TCR (T cell receptor) of targeted polypeptide-HLA-A1101 compound or antigen binding fragment of TCR and application of TCR or antigen binding fragment

Abstract

The invention belongs to the technical field of tumor drugs, and particularly relates to a TCR (T cell receptor) targeting a polypeptide-HLA-A1101 compound or an antigen binding fragment of the TCR and application of the TCR or the antigen binding fragment. A variable region of the TCR is obtained by mutating a first TCR variable region, and a mutation position and a mutation type are selected from any one of the following: (1) the mutation position is 29th to 31st sites of an alpha chain variable region, and the mutation type is that DTT mutates into LCL, NSL, MFL, LHL, HSA, IQT or NDN; and (2) the mutation position is 98th to 101st sites of a beta chain variable region, and the mutation type is that 'GQNN' is mutated into 'ARHN' or 'SEHS'. According to the present invention, the TCR does not specifically recognize the self epitope peptide SMC1A29-38 besides the excellent functional affinity and the strong recognition ability on the KRASG12D;

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor drugs, and specifically relates to a TCR or an antigen binding fragment thereof targeting a polypeptide-HLA-A1101 complex and an application thereof. Background Art

[0002] T cell therapy is one of the current means of tumor treatment, which mainly includes TCR-T and CAR-T treatment methods. Currently, 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.

[0003] 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. TCR-T technology is not limited by specific targets and has great application prospects. Specifically, 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.

[0004] However, in addition to recognizing tumor antigens, TCRs also cross-recognize self-antigens, which may lead to serious clinical toxicity. Therefore, it is necessary to develop a TCR that does not cross-recognize self-antigens on the basis of recognizing tumor antigens. Summary of the invention

[0005] The present invention obtains a TCR that does not specifically recognize the self-epitope peptide SMC1A29-38 by mutating the CDR region of the KT13-TCR in the patent publication CN115850444A under structure guidance, which is the TCR in the present invention.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] In one aspect, the present invention provides a TCR targeting a polypeptide-HLA-A1101 complex, wherein the variable region of the TCR is obtained by mutation of a first TCR variable region, the α chain variable region sequence of the first TCR is shown in SEQ ID NO: 1, and the β chain variable region sequence is shown in SEQ ID NO: 2; the mutation position and mutation type are selected from any one of the following:

[0008] (1) The mutation position is from position 29 to 31 of the α chain variable region, and the mutation type is from "DTT" to "LCL", "NSL", "MFL", "LHL", "HSA", "IQT" or "NDN";

[0009] (2) The mutation position is from position 98 to 101 in the variable region of the β chain, and the mutation type is from "GQNN" to "ARHN" or "SEHS".

[0010] Another aspect of the present invention provides an antigen-binding fragment comprising the TCR of the present invention.

[0011] In another aspect, the present invention provides a polynucleotide encoding the TCR of the present invention.

[0012] In another aspect, the present invention provides an expression vector comprising the polynucleotide of the present invention.

[0013] In another aspect, the present invention provides an engineered cell comprising the expression vector of the present invention.

[0014] In another aspect, the present invention provides a pharmaceutical composition comprising the TCR of the present invention and / or the antigen binding fragment of the present invention and / or the polynucleotide of the present invention and / or the expression vector of the present invention and / or the engineered cell of the present invention.

[0015] In another aspect, the present invention provides a pharmaceutical preparation, which comprises the TCR of the present invention and / or the antigen binding fragment of the present invention and / or the polynucleotide of the present invention and / or the expression vector of the present invention and / or the engineered cell of the present invention and / or the pharmaceutical composition of the present invention; and a pharmaceutically acceptable carrier and / or diluent.

[0016] In another aspect, the present invention provides a use of the TCR of the present invention and / or the antigen binding fragment of the present invention and / or the polynucleotide of the present invention and / or the expression vector of the present invention and / or the engineered cell of the present invention and / or the pharmaceutical composition of the present invention in the preparation of a pharmaceutical preparation for treating cancer.

[0017] Preferably, the pharmaceutical preparation in the above application may include a protein drug, an ADC drug or a TCR and antigen combination drug.

[0018] Preferably, the cancer in the above application includes pancreatic cancer, colon cancer, endometrial cancer, lung cancer, bile duct cancer, cervical cancer or bladder cancer.

[0019] The beneficial effects of the present invention include at least: in addition to having excellent functional affinity and strong recognition ability for KRASG12D, the TCR provided by the present invention does not specifically recognize the self-epitope peptide SMC1A29-38, thereby reducing the safety risks of clinical toxicity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1A It is a visual quantitative display of IFN-γ secretion;

[0021] Figure 1B Schematic diagram of mini-gene clusters expressing homologous peptides;

[0022] Figure 1C The activation of KT13T cells was moderately induced by K562 cells expressing MC-1;

[0023] Figure 1D Induction of CD137 by homologous peptides derived from the protein SMC1A + Increased proportion of T cells;

[0024] Figure 1E The activation of KT13T cells induced by K562 cells loaded with peptides; Figure 1F HLA-A*11:01 + SMC1A+COS-7 cells significantly increased the proportion of CD137+T cells;

[0025] Figure 2A The software predicts the structure of the KT13TCR-peptide-MHC complex;

[0026] Figure 2B for the interaction between CDR1A and CDR3B of KT13TCR and pHLA;

[0027] Figure 2C The hydrogen bond formation between KT13 CDR-1AT30 and HLA;

[0028] Figure 2D The changes in the functional affinity of KT13TCR to KRASG12D;

[0029] Figure 2E There is a structural correspondence between Q112.1 and position 8 of the KRASG12D peptide;

[0030] Figure 2FThe Q112.1 site mutation affects the TCR clone's ability to recognize SMC1A29-38 and KRASG12D;

[0031] Figure 3A KT13Lib1 is in close contact with the peptide The four consecutive sites of

[0032] Figure 3B KT13Lib2 consists of three consecutive sites closest to HLA interactions;

[0033] Figure 3C Four rounds of flow cytometry sorting were performed to enrich for TCR clones that could bind to KRASG12D tetramers and exhibit activation when exposed to KRASG12D peptides;

[0034] Figure 3D The staining enrichment of SMC1A29-38 tetramer and KRASG12D tetramer;

[0035] Figure 4A To analyze the functional affinity of all TCR clones to KRASG12D;

[0036] Figure 4B To detect the binding ability of TCR clones using SMC1A29-38 tetramer;

[0037] Figure 4C To study TCR activation in response to off-target stimuli;

[0038] Figure 4D To test whether the artificially engineered TCR clones have peptide-independent HLA reactivity;

[0039] Figure 4E X-scan analysis of the peptide specificity profiles of 9 TCR clones; DETAILED DESCRIPTION

[0040] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0041] 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.

[0042] An embodiment of the present invention provides a TCR targeting a polypeptide-HLA-A1101 complex, wherein the variable region of the TCR is obtained by mutation of a first TCR variable region, the α chain variable region sequence of the first TCR is shown in SEQ ID NO: 1, and the β chain variable region sequence is shown in SEQ ID NO: 2; the mutation position and mutation type are selected from any one of the following: (1) the mutation position is from position 29 to 31 of the α chain variable region, and the mutation type is "DTT" mutated to "LCL", "NSL", "MFL", "LHL", "HSA", "IQT" or "NDN"; (2) the mutation position is from position 98 to 101 of the β chain variable region, and the mutation type is "GQNN" mutated to "ARHN" or "SEHS".

[0043] It should be noted that the first TCR in the present invention is the KT13TCR recorded in the example of the patent CN115850444A, that is, the variable region of the TCR of the present invention is modified on the basis of the variable region of KT13TCR, and the modified TCR variable region reduces its reactivity to its own peptide SMC1A29-38 while maintaining excellent functional affinity and recognition ability for KRASG12D, thereby eliminating nonspecific recognition of SMC1A29-38.

[0044] It should also be noted that the TCR of the targeting polypeptide-HLA-A1101 complex of the present invention contains, in addition to the above-mentioned α chain variable region and β chain variable region, the α chain also contains the α chain constant region, and the β chain also contains the 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 are almost no mutation regions. The sequence of the constant region can preferably be the constant region of KT13TCR; that is, the TCR of the targeting polypeptide-HLA-A1101 complex of the present invention is preferably the same as KT13TCR except that the mutation region is different from KT13TCR.

[0045] It should also be noted that the α chain and β chain of the TCR of the targeting polypeptide-HLA-A1101 complex of the present invention may also include a signal peptide, which may help the TCR to penetrate the membrane; the signal peptide may be a signal peptide known in the art.

[0046] An embodiment of the present invention further provides an antigen binding fragment, which comprises the TCR of the present invention.

[0047] An embodiment of the present invention also provides a polynucleotide encoding the TCR of the present invention.

[0048] It should be noted that the polynucleotides in the present invention include all nucleotides that can encode the TCR in the present invention, which may be codon-optimized nucleotides or non-codon-optimized nucleotides. The nucleotide sequence in the same region as KT13TCR can refer to the sequence disclosed in patent CN115850444A, and the nucleotides in the mutation region can be translated accordingly according to methods known in the art.

[0049] An embodiment of the present invention further provides an expression vector comprising the polynucleotide of the present invention.

[0050] It should be noted that 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. 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).

[0051] The embodiment of the present invention further provides an engineered cell, which comprises the expression vector of the present invention.

[0052] It should be noted that the engineered cells can be host cells, into which the above expression vector is introduced to encode a TCR polypeptide; or they can be T cells, into which the above expression vector (loaded with the target gene) is transfected to obtain TCR-T cells for the recognition and killing of KRAS-G12D mutant polypeptides.

[0053] An embodiment of the present invention further provides a pharmaceutical composition comprising the TCR of the present invention and / or the antigen binding fragment of the present invention and / or the polynucleotide of the present invention and / or the expression vector of the present invention and / or the engineered cell of the present invention.

[0054] It should be noted that the TCR in the present invention and / or the antigen-binding fragment in the present invention and / or the polynucleotide in the present invention and / or the expression vector in the present invention and / or the engineered cell in the present invention can be used in combination with other active ingredients to prepare a pharmaceutical composition, for example, in combination with some chemotherapeutic drugs, such as alkylating agents, antimetabolites, antitumor antibiotics, plant-based anticancer drugs, hormones, immune preparations, etc.

[0055] An embodiment of the present invention also provides a pharmaceutical preparation, which comprises the TCR of the present invention and / or the antigen binding fragment of the present invention and / or the polynucleotide of the present invention and / or the expression vector of the present invention and / or the engineered cell of the present invention and / or the pharmaceutical composition of the present invention; and a pharmaceutically acceptable carrier and / or diluent.

[0056] It should be noted that pharmaceutically acceptable carriers and / or diluents refer to 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 cells 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.

[0057] It should also be noted that the pharmaceutical preparation of the present invention can also be combined with surgery for treatment, and the specific situation is selected according to the tumor condition.

[0058] The embodiments of the present invention also provide a use of the TCR of the present invention and / or the antigen binding fragment of the present invention and / or the polynucleotide of the present invention and / or the expression vector of the present invention and / or the engineered cell of the present invention and / or the pharmaceutical composition of the present invention in the preparation of a pharmaceutical preparation for treating cancer.

[0059] In some specific examples, the drug preparations in the above applications may include protein drugs, ADC drugs, or TCR and antigen combination drugs.

[0060] In some specific examples, the cancer in the above applications includes pancreatic cancer, colon cancer, endometrial cancer, lung cancer, bile duct cancer, cervical cancer or bladder cancer.

[0061] 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.

[0062] In the following experiments, the KT13TCR used is the KT13TCR described in the example of the patent publication CN115850444A.

[0063] In the following experiments, ELISA quantification of IFN-γ secretion levels uses the recommended antibody pair for IFN-γ cytokine quantification, including IFN-γ capture antibody (clone: ​​MD-1, Biolegend) and IFN-γ detection antibody (clone: ​​4S.B3, Biolegend); the specific quantitative method is as follows:

[0064] (1) ELISA plates were coated with 50 μl / well of 2 μg / ml capture antibody at 4°C overnight;

[0065] (2) After coating, the plates were washed three times with PBS containing 0.05% Tween-20 (PBS-T) and blocked with 3% bovine serum albumin (BSA) in PBS for 1 h at room temperature to prevent nonspecific binding;

[0066] (3) Add samples (supernatants collected from T cell and target cell co-cultures) to the prepared plates at a volume of 50 μl per well and incubate at room temperature for 2 h to allow cytokine binding;

[0067] (4) After incubation, wash the plate three times with PBS-T to remove unbound substances;

[0068] (5) Then, the detection antibody diluted to 1 μg / mL was added at a concentration of 50 μL / well and incubated at room temperature for 1 hour;

[0069] (6) After washing the plate five times with PBS-T, streptavidin alkaline phosphatase (Mabtech) diluted 1:1000 with PBS was added to each well and incubated at room temperature in the dark for 1 h;

[0070] (7) After incubation, the plate was washed six times, and p-nitrophenyl phosphate (pNPP) substrate (Thermo) was added at a concentration of 50 μl / well. The enzymatic reaction was carried out at 37°C in the dark for 30 min.

[0071] (8) Then add 50 μl of stop solution to each well to terminate the reaction;

[0072] (9) The plate was read at a wavelength of 405 nm (OD405) using a VarioskanLUX multifunctional microplate reader (Thermo); the amount of IFN-γ in the sample was determined by comparing the optical density of the sample with the optical density of a standard curve prepared with known concentrations of recombinant human IFN-γ.

[0073] The method for peptide-specific analysis using X-scan analysis in the following experiments includes:

[0074] The first step is to detect the reactivity of T cells to the peptide library. In order to study TCR specificity using TCR T cells, GenScript designed and synthesized a KRAS G12D peptide single-site saturation mutation peptide library with reference to the KRAS G12D peptide (VVVGADGVGK) peptide sequence; each library member (n = 200) was presented to HLA-A*11:01 at a final concentration of 10 μM. + K562 cells. When TCRT cells were used as effector cells, K562 cells loaded with polypeptides were co-cultured with TCR-transduced T cells; after overnight incubation, IFN-γ secretion levels were quantified by ELISA. When TCRJurkat cells were used as effector cells, K562 cells loaded with polypeptides were co-cultured with TCR Jurkat cells. After 6 hours, the cells were stained with APC anti-human CD69 antibody for 10 minutes at 4°C in the dark. After washing twice with PBS, flow cytometry was performed to analyze the expression of CD69.

[0075] Step 2: T cell homologous peptide prediction. The background activation level determined from the DMSO-treated control was subtracted from all measured responses and then normalized to 0% for the DMSO group and 100% for the KRASG12D group. The 20% reactivity threshold relative to the KRASG12D-induced response was used as the critical value for defining the TCR recognition motif, which helped to identify the allowed amino acids at each position in the peptide sequence; this TCR recognition motif was then used to scan the UniProtKB human proteome database using the ScanProsite tool to search for homologous peptide sequences that could be cross-recognized by TCR. Peptides that could bind to HLA were filtered out using NetMHCpan-4.1, which were then used for validation of subsequent minigene experiments.

[0076] Step 3: Verification of T cell reactivity to homologous peptides. Each minigene encodes a 10-mer homologous peptide flanked by 10 natural amino acids from the corresponding protein. A total of 10 such minigenes were strung together into a minigene cluster (MC) and then fused to the NGFR gene (Uniprot ID P08138, residues 29-275) via a P2A linker to generate a bicistronic MC-NGFR construct; the codon-optimized MC-NGFR construct was cloned into the pWPXL vector for the production of lentiviral particles. These lentiviral particles were used to transduce HLA-A*11:01 +K562 cells were engineered to express minigene clusters, and NGFR protein was used as a marker for quality control of viral transduction efficiency; TCR T cells were co-cultured with these engineered K562 cells, and CD137 expression was analyzed by flow cytometry after 24 hours; the P64 minigene encoding an HLA-A*11:01 restricted epitope from the COVID-19 N protein (KTFPPTEPK) and known to activate the P64 TCR was included in each minigene cluster as a positive control. This setup ensures the correct intracellular processing and presentation of the minigene.

[0077] In the following experiments, the design and screening of the TCR library included three stages, as follows:

[0078] Phase I: Design of TCR libraries. Based on structural data from TCR JDI, amino acid residues within the CDR region that are critical for interaction with pHLA were identified. Two regions of the CDR were selected for the construction of mutation libraries. The first library, named TCR1 Lib1, contained a saturation mutation library targeting four consecutive sites (G111 to N113) proximal to the peptide interaction within CDR-3B. The second library, TCR1 Lib2, consisted of a saturation mutation library of three consecutive sites (D29 to T36) that are closest to the HLA interaction within CDR-1A. DNA of both TCR libraries was synthesized and subsequently cloned into the pWPXL plasmid by GenScript. Extracts of the TCR library plasmids were also prepared by GenScript. The IMGT numbering scheme was used to describe the position of amino acids in the TCR. This facilitates direct comparisons between different T cell receptors.

[0079] Phase II: TCR library display in Jurkat cells. Recombinant lentivirus encoding the TCR library was generated by transfecting HEK293T cells with the TCR library plasmid and packaging plasmids (psPAX2 and pMD2.G). The viral titer was determined to achieve approximately 10% positive expression of the mouse TCR in CD8+ Jurkat cells, ensuring that only one copy of the TCR variant was expressed per cell. The saturated mutation library of four sites contained 1.6E5 TCR variants. In order to prepare a TCR library Jurkat cell covering 100 times the library capacity, an initial cell count of 1.6E8 CD8 + Jurkat cells were infected with lentivirus. 24 hours after transfection, TCR library Jurkat cells were harvested and resuspended in G-Rex 100 (Wilson Wolf) at a concentration of 2E5 / ml. Two days after transfection, 50% of the culture medium volume was replaced with fresh culture medium. Three days after transfection, TCR library Jurkat cells were harvested for downstream screening procedures.

[0080] Phase III: Screening of the TCR Library. The screening of the TCR library was performed in five rounds to ensure enrichment and selection of functional TCR clones specific for the KRASG12D mutation. Round 01: Enrichment of TCR expression. 2.0E7 TCR library Jurkat cells were stained with APC anti-mouse TCR β chain antibody. Using EasySep TM APC Positive Selection Kit (STEMCELL Technologies) was used to enrich TCR clones that showed normal TCR expression on the cell membrane according to the manufacturer's protocol. Round 2: KRAS G12D Tetramer specificity. Jurkat cells from the 2.0E7 TCR library were stained with APC anti-mouse TCRβ chain antibody and PE-KRAS G12D tetramer. + KRASG12D Tet+TCR clones were sorted. Round 3 and 04: Enrichment of functional clones. HLA-A*11:01+K562 cells were pulsed with 10μM KRASG12D peptide and then co-cultured with TCR library Jurkat cells. 6h after co-culture, cells were stained with PE anti-mouse TCRβ chain antibody, APC anti-human CD69 antibody, and BV421 anti-human CD62L antibody. This panel can distinguish between specifically activated (CD62L-CD69+) and non-specifically activated (CD62L+CD69+) cells. The top 10% of CD62L-CD69+ cells showing the highest MFI of CD69 were sorted. Each enrichment process takes 10 to 14 days to propagate enough cells for subsequent rounds. Round 5: Isolation of specific clones. Clones were stained with PE-SMC1A29-38 tetramer and APC-KRASG12D tetramer, and KRASG12D Tetra+SMC1ATetra-TCR clones were sorted by single-cell FACS into 96-well PCR plates to determine TCR sequences.

[0081] 1. KT13TCR recognizes the self-antigen peptide SMC1A29-38

[0082] In order to evaluate the potential off-target recognition of KT13TCR, the present invention uses X-scan analysis to evaluate the specificity of TCR recognition polypeptides, specifically including:

[0083] (1) KT13TCR-T cells were incubated with HLA-A*11:01 loaded with the KRAS G12D peptide single-site saturation mutant peptide library designed and synthesized by GenScript at a final concentration of 10 μM. + K562 cells were co-cultured and IFN-γ release was measured 24 hours later. Visual quantification of IFN-γ secretion showed that changes in N-terminal positions 4 to 10 within the KRAS G12D peptide affected peptide recognition by KT13 ( Figure 1A ).

[0084] (2) A KT13 recognition motif ([ACDEFGHIKLMNPQRSTVWY]-[ACDEFGHILMNPRSTV]-[ACDEGIKLMNPQRSTV]-[AGSW]-[APS]-[DN]-[ACFGMPQSTV]-[ACEHIKLMNQRSTVWY]-[GN]-[DIKLY]) was constructed using a peptide with a reactivity rate greater than 20% of that of the KRASG12D peptide. The motif was scanned against the human reference proteome (ProteomeID: UP000005640) using the ScanProsite tool, and a total of 48 KT13 cross-recognition candidate peptides were identified.

[0085] (3) Five mini-gene clusters (MC-1 to MC-5) were designed, each expressing 10 homologous peptides ( Figure 1B ), the homologous peptide information contained in each mini-gene cluster is shown in Table 1.

[0086] Table 1 Minigene clusters contain homologous peptide information

[0087]

[0088]

[0089]

[0090] Accordingly, five groups of HLA-A*11:01+K562 cells expressing 1 MC were added to each group ( Figure 1C ) were co-cultured with KT13T cells; flow cytometric analysis of the proportion of CD137+ cells showed that only K562 cells expressing MC-1 moderately induced KT13T cell activation ( Figure 1D ).

[0091] (4) Subsequently, 10 short peptides encoded by MC-1 were synthesized and loaded into HLA-A*11:01+K562 cells individually. Co-culture with KT13T cells showed that the homologous peptides derived from the protein SMC1A induced CD137 + A strong increase in the proportion of T cells ( Figure 1C and 1D The peptide corresponds to the 29th to 38th amino acids of the N-terminus of the protein SMC1A (i.e., SMC1A29-38). The full-length cDNA of SMC1A was transduced into HLA-A*11:01+COS-7 cells; co-culture experiments with KT13T cells showed that compared with the vector-transduced control group, HLA-A*11:01 + SMC1A+COS-7 cells significantly increased CD137+ The proportion of T cells ( Figure 1E ). This result confirmed that the SMC1A29-38 epitope can be endogenously processed by HLA-A*11:01 and presented on the cell membrane.

[0092] The above results indicate that KT13TCR is a functional TCR that can recognize the KRASG12D epitope, but it exhibits cross-reactivity with its own epitope SMC1A29-38.

[0093] 2. Prediction and analysis of the KT13TCR-peptide-MHC complex structure

[0094] The TCR sequence information was input through the TCRpMHCmodels-1.0 software (https: / / services.healthtech.dtu.dk / services / TCRpMHCmodels-1.0 / ) to predict the KT13TCR-peptide-MHC complex structure ( Figure 2A ), the structure shows that CDR1α and CDR3β of KT13TCR are involved in the interaction with pHLA ( Figure 2B ). Among them, CDR1AT30 of KT13 TCR forms a hydrogen bond with HLA ( Figure 2C ), and a point mutation analysis was performed on this amino acid residue. The T30Q mutation reduced the pMut-HLA binding positivity from 91% to 60% and reduced the functional affinity of KT13 TCR for KRASG12D from 15nM to 53nM ( Figure 2D ); This indicates that the T30 site is indeed involved in HLA recognition.

[0095] In addition, there is a structural correspondence between amino acid Q112.1 of TCRCDR3B of KT13TCR and position 8 of KRASG12D peptide ( Figure 2E ), so a single-point saturation mutation was performed on this residue. After Jurat cells expressed the mutant TCR, 19 TCR mutant clones were stained with HLA-A1101-KRASG12D tetramer and HLA-A1101-SMC1A29-38 tetramer (tetramer provided by Beijing Borma Biotechnology Co., Ltd.), and the binding strength of TCR and HLA-peptide pairs was detected by flow cytometry. The results are shown in Figure 2f. The mutation at the Q112.1 site affects the recognition ability of TCR clones for SMC1A29-38 and KRASG12D.

[0096] 3. Design and screening of KT13TCR display library

[0097] Based on the above results, the range of amino acid residues to be mutated was expanded based on the TCR-pHLA interaction region to construct a TCR library covering; KT13Lib1 is closely in contact with the peptide The saturation mutation library formed by four consecutive sites (CDR-3B: G111 to N113) Figure 3A ), KT13Lib2 consists of a saturation mutation library formed by three consecutive sites closest to HLA interaction (CDR-1A: D29 to T36) ( Figure 3B ); After expressing the TCR library in CD8+ Jurkat cells, four rounds of flow cytometry sorting were performed to enrich for TCR clones that could bind to KRASG12D tetramers (tetramer binding positive cell population) and exhibit activation when exposed to KRASG12D peptide (cell population expressing the activation molecule CD69) ( Figure 3C ); After four rounds of enrichment, the TCR library was stained with SMC1A29-38 tetramer and KRASG12D tetramer to enrich TCR clones that only bind to KRASG12D tetramer alone ( Figure 3D ); The above flow staining conditions are: 100ul resuspended 1E7 cells, adding 1ug / ml final concentration of antibody or tetramer, protected from light at 4 degrees, 25 minutes. After TCR gene sequencing, a total of 24 TCR clones were obtained, see Table 2 below.

[0098] Table 224 TCR clones

[0099]

[0100] 4. Functional affinity and safety evaluation of KT13TCR mutants

[0101] As can be seen above, 24 new TCR clones were established from the two KT13 libraries, and their characteristics and functions were evaluated in four steps, as follows:

[0102] First, the functional affinity of all TCR clones to KRASG12D was analyzed using TCR-T cells prepared from T cells of human PBMC, specifically including: co-culturing TCR-T cells with HLA-A*11:01-positive K562 cells loaded with different concentrations of KRASG12D peptide for 24 hours; then, the cells were stained with 20ul PBS containing PE-labeled anti-human CD137 antibody (antibody final concentration 1ug / ml) at 25°C in the dark for 10 minutes; after two PBS washes, the expression of CD137 was analyzed by flow cytometry, and the EC50 value (the peptide concentration that can cause 50% of TCR-T cells to occur is the EC50 value) was calculated. The results are shown in Tables 3 and 4. Figure 4AAs shown, the results showed that most of these TCR clones showed unchanged or increased EC50, indicating that the recognition ability of the obtained mutant strains for KRASG12D was not affected.

[0103] Table 3 EC50 values ​​of different antibodies

[0104]

[0105] Secondly, the binding ability of TCR clones was detected using SMC1A29-38 tetramers, specifically including: taking 1*10^6 TCR-Jurkat cells, centrifuging to remove the supernatant, resuspending the cells with 20ul PBS containing APC-labeled SMC1A29-38 tetramers (tetramer final concentration 1ug / ml), and staining at 4°C in the dark for 25 minutes; after two PBS washes, the expression of the binding ability of TCR and tetramers was analyzed by flow cytometry; the results are as follows Figure 4B As shown, the results showed that six TCR clones from KT13 as well as Lib1 and all clones from Lib2 showed negligible tetramer binding levels.

[0106] Third, functional confirmation was performed to study TCR activation in response to off-target stimulation, including: co-culturing TCR-Jurkat cells with HLA-A*11:01-positive K562 cells loaded with 100nM SMC1A29-38 peptide for 6 hours, with 5*10^5 cells of each type; then, resuspending the cells with 20ul PBS containing PE-labeled anti-human CD69 antibody (final antibody concentration 1ug / ml), staining at 25℃ in the dark for 10 minutes; after two PBS washes, analyzing the expression of CD69 by flow cytometry. The results are shown in Figure 4C As shown, the results showed that 12 TCR clones remained activated when stimulated with 100 nM SMC1A29-38 peptide.

[0107] Finally, the above TCR clones were checked for peptide-independent HLA reactivity, specifically including: co-culturing all remaining clones with K562, HLA-A*11:01+K562 or HLA-A*11:01+K562 loaded with KRASG12D peptide; co-culturing TCR-T cells with K562, HLA-A*11:01-positive K562, and HLA-A*11:01-positive K562 cells loaded with 10uM KRASG12D peptide for 24 hours. Subsequently, the cells were stained with 20ul PBS containing PE-labeled anti-human CD137 antibody (final antibody concentration 1ug / ml) at 25°C in the dark for 10 minutes. After two PBS washes, the expression of CD137 was analyzed by flow cytometry. Results Figure 4DTherefore, the results showed that the three TCR clones showed a significant increase in the CD69 + proportion when stimulated with HLA-A*11:01+K562 alone, and therefore no further testing was required.

[0108] Through this four-step functional evaluation, a total of 9 TCR clones were found to have sufficient KRASG12D recognition affinity and lost reactivity to the SMC1A29-38 peptide, see Table 4 below.

[0109] Table 4 Nine TCR clones that lost reactivity to SMC1A29-38 peptide

[0110]

[0111] In addition, the peptide specificity profiles of these nine TCR clones were determined by X-ray scanning analysis. Figure 4E As shown, the TCR cloned after modification exhibits recognition characteristics similar to those of KT13TCR. Therefore, the present invention adjusts the recognition of KT13TCR to pHLA so that it loses recognition of the homologous peptide SMC1A29-38.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A TCR targeting a polypeptide-HLA-A1101 complex, characterized in that: The variable region of TCR is obtained by mutation of the variable region of the first TCR, the sequence of the α chain variable region of the first TCR is shown in SEQ ID NO: 1, and the sequence of the β chain variable region is shown in SEQ ID NO: 2; the mutation position and mutation type are selected from any one of the following: (1) The mutation position is from position 29 to 31 of the α chain variable region, and the mutation type is from "DTT" to "LCL", "NSL", "MFL", "LHL", "HSA", "IQT" or "NDN"; (2) The mutation position is from position 98 to 101 in the variable region of the β chain, and the mutation type is from "GQNN" to "ARHN" or "SEHS".

2. An antigen-binding fragment, characterized in that Comprising the TCR of claim 1.

3. A polynucleotide, characterized in that Encoding the TCR of claim 1.

4. An expression vector, characterized in that Comprising the polynucleotide of claim 3.

5. An engineered cell, characterized in that Comprising the expression vector according to claim 4.

6. A pharmaceutical composition, characterized in that Comprising the TCR of claim 1 and / or the antigen binding fragment of claim 2 and / or the polynucleotide of claim 3 and / or the expression vector of claim 4 and / or the engineered cell of claim 5.

7. A pharmaceutical preparation, characterized in that Comprising the TCR of claim 1 and / or the antigen binding fragment of claim 2 and / or the polynucleotide of claim 3 and / or the expression vector of claim 4 and / or the engineered cell of claim 5 and / or the pharmaceutical composition; and a pharmaceutically acceptable carrier and / or diluent.

8. Use of the TCR according to claim 1 and / or the antigen binding fragment according to claim 2 and / or the polynucleotide according to claim 3 and / or the expression vector according to claim 4 and / or the engineered cell and / or the pharmaceutical composition according to claim 5 in the preparation of a pharmaceutical preparation for treating cancer.

9. The use according to claim 8, characterized in that Drug preparations include protein drugs, ADC drugs or TCR and antigen combination drugs.

10. Use according to claim 8 or 9, characterized in that Cancers include pancreatic cancer, colon cancer, endometrial cancer, lung cancer, bile duct cancer, cervical cancer, or bladder cancer.

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