CN114835800B - TCR or its antigen-binding fragment and its application - Google Patents

Abstract

The 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 its application. The TCR includes a sequence shown in SEQ ID NO: 15, SEQ ID NO: 36 or SEQ ID NO: 53. Encoded fragments. This TCR has strong specificity for KRAS‑G12V mutations and/or KRAS‑G12C mutations, and can mediate T cells to secrete large amounts of IFN‑γ and Granzyme‑B cytokines and kill KRAS‑G12V and/or KRAS ‑G12C gene mutated tumor cells.

Description

TCR or its antigen-binding fragment and its application

Technical field

The invention belongs to the technical field of T cell immunotherapy drugs, and specifically relates to a TCR or its antigen-binding fragment and its application.

Background technique

With the progress of research in the field of biomedicine, the selection of targets for T cell immunotherapy targeting malignant solid tumors continues to evolve, from the initial lineage antigens to viral antigens. With the emergence of high-throughput sequencing technology in recent years, , targeting individual mutations in tumors has become the focus of the new generation of T cell therapy.

T cell therapy currently mainly includes TCR-T and CAR-T treatments. At present, CAR-T technology treatment has played a significant role in the treatment of hematological 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 are limited by their current specific targets. Limited, 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. It binds to CD3 through 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 generate an immune response against tumor cells. Currently, in human cancers, 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 cancers. , 30%-40% of colon cancers, 17% of endometrial cancers, 15%-20% of lung cancers including lobular lung cancers, as well as bile duct cancer, cervical cancer, bladder cancer, etc.

Based on the above situation, there is a need for a TCR that can specifically bind to KRAS mutated polypeptides and mediate T cells to kill KRAS mutated tumor cells.

Contents of the invention

One of the objects of the present invention is to provide a TCR that can target and specifically recognize one or two sites among the KRAS-G12C and KRAS-G12V mutation sites, and a polypeptide with a KRAS-G12V mutation or a KRAS-G12C mutation. It specifically binds to the HLA complex, thereby stimulating T cell activation, mediating T cells to secrete IFN-γ and other cytokines, thereby killing tumor cells expressing KRAS-G12V mutation and/or KRAS-G12C mutation gene. The TCR or its antigen-binding fragment has strong specificity for polypeptides with KRAS-G12V mutations and/or KRAS-G12C mutations, and can mediate T cells to secrete a large amount of IFN-γ cytokines to kill KRAS-G12V mutations and/or KRAS -G12C mutated tumor cells.

On the one hand, the present invention provides a TCR, which can be selected from the following TCRs:

Type 1: The α-chain variable region of the TCR may include the α-chain CDR3 encoded by the sequence shown in SEQ ID NO:3, and the β-chain variable region of the TCR may include the β-chain encoded by the sequence shown in SEQ ID NO:6. CDR3.

Second: The α chain variable region of the TCR can include the α chain CDR3 encoded by the sequence shown in SEQ ID NO:24, and the β chain variable region of the TCR can include the β chain encoded by the sequence shown in SEQ ID NO:27. CDR3.

The third type: the α chain variable region of the TCR can include the α chain CDR3 encoded by the sequence shown in SEQ ID NO:41, and the β chain variable region of the TCR can include the β chain encoded by the sequence shown in SEQ ID NO:44. CDR3.

Type 4: TCR may include fragments encoded by the sequence shown in SEQ ID NO:15, SEQ ID NO:36 or SEQ ID NO:53.

Another aspect of the invention provides an antigen-binding fragment, which may comprise any one of the above four TCRs.

In yet another aspect, the present invention provides a polynucleotide, which can be selected from the following polynucleotides:

The first type: may include the sequences shown in SEQ ID NO:3 and SEQ ID NO:6.

The second type: may include the sequences shown in SEQ ID NO:24 and SEQ ID NO:27.

The third type: may include the sequences shown in SEQ ID NO:41 and SEQ ID NO:44.

The fourth type: may include the sequence shown in SEQ ID NO:15, SEQ ID NO:36 or SEQ ID NO:53.

Another aspect of the present invention provides an expression vector, which may contain any one of the above four polynucleotides.

Another aspect of the present invention provides an engineered cell, which can contain the above-mentioned expression vector.

Another aspect of 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 thinner.

In another aspect, the present invention provides 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 for use in preparing T cells to secrete IFN-γ and/or Granzyme- Application of B-cell factor level drugs.

