TCR or antigen binding fragment thereof and application thereof

Abstract

The present invention belongs to the technical field of T cell immunotherapy drugs, and specifically relates to a T cell receptor (TCR) or an antigen binding fragment thereof and an application thereof, wherein the TCR comprises a sequence as shown in SEQ ID NO: 37. The TCR has a strong specificity for the KRAS-G12D mutation, and can mediate T cells to secrete a large amount of IFN-γ or IFN-α cytokines and kill tumor cells with KRAS-G12D gene mutations.

Description

Technical Field

[0001] The present invention belongs to the technical field of T cell immunotherapy drugs, and specifically relates to a TCR or an antigen binding fragment thereof and applications thereof. Background Art

[0002] With the progress of research in the biomedical field, the target selection of T cell immunotherapy for malignant solid tumors has been constantly evolving, from the initial lineage antigens to viral antigens. With the emergence of high-throughput sequencing technology in recent years, targeting individual tumor mutations has become the focus of the new generation of T cell therapy.

[0003] T cell therapy currently mainly includes TCR-T and CAR-T treatment methods. At present, CAR-T technology treatment has played a significant role in the treatment of blood tumors such as leukemia and lymphoma, greatly improving the survival rate and quality of life of patients. However, for solid tumors, CAR-T treatment methods have limited specific targets, which greatly limits its application prospects. TCR-T technology is different from CAR-T cell technology. TCR is a characteristic marker on the surface of all T cells, which binds to CD3 with non-covalent bonds to form a TCR-CD3 complex. In peripheral blood, 90%-95% of T cells express TCR. T cells with genetically modified TCR can specifically recognize antigen molecules on the surface of tumor cells, and then produce immune responses against tumor cells. Currently, in human cancer, the KRAS gene is one of the most famous oncogenes in the field of oncology. KRAS gene mutations (KRAS-G12C mutation, KRAS-G12D mutation, KRAS-G12V mutation, etc.) appear in nearly 90% of pancreatic cancer, 30%-40% of colon cancer, 17% of endometrial cancer, 15%-20% of lung cancer including lobular lung cancer, as well as bile duct cancer, cervical cancer, bladder cancer, etc.

[0004] Based on the above situation, there is a need for a TCR that can specifically bind to the KRAS mutant polypeptide and mediate T cells to kill KRAS mutant tumor cells. Summary of the invention

[0005] One of the purposes of the present invention is to provide a TCR that can specifically target and recognize the KRAS-G12D mutation site, specifically bind to the complex of the KRAS-G12D mutant polypeptide and HLA, thereby stimulating T cell activation, mediating T cells to secrete cytokines such as IFN-γ, and then killing tumor cells expressing the KRAS-G12D mutant gene. The TCR or its antigen-binding fragment has strong specificity for the KRAS-G12D mutant polypeptide, and can mediate T cells to secrete a large amount of IFN-γ cytokines to kill KRAS-G12D mutant tumor cells.

[0006] In one aspect, the present invention provides a TCR, which can be selected from the following two TCRs:

[0007] The first type: the α chain variable region of TCR may include an α chain CDR3 with a sequence as shown in SEQ ID NO:3, and the β chain variable region of TCR may include a β chain CDR3 with a sequence as shown in SEQ ID NO:12.

[0008] The second type: TCR may include a fragment obtained from the sequence shown in SEQ ID NO:37.

[0009] Another aspect of the present invention provides an antigen-binding fragment, which may comprise any one of the two TCRs mentioned above.

[0010] In another aspect, the present invention provides a polynucleotide that can be selected from the following two polynucleotides:

[0011] The first type may include sequences shown as SEQ ID NO:21 and SEQ ID NO:30.

[0012] The second type: may contain the sequence shown in SEQ ID NO:38.

[0013] In another aspect, the present invention provides an expression vector, which may contain any one of the two polynucleotides mentioned above.

[0014] In another aspect, the present invention provides an engineered cell, which may contain the above-mentioned expression vector.

[0015] In another aspect, the present invention provides a pharmaceutical composition, which may include the above-mentioned TCR or the above-mentioned antigen-binding fragment or the above-mentioned polynucleotide or the above-mentioned expression vector or the above-mentioned engineered cell, and a pharmaceutically acceptable carrier and / or diluent.

[0016] In another aspect, the present invention provides a use of the above-mentioned TCR or the above-mentioned antigen-binding fragment or the above-mentioned polynucleotide or the above-mentioned expression vector or the above-mentioned engineered cell in the preparation of a drug for increasing the level of IFN-γ and / or TNF-α cytokine secretion by T cells.

