Tcrs or antigen-binding fragments thereof targeting polypeptide-hla-a1101 complexes and uses thereof

Abstract

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

Description

Technical Field

[0001] This invention belongs to the field of tumor drug technology, specifically relating to a TCR of a targeted polypeptide-HLA-A1101 complex or its antigen-binding fragment and its application. Background Technology

[0002] T-cell therapy is one of the current methods for cancer treatment, mainly including TCR-T and CAR-T therapies. Currently, CAR-T technology has shown significant effects in the treatment of hematological malignancies such as leukemia and lymphoma, greatly improving patient survival rates and quality of life. However, for solid tumors, the limited specific targets of CAR-T therapy currently restrict its application prospects.

[0003] TCR-T technology differs from CAR-T cell technology. TCR is a characteristic marker on the surface of all T cells, binding nonvalently to CD3 to form the TCR-CD3 complex. In peripheral blood, 90%-95% of T cells express TCR. Genetically modified T cells with TCR can specifically recognize antigen molecules on the surface of tumor cells, thereby generating an immune response against them. TCR-T technology is not limited by specific targets and has significant application potential. Specifically, TCR is a characteristic marker on the surface of all T cells, binding nonvalently to CD3 to form the TCR-CD3 complex. In peripheral blood, 90%-95% of T cells express TCR. Genetically modified T cells with TCR can specifically recognize antigen molecules on the surface of tumor cells, thereby generating an immune response against them.

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

[0005] This invention obtains a TCR that does not specifically recognize its own epitope peptide SMC1A29-38 by performing structure-guided mutations on the CDR- region of the KT13-TCR in the published patent CN115850444A, which is the TCR in this invention.

[0006] To achieve the above objectives, the present invention can adopt the following technical solutions:

[0007] This invention provides a TCR targeting the peptide-HLA-A1101 complex, wherein the variable region of the TCR is obtained by mutation of the variable region of a first TCR, 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 location and mutation type are selected from any of the following:

[0008] (1) The mutation location is the 29th to 31st position of the variable region of the α chain, and the mutation type is “DTT” mutated to “LCL”, “NSL”, “MFL”, “LHL”, “HSA”, “IQT” or “NDN”.

[0009] (2) The mutation location is the 98th to 101st position of the variable region of the β chain, and the mutation type is “GQNN” mutated into “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 that encodes the TCR of the present invention.

[0012] In another aspect, the present invention provides an expression vector comprising the polynucleotides described herein.

[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 formulation 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 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 the use of the TCR and / or the antigen-binding fragment and / or the polynucleotide and / or the expression vector and / or the engineered cell and / or the pharmaceutical composition of the present invention in the preparation of a pharmaceutical formulation for treating cancer.

[0017] Preferably, the pharmaceutical formulation used in the above applications may include protein drugs, ADC drugs, or TCR-antigen combination drugs.

[0018] Preferably, the cancers used in the above applications include pancreatic cancer, colon cancer, endometrial cancer, lung cancer, bile duct cancer, cervical cancer, or bladder cancer.

[0019] The beneficial effects of this invention include at least the following: In addition to having excellent functional affinity and a strong ability to recognize KRASG12D, the TCR provided by this invention does not specifically recognize its own epitope peptide SMC1A29-38, thus reducing the safety risks of clinical toxicity. Attached Figure Description

[0020] Figure 1A This provides a visible quantitative representation of IFN-γ secretion.

[0021] Figure 1B A schematic diagram illustrating the expression of homologous peptides by a miniature gene cluster;

[0022] Figure 1C To determine the moderate induction of KT13T cell activation in K562 cells expressing MC-1;

[0023] Figure 1D CD137 is induced by homologous peptides derived from protein SMC1A. + Increased T cell ratio;

[0024] Figure 1E To illustrate the effect of K562 cells loaded with peptides on the activation of KT13T cells;

[0025] Figure 1F HLA-A*11:01 + SMC1A+COS-7 cells significantly increased the proportion of CD137+ T cells;

[0026] Figure 2A To predict the structure of the KT13TCR-peptide-MHC complex using software;

[0027] Figure 2B The interaction between CDR1A, CDR3B of KT13TCR and pHLA;

[0028] Figure 2C The hydrogen bond formation between KT13 CDR-1AT30 and HLA;

[0029] Figure 2D The changes in the functional affinity of KT13TCR for KRASG12D;

[0030] Figure 2E There is a structural correspondence between Q112.1 and the 8th position of the KRASG12D peptide;

[0031] Figure 2FTo investigate the impact of the Q112.1 site mutation on the TCR clone's ability to recognize SMC1A29-38 and KRASG12D;

[0032] Figure 3A KT13Lib1 is composed of peptides in close contact The situation of four consecutive sites;

[0033] Figure 3B For KT13Lib2, consider the case of three consecutive sites most closely interacting with HLA;

[0034] Figure 3C The TCR clones that bind to KRASG12D tetramer and exhibit activation upon exposure to KRASG12D peptide were sorted and enriched using four-round cytometry.

[0035] Figure 3D The staining enrichment of SMC1A29-38 tetramer and KRASG12D tetramer;

[0036] Figure 4A The functional affinity of all TCR clones for KRASG12D was analyzed.

[0037] Figure 4B The binding affinity of TCR clones was assessed using SMC1A29-38 tetramer.

[0038] Figure 4C To investigate TCR activation in response to off-target stimuli;

[0039] Figure 4D To determine whether artificially engineered TCR clones exhibit peptide-independent HLA reactivity;

[0040] Figure 4E X-ray scanning was used to analyze the peptide-specific profiles of nine TCR clones; Detailed Implementation

[0041] The embodiments described are provided to better illustrate the present invention, but are not intended to limit the scope of the invention to the embodiments described. Therefore, non-essential improvements and adjustments made to the embodiments by those skilled in the art based on the above description are still within the scope of protection of the present invention.

[0042] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. Singular expressions include plural expressions unless they have a distinct meaning in the context. As used herein, it should be understood that terms such as “comprising,” “having,” “including,” are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations thereof. The terminology of the invention is disclosed in the specification and is not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials, or combinations thereof may be present or added. As used herein, “ / ” may be interpreted as “and” or “or,” depending on the context.

[0043] This invention provides a TCR targeting the peptide-HLA-A1101 complex. The variable region of the TCR is obtained by mutation of the variable region of the first TCR. 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 of the following: (1) The mutation position is the 29th to 31st position 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 the 98th to 101st position of the β chain variable region, and the mutation type is “GQNN” mutated to “ARHN” or “SEHS”.

[0044] It should be noted that the first TCR in this invention is the KT13TCR described in the embodiment of the published patent CN115850444A. That is, the variable region of the TCR in this invention is obtained by modifying the variable region of KT13TCR. The modified TCR variable region maintains excellent functional affinity and recognition ability of KRASG12D, while reducing its reactivity to its own peptide SMC1A29-38 and eliminating non-specific recognition of SMC1A29-38.

[0045] It should also be noted that, in addition to the aforementioned α-chain variable region and β-chain variable region, the TCR of the targeted peptide-HLA-A1101 complex in this invention also includes an α-chain constant region and a β-chain constant region. The α-chain and β-chain constant regions can be derived from mice, humans, or human-mouse chimeras (but other sources, such as rabbits or pigs, are not excluded), and are regions where mutations are almost impossible. The sequence of the constant region can preferably be the constant region of KT13TCR; that is, the TCR of the targeted peptide-HLA-A1101 complex in this invention is preferably identical to KT13TCR except for the mutation region.

[0046] It should also be noted that the α and β chains of the TCR of the targeted polypeptide-HLA-A1101 complex in this invention may also include signal peptides, which can help the TCR to penetrate the membrane; the signal peptides may be signal peptides known in the art.

[0047] This invention also provides an antigen-binding fragment comprising the TCR described in this invention.

[0048] This invention also provides a polynucleotide encoding the TCR of this invention.

[0049] It should be noted that the polynucleotides in this invention include all nucleotides that can encode the TCR in this invention. They can be nucleotides with optimized codons or nucleotides without optimized codons. The nucleotide sequence of the same region as KT13TCR can refer to the sequence disclosed in patent CN115850444A. The nucleotides in the mutation region can be translated according to the methods known in the art.

[0050] This invention also provides an expression vector comprising the polynucleotides described in this invention.

[0051] It should be noted that the expression vector can be any one of lentiviral expression vectors, retroviral expression vectors, adenoviral expression vectors, adeno-associated virus expression vectors, DNA vectors, RNA vectors, and plasmids. Specifically, lentiviral vectors can be selected from the following groups: human immunodeficiency virus 1 (HIV-1), human immunodeficiency virus 2 (HIV-2), visna-maedivirus (VMV), caprine arthritis-encephalitis virus (CAEV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), and simian immunodeficiency virus (SIV).

[0052] This invention also provides an engineered cell that includes the expression vector described in this invention.

[0053] It should be noted that the engineered cells can be host cells, into which the above expression vector is introduced to encode the TCR polypeptide; or they can be T cells, into which the above expression vector (loaded with the target gene) is transfected into T cells to obtain TCR-T cells for the recognition and killing of the KRAS-G12D mutant polypeptide.

[0054] This invention also provides a pharmaceutical composition comprising the TCR of this invention and / or the antigen-binding fragment of this invention and / or the polynucleotide of this invention and / or the expression vector of this invention and / or the engineered cell of this invention.

[0055] It should be noted that the TCR and / or antigen-binding fragment and / or polynucleotide and / or expression vector and / or engineered cell in this invention can be used in combination with other active ingredients to prepare a pharmaceutical composition, such as in combination with some chemotherapeutic drugs, such as alkylating agents, antimetabolites, antitumor antibiotics, herbal anticancer drugs, hormones, immunomodulators, etc.

[0056] This invention also provides a pharmaceutical formulation comprising the TCR of this invention and / or the antigen-binding fragment of this invention and / or the polynucleotide of this invention and / or the expression vector of this invention and / or the engineered cell of this invention and / or the pharmaceutical composition of this invention; and a pharmaceutically acceptable carrier and / or diluent.

[0057] It should be noted that pharmaceutically acceptable carriers and / or diluents refer to those that can be used to prepare the aforementioned TCR, antigen-binding fragments, polynucleotides, expression vectors, or engineered cells into various desired dosage forms. Examples include tablets, powders, pills, granules, fine granules, soft / hard capsules, film-coated tablets, small pellets, sublingual tablets, and ointments as oral dosage forms, and injections, suppositories, transdermal preparations, ointments, plasters, and topical liquids as non-oral dosage forms. Those skilled in the art can select appropriate dosage forms based on the route of administration and the target population.

[0058] It should also be noted that the pharmaceutical preparations in this invention can be used in combination with surgery. The specific approach depends on the condition of the tumor.

[0059] The present invention also provides the use of the TCR and / or the antigen-binding fragment and / or the polynucleotide and / or the expression vector and / or the engineered cell and / or the pharmaceutical composition of the present invention in the preparation of a pharmaceutical formulation for treating cancer.

[0060] In some specific examples, the pharmaceutical formulations used in the above applications may include protein drugs, ADC drugs, or TCR-antigen combination drugs.

[0061] In some specific examples, the cancers mentioned above include pancreatic cancer, colon cancer, endometrial cancer, lung cancer, bile duct cancer, cervical cancer, or bladder cancer.

[0062] To better understand the present invention, specific examples are provided below to further illustrate the content of the present invention, but the content of the present invention is not limited to the examples below.

[0063] In the following experiments, the KT13TCR used is the KT13TCR described in the embodiment of the published patent CN115850444A.

[0064] In the following experiments, ELISA was used to quantify IFN-γ secretion levels using recommended antibody pairs for IFN-γ cytokine quantification. Recommended antibodies include IFN-γ capture antibody (clone: ​​MD-1, Biolegend) and IFN-γ detection antibody (clone: ​​4S.B3, Biolegend). The specific quantification method is as follows:

[0065] (1) The ELISA plate was coated overnight at 4°C with 50 μl / well of capture antibody at a concentration of 2 μg / ml;

[0066] (2) After coating, the plate was washed three times with PBS containing 0.05% Tween-20 (PBS-T) and blocked for 1 hour at room temperature with PBS solution containing 3% bovine serum albumin (BSA) to prevent nonspecific binding.

[0067] (3) Add the sample (supernatant collected from the co-culture of T cells and target cells) to the prepared plate at a volume of 50 μl per well and incubate at room temperature for 2 hours to allow cytokine binding;

[0068] (4) After incubation, wash the plate three times with PBS-T to remove unbound material;

[0069] (5) Then add the detection antibody diluted to 1 μg / mL at a concentration of 50 μL / well and incubate at room temperature for 1 hour;

[0070] (6) After washing the plate 5 times with PBS-T, add streptavidin alkaline phosphatase (Mabtech) diluted with PBS at a ratio of 1:1000 to each well and incubate at room temperature in the dark for 1 hour.

[0071] (7) After incubation, wash the plate 6 times and add p-nitrophenyl phosphate (pNPP) substrate (Thermo) at a concentration of 50 μl / well. Incubate the enzymatic reaction at 37°C in the dark for 30 minutes.

[0072] (8) Then add 50 μl of stop solution to each well to terminate the reaction;

[0073] (9) The plate was read at a wavelength of 405 nm (OD405) using a Varioskan LUX multi-plate 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 a known concentration of recombinant human IFN-γ.

[0074] The following experiments used X-ray scanning analysis for peptide-specific analysis:

[0075] Step 1: Detecting T cell responsiveness to the peptide library. To study TCR specificity using TCR T cells, a single-point saturation mutant peptide library of KRAS G12D peptide was designed and synthesized by GenScript, referencing the KRAS G12D peptide (VVVGADGVGK) polypeptide sequence. Each library member (n=200) was individually presented to HLA-A*11:01 at a final concentration of 10 μM. + K562 cells. When using TCRT cells as effector cells, K562 cells loaded with the peptide were co-cultured with TCR-transduced T cells; after overnight incubation, IFN-γ secretion levels were quantified by ELISA. When using TCRJurkat cells as effector cells, K562 cells loaded with the peptide were co-cultured with TCRJurkat cells. After 6 hours, 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 CD69 expression.

[0076] The second step was T-cell homologous peptide prediction. Background activation levels determined from the DMSO-treated control were subtracted from all measured responses, and then normalized (0% for the DMSO group and 100% for the KRASG12D group). A 20% reactivity threshold relative to the KRASG12D-induced response was used as a critical value to define the TCR recognition motif, which helps 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 might be cross-recognized by the TCR. Peptides capable of binding to HLA were filtered using NetMHCpan-4.1 and then used for validation in subsequent small gene experiments.

[0077] Step 3: Verification of T cell responsiveness to homologous peptides. Each minigene encodes a 10-mer homologous peptide flanked by 10 native amino acids from the corresponding protein. A total of 10 such minigenes were strung together into a minigene cluster (MC), and then fused with 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 used to express small gene clusters, with NGFR protein serving 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 small gene, encoding the HLA-A*11:01 restriction epitope from the COVID-19 N protein (KTFPPTEPK) and known to activate the P64 TCR, was included as a positive control in each small gene cluster. This setup ensured proper intracellular processing and presentation of the small genes.

[0078] In the following experiments, the design and screening of the TCR library included three stages, as detailed below:

[0079] Phase 1: TCR Library Design. Based on structural data from TCR JDI, amino acid residues within the CDR region crucial for pHLA interaction were identified. Two regions of the CDR were selected for mutant library construction. The first library, named TCR1 Lib1, contained a saturated mutant library targeting four consecutive sites (G111 to N113) proximal to the peptide interaction site of CDR-3B. The second library, TCR1 Lib2, consisted of a saturated mutant library with three consecutive sites (D29 to T36), closest to the HLA interaction site of CDR-1A. DNA from both TCR libraries was synthesized and subsequently cloned into the pWPXL plasmid using GenScript. TCR library plasmid extraction was also performed using GenScript. The IMGT numbering scheme was used to describe the amino acid positions within the TCR, facilitating direct comparison between different T cell receptors.

[0080] Phase 2: TCR Library Display in Jurkat Cells. Recombinant lentivirus encoding the TCR library was generated by transfecting HEK293T cells with a TCR library plasmid and packaging plasmids (psPAX2 and pMD2.G). Viral titers were 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 mutant library at four sites contained 1.6E5 TCR variants. To prepare Jurkat cells with a TCR library covering 100-fold library capacity, an initial cell count of 1.6E8 CD8+ cells was used. + Jurkat cells were infected with lentiviruses. Twenty-four hours post-transfection, Jurkat cells containing the TCR library were harvested and resuspended in G-Rex 100 (Wilson Wolf) at a concentration of 2E5 / ml. Two days post-transfection, 50% of the culture medium volume was replaced with fresh medium. Three days post-transfection, Jurkat cells containing the TCR library were harvested for downstream selection procedures.

[0081] Phase 3: TCR library screening. TCR library screening was conducted in five rounds to ensure enrichment and selection of functional TCR clones specific for the KRASG12D mutation. Round 1: TCR expression enrichment. 2.0E7 TCR library Jurkat cells were stained with APC anti-mouse TCR β-chain antibody. EasySep was used. TM APC positive selection kit (STEMCELL Technologies) enriched TCR clones exhibiting normal TCR expression on the cell membrane according to the manufacturer's protocol. Round 2: KRAS G12D Tetramer specificity. Jurkat cells containing a 2.0E7 TCR library were stained with APC anti-mouse TCR β-chain antibody and PE-KRAS G12D tetramer. mTCR... + KRASG12D Tet+TCR clones were classified. Rounds 3 and 4: Enrichment of functional clones. HLA-A*11:01+K562 cells were pulsed with 10 μM KRASG12D peptide and then co-cultured with Jurkat cells containing the TCR library. After 6 h of 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 could 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 required 10 to 14 days to proliferate enough cells for subsequent rounds. Round 5: Isolation of specific clones. Clones were stained with PE-SMC1A29-38 tetramer and APC-KRASG12D tetramer. KRASG12D Tetra+SMC1ATetra-TCR clones were sorted into 96-well PCR plates by single-cell FACS and the TCR sequences were determined.

[0082] I. KT13TCR recognizes autoantigen peptide SMC1A29-38

[0083] To assess the potential off-target recognition of KT13TCR, this invention employs X-scan analysis to evaluate the specificity of TCR-recognized peptides, specifically including:

[0084] (1) KT13TCR-T cells were inoculated with HLA-A*11:01 from the KRAS G12D peptide single-point saturated mutant peptide library designed and synthesized by GenScript, loaded with a final concentration of 10 μM. + K562 cells were co-cultured, and IFN-γ release was measured after 24 hours. Quantitative analysis of IFN-γ secretion showed that changes in the N-terminal position (4-10) of the KRAS G12D peptide affected peptide recognition by KT13. Figure 1A ).

[0085] (2) A KT13 recognition motif ([ACDEFGHIKLMNPQRSTVWY]-[ACDEFGHILMNPRSTV]-[ACDEGIKLMNPQRSTV]-[A GSW]-[APS]-[DN]-[ACFGMPQSTV]-[ACEHIKLMNQRSTVWY]-[GN]-[DIKLY]) was constructed using peptides with a reactivity of more than 20% of the KRASG12D peptide reactivity. The motif was scanned against the human reference proteome (ProteomeID: UP000005640) using the ScanProsite tool, and a total of 48 candidate peptides for KT13 cross-recognition were identified.

[0086] (3) Five miniature gene clusters (MC-1 to MC-5) were designed, each expressing 10 homologous peptides. Figure 1B Each microgene cluster contains homologous peptide information, which can be found in Table 1.

[0087] Table 1 contains information on homologous peptides in mini-gene clusters.

[0088]

[0089]

[0090]

[0091] Accordingly, five groups of HLA-A*11:01+K562 cells, each expressing 1 MC, were used. Figure 1C K562 cells were co-cultured with KT13T cells; flow cytometry analysis of the proportion of CD137+ cells showed that only K562 cells expressing MC-1 moderately induced KT13T cell activation. Figure 1D ).

[0092] (4) Subsequently, 10 short peptides encoded by MC-1 were synthesized and individually loaded into HLA-A*11:01+K562 cells. Co-culture with KT13T cells showed that homologous peptides derived from the protein SMC1A induced CD137 + A strong increase in the proportion of T cells ( Figure 1C and 1D This peptide corresponds to amino acids 29 to 38 of the N-terminus of the protein SMC1A (i.e., SMC1A29-38). Full-length SMC1A cDNA was transduced into HLA-A*11:01+COS-7 cells; co-culture experiments with KT13T cells showed that, compared with the vector transduction control group, HLA-A*11:01... + SMC1A+COS-7 cells significantly increased CD137+ The proportion of T cells ( Figure 1E This result confirms that the SMC1A29-38 epitope can be endogenously processed and presented on the cell membrane via HLA-A*11:01.

[0093] The above results indicate that KT13TCR is a functional TCR capable of recognizing the KRASG12D epitope; however, it exhibits cross-reactivity with its own epitope SMC1A29-38.

[0094] II. Structural Prediction and Analysis of KT13TCR-Peptide-MHC Complex

[0095] The structure of the KT13TCR-peptide-MHC complex was predicted by inputting TCR sequence information using the TCRpMHCmodels-1.0 software (https: / / services.healthtech.dtu.dk / services / TCRpMHCmodels-1.0 / ). 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 Point mutation analysis was performed on this amino acid residue. The T30Q mutation reduced the pMut-HLA binding positivity rate from 91% to 60% and decreased the functional affinity of KT13 TCR for KRASG12D from 15 nM to 53 nM. Figure 2D This indicates that the T30 site is indeed involved in HLA recognition.

[0096] Furthermore, there is a structural correspondence between amino acid Q112.1 of TCRCDR3B in KT13TCR and position 8 of the KRASG12D peptide. Figure 2E Therefore, 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 (tetramers were provided by Beijing Bomei Biotechnology Co., Ltd.). Flow cytometry was used to detect the binding strength of TCR to HLA-peptide pairs. The results are shown in Figure 2f. The mutation at the Q112.1 site affected the TCR clone's ability to recognize SMC1A29-38 and KRASG12D.

[0097] III. KT13TCR Display Library Design and Selection

[0098] Based on the above results, and based on the TCR-pHLA interaction region, the range of mutated amino acid residues was expanded, and a TCR library was constructed to cover the region; KT13Lib1 is tightly bound to the peptide. The saturated mutant library consists of four consecutive sites (CDR-3B: G111 to N113) Figure 3A KT13Lib2 consists of a saturated mutant library formed by three consecutive sites (CDR-1A: D29 to T36) closest to HLA interactions. Figure 3B After expressing the TCR library in CD8+ Jurkat cells, four-round cytometry was used to enrich TCR clones that could bind to the KRASG12D tetramer (tetramer-positive cell population) and showed activation upon exposure to the KRASG12D peptide (cell population expressing the activating 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 bind only to KRASG12D tetramer. Figure 3D The flow cytometry staining conditions were as follows: 1E7 cells were resuspended in 100 μL, and antibody or tetramer was added at a final concentration of 1 μg / ml. The staining was carried out at 4°C in the dark for 25 minutes. After TCR gene sequencing, a total of 24 TCR clones were obtained, as shown in Table 2 below.

[0099] Table 224 TCR clones

[0100]

[0101] IV. Functional affinity and safety evaluation of KT13TCR mutant strain

[0102] From the above, we can see that 24 new TCR clones were established from the two KT13 libraries. Their characteristics and functions were evaluated in four steps, as follows:

[0103] First, the functional affinity of all TCR clones for KRASG12D was analyzed using T cells prepared from human PBMCs. Specifically, TCR-T cells were co-cultured with HLA-A*11:01-positive K562 cells loaded with different concentrations of KRASG12D peptide for 24 hours. Subsequently, the cells were stained at 25°C in the dark with 20 μL of PBS containing PE-labeled anti-human CD137 antibody (final antibody concentration 1 μg / mL) for 10 minutes. After two PBS washes, CD137 expression was analyzed by flow cytometry, and the EC50 value (the peptide concentration that induces 50% TCR-T cell development is defined as the EC50 value) was calculated. The results are shown in Tables 3 and 4. Figure 4AAs shown, the results indicate that most of these TCR clones showed no change or an increase in EC50, indicating that the recognition ability of the obtained mutants for KRASG12D was not affected.

[0104] Table 3 EC50 values ​​of different antibodies

[0105]

[0106] Secondly, the binding capacity of TCR clones was detected using SMC1A29-38 tetramer. Specifically, 1*10^6 TCR-Jurkat cells were collected, centrifuged to remove the supernatant, and resuspended in 20 μL of PBS containing APC-labeled SMC1A29-38 tetramer (final tetramer concentration 1 μg / mL). The cells were stained at 4°C in the dark for 25 minutes. After two PBS washes, the expression of TCR binding to the tetramer was analyzed by flow cytometry. The results are as follows: Figure 4B As shown, the results indicate that six TCR clones from KT13 and Lib1, and all clones from Lib2, exhibited negligible tetramer binding levels.

[0107] Third, functional confirmation was performed to study TCR activation in response to off-target stimuli. Specifically, TCR-Jurkat cells were co-cultured with HLA-A*11:01-positive K562 cells loaded with 100 nM SMC1A29-38 peptide for 6 hours, with each cell type accounting for 5*10^5 cells. Subsequently, the cells were resuspended in 20 μL of PBS containing PE-labeled anti-human CD69 antibody (final antibody concentration 1 μg / mL) and stained at 25°C in the dark for 10 minutes. After two PBS washes, CD69 expression was analyzed by flow cytometry. Results are as follows: Figure 4C As shown, the results indicate that 12 TCR clones remained activated when stimulated with 100 nM SMC1A29-38 peptide.

[0108] Finally, the presence of peptide-independent HLA reactivity in the TCR clones was examined. Specifically, all remaining clones were co-cultured with K562 cells loaded with KRASG12D peptide, HLA-A*11:01+K562 cells, or HLA-A*11:01+K562 cells. TCR-T cells were co-cultured with K562 cells, HLA-A*11:01-positive K562 cells, and HLA-A*11:01-positive K562 cells loaded with 10 μM KRASG12D peptide for 24 hours. Subsequently, the cells were stained with 20 μL of PBS containing PE-labeled anti-human CD137 antibody (final antibody concentration 1 μg / ml) for 10 minutes. After two PBS washes, CD137 expression was analyzed by flow cytometry. Results Figure 4DTherefore, the results showed that the three TCR clones exhibited a significant increase in CD69+ ratio when stimulated alone by HLA-A*11:01+K562, thus no further testing was required.

[0109] Through these four steps of functional evaluation, a total of 9 TCR clones were found to have sufficient KRASG12D recognition affinity and lost responsiveness to the SMC1A29-38 peptide, as shown in Table 4 below.

[0110] Table 4. Nine TCR clones that lost responsiveness to SMC1A29-38 peptide

[0111]

[0112] In addition, the peptide-specific profiles of these nine TCR clones were determined by X-ray scanning analysis, and the results are as follows: Figure 4E As shown, the modified cloned TCR exhibits recognition characteristics similar to KT13TCR. Therefore, this invention adjusts the recognition of pHLA by KT13TCR, causing it to lose its recognition of the homologous peptide SMC1A29-38.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A TCR targeting the peptide-HLA-A1101 complex, characterized in that, The variable region of the 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 location and mutation type are selected from any of the following: (1) The mutation location is at position 29 to 31 of the variable region of the α chain, and the mutation type is "DTT" mutated to "MFL"; (2) The mutation location is the 29th to 31st position of the variable region of the α chain, and the mutation type is "DTT" to "LHL".

2. An antigen-binding fragment, characterized in that, It includes the TCR as described in claim 1.

3. A polynucleotide, characterized in that, Encode the TCR as described in claim 1.

4. An expression carrier, characterized in that, It comprises the polynucleotide of claim 3.

5. Engineered cells, characterized in that, It includes the expression vector as described in claim 4.

6. A pharmaceutical composition, characterized in that, 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, Containing 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 cells of claim 5 and / or the pharmaceutical composition of claim 6; and Pharmaceutically acceptable carriers and / or diluents.

8. The use of 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 of claim 6 in the preparation of a pharmaceutical formulation for treating cancer; the cancer being pancreatic cancer, colon cancer, endometrial cancer, lung cancer, bile duct cancer, cervical cancer, or bladder cancer.

9. The application according to claim 8, characterized in that, Pharmaceutical formulations include protein drugs, ADC drugs, or TCR-antigen combination drugs.

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