Another aspect of the present invention provides 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 for preparation and detection of expression of KRAS-G12V mutation and/or KRAS-G12C. Mutated tumor cells for use in reagents or kits.

Another aspect of the present invention provides 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 for preparation and use in treating patients carrying KRAS-G12V mutation and/or KRAS-G12C. Applications in drugs for diseases caused by mutations.

The beneficial effects of the present invention are: the KT2TCR and KT3TCR provided by the present invention have strong specificity for KRAS-G12V mutant polypeptide and KRAS-G12C mutant polypeptide, and both can mediate T cells to secrete a large amount of IFN-γ and Granzyme-B Cytokines kill tumor cells with KRAS-G12V mutations and/or KRAS-G12C mutations; KT8TCR has strong specificity for KRAS-G12C mutant peptides and can mediate T cells to secrete large amounts of IFN-γ and Granzyme-B cells Factor kills KRAS-G12C mutated tumor cells.

Description of the drawings

Figure 1 is a diagram showing the flow cytometric detection results of KT2 specificity for KRAS-G12V and KRAS-G12C mutant polypeptides in the Example.

Figure 2 is a diagram showing the flow cytometric detection results of KT3 specificity for KRAS-G12V and KRAS-G12C mutant polypeptides in the Example.

Figure 3 is a diagram showing the flow cytometric detection results of KT8 specificity for KRAS-G12C mutant polypeptide in the Example.

Figure 4 shows the KT2-mediated secretion of Granzyme-B cytokines by T cells in the Example.

Figure 5 shows the KT2-mediated secretion of IFN-γ cytokines by T cells in the Example.

Figure 6 shows the KT3-mediated secretion of Granzyme-B cytokines by T cells in the Example.

Figure 7 shows the KT3-mediated secretion of IFN-γ cytokines by T cells in the Example.

Figure 8 shows the KT8-mediated secretion of Granzyme-B cytokines by T cells in the Example.

Figure 9 shows the KT8-mediated secretion of IFN-γ cytokines by T cells in the Example.

Detailed ways

The terminology used herein is used to describe particular embodiments only and is not intended to limit the disclosure. Expressions in the singular include expressions in the plural unless there is a clearly different meaning in the context. As used herein, it should be understood that terms such as "includes," "has," "comprises" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials, or combinations. 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, assemblies, parts, elements, materials or combinations thereof may be present or may be added. As used herein, "/" may be interpreted as "and" or "or" depending on the context.

In the present invention, the term "antigen-binding fragment" refers to the antigen-binding fragment of TCR and TCR analogs, which usually include at least part of the antigen-binding region or variable region of the parent TCR, such as one or more CDRs. Fragments of a TCR retain at least some of the binding specificity of the parent TCR.

The object of the present invention is to provide a TCR. One of the objects of the present invention is to provide a TCR that can target and specifically recognize the KRAS-G12C and/or KRAS-G12V mutation site and interact with the KRAS-G12V and/or KRAS-G12C mutation. The polypeptide specifically binds to the HLA complex, thereby stimulating T cell activation, mediating T cells to secrete IFN-γ and other cytokines, thereby killing tumor cells expressing KRAS-G12V mutations and/or KRAS-G12C mutations. The TCR or its antigen-binding fragment has strong specificity for KRAS-G12V mutated and/or KRAS-G12C mutated polypeptides, and can mediate T cells to secrete a large amount of IFN-γ cytokines.

On the one hand, the present invention provides a TCR. The TCR includes an alpha chain variable region and a beta chain variable region. The TCR in the present invention can be selected from the following TCRs:

The first TCR:

In the first TCR, the α chain variable region at least includes the α chain CDR3 encoded by the sequence shown in SEQ ID NO:3, and the β chain variable region at least includes the β chain CDR3 encoded by the sequence shown in SEQ ID NO:6.

Further, the α chain variable region in the first TCR may also include: α chain CDR1 encoded by the sequence shown in SEQ ID NO:1 and/or α chain CDR2 encoded by the sequence shown in SEQ ID NO:2; The β-chain variable region may also include: β-chain CDR1 encoded by the sequence shown in SEQ ID NO:4 and/or β-chain CDR2 encoded by the sequence shown in SEQ ID NO:5.