[0017] In another aspect, the present invention provides a use of the above-mentioned TCR or the above-mentioned antigen-binding fragment or the above-mentioned polynucleotide or the above-mentioned expression vector or the above-mentioned engineered cell in the preparation of a reagent or a kit for detecting tumor cells expressing KRAS-G12D mutation.

[0018] In another aspect, the present invention provides a use of the above-mentioned TCR or the above-mentioned antigen-binding fragment or the above-mentioned polynucleotide or the above-mentioned expression vector or the above-mentioned engineered cell in the preparation of a drug for treating a disease caused by carrying the KRAS-G12D mutation.

[0019] The beneficial effects of the present invention are as follows: the TCR provided by the present invention has strong specificity for the KRAS-G12D mutant polypeptide, and can mediate T cells to secrete a large amount of IFN-γ or IFN-α cytokines to kill KRAS-G12D mutant tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a flow cytometry test result of KT13 specifically binding to KRAS-G12D mutant polypeptide in the Example;

[0021] Figure 2 The figure is a graph showing the specific activation results of KT13 in the examples on KRAS-G12D mutant polypeptides at different concentrations;

[0022] Figure 3 The KT13 in the embodiment mediates the secretion of IFN-γ cytokine by T cells;

[0023] Figure 4 The KT13 in the embodiment mediates the secretion of IFN-α cytokine by T cells;

[0024] Figure 5 This is the killing effect of KT1 on KRAS-G12D mutant cells in the example. DETAILED DESCRIPTION

[0025] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0026] In the present invention, the term "antigen binding fragment" refers to antigen binding fragments of TCR and TCR analogs, which generally include at least part of the antigen binding region or variable region of the parent TCR, such as one or more CDRs. The fragment of TCR retains at least some of the binding specificity of the parent TCR.

[0027] The present invention aims to provide a TCR, one of the purposes of the present invention is to provide a TCR that can specifically recognize the KRAS-G12D mutation site, specifically bind to the complex of the KRAS-G12D mutant polypeptide and HLA, thereby stimulating T cell activation, mediating T cells to secrete cytokines such as IFN-γ, and then killing tumor cells expressing KRAS-G12D mutations. The TCR or its antigen-binding fragment has strong specificity for KRAS-G12D mutant polypeptides and can mediate T cells to secrete a large amount of IFN-γ cytokines.

[0028] In one aspect, the present invention provides a TCR, wherein the TCR comprises an α chain variable region and a β chain variable region. The TCR in the present invention can be selected from the following TCRs:

[0029] The first TCR:

[0030] In the first TCR, the α chain variable region comprises at least the α chain CDR3 having the sequence shown in SEQ ID NO:3, and the β chain variable region comprises at least the β chain CDR3 having the sequence shown in SEQ ID NO:12.

[0031] Furthermore, the α chain variable region in the first TCR may also include: an α chain CDR1 with a sequence as shown in SEQ ID NO:1 and / or an α chain CDR2 with a sequence as shown in SEQ ID NO:2; the β chain variable region may also include: a β chain CDR1 with a sequence as shown in SEQ ID NO:10 and / or a β chain CDR2 with a sequence as shown in SEQ ID NO:11.

[0032] Furthermore, the α chain variable region in the first TCR may also include: one or more of: an α chain FR1 with a sequence as shown in SEQ ID NO:4, an α chain FR2 with a sequence as shown in SEQ ID NO:5, an α chain FR3 with a sequence as shown in SEQ ID NO:6, and an α chain FR4 with a sequence as shown in SEQ ID NO:7; the β chain variable region may also include: one or more of: a β chain FR1 with a sequence as shown in SEQ ID NO:13, a β chain FR2 with a sequence as shown in SEQ ID NO:14, a β chain FR3 with a sequence as shown in SEQ ID NO:15, and a β chain FR4 with a sequence as shown in SEQ ID NO:16.

[0033] Specifically, the CDR3 of the α chain and β chain in the above-mentioned TCR belongs to the hypervariable region of the variable region. When the TCR recognizes the MHC-antigen peptide complex, CDR3 directly binds to the antigen peptide, which directly affects the TCR's ability to recognize the KRAS-G12D mutation. The hypervariable regions of the α chain and β chain of the TCR also include two hypervariable regions, CDR1 and CDR2. The CDR1 and CDR2 hypervariable regions are relatively stable relative to CDR3. When the TCR recognizes the MHC-antigen peptide complex, the CDR1 and CDR2 hypervariable regions recognize and bind to the side walls of the antigen binding groove of the MHC molecule.

