CD8抗体一起染色从免疫后小鼠脾细胞中选取CD3 +CD8 +T细胞并分选而得到了其中的KRAS-G12突变多肽特异性T细胞。
1.KRAS-G12突变多肽的HLA限制性表位预测
通过NetMHC-4.0在线预测系统，对发生了KRAS-G12突变的多肽进行HLA-A11限制性T细胞表位多肽预测。结果发现KRAS-G12V表位与HLA-A11具有较强的亲和力。
委托公司(中科亚光公司)合成了KRAS-G12野生型多肽 8-16(序列如SEQ ID NO:34所示)、G12V 8-16(序列如SEQ ID NO:35所示)、G12D 8-16(序列如SEQ ID NO:36所示)、G12C 8-16(序列如SEQ ID NO:37所示)突变多肽、KRAS-G12野生型多肽 7-16(序列如SEQ ID NO:38所示)、G12V 7-16(序列如SEQ ID NO:39所示)、G12D 7-16(序列如SEQ ID NO:40所示)、G12C 7-16(序列如SEQ ID NO:41所示)。
2.KRAS-G12V/HLA-A11四聚体的制备
对这些多肽与HLA-A11的结合特性及特异性T细胞反应，以及特异性TCR进行了评价和筛选。
按照常规方法对β 2m(β2-微球蛋白，HLA-A11的轻链基因)(Uniprot:P61769)及HLA-A11重链基因(IMGT/HLA Acc No:HLA00043)进行了原核密码子优化，得到的β 2m核酸序列如SEQ ID NO:48所示，其编码的氨基酸序列如SEQ ID NO:47所示，得到的HLA-A11重链基因如SEQ ID NO:44所示，其编码的氨基酸序列如SEQ ID NO:43所示。对于HLA-A11重链基因，在序列SEQ ID NO:44所示的重链基因的C端加入 表达生物素特异性结合多肽的序列(Biotin-tag，氨基酸序列如SEQ ID NO:33所示)。
委托公司分别合成这些DNA序列(南京金斯瑞公司)，并分别引入酶切位点Nde I和Xho I，其中Nde I酶切位点位于序列的5’端，酶切位点Xho I位于序列的3’端。利用酶切位点Nde I和Xho I，将合成的β 2m和HLA-A11重链基因的DNA序列分别克隆入表达载体pET-21a(Invitrogen公司)，建立β 2m和HLA-A11重链蛋白的原核重组表达质粒β2m-pET 21a和HLA-A11-pET 21a。
将两种表达质粒分别用热激发转入E.coli.BL21(DE3)感受态细胞(购自天恩泽生物)，加入IPTG进行诱导表达，破碎大肠杆菌并匀浆提取包涵体，获得包涵体状态的β2m和HLA-A11重链的包涵体蛋白。
将1mlβ2m包涵体(30mg/ml溶于含6M Gua-HCl、50mM Tris pH8.0、100mM NaCl、10mM EDTA和10mM DTT的溶解液中)缓慢滴加到分别含5mg上述制备的KRAS-G12野生型多肽8-16、G12V8-16、G12D8-16、G12C8-16突变多肽、KRAS-G12野生型多肽7-16、G12V7-16、G12D7-16、G12C7-16多肽(北京中科亚光公司合成)的1L复性液(20mM Tris-HCL，400mM L-精氨酸，EDTA 2mM，GSH/GSSG 5mM/1mM)中，1小时后按照β2m：HLA-A11重链＝1:1的摩尔比将HLA-A11的重链包涵体缓慢滴加到上述复性液中，复性8小时以上。
使用超滤杯使复性后的样品通过10kDa滤膜以浓缩样品，并通过浓缩换液为20mM Tris-Cl，50mM NaCl，pH8.0的缓冲液；两次换液：将样品浓缩至约20ml后，加入至200ml含20mM Tris-Cl，50mM NaCl，pH8.0的缓冲液；之后浓缩至约20ml后再次加入20mM Tris-Cl，50mM NaCl，pH8.0的缓冲液至100ml，并最终浓缩至约10-20ml体积。将样品取出后4℃12000rpm离心10min，将上清转移到超滤管中浓缩到约0.5-1ml，过superdex200分子筛(购自GE Healthcare)进行KRAS-G12野生型或突变多肽/HLA-A11复合物纯化。根据280nm的光吸收值收集KRAS-G12野生型或突变多肽//HLA-A11复合物蛋白峰(峰值约在15.8mL)。
将经过分子筛纯化后的KRAS-G12野生型或突变多肽//HLA-A11复合物蛋白样品收集于超滤浓缩管中，浓缩至约500μL，之后4℃下离心去除沉淀，得到KRAS-G12野生型或突变多肽/HLA-A11复合物蛋白样品。
(2)生物素化反应
使用步骤(1)得到的KRAS-G12V/HLA-A11复合物蛋白样品，分别配制如下的生物素化反应体系(500μl)。
生物素化反应体系(购自AVIDITY公司)：总计500μl
KRAS-G12野生型和突变多肽/HLA-A11复合物蛋白样品1mg/ml200μl，
Buffer A(N-二(羟乙基)甘氨酸缓冲液)50μl，
Buffer B(ATP，生物素)50μl，
200μM生物素，Bir-A酶(3mg/ml)20μl，
用20mM Tris-Cl，50mM NaCl，pH8.0补足到500μl。
配完后混匀，置冰上，于4℃冷库孵育过夜。
将上述生物素化反应体系样品过Superdex200分子筛，进行生物素化后的复合物纯化，以去除多余的生物素，同上操作，得到生物素化的KRAS-G12突变多肽/HLA-A11复合物蛋白约0.1-0.2mg，KRAS-G12野生型或突变多肽//HLA-A11复合物蛋白峰的峰值约在15.8mL(示于图1)。
生物素化效率检测：
将上述生物素化的KRAS-G12/HLA-A11复合物浓缩到约500μl，取样进行SDS-PAGE shift试验验证生物素化效果。
设一个样品及两个对照：
A.生物素化的KRAS-G12/HLA-A11复合物样品8μl+分子筛缓冲液2μl；
B.生物素化的KRAS-G12/HLA-A11复合物样品8μl+链霉亲和素2μl(20mg/ml)；
C.链霉亲和素2μl+分子筛缓冲液8μl。
将上述三支样品置冰上孵育30min后进行SDS-PAGE鉴定，结果示于图1中。
结果显示：由于生物素化的KRAS-G12/HLA-A11复合物能够与链霉亲和素结合成为大分子，从而其在SDS-PAGE中的条带滞后。通过比较各组多肽生物素化前后(KRAS-G12/HLA-A11复合物原始蛋白SDS-PAGE条带灰度链霉亲和素加入KRAS-G12/HLA-A11复合物条带灰度后)/KRAS-G12/HLA-A11复合物原始蛋白SDS-PAGE条带灰度的比值，可以判断，这些KRAS-G12野生型或突变多肽均能够高效的生物素化(如图1所示)。
(3)KRAS-G12野生型和突变多肽/HLA-A11-PE四聚体的制备
将生物素化的KRAS-G12野生型和突变多肽/HLA-A11复合物分子进行超滤浓缩，按照链霉亲和素-PE：KRAS-G12野生型和突变多肽/HLA-A11复合物＝1:5的摩尔比将生物素化的KRAS-G12野生型或突变多肽/HLA-A11复合物分子中加入链霉亲和素-PE进行四聚化，最后置4℃孵育过夜，分别得到KRAS-G12野生型 8-16(VVGA GGVGK)多肽、G12V 8-16(VVGA VGVGK)、G12D 8-16(VVGA DGVGK)、G12C 8-16(VVGA CGVGK)突变多肽、KRAS-G12野生型 7-16(VVVGA GGVGK)多肽、G12V 7-16(VVVGA VGVGK)、G12D 7-16(VVVGA DGVGK)、G12C 7-16(VVVGA CGVGK)/HLA-A11-PE四聚体备用。
3.KRAS-G12V多肽免疫HLA-A11转基因小鼠
在本步骤中，使用了KRAS-G12V 8-16(VVGA VGVGK)突变多肽免疫HLA-A11转基因小鼠(委托北京百奥赛图公司制备)，诱导小鼠体内产生针对KRAS-G12V 8-16突变多肽的特异性T细胞，以便进一步获得KRAS-G12V 8-16突变多肽的特异性的TCR。
具体地，取化学合成的KRAS-G12V 8-16突变多肽(中科亚光)100μg溶于100μL PBS，并与等体积的弗氏完全佐剂混合并进行乳化。将乳化的多肽和弗氏完全佐剂混合液采用背部皮下多点注射方法，进行HLA-A11转基因小鼠免疫。
第一次免疫起一周后，利用同样方法取KRAS-G12V 8-16突变多肽100μg溶于100μL PBS，并与等体积的弗氏不完全佐剂混合乳化，以同样的方法进行注射而实施了加强免疫。一周以后处死小鼠，取脾脏，并研磨获得小鼠脾细胞。
4.KRAS-G12V 8-16(VVGA VGVGK)/HLA-A11-PE四聚体特异性T细胞分选及单细胞TCR基因扩增及测序
经PBS清洗重悬，步骤1中经过KRAS-G12V 8-16突变多肽免疫后获得的小鼠脾细胞约1 x 10 7，在200-250g离心10min；用含有0.5％BSA的PBS洗三次，200-250g离心10min；将KRAS-G12V 8-16(VVGA VGVGK)/HLA-A11-PE四聚体(由上述四聚体制备中获得)、PerCP-Cy5-CD8(购自BD公司)和FITC-CD3荧光抗体(购自BD公司)按照1:1:1的摩尔比与小鼠脾细胞在25℃条件下共同孵育20分钟；用含0.5％BSA的PBS洗三次，200-250g离心10min；用含0.5％BSA的PBS重悬细胞。
之后对上述细胞进行流式细胞技术单细胞分选。选取淋巴细胞亚群，选择CD3 +CD8 +T细胞，分选得到KRAS-G12V 8-16(VVGA VGVGK)/HLA-A11-PE四聚体阳性的CD8+T细胞(示于图2，方框中示出四聚体阳性的细胞及比例)。
将单个阳性细胞分选至含有细胞裂解液(购自天根生物)和RNA酶抑制剂(购自康为世纪生物)的96孔板中。之后对每个孔内的KRAS-G12V 8-16(VVGA VGVGK)/HLA-A11-PE四聚体阳性T细胞提取总RNA，进行5’RACE TCR基因扩增，具体如下所述。
5’RACE分为三个步骤：反转录(RT-PCR)、第一轮PCR扩增和第二轮PCR扩增。以下使用了Takara公司D315-FullRACE Kit并按照说明书进行操作。
(1)RT-PCR：使用的下游引物是TCR基因恒定区特异性引物GSP1(购自Takara公司)，上游引物为3’末端带有寡聚鸟嘌呤脱氧核糖核酸(Oligo dG)的靶开关引物(target-switching primer)(Takara公司)。
(2)第一轮PCR：以上述(1)得到的TCR的cDNA为模板，上游引物为外层接头引物1(5’RACE outer Primer,Takara公司)，下游引物 为在恒定区GSP1上游的一段TCR恒定区的特异性引物(购自Takara公司)，得到TCR的α链或β链第一轮PCR产物。
(3)第二轮PCR：以上述(2)得到的TCR的α链或β链第一轮PCR产物为模板，上游引物为内层的接头引物2(5’RACE inner Primer,Takara公司))，下游引物为恒定区GSP2上游的一段TCR恒定区特异性引物(购自Takara公司D315-FullRACE Kit)，分别得到TCR的α链或β链第二轮PCR产物。
将扩增之后的含有TCRα链和β链可变区基因的第二轮PCR扩增产物进行琼脂糖凝胶电泳，在500bp位置分别获得了TCRα链或β链可变区目的基因。将目的条带进行回收，并用T4连接酶将目的基因片段连接到T载体(pMD18T，Takara)。之后将连接产物转化DH5α细胞(购自天根生物)，并进行单克隆基因测序(委托睿博兴科)。
经过上述过程对所获得的KRAS-G12V 8-16特异性T细胞进行单细胞TCR基因扩增测序，并结果分析后，选取其中高频率出现的α链和β链组合为新的TCR，命名为1-2C和3-2E TCR，并进一步对其进行了结合及功能验证。
其中，1-2C TCR具有如SEQ ID NO:2的序列所示的α链可变区，和如SEQ ID NO:7的序列所示的β链可变区。3-2E TCR具有如SEQ ID NO:12的序列所示的α链可变区，和如SEQ ID NO:17的序列所示的β链可变区。
实施例2.KRAS-G12V 8-16(VVGAVGVGK)/HLA-A11四聚体与表达1-2C和3-2E TCR的细胞结合实验
在本实施例中，发明人进一步确认了所筛选的1-2C和3-2E TCR具有针对KRAS-G12V 8-16(VVGA VGVGK)/HLA-A11的特异性识别。除此之外，本实施例还发现，1-2C和3-2E除能够与HLA-A11呈递的KRAS-G12V 8-16(VVGA VGVGK)结合以外，也能够与HLA-A03呈递的KRAS-G12V 8-16(VVGA VGVGK)结合。
1. 1-2C和3-2E TCR结合特异性验证
首先，将1-2C和3-2E TCR的α链和β链可变区(V区)(1-2C：核酸序列如SEQ ID NO:1所示，氨基酸序列如SEQ ID NO:6所示，及3-2E：核酸序列如SEQ ID NO:7所示，氨基酸序列如SEQ ID NO:11所示)基因与人TCR的α链和β链恒定区(C区)基因(泓讯生物公司合成)连接，得到1-2C和3-2E嵌合TCRα和β链序列。嵌合序列的具体序列示于以下表1。
表1 1-2C和3-2E嵌合TCRα和β链序列
Figure PCTCN2021121576-appb-000001
并将1-2C和3-2E TCR的α链和β链中间以T2A序列(T2A序列的氨基酸序列如SEQ ID NO:42所示)连接的方式，以慢病毒表达质粒pCDH(购自Invitrogen)为出发质粒，分别构建了嵌合1-2C和3-2E TCR慢病毒表达载体，即慢病毒表达载体1-2C-pCDH和慢病毒表达载体3-2E-pCDH。
分别将1-2C或3-2E-pCDH慢病毒表达载体与表达CD3和CD8的CD3-CD8-pCDH质粒(购自南京金斯瑞公司)按照数量1:1的比例共转染HEK-293T细胞(购自ATCC)。共转染24小时后，将细胞在200-250g离心10min；用含有0.5％BSA的PBS洗三次，在200-250g离心10min，得到了表达1-2C或3-2E TCR的HEK-293T细胞。
然后，本发明人为了进一步验证1-2C或3-2E TCR与KRAS-G12野生型多肽及其他突变多肽的结合，分别将上述制备的KRAS-G12野生型多肽 8-16(VVGA GGVGK)、G12V 8-16(VVGA VGVGK)、G12D 8-16(VVGA DGVGK)、G12C 8-16(VVGA CGVGK)突变多肽/HLA-A11的四聚体与表达1-2C或3-2E TCR的293T细胞进行染色分析，以评价其结合特异性。
具体而言，将该共转染得到的HEK-293T细胞与上述制备的KRAS-G12V 8-16(VVGA VGVGK)/HLA-A11-PE四聚体，以及PerCP-Cy5-CD8和FITC-CD3抗体(BD公司)按照1:1:1摩尔比共同孵育30min；用含0.5％BSA的PBS洗三次，200-250g离心10min；用含0.5％BSA的PBS重悬细胞，用于检测KRAS-G12V 8-16(VVGA VGVGK)/HLA-A11阳性的T细胞频率。采用流式细胞技术进行了分析(示于图3A)。
图3A中的第1、2行(横向)为通过KRAS-G12V 8-16(VVGA VGVGK)/HLA-A11-PE四聚体对共转染的HEK-293T细胞染色分析的流式细胞图。结果显示，转染1-2C或3-2E TCR的293T细胞中能够结合KRAS-G12V 8-16(VVGA VGVGK)/HLA-A11-PE四聚体的细胞约占CD8阳性细胞的比例为25.9％。
通过对KRAS-G12野生型多肽及其他突变多肽/HLA-A11四聚体结合特异性的检测，证实表达1-2C或3-2E TCR的293T细胞不能够结合KRAS-G12野生型多肽(VVGA GGVGK)或G12D 8-16(VVGA DGVGK)与HLA-A11形成的四聚体；但能够结合G12C 8-16(VVGA CGVGK)与HLA-A11形成的四聚体(图3A)。
由此可知，细胞水平的结合实验表明，1-2C或3-2E TCR除了能够特异性结合G12V 8-16(VVGA VGVGK)/HLA-A11外，还能够特异性结合G12C 8-16(VVGA CGVGK)/HLA-A11复合物。
2. 1-2C和3-2E TCR与KRAS-G12 10肽结合特异性检测
本发明人考虑到CD8T细胞表位多肽的长度一般为9-10个氨基酸，鉴于上述检测的多肽均为9个氨基酸的多肽(9肽)，不能排除1-2C和3-2E TCR是否能够结合10个氨基酸的多肽(10肽)的可能性。
根据HLA-A11结合多肽的基序，发现其N端前移一个氨基酸仍可能成为T细胞表位，即KRAS-G12野生型多肽 7-16(如SEQ ID NO:38所示)、G12V 7-16(如SEQ ID NO:39所示)、G12D 7-16(如SEQ ID NO:40所示)、G12C 7-16(如SEQ ID NO:41所示)。本实施例中，使用同实施例1中的条件和操作进一步将这些长度为10的多肽制备成四聚体，与表达 1-2C或3-2E TCR的293T细胞进行了细胞结合实验分析，各操作及条件同上述G12D 8-16多肽，并将流式细胞的结果示于图3B。
结果显示，根据流式图的右上象限，可知10肽KRAS野生型及突变体多肽制备的四聚体均不能够结合表达1-2C或3-2E TCR的293T细胞。因此，进一步证明了本发明的1-2C或3-2E TCR与KRAS-G12V 8-16(VVGA VGVGK)和KRAS-G12C 8-16(VVGA CGVGK)多肽表位为高度特异性的识别。
3. 1-2C和3-2E TCR与HLA-A03呈递的KRAS-G12V 8-16(VVGAVGVGK)的交叉识别检测
已知HLA-A11属于HLA-A3超家族分子成员，HLA-A3超家族分子成员还包括HLA-A03、HLA-A31、HLA-A33及HLA-A68等，HLA-A3超家族分子具有类似的抗原呈递特征。HLA-A3超家族分子的抗原呈递特征在于，其所呈递的多肽C端一般为赖氨酸(K)或精氨酸(R)，而多肽N端起始的两个氨基酸和C端的氨基酸插入到HLA分子的多肽结合槽内，多肽中间部分氨基酸暴露而被TCR所识别。其次，HLA-A03、HLA-A31、HLA-A33及HLA-A68等HLA-A3超家族分子能够与TCR相互作用的α1和α2螺旋保守性超过70％。
因此，实施例1所筛选获得的TCR也能够具有识别HLA-A03、HLA-A31、HLA-A33及HLA-A68等HLA-A3超家族分子与KRAS-G12V 8-16(VVGAVGVGK)或其他突变多肽所形成的复合物的能力。本实施例中，使用HLA-A03与KRAS-G12V 8-16(VVGAVGVGK)制备四聚体，通过流式细胞检测方法对其与上述筛选出的1-2C和3-2E TCR的结合能力进行了检测，以确定其与其他HLA-A3超家族分子所呈递的KRAS-G12突变多肽的特异性结合能力。
因此，本实施例对1-2C和3-2E TCR与HLA-A03所呈递的KRAS-G12V 8-16(VVGAVGVGK)的交叉识别能力进行了检测。
其中使用的HLA-A03重链基因如SEQ ID NO:46所示，其编码的氨基酸序列如SEQ ID NO:45所示。其余使用的材料和操作均与HLA-A11中使用的相同。
本实施例利用上述实施例1所示方法同样制备了HLA-A03(IMGT/HLA Acc No:HLA00037)与KRAS-G12V 8-16(VVGAVGVGK)多肽的四聚体蛋白，并与KRAS-G12V 8-16(VVGAVGVGK)/HLA-A11四聚体一起，分别对表达1-2C或3-2E TCR的293T细胞进行染色分析(图4)。
结果表明，KRAS-G12V 8-16(VVGAVGVGK)/HLA-A03四聚体能够显著结合表达1-2C或3-2E TCR的293T细胞(图4B，右上象限)。
因此，1-2C或3-2E TCR除了能够结合HLA-A11呈递的KRAS-G12V 8-16(VVGAVGVGK)多肽，还能够结合HLA-A03呈递的KRAS-G12V 8-16(VVGAVGVGK)多肽，提示其能够在更广的人群中具有广泛的应用价值。
实施例3. 1-2C和3-2E TCR-T细胞制备及其与KRAS不同突变多肽的反应
本实施例中，将1-2C或3-2E TCR基因导入从HLA-A11遗传背景的健康志愿者中分离的外周血单个核细胞(PBMC)或分离的CD8T细胞作为TCR-T效应细胞。将KRAS-G12野生型多肽 8-16(VVGA GGVGK)、G12V 8-16(VVGA VGVGK)、G12D 8-16(VVGA DGVGK)、G12C 8-16(VVGA CGVGK)突变多肽，分别加入到上述TCR-T效应细胞的体系共同培养，检测了效应细胞和呈递KRAS野生型及突变体多肽的靶细胞作用后分泌的IFN-γ的水平，对1-2C或3-2E TCR与表达KRAS野生型及突变体多肽/HLA-A11的靶细胞的作用进行评价。具体操作如下。
1.表达1-2C和3-2E TCR的慢病毒的制备
将实施例2中的1-2C和3-2E TCR慢病毒表达质粒(1-2C–pCDH和3-2E–pCDH)与慢病毒包装质粒PLP1、PLP2和VSVG(购自Addgene公司)按照PLP1:PLP2:VSVG:TCR-pCDH＝20:13:5:20的比例混合，并取20ul稀释到DMEM培养基(1.25ml)中，作为DNA溶液。取20ul聚醚酰亚胺(PEI，1μg/μl)加入到DMEM(1.25ml)中，并将上述PEI/DMEM溶液全部加入到已经配好的DNA溶液中，室温孵育15分钟后加入15cm盘培养的293T细胞(上海细胞库)中并混匀。6小时后， 小心吸出培养液，加入25ml新的培养液继续培养。72小时后，收集含有病毒的上清液，即为表达1-2C或3-2E TCR的慢病毒上清。
2. 1-2C和3-2E TCR-T细胞制备及TCR表达效率检测
采集两名健康志愿者(D1和D2)的外周血淋巴细胞，得到PBMC，并取部分PBMC通过磁珠(Biolegend公司)负选分离其中的CD8T细胞。用包被有anti-CD3/anti-CD28的微球(ThermoFisher)按照1:1的数量比例加入到PBMC或CD8T细胞中活化培养过夜，之后将1-2C或3-2E TCR慢病毒按照1:1体积比加入到PBMC或CD8T细胞中，混匀，设无病毒感染孔做对照，置于37℃、5％CO2孵箱中培养。24小时后，换成完全培养基继续培养至第10天。
将anti-CD3/anti-CD28的微球在磁场条件下除掉，并用与细胞培养相同的培养基进行两次清洗，得到本实施例的1-2C或3-2E TCR-T效应细胞。
将上述利用PBMC或CD8T细胞制备的，表达1-2C或3-2E TCR-T细胞(已培养至10天)进行培养，利用KRAS-G12V 8-16(VVGA VGVGK)/HLA-A11四聚体染色与实施例2相同地进行了流式细胞检测，确认了1-2C或3-2E TCR的表达(图5，右上象限)。
结果表明，利用PBMC或CD8+T细胞制备的来自志愿者D1的1-2C、3-2E及来自志愿者D2的D1、3-2E-D2TCR-T细胞中均能够检测到具有0.26％-2.38％的不同阳性率的四聚体阳性T细胞，其中，PBMC中的分别为1-2C：0.26％，0.77％，3-2E：1.03％，0.55％，而CD8+T细胞的中的分别为1-2C：0.68％，0.91％，3-2E：2.38％，2.37％。
可知这些本发明的TCR-T效应细胞能够特异性结合KRAS-G12V 8-16(VVGA VGVGK)/HLA-A11。
3. 1-2C和3-2E TCR-T细胞与KRAS野生型及突变体多肽的免疫反应检测
之后，分别利用不同的T细胞检测方法(IFN-γ-ELISPOT和IFN-γ-ELISA)对1-2C或3-2E TCR-T细胞与呈递KRAS野生型及突变体多肽的靶细胞作用后分泌的IFN-γ的水平进行检测。
将利用D1和D2两名志愿者的PBMC或CD8T细胞制备的1-2C或3-2E TCR-T细胞，各自来源对应地与D1和D2两名志愿者的PBMC按照1:1的比例混合作为抗原递呈细胞，并按照100μl体积1×10 5细胞数/孔加入到预包被有anti-IFN-γ抗体的ELISPOT板中，同时加入KRAS野生型及突变体多肽(100μl体积，10μg/ml)，置37℃100％湿度5％CO2细胞培养箱中继续培养18小时，将施加了PMA/伊屋诺霉素(ION)(100μl体积，1μg/ml)刺激或仅100μl培养基的样品分别作为阳性和阴性对照。对产生IFN-γ的特异性T细胞进行了ELISPOT斑点分析，将结果示于图6A-B,D-E。
进一步，将利用D1和D2两名志愿者的PBMC或CD8T细胞制备的1-2C或3-2E TCR-T细胞，与D1和D2两名志愿者的PBMC按照1:1的比例混合，以作为抗原递呈细胞。将准备好的细胞悬液铺至96孔板内，每孔100μl细胞悬液(2×10 5个细胞)，每孔做三个重复，其中PMA/伊屋诺霉素(ION)组按照每250μl细胞培养基中加入1μl PMA/伊屋诺霉素混合液(250×)提前稀释成工作液，并作为阳性刺激对照。未感染1-2C或3-2E TCR慢病毒的T细胞(D1mock和D2mock)平行加入，作为阴性对照。在37℃下培养20h后，取96孔板培养孔的上清，并于500g转速离心5min去掉残留的细胞，将上清加入包被有抗IFN-γ抗体(BD公司)的ELISA检测板(BD公司)，检测上清中的IFN-γ水平，将结果示于图6C和F。
ELISPOT实验结果表明，利用D1和D2志愿者的PBMC制备的1-2C或3-2E TCR-T细胞均能够对KRAS-G12V突变多肽产生较强的T细胞免疫反应，对KRAS-G12C突变多肽也具有一定水平的交叉反应。另一方面，未检测到对KRAS野生型和KRAS-G12D突变多肽的T细胞反应(图6A-B)。在利用D1和D2志愿者的CD8T细胞制备的1-2C或3-2E TCR-T细胞中，能够检测到与PBMC制备的TCR-T细胞类似的T细胞免疫反应(图6D-E)。
IFN-γ水平的ELISA实验结果表明，利用D1和D2志愿者的PBMC或CD8T细胞制备的1-2C或3-2E TCR-T细胞均能够对KRAS-G12V突 变多肽产生较强的T细胞免疫反应，IFN-γ水平升高，而未检测到对KRAS野生型和KRAS-G12D突变多肽的T细胞反应(图6C和F)。
因此，可知1-2C和3-2E TCR-T细胞能够特异性识别呈递KRAS-G12V 8-16(VVGA VGVGK)/HLA-A11的靶细胞，并对呈递KRAS-G12C 8-16(VVGA CGVGK)的靶细胞具有一定的交叉反应，能够特异性分泌细胞因子IFN-γ。鉴于CD8+T细胞对靶细胞的潜在细胞毒性作用，提示本发明的1-2C和3-2E TCR-T细胞具有潜在的靶细胞杀伤活性和肿瘤治疗价值。
实施例4. 1-2C和3-2E TCR对KRAS突变体多肽表位的结合特性分析
为了准确测定1-2C和3-2E TCR与KRAS不同突变体多肽/HLA-A11复合物蛋白的结合特性和亲和力，发明人进一步利用表面等离子共振实验(SPR)在蛋白水平的亲和力进行了检测。由于1-2C或3-2E TCR的功能区是胞外区，且不含跨膜区的胞外区是可溶蛋白，所以合成了1-2C或3-2E TCR的胞外区。具体操作如下。
1.TCR蛋白表达与纯化
将1-2C或3-2E TCRα链和β链的胞外区基因按照原核密码子进行优化，分别合成1-2C和3-2E TCR嵌合α链和β链的胞外区的DNA序列(1-2C：α链，SEQ ID NO:29；β链，SEQ ID NO:30；3-2E:α链，SEQ ID NO:31；β链SEQ ID NO:32)，其中，1-2C TCR具有如SEQ ID NO:2的序列所示的α链可变区，和如SEQ ID NO:7的序列所示的β链可变区。3-2E TCR具有如SEQ ID NO:12的序列所示的α链可变区，和如SEQ ID NO:17的序列所示的β链可变区。分别引入酶切位点Nde I和Xho I，其中Nde I酶切位点位于序列的5’端，酶切位点Xho I位于序列的3’端。利用酶切位点Nde I和Xho I将合成的1-2C或3-2E TCRα链和β链的胞外区的DNA序列分别克隆入表达载体pET21a(Invitrogen公司)，建立了1-2C或3-2E TCRα链和β链的胞外区蛋白的原核重组表达质粒。
将表达质粒用热激法转入E.coli.BL21(DE3)感受态细胞，加入IPTG进行诱导表达，获得包涵体状态的1-2C或3-2E TCRα链和β链的胞外区蛋白。
将6ml 1-2C或3-2E TCRα链和β链的胞外区的包涵体(各包涵体均以30mg/ml溶于含6M Gua-HCl、50mM Tris pH8.0、100mM NaCl、10mM EDTA和10mM DTT的溶解液中)按照2:1的质量比滴在1L配好的复性液(5M脲，20mM Tris-HCL，400mM L-精氨酸，EDTA 2mM，GSH/GSSG 5mM/1mM)中，分两次滴加，每次滴加3mL，两次滴加之间间隔最少8h，然后用浓缩杯(Millipore公司)浓缩。
浓缩后分别置于4L去离子水和4L 10mM Tris，pH 8.0的透析液中，各透析24h。之后用Source 15Q离子交换层析进行初步纯化，通过SDS-PAGE鉴定目的蛋白。
具体而言，将目的蛋白用浓缩杯(Millipore公司)浓缩，并用20mM Tris-HCL，150mM NaCL，pH 8.0的缓冲液换液，浓缩后用Superdex200pg分子筛(GE Healthcare)纯化，得到1-2C或3-2E TCR蛋白约2-3mg，还原性(含二硫苏糖醇(DTT))和非还原性(不含二硫苏糖醇(DTT))SDS-PAGE检测目的蛋白(图7)。
结果表明，1-2C TCR在18.19mS/cm条件下洗脱，3-2E TCR在3-2E21.19mS/cm条件下洗脱，二者分子筛层析在15mL洗脱体积时出现了蛋白目的峰，且通过SDS-PAGE鉴定发现，1-2C或3-2E呈现αβ异源二聚体，其在不加DTT的非还原条件的SDS-PAGE中条带大小约为52KD，加入DTT的还原条件的SDS-PAGE中α链和β链间二硫键被打开，分别显示为24KD和28KD左右大小的条带(图7)。
2.SPR检测分析
将如实施例1中同样的操作进行了体外复性实验而制备的1-2C和3-2E TCR蛋白、以及实施例1中制备的生物素化的KRAS野生型和不同突变体9肽/HLA-A11复合物蛋白换液至SPR缓冲液中(10mM HEPES-HCl，150mM Na-Cl，0.005％Tween-20，pH 7.4)。将KRAS野生型和不同突变体9肽/HLA-A11复合物蛋白稀释到20μg/ml固定到SA 芯片(GE Health)上，之后将梯度(0μM、6.25μM、12.5μM、25μM、50μM、100μM)稀释的1-2C和3-2E TCR蛋白分别流过SA芯片各通道，利用BIA评价软件分析结合动力学参数，并计算亲和力常数。进行了对1-2C和3-2E TCR与野生型及KRAS野生型和不同突变体9肽/HLA-A11复合物蛋白亲和力的检测(图8)。
结果表明，从图8中曲线可知，1-2C TCR结合KRAS-G12V 8-16(VVGA VGVGK)/HLA-A11的结合呈现快结合快解离的模式，结合亲和力(KD)为7.21μM,与KRAS-G12C 8-16(VVGA CGVGK)/HLA-A11结合亲和力(KD)为46.2μM,显著低于KRAS-G12V 8-16表位。
而1-2C不能结合KRAS野生型KRAS-G12 8-16(VVGA GGVGK)/HLA-A11和KRAS-G12D 8-16(VVGA DGVGK)/HLA-A11。3-2E TCR与KRAS-G12V 8-16(VVGA VGVGK)/HLA-A11的结合亲和力(KD)为35.9μM,显著低于1-2C的亲和力；与KRAS-G12C 8-16(VVGA CGVGK)/HLA-A11结合亲和力(KD)为50.5μM,与KRAS-G12V 8-16在同一水平。
与1-2C类似，3-2E TCR不能结合KRAS野生型KRAS-G12 8-16(VVGA GGVGK)/HLA-A11和KRAS-G12D 8-16(VVGA DGVGK)/HLA-A11。
因此，从SPR结果可以得出，1-2C TCR和3-2E TCR能够特异性结合KRAS-G12V 8-16(VVGA VGVGK)/HLA-A11，1-2C TCR与KRAS-G12C 8-16(VVGA CGVGK)/HLA-A11存在一定的交叉反应，而1-2C TCR与KRAS-G12C 8-16(VVGA CGVGK)/HLA-A11亲和力与KRAS-G12V在同一水平。因此，1-2C和3-2E TCR具有良好的结合特性和亲和力，可以推测当将1-2C和3-2E TCR用于抗肿瘤治疗中，能够对携带基因KRAS的G12V和G12C突变的肿瘤细胞产生IFN-γ，进而杀伤肿瘤细胞，达到治疗肿瘤的作用。
实施例5. 1-2C TCR-T细胞在肿瘤小鼠模型中的肿瘤抑制活性
本实施例应用NCG免疫缺陷小鼠PANC-1肿瘤模型评价1-2C TCR-T细胞的肿瘤抑制效果。
TCR-T细胞的NCG小鼠肿瘤抑制实验步骤包括：
1.NCG小鼠PANC-1肿瘤模型建立
NCG小鼠来源于南京大学-南京生物医药研究院，向每只NCG小鼠皮下接种携带KRAS-G12V突变基因的PANC-1肿瘤细胞(北京协和细胞资源中心)，在NCG小鼠中建立具有人源免疫系统的小鼠：
a)接种PANC-1细胞数量：4×10 6个细胞/200μL/只；
b)接种部位：背部皮下；
2. 1-2C TCR-T细胞治疗
NCG小鼠PANC-1肿瘤细胞接种第7天，肿瘤体积生长至约200mm 3，将实施例3中以D1和D2两名志愿者PBMC制备的1-2C TCR-T细胞瘤内多点注射到NCG小鼠的PANC-1肿瘤内部：
a)接种1-2C TCR-T细胞数量：按照三个剂量：分别为1×10 7、1×10 6、1×10 5细胞/200μL/只；
b)接种部位：瘤内；
3.分组及处理：
肿瘤细胞注射后约1周后选择成瘤较为均一的小鼠进行分组，之后进行1-2C TCR-T细胞瘤内注射治疗。本实施例以未转入TCR的T细胞(1×10 7)注射组为阴性对照，每组6只小鼠，其中D1和D2制备的TCR-T细胞治疗组各3只。各分组信息及处理如下表所示：
表2.小鼠分组及治疗处理
小鼠分组 细胞注射及剂量 小鼠数量
阴性对照组 1×10 7，200μL 6只
1-2C TCR-T细胞高剂量治疗组 1×10 7，200μL 6只
1-2C TCR-T细胞中剂量治疗组 1×10 6，200μL 6只
1-2C TCR-T细胞低剂量治疗组 1×10 5，200μL 6只
成瘤后每三到四天检测一次肿瘤大小，至小鼠最大肿瘤体积长4000mm 3时实验结束，将小鼠处死并分离肿瘤称重。
4.治疗效果观察：
1)肿瘤大小检测：
a)在TCR-T细胞注射后用卡尺进行直径测量，单位为mm，计算公式为：v＝1/2×a×b×b(a为长径，b为短径)；
b)最后一次观察后实验终止，分离肿瘤组织直接称重；
结果表明，注射1×10 7高剂量和1×10 6中剂量组的1-2C TCR-T细胞治疗组肿瘤体积显著小于阴性对照组(T检验，p<0.01)(图9C),而1×10 5低剂量组与阴性对照组肿瘤体积无显著差异(T检验，p>0.05)。实验终止时对每组肿瘤重量的分析表明，高剂量组瘤重显著低于阴性对照组(图9A)。本实施例结果表明，1-2C TCR-T细胞能够有效抑制肿瘤生长，且抑瘤活性与TCR-T细胞数量呈现显著的剂量依赖效应，具有潜在的肿瘤治疗价值(如图9)。

Claims (11)

  1. T细胞受体(TCR)或其抗原结合片段，所述TCR或其抗原结合片段能够与KRAS-G12V 8-16(VVGA VGVGK)表位和HLA-A11复合物，或KRAS-G12C 8-16(VVGA CGVGK)表位和HLA-A11复合物，或KRAS-G12V 8-16(VVGA VGVGK)表位和HLA-A03复合物结合，并且所述TCR包含α链可变区和β链可变区，其特征在于，所述TCR或其抗原结合片段包含以下的α链互补决定区(CDR)和β链互补决定区(CDR)：
    如SEQ ID NO:3所示的α链互补决定区CDR1；
    如SEQ ID NO:4所示的α链互补决定区CDR2；
    如SEQ ID NO:5所示的α链互补决定区CDR3；
    如SEQ ID NO:8所示的β链互补决定区CDR1；
    如SEQ ID NO:9所示的β链互补决定区CDR2；和
    如SEQ ID NO:10所示的β链互补决定区CDR3，
    或
    如SEQ ID NO:13所示的α链互补决定区CDR1；
    如SEQ ID NO:14所示的α链互补决定区CDR2；
    如SEQ ID NO:15所示的α链互补决定区CDR3；
    如SEQ ID NO:18所示的β链互补决定区CDR1；
    如SEQ ID NO:19所示的β链互补决定区CDR2；和
    如SEQ ID NO:20所示的β链互补决定区CDR3。
  2. 如权利要求1所述的T细胞受体(TCR)或其抗原结合片段，其包含：
    如SEQ ID NO:2的序列所示的α链可变区，和
    如SEQ ID NO:7的序列所示的β链可变区；
    或
    如SEQ ID NO:12的序列所示的α链可变区，和
    如SEQ ID NO:17的序列所示的β链可变区。
  3. 根据权利要求1或2所述的TCR或其抗原结合片段，其中所述TCR为鼠源TCR、人鼠嵌合TCR或人源化TCR。
  4. 多核苷酸，其编码权利要求1-3中任一项所述的TCR或其抗原结合片段，其为选自由SEQ ID NO:1，SEQ ID NO:6，SEQ ID NO:11，SEQ ID NO:16组成的组中的一个或多个序列。
  5. 表达载体，其包含权利要求4所述的多核苷酸，所述表达载体为慢病毒载体。
  6. 宿主细胞，其包含权利要求5所述的表达载体。
  7. 制备权利要求1-3中任一项所述的TCR或其抗原结合片段的方法，所述方法包括：
    1)培养权利要求6所述的宿主细胞；
    2)从所述宿主细胞或其培养基中回收权利要求1-3中任一项所述的TCR或其抗原结合片段。
  8. 药物组合物，其包含权利要求1-3中任一项所述的TCR或其抗原结合片段，和药学上可接受的载体。
  9. 权利要求1-3中任一项所述的TCR或其抗原结合片段在制备用于提高T细胞分泌IFN-γ的细胞因子水平的药物中的用途，其中所述药物例如为蛋白类药物、ADC药物或TCR与抗原组合的药物。
  10. 权利要求1-3中任一项所述的TCR或其抗原结合片段在制备表达KRAS-G12V 8-16(VVGA VGVGK)或KRAS-G12C 8-16(VVGA CGVGK)突变的肿瘤细胞的检测试剂中的用途，或在制备检测或诊断肿瘤的试剂 中的用途，优选地，所述TCR或其抗原结合片段与KRAS-G12V 8-16(VVGA VGVGK)/HLA-A11，或KRAS-G12C 8-16(VVGA CGVGK)/HLA-A11，或KRAS-G12V 8-16(VVGA VGVGK)表位和HLA-A03特异性结合，或
    所述TCR或其抗原结合片段与具有与HLA-A11或HLA-A03类似的抗原结合特性的HLA分子，与KRAS-G12V 8-16(VVGA VGVGK)或KRAS-G12C 8-16(VVGA CGVGK)突变多肽特异性结合，其中，所述HLA分子优选为HLA-A31,HLA-A33,HLA-A68，HLA-A30。
  11. 权利要求1-3中任一项所述的TCR或其抗原结合片段在制备用于治疗携带KRAS基因的G12V和G12C突变的肿瘤的患者的抗肿瘤药物中的用途，所述肿瘤优选胰腺癌、结直肠癌、肺癌，所述肺癌优选为非小细胞肺癌，优选地，所述KRAS基因的G12V和G12C突变为KRAS基因的KRAS-G12V 8-16(VVGA VGVGK)突变或KRAS-G12C 8-16(VVGA CGVGK)突变。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114853880A (zh) * 2022-04-24 2022-08-05 中国科学院微生物研究所 Wt1抗原特异性t细胞受体及其抗肿瘤用途

Families Citing this family (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112300269B (zh) * 2020-09-29 2022-12-09 中国科学院微生物研究所 Kras突变特异性t细胞受体筛选及抗肿瘤用途
CN116063511B (zh) * 2021-09-30 2023-10-03 北京可瑞生物科技有限公司 抗原结合蛋白及其应用
WO2023102418A1 (en) * 2021-12-01 2023-06-08 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Hla-a3-restricted t cell receptors against ras with g12v mutation
KR20230101287A (ko) * 2021-12-29 2023-07-06 의료법인 명지의료재단 K-ras 특이적 활성화 T 세포를 포함하는 흑색종의 예방 및 치료용 약제학적 조성물 및 이의 제조방법
KR20230101284A (ko) * 2021-12-29 2023-07-06 의료법인 명지의료재단 K-ras 특이적 활성화 T 세포를 포함하는 대장암의 예방 및 치료용 약제학적 조성물 및 이의 제조방법
KR20230101285A (ko) * 2021-12-29 2023-07-06 의료법인 명지의료재단 K-ras 특이적 활성화 T 세포를 포함하는 유방암의 예방 및 치료용 약제학적 조성물 및 이의 제조방법
KR20230101283A (ko) * 2021-12-29 2023-07-06 의료법인 명지의료재단 K-ras 특이적 활성화 T 세포를 포함하는 폐 유두상 선암종의 예방 및 치료용 약제학적 조성물 및 이의 제조방법
KR20230101286A (ko) * 2021-12-29 2023-07-06 의료법인 명지의료재단 K-ras 특이적 활성화 T 세포를 포함하는 폐 선암종의 예방 및 치료용 약제학적 조성물 및 이의 제조방법
CN115109139B (zh) * 2022-04-01 2023-10-24 重庆医科大学 Tcr或其抗原结合片段及其应用
CN114920823B (zh) * 2022-05-27 2023-10-17 重庆医科大学 Tcr或其抗原结合片段及其应用
CN114835800B (zh) * 2022-05-27 2023-10-13 重庆医科大学 Tcr或其抗原结合片段及其应用
CN114920824B (zh) * 2022-05-27 2023-12-05 重庆医科大学 Tcr或其抗原结合片段及其应用
CN117264043B (zh) * 2022-06-14 2024-05-10 上海镔铁生物科技有限责任公司 靶向kras g12v突变多肽的t细胞受体及其用途
WO2024081674A1 (en) 2022-10-11 2024-04-18 Aadi Bioscience, Inc. Combination therapies for the treatment of cancer
CN117430689A (zh) * 2022-11-04 2024-01-23 新景智源生物科技(苏州)有限公司 Kras_g12v突变抗原特异性tcr及其与cd8共表达重定向cd4 t细胞

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170319638A1 (en) * 2016-05-06 2017-11-09 Virttu Biologics Limited Treatment of cancer
CN108137685A (zh) * 2015-03-23 2018-06-08 约翰·霍普金斯大学 由体细胞突变基因编码的hla限制性表位
WO2020041501A1 (en) * 2018-08-22 2020-02-27 Fred Hutchinson Cancer Research Center Immunotherapy targeting kras or her2 antigens
CN112300269A (zh) * 2020-09-29 2021-02-02 中国科学院微生物研究所 Kras突变特异性t细胞受体筛选及抗肿瘤用途

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4286407A3 (en) * 2014-11-26 2024-03-06 The United States of America, as represented by The Secretary, Department of Health and Human Services Anti-mutated kras t cell receptors
IL273254B1 (en) * 2017-09-20 2024-05-01 Us Health HLA type II-restricted T cell receptors against RAS
CN109957582A (zh) * 2017-12-26 2019-07-02 上海尚泰生物技术有限公司 一种靶向肿瘤多种kras突变抗原表位的细胞毒性t淋巴细胞的制备方法
CN109293739B (zh) * 2018-01-24 2021-03-30 中国疾病预防控制中心病毒病预防控制所 一种a3超家族通用肿瘤抗原多肽及其应用
WO2020172332A1 (en) * 2019-02-20 2020-08-27 Fred Hutchinson Cancer Research Center Binding proteins specific for ras neoantigens and uses thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108137685A (zh) * 2015-03-23 2018-06-08 约翰·霍普金斯大学 由体细胞突变基因编码的hla限制性表位
US20170319638A1 (en) * 2016-05-06 2017-11-09 Virttu Biologics Limited Treatment of cancer
WO2020041501A1 (en) * 2018-08-22 2020-02-27 Fred Hutchinson Cancer Research Center Immunotherapy targeting kras or her2 antigens
CN112300269A (zh) * 2020-09-29 2021-02-02 中国科学院微生物研究所 Kras突变特异性t细胞受体筛选及抗肿瘤用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LILI QIN;YIJIAN LI;ZHAODUAN LIANG;LEI CHEN;WENHUI LI;CHAO CHEN;YALING HUANG;LE ZHANG;SONGMING LIU;SI QIU;YUPING GE;WENTING PENG;XI: "A Method of Screening Highly Common Neoantigens with Immunogenicity in Colorectal Cancer based on Public Somatic Mutation Library", HEREDITAS, vol. 42, no. 6, 20 May 2020 (2020-05-20), pages 599 - 612, XP055917602, ISSN: 0253-9772, DOI: 10.16288/j.yczz.20-032 *
Q. J. WANG, Z. YU, K. GRIFFITH, K.-I. HANADA, N. P. RESTIFO, J. C. YANG: "Identification of T-cell Receptors Targeting KRAS-Mutated Human Tumors", CANCER IMMUNOLOGY RESEARCH, vol. 4, no. 3, 1 March 2016 (2016-03-01), US , pages 204 - 214, XP055314168, ISSN: 2326-6066, DOI: 10.1158/2326-6066.CIR-15-0188 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114853880A (zh) * 2022-04-24 2022-08-05 中国科学院微生物研究所 Wt1抗原特异性t细胞受体及其抗肿瘤用途
CN114853880B (zh) * 2022-04-24 2023-11-10 中国科学院微生物研究所 Wt1抗原特异性t细胞受体及其抗肿瘤用途

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