Furthermore, the α chain variable region in the first TCR may also include: α chain FR1 encoded by the sequence shown in SEQ ID NO:7, α chain FR2 encoded by the sequence shown in SEQ ID NO:8, and α chain FR2 encoded by the sequence shown in SEQ ID NO:8. One or more of the α chain FR3 encoded by the sequence shown in ID NO:9 and the α chain FR4 encoded by the sequence shown in SEQ ID NO:10; the β chain variable region may also include: as shown in SEQ ID NO:11 β chain FR1 encoded by the sequence shown in SEQ ID NO:12, β chain FR2 encoded by the sequence shown in SEQ ID NO:13, β chain FR3 encoded by the sequence shown in SEQ ID NO:13 and β chain encoded by the sequence shown in SEQ ID NO:14 One or more of FR4.

The second type of TCR:

In the second type of TCR, the α chain variable region at least includes the α chain CDR3 encoded by the sequence shown in SEQ ID NO:24, and the β chain variable region at least includes the β chain CDR3 encoded by the sequence shown in SEQ ID NO:27.

Further, the α chain variable region in the second TCR may also include: α chain CDR1 encoded by the sequence shown in SEQ ID NO:22 and/or α chain CDR2 encoded by the sequence shown in SEQ ID NO:23; The β-chain variable region may also include: β-chain CDR1 encoded by the sequence shown in SEQ ID NO:25 and/or β-chain CDR2 encoded by the sequence shown in SEQ ID NO:26.

Furthermore, the α-chain variable region in the second TCR may also include: α-chain FR1 encoded by the sequence shown in SEQ ID NO:28, α-chain FR2 encoded by the sequence shown in SEQ ID NO:29, and α-chain FR2 encoded by the sequence shown in SEQ ID NO:29. One or more of the α chain FR3 encoded by the sequence shown in ID NO:30 and the α chain FR4 encoded by the sequence shown in SEQ ID NO:31; the β chain variable region may also include: as shown in SEQ ID NO:32 β chain FR1 encoded by the sequence shown in SEQ ID NO:33, β chain FR2 encoded by the sequence shown in SEQ ID NO:34, β chain FR3 encoded by the sequence shown in SEQ ID NO:34 and β chain encoded by the sequence shown in SEQ ID NO:35 One or more of FR4.

The third type of TCR:

In the third type of TCR, the α chain variable region at least includes the α chain CDR3 encoded by the sequence shown in SEQ ID NO:41, and the β chain variable region at least includes the β chain CDR3 encoded by the sequence shown in SEQ ID NO:44.

Further, the α chain variable region in the third TCR may also include: α chain CDR1 encoded by the sequence shown in SEQ ID NO:39 and/or α chain CDR2 encoded by the sequence shown in SEQ ID NO:40; The β-chain variable region may also include: β-chain CDR1 encoded by the sequence shown in SEQ ID NO:42 and/or β-chain CDR2 encoded by the sequence shown in SEQ ID NO:43.

Furthermore, the α chain variable region in the third TCR may also include: α chain FR1 encoded by the sequence shown in SEQ ID NO:45, α chain FR2 encoded by the sequence shown in SEQ ID NO:46, and α chain FR2 encoded by the sequence shown in SEQ ID NO:46. One or more of the α chain FR3 encoded by the sequence shown in ID NO:47 and the α chain FR4 encoded by the sequence shown in SEQ ID NO:48; the β chain variable region may also include: as shown in SEQ ID NO:49 β chain FR1 encoded by the sequence shown in SEQ ID NO:50, β chain FR2 encoded by the sequence shown in SEQ ID NO:51, β chain FR3 encoded by the sequence shown in SEQ ID NO:51 and β chain encoded by the sequence shown in SEQ ID NO:52 One or more of FR4.

Specifically, the CDR3 of the α chain and β chain of the above three TCRs belong to the hypervariable region of the variable region. When the TCR recognizes the MHC-antigen peptide complex, CDR3 directly binds to the antigen peptide, directly affecting the TCR's effect on KRAS. -Recognition ability of G12V mutations. 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 compared to CDR3. When the TCR recognizes the MHC-antigen peptide complex, The two hypervariable regions, CDR1 and CDR2, recognize and bind to the side walls of the antigen-binding groove of MHC molecules.