[0034] Specifically, the FR (FR1, FR2, FR3, FR4) in the above-mentioned TCR is a framework region, which is used to connect the CDR region and is relatively stable. In addition to selecting the above-mentioned sequences, the FR region in the above-mentioned first TCR can also select sequences with a sequence identity of ≥80% or more with the above-mentioned FR1, FR2, FR3 and FR4, such as ≥80%, 85%, 90% or 95%, and the source can be mouse or human (but other sources, such as rabbit, pig and other animal sources are not excluded). In some specific implementations, the sequences of the FR regions of the above-mentioned α chain and β chain and the sequences of the CDR regions can be arranged according to FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, respectively constituting the α chain variable region and the β chain variable region of the two TCRs, the α chain variable region comprises the sequence shown in SEQ ID NO: 8, and the encoding β chain variable region comprises the sequence shown in SEQ ID NO: 17.

[0035] Specifically, in addition to the above-mentioned α chain variable region and β chain variable region, the α chain also includes an α chain constant region, and the β chain also includes a β chain constant region. The sources of the α chain constant region and the β chain constant region can be mouse, human or human-mouse chimera (but other sources, such as rabbit, pig and other animal sources are not excluded), which is almost a region that will not mutate. In addition, the α chain and β chain of TCR can also include a signal peptide, which can help TCR penetrate the membrane; the signal peptide can be a signal peptide known in the art. In some specific embodiments, the α chain can include a sequence as shown in SEQ ID NO:9, and the β chain can include a sequence as shown in SEQ ID NO:18.

[0036] The second TCR:

[0037] The second TCR comprises the sequence shown in SEQ ID NO:37.

[0038] Specifically, the second TCR comprises the sequence shown in SEQ ID NO: 37, which comprises the α chain variable region and the β chain variable region in the first TCR.

[0039] Specifically, in the second TCR, the TCR sequence can also be a sequence having an identity ≥80% with the sequence shown in SEQ ID NO:37, such as a sequence having an identity ≥80%, 85%, 90%, or 95%.

[0040] Specifically, in the above-mentioned TCR, regarding the sources of the variable region and constant region of the α chain or β chain, the α chain variable region or β chain variable region is from mouse or human (but other sources, such as rabbit, pig and other animal sources are not excluded), and the source of the α chain or β chain constant region may be the same as or different from the source of the α chain or β chain variable region.

[0041] In another aspect, the present invention provides an antigen-binding fragment comprising any one of the two TCRs described above.

[0042] In another aspect, the present invention provides a polynucleotide that can be selected from the following polynucleotides:

[0043] First polynucleotide:

[0044] The first polynucleotide comprises at least the sequences shown in SEQ ID NO:21 and SEQ ID NO:30.

[0045] Further, the first polynucleotide may also include one or more of a sequence as shown in SEQ ID NO:19, a sequence as shown in SEQ ID NO:20, a sequence as shown in SEQ ID NO:22, a sequence as shown in SEQ ID NO:23, a sequence as shown in SEQ ID NO:24, a sequence as shown in SEQ ID NO:25, a sequence as shown in SEQ ID NO:28, a sequence as shown in SEQ ID NO:29, a sequence as shown in SEQ ID NO:31, a sequence as shown in SEQ ID NO:32, a sequence as shown in SEQ ID NO:33 and a sequence as shown in SEQ ID NO:34. In some specific embodiments, the first polynucleotide may also include a sequence as shown in SEQ ID NO:26 and / or a sequence as shown in SEQ ID NO:27; in some specific embodiments, the first polynucleotide may also include a sequence as shown in SEQ ID NO:35 and / or a sequence as shown in SEQ ID NO:36; in some specific embodiments, the first polynucleotide may also include a sequence as shown in SEQ ID NO:26 and / or a sequence as shown in SEQ ID NO:27 and / or a sequence as shown in SEQID NO:35 and / or a sequence as shown in SEQ ID NO:36.

[0046] Second polynucleotide:

[0047] The second polynucleotide comprises the sequence shown in SEQ ID NO:38.

[0048] Specifically, the second polynucleotide comprises the sequence of the first polynucleotide, that is, comprises the sequences shown in SEQ ID NO:21 and SEQ ID NO:30.

[0049] Specifically, the above-mentioned polynucleotide may also contain any other nucleotides (in addition to the above-mentioned polynucleotides) encoding any of the above-mentioned TCRs, such as a sequence after codon optimization as shown in SEQ ID NO:21, a sequence after codon optimization as shown in SEQ ID NO:30, and a sequence after codon optimization as shown in SEQ ID NO:38.

[0050] In another aspect, the present invention provides an expression vector, which may contain any one of the two polynucleotides mentioned above.

[0051] Furthermore, the expression vector is selected from any one of a lentiviral expression vector, a retroviral expression vector, an adenoviral expression vector, an adeno-associated viral expression vector, a DNA vector, an RNA vector and a plasmid.

[0052] Specifically, the lentiviral vector can be selected from the following groups: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedivirus (VMV) virus, caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV) and simian immunodeficiency virus (SIV).

[0053] In another aspect, the present invention provides an engineered cell comprising the above-mentioned expression vector. Specifically, the engineered cell can be a host cell, into which the above-mentioned expression vector is introduced to encode a TCR polypeptide; or it can be a T cell, into which the above-mentioned expression vector (loaded with the target gene) is transfected to obtain a TCR-T cell for the recognition and killing of the KRAS-G12D mutant polypeptide.

[0054] In another aspect, the present invention provides a pharmaceutical composition, which comprises the above-mentioned TCR or the above-mentioned antigen-binding fragment or the above-mentioned polynucleotide or the above-mentioned expression vector or the above-mentioned engineered cell, and a pharmaceutically acceptable carrier and / or diluent.

[0055] Specifically, the pharmaceutically acceptable carrier and / or diluent refers to the TCR or the antigen binding fragment or the polynucleotide or the expression vector or the engineered cell that can be prepared into various desired dosage forms. For example, tablets, powders, pills, powders, granules, fine granules, soft / hard capsules, film coatings, pellets, sublingual tablets, ointments, etc. can be listed as oral agents, and injections, suppositories, transdermal agents, ointments, plasters, external liquids, etc. can be listed as non-oral agents. Those skilled in the art can select appropriate dosage forms according to the route of administration and the subject of administration.

[0056] In another aspect, the present invention provides a use of the above-mentioned TCR or the above-mentioned antigen binding fragment or the above-mentioned polynucleotide or the above-mentioned expression vector or the above-mentioned engineered cell in the preparation of a drug for increasing the level of IFN-γ and / or TNF-α cytokine secretion by T cells.

[0057] Furthermore, drugs for increasing the level of IFN-γ and / or TNF-α cytokine secretion by T cells include cell drugs, protein drugs, ADC drugs or TCR and antigen combination drugs.

[0058] In another aspect, the present invention provides a use of the above-mentioned TCR or the above-mentioned antigen-binding fragment or the above-mentioned polynucleotide or the above-mentioned expression vector or the above-mentioned engineered cell in the preparation of a reagent or a kit for detecting tumor cells expressing KRAS-G12D mutation.

[0059] Specifically, the kit involved can be divided into various small boxes, and then filled with various detection reagents. The detection reagents and detection kits can be indirectly or directly applied to various malignant tumors expressing KRAS-G12D mutations, such as pancreatic tumors, colorectal malignancies, endometrial malignancies, lung malignancies, bile duct malignancies, cervical malignancies, etc.

[0060] In another aspect, the present invention provides a use of the above-mentioned TCR or the above-mentioned antigen-binding fragment or the above-mentioned polynucleotide or the above-mentioned expression vector or the above-mentioned engineered cell in the preparation of a drug for treating a disease caused by carrying the KRAS-G12D mutation.

[0061] Furthermore, diseases caused by carrying the KRAS-G12D mutation include pancreatic cancer, colorectal cancer, endometrial cancer, lung cancer, bile duct cancer, cervical cancer or bladder cancer.

[0062] Specifically, in the above application, for the HLA-restricted T cell epitope polypeptide of the KRAS-G12D mutation, HLA-A1101 with stronger affinity is selected, that is, the polypeptide after the KRAS-G12D mutation is presented by the HLA-A1101 molecule, but the selection of other HLA molecules, such as other small molecules in the HLA-A11 series, is not excluded.

[0063] Specifically, in the above application, TCR selects any one of the above two TCRs; polynucleotide selects any one of the above two polynucleotides. In the above application, TCR or antigen binding fragment or polynucleotide or expression vector or engineered cell can be added with excipients to form a drug, or can be used in combination with other active drugs or detection reagents, such as some existing chemotherapy drugs, such as alkylating agents, antimetabolites, antitumor antibiotics, plant anticancer drugs, hormones, immune preparations, etc.; it can also be combined with surgery. The specific situation is based on the tumor situation. Medication or combined medication.

[0064] Specifically, in the above application, the polypeptides of the KRAS-G12D mutation can be 9 polypeptides, 10 polypeptides or other number of polypeptides, and the amino acid sequence of the 10 polypeptides of the KRAS-G12D mutation is shown in SEQ ID NO:41.

[0065] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0066] In the following examples, the KRAS-G12 wild-type polypeptide (abbreviated as KRAS-G12) involved in the verification is shown in SEQ ID NO: 39; the KRAS-G12C mutant polypeptide (abbreviated as KRAS-G12C) involved in the verification is shown in SEQ ID NO: 40; the KRAS-G12D mutant polypeptide (abbreviated as KRAS-G12D) involved in the verification is shown in SEQ ID NO: 41; the KRAS-G12V mutant polypeptide (abbreviated as KRAS-G12V) involved in the verification is shown in SEQ ID NO: 42.

[0067] In the following examples, the gene amplification, sequencing and analysis methods involved were performed according to the following steps:

[0068] (1) RT-PCR: The downstream primers used were TCR gene constant region specific primers (Ca_RV1primer, Cb_RV1primer), and the upstream primers were sequences containing the outer linker and the initial 20 bp of the TCR signal peptide (AL primers, BL primers) (specifically, according to the content and embodiment described in the document "Hamana H, Shitaoka K, Kishi H, Ozawa T, Muraguchi A. A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells. Biochem Biophys Res Commun. 2016 Jun 10; 474(4): 709-714. doi: 10.1016 / j.bbrc.2016.05.015. Epub 2016 May 4. PMID: 27155153");

[0069] (2) Second round of PCR: The TCR PCR product obtained in (1) above was used as a template, the upstream primer was an outer linker primer (P2A-Cprimer), and the downstream primer was a specific primer for a segment of the TCR constant region upstream of the α chain constant region (Ca_RV2primer) to obtain the second round of PCR product of the TCR α chain; the upstream primer was an outer linker primer (BES-AP primer), and the downstream primer was a specific primer for a segment of the TCR constant region upstream of the β chain constant region (Cb_RV2 primer) to obtain the second round of PCR product of the TCR β chain (specifically according to the literature "Hamana H, Shitaoka K, Kishi H, Ozawa T, Muraguchi A. A novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells. Biochem Biophys Res Commun. 2016 Jun 2016). 10; 474(4):709-714.doi:10.1016 / j.bbrc.2016.05.015.Epub 2016May4.PMID:27155153)

[0070] (3) The amplified second-round PCR products containing the TCR α chain and β chain variable region genes were subjected to agarose gel electrophoresis to obtain the TCR α chain or β chain variable region target gene at the 500 bp-750 bp position;

[0071] (4) The target band was subjected to first-generation sequencing (Beijing Qingke Company). After TCR sequence analysis, a TCR clone with a high frequency was selected and named KT13.

[0072] The amplification system used in the above-mentioned implementation steps of the gene amplification, sequencing and analysis methods is shown in Table 1 below (specifically implemented according to the contents of the document "Hamana H, Shitaoka K, Kishi H, Ozawa T, Muraguchi AA novel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells. Biochem Biophys Res Commun. 2016 Jun 10; 474 (4): 709-714. doi: 10.1016 / j.bbrc.2016.05.015. Epub 2016 May 4. PMID: 27155153").

[0073] Table 1 Amplification system

[0074]

[0075]

[0076] In the following examples, the human PBMC cells and human peripheral blood involved were all derived from volunteers.

[0077] In the following embodiments, the nucleic acid sequences or amino acid sequences of the materials involved are shown in Table 2 below, wherein the KT13TCR α chain variable region refers to the fragment composed of the sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 in the above-mentioned first TCR in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the KT13TCR β chain variable region refers to the fragment composed of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15 and SEQ ID NO:16 in the above-mentioned first TCR. The sequence shown in NO:16 is a fragment composed of the arrangement order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the full-length KT13TCR sequence refers to the TCR with a complete structure after the KT13 α chain variable region and the β chain variable region are respectively connected to other structures, which is the second TCR mentioned above, comprising the sequence shown in SEQ ID NO:37, which is encoded by the sequence shown in SEQ ID NO:38.

[0078] Table 2 Amino acid sequence and nucleic acid sequence of the materials involved in the examples

[0079] materials Amino acid sequence Nucleic acid sequence KRAS-WT SEQ ID NO:39 - KRAS-G12C SEQ ID NO:40 - KRAS-G12D SEQ ID NO:41 - KRAS-G12V SEQ ID NO:42 - KT13TCR α chain variable region (αV region) SEQ ID NO:8 SEQ ID NO:26 KT13TCRβ chain variable region (βV region) SEQ ID NO:17 SEQ ID NO:35 Full-length KT13TCR sequence SEQ ID NO:37 SEQ ID NO:38

[0080] In the following examples, KRAS-G12, KRAS-G12V, KRAS-G12C, and KRAS-G12D polypeptides were commissioned to be synthesized by Kingruis Biotech Co., Ltd.

[0081] In the following examples, the tetramers of KRAS-G12 / HLA-A1101, KRAS-G12C / HLA-A1101, KRAS-G12D / HLA-A1101 and KRAS-G12V / HLA-A1101 were all prepared using QuickSwitch TMThe reagent is prepared by Quant Tetramer Kit, which can be easily prepared in the laboratory. The specific preparation process is as follows: 1ul KRAS-G12 peptide and 1ul KRAS-G12V peptide displacer, 1ul KRAS-G12 peptide and 1ul KRAS-G12C peptide displacer, 1ul KRAS-G12 peptide and 1ul KRAS-G12D peptide displacer are added to 50ul QuickSwitchTetramer to obtain KRAS-G12 / HLA-A1101 tetramer, KRAS-G12C / HLA-A1101 tetramer, KRAS-G12D / HLA-A1101 tetramer and KRAS-G12V / HLA-A1101 tetramer, respectively, and placed at room temperature for 4 hours and stored at 4°C for use.

[0082] Example 1 Specific TCR Screening

[0083] (1) The immunogenicity of the KRAS-G12D mutant protein was predicted by prediction software (HLAthena), and the antigenic peptide sequence with immunogenicity was obtained by analysis. The sequence of the KRAS-G12D mutant is shown in SEQ ID NO: 20;

[0084] (2) synthesizing relevant short peptides with a purity of 85%, and dissolving the synthesized peptides in DMSO to form a 10 mM solution;

[0085] (3) In vitro stimulation and expansion of KRAS-specific T cells: peripheral blood was collected from healthy subjects, and the HLA gene subtype was detected. HLA-A1101-positive peripheral blood samples were selected; monocytes and initial T cells were separated from HLA-A1101-positive peripheral blood, and 800U / ml IL-4 and 800U / ml GM-CSF were added to the monocyte culture medium, and cultured for 4 days to differentiate them into DC cells. Then, the above-mentioned KRAS-G12D mutant polypeptide (at a concentration of 10uM) was added to the DC cell culture medium, and the cells were placed in an incubator for static culture for 16 hours; then, the DC cells loaded with the above-mentioned mutant peptide were mixed with the initial T cells and cultured for another 3 weeks; the content of specific T cells in the cultured T cell products was detected by flow cytometry;

[0086] (4) Take 1×10 7The amplified T cells were centrifuged at 250g for 10 minutes, the supernatant was discarded, 100ul of antibody staining solution (PBS containing 1ug / ml KRAS-G12D / HLA-A1101 tetramer and 1ug / ml APC-CD3 antibody) was added, and incubated at room temperature for 30min; washed three times with 5ml PBS containing 0.5% BSA, and centrifuged at 250g for 10min; resuspended the cells with 1ml PBS containing 0.5% BSA, and single specific T cells were sorted using a flow cytometer;

[0087] (5) Total RNA was extracted from the sorted specific T cells, and then RT-PCR amplification was performed to obtain the TCR gene sequence. The sequence structure of TCR was analyzed by first-generation sequencing and comparison with the IMGT database, and it was found that the KT13 variable region consisted of an α chain variable region encoded by the sequence shown in SEQ ID NO:26 and a β chain variable region encoded by the sequence shown in SEQ ID NO:35.

[0088] Example 2 KT13 binding specificity verification

[0089] The variable regions (V regions) of the KT13 α chain and β chain were connected to the constant region (C region) genes of the α chain and β chain of TCR, and then the α chain and β chain were connected to obtain the full-length KT13TCR sequence, and the full-length KT13TCR sequence gene is shown in SEQ ID NO:38; using the lentiviral expression plasmid pWPXL (purchased from Addegene) as a backbone vector, KT13 lentiviral expression vectors were constructed, namely, lentiviral expression vectors pWPXL-KT13TCR; the pWPXL-KT13TCR lentiviral expression vectors were co-transfected with lentiviral packaging plasmids pMD2.G and psPAX2 plasmids at a mass ratio of 1:0.5:1 into 293T cells, and the viral supernatant was collected two days later; the viral supernatant was used to infect Jurkat cells, the medium was changed 24 hours after infection, and after culturing for 2-3 days, Jurkat cells expressing KT13TCR were obtained for downstream experiments.

[0090] (1) Jurkat cell activation CD69

[0091] The above-mentioned Jurkat cells expressing KT13TCR and K562 cells expressing A1101 gene were mixed at a cell number ratio of 1:1, and KRAS-G12D polypeptide with a final concentration of 10uM was added respectively. After 6 hours, the cells were recovered; then 50ul of cell staining solution containing PE-CD69 antibody was added and incubated at room temperature for 30min; then washed three times with PBS containing 0.5% BSA and centrifuged at 200g-250g for 10min; then resuspended the cells with PBS containing 0.5% BSA for downstream detection; Jurkat cell activation analysis was performed using flow cytometry to detect the expression of CD69 in Jurkat cell activation analysis;

[0092] The results of the analysis of Jurkat cells expressing KT13TCR are shown in Figure 1 As shown, the results show that KT13TCR is specifically activated after stimulation by KRAS-G12D, so it is a KRAS-G12D-specific TCR.

[0093] (2) Activation degree of KRAS-G12D peptide at different concentrations

[0094] The above-mentioned Jurkat cells expressing KT13TCR and K562 cells expressing A1101 gene were mixed at a cell number ratio of 1:1, and KRAS-G12D polypeptides with final concentrations of 10μM, 1μM, 0.1μM, 0.01μM, 0.001μM and 0.0001μM were added respectively, and the cells were recovered after 6 hours; then 50ul of cell staining solution containing PE-CD69 antibody was added and incubated at room temperature for 30min; then washed three times with PBS containing 0.5% BSA and centrifuged at 200g-250g for 10min; then resuspended the cells with PBS containing 0.5% BSA for downstream detection; Jurkat cell activation analysis was performed using flow cytometry to detect the expression of CD69 in Jurkat cell activation analysis;

[0095] The results of the analysis of Jurkat cells expressing KT13TCR are shown in Figure 2 As shown, the results show that as the peptide concentration decreases, the degree of activation of KT13TCR changes after stimulation with KRAS-G12D, so that its affinity is higher.

[0096] Example 3 Test of secretion levels of IFN-γ and TNF-α

[0097] KT13-T cells prepared from human PBMC T cells were mixed 1:1 with A1101-K562 cells loaded with KRAS-G12, KRAS-G12C, KRAS-G12D, and KRAS-G12V peptides, respectively, and 2 × 10 5The number of cells / well was added to a 96-well plate, and each sample was repeated three times. The PMA / ionomycin (ION) group was diluted in advance into a working solution by adding 1 μl of PMA / ionomycin mixture (250×) to every 250 μl of cell culture medium, and used as a positive stimulation control; after culturing at 37°C for 24 hours, the supernatant of the 96-well plate culture wells was taken and centrifuged at 500g for 5 minutes to remove the residual cells, and the supernatant was added to an ELISA plate coated with anti-IFN-γ antibody or anti-TNF-α antibody to detect the IFN-γ level and TNF-α level in the supernatant. The results are shown in Figure 3 and Figure 4 As shown, KT13-T cells secrete IFN-γ or TNF-α after stimulation by KRAS-G12D, so KT13 is a specific TCR for KRAS-G12D.

[0098] Example 4 Cell Killing Experiment

[0099] KT13-T cells prepared from human PBMC T cells were mixed with A1101-K562 cells loaded with KRAS-G12 and KRAS-G12D peptides at different ratios (1:1, 2:1, 5:1, and 10:1) and incubated at 2 × 10 cells / mL in a volume of 100 μl. 4 , 4×10 4 , 1×10 5 and 2×10 5 The number of cells / well was added to a 96-well plate, and each sample was repeated three times. The PMA / ionomycin (ION) group was diluted in advance into a working solution by adding 1 μl of PMA / ionomycin mixture (250×) to every 250 μl of cell culture medium, and used as a positive stimulation control; after culturing at 37°C for 24 hours, the supernatant of the 96-well plate culture wells was taken and centrifuged at 500g for 5 minutes to remove the residual cells, and the LDH content in the supernatant was detected. The results are shown in Figure 5 As shown, KT13 has the specific killing ability of KRAS-G12D.

Claims

1. A TCR targeting a polypeptide-HLA-A1101 complex, characterized in that It comprises an α chain variable region and a β chain variable region; the α chain variable region comprises an α chain CDR1 with a sequence as shown in SEQ ID NO:1, an α chain CDR2 with a sequence as shown in SEQ ID NO:2, and an α chain CDR3 with a sequence as shown in SEQ ID NO:3; the β chain variable region comprises a β chain CDR1 with a sequence as shown in SEQ ID NO:10, a β chain CDR2 with a sequence as shown in SEQ ID NO:11, and a β chain CDR3 with a sequence as shown in SEQ ID NO:12; the amino acid sequence of the polypeptide is shown in SEQ ID NO:

41.

2. The TCR according to claim 1, characterized in that The α chain variable region further comprises: one or more of an α chain FR1 with a sequence as shown in SEQ ID NO:4, an α chain FR2 with a sequence as shown in SEQ ID NO:5, an α chain FR3 with a sequence as shown in SEQ ID NO:6, and an α chain FR4 with a sequence as shown in SEQ ID NO:7; the β chain variable region further comprises: one or more of a β chain FR1 with a sequence as shown in SEQ ID NO:13, a β chain FR2 with a sequence as shown in SEQ ID NO:14, a β chain FR3 with a sequence as shown in SEQ ID NO:15, and a β chain FR4 with a sequence as shown in SEQ ID NO:

16.

3. A TCR targeting a polypeptide-HLA-A1101 complex, characterized in that It comprises a fragment encoded by the sequence shown in SEQ ID NO:37; the amino acid sequence of the polypeptide is shown in SEQ ID NO:

41.

4. An antigen-binding fragment, characterized in that Comprising the TCR according to any one of claims 1 to 3.

5. The polynucleotide encoding the TCR according to claim 1, characterized in that It comprises the sequences shown in SEQ ID NO:21 and SEQ ID NO:

30.

6. The polynucleotide according to claim 5, characterized in that It also includes one or more of the sequence shown in SEQ ID NO:19, the sequence shown in SEQ ID NO:20, the sequence shown in SEQ ID NO:22, the sequence shown in SEQ ID NO:23, the sequence shown in SEQ ID NO:24, the sequence shown in SEQ ID NO:25, the sequence shown in SEQ ID NO:28, the sequence shown in SEQ ID NO:29, the sequence shown in SEQ ID NO:31, the sequence shown in SEQ ID NO:32, the sequence shown in SEQ ID NO:33 and the sequence shown in SEQ ID NO:

34.

7. The polynucleotide encoding the TCR according to claim 1, characterized in that It comprises the sequence shown in SEQ ID NO:

38.

8. An expression vector, characterized in that Comprising the polynucleotide according to any one of claims 5 to 7.

9. An engineered cell, characterized in that Comprising the expression vector according to claim 8.

10. A pharmaceutical composition, characterized in that Comprising the TCR according to any one of claims 1 to 3, the antigen-binding fragment according to claim 4, the polynucleotide according to any one of claims 5 to 7, the expression vector according to claim 8, or the engineered cell according to claim 9, and a pharmaceutically acceptable carrier and / or diluent.

11. Use of the TCR according to any one of claims 1 to 3, the antigen-binding fragment according to claim 4, the polynucleotide according to any one of claims 5 to 7, the expression vector according to claim 8, or the engineered cell according to claim 9 in the preparation of a medicament for treating a tumor positive for polypeptide-HLA-A1101 expression; the amino acid sequence of the polypeptide is shown in SEQ ID NO:41; the tumor is pancreatic cancer, colon cancer, endometrial cancer, lung cancer, bile duct cancer, cervical cancer or bladder cancer.

12. The use according to claim 11, characterized in that: The drugs include protein drugs, ADC drugs or TCR and antigen combination drugs.

13. Use of the TCR according to any one of claims 1 to 3, the antigen-binding fragment according to claim 4, the polynucleotide according to any one of claims 5 to 7, the expression vector according to claim 8, or the engineered cell according to claim 9 in the preparation of a reagent or kit for detecting tumor cells that are positive for polypeptide-HLA-A1101 expression and express KRAS-G12D mutation; the amino acid sequence of the polypeptide is shown in SEQ ID NO:

41.

14. Use of the TCR according to any one of claims 1 to 3, the antigen-binding fragment according to claim 4, the polynucleotide according to any one of claims 5 to 7, the expression vector according to claim 8, or the engineered cell according to claim 9 in the preparation of a medicament for treating a tumor caused by a KRAS-G12D mutation; the tumor is pancreatic cancer, colon cancer, endometrial cancer, lung cancer, bile duct cancer, cervical cancer, or bladder cancer.

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