Specifically, the FRs (FR1, FR2, FR3, FR4) in the above three TCRs are skeleton regions, which are used to connect the CDR regions and are relatively stable. In addition to selecting the above-mentioned sequences, the FR regions in the above-mentioned first, second and third TCRs can also select FR1, FR2, FR3 and FR4 sequence identity of ≥80% with the above-mentioned three TCRs. The above sequences, such as ≥80%, 85%, 90% or 95% of the sequences, can be of mouse or human origin (but other sources are not excluded, such as rabbit, pig and other animal sources). In some specific implementations, the sequences of the FR regions and the CDR regions of the above-mentioned α chain and β chain can be arranged according to FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, respectively constituting the α chain variable region of the TCR and the β chain variable region. For example, in the first TCR, the sequence encoding the α chain variable region is shown in SEQ ID NO: 16, and the sequence encoding the β chain variable region is shown in SEQ ID NO: 17; In the two types of TCR, the sequence encoding the variable region of the α chain is shown in SEQ ID NO: 37, and the sequence encoding the variable region of the β chain is shown in SEQ ID NO: 38; in the third TCR, the sequence encoding the variable region of the α chain is shown in SEQ ID NO: 37. The sequence of the region is shown in SEQ ID NO:54, and the sequence encoding the β chain variable region is shown in SEQ ID NO:55.

Specifically, in addition to the above-mentioned α chain variable region and β chain variable region, the TCR also contains the α chain constant region, the β chain also contains the β chain constant region, and the sources of the α chain constant region and the β chain constant region It can be mouse, human or human-mouse chimeric (but other sources are not excluded, such as rabbit, pig and other animal sources). It is an area where mutations will almost never occur.

The fourth type of TCR:

The fourth TCR includes a fragment encoded by the sequence shown in SEQ ID NO:15, SEQ ID NO:36 or SEQ ID NO:53.

Specifically, in the fourth TCR, the fragment encoded by the sequence shown in SEQ ID NO: 15 includes the α chain variable region and the β chain variable region in the first TCR; it can be encoded by the sequence shown in SEQ ID NO: 15: The fragment encoded by the sequence shown in 36 includes the α chain variable region and the β chain variable region in the first TCR; the fragment encoded by the sequence shown in SEQ ID NO:53 includes the α chain in the first TCR chain variable region and beta chain variable region.

Specifically, in the second TCR, the encoding TCR sequence can also be a sequence with an identity of ≥80% to the sequence shown in SEQ ID NO:15, SEQ ID NO:36 or SEQ ID NO:53, such as an identity of ≥80%. , 85%, 90%, 95% of the sequences.

Specifically, in the above-mentioned TCR, regarding the source of the variable region and constant region of the α chain or β chain, the α chain variable region or the β chain variable region comes from mouse or human sources (but other sources are not excluded, such as rabbits). source, pig source and other animal sources), the source of the α chain or β chain constant region can be the same as the source of the α chain or β chain variable region, or it can be different.

In yet another aspect, the present invention provides an antigen-binding fragment, which may include any one of the above four TCRs.

In yet another aspect, the present invention provides a polynucleotide, which can be selected from the following polynucleotides:

The first polynucleotide:

The first polynucleotide includes at least the sequence shown in SEQ ID NO:3 and SEQ ID NO:6.

Further, the first polynucleotide may further include a sequence shown in SEQ ID NO:1, a sequence shown in SEQ ID NO:2, a sequence shown in SEQ ID NO:4, a sequence shown in SEQ ID NO:5 Sequence, as shown in SEQ ID NO:7, sequence as shown in SEQ ID NO:8, sequence as shown in SEQ ID NO:9, sequence as shown in SEQ ID NO:10, sequence as shown in SEQ ID NO:11 Sequence, one or more of the sequence shown in SEQ ID NO:12, the sequence shown in SEQ ID NO:13 and the sequence shown in SEQ ID NO:14.

Second polynucleotide:

The second polynucleotide includes at least the sequence set forth in SEQ ID NO:24 and SEQ ID NO:27.

Further, the second polynucleotide may further include a sequence shown in SEQ ID NO:22, a sequence shown in SEQ ID NO:23, a sequence shown in SEQ ID NO:25, a sequence shown in SEQ ID NO:26 Sequence, as shown in SEQ ID NO:27, as shown in SEQ ID NO:28, as shown in SEQ ID NO:29, as shown in SEQ ID NO:30, as shown in SEQ ID NO:31 , one or more of the sequence shown in SEQ ID NO:32, the sequence shown in SEQ ID NO:33, and the sequence shown in SEQ ID NO:35.

The third polynucleotide:

The third polynucleotide includes at least the sequence set forth in SEQ ID NO:41 and SEQ ID NO:44.

Further, the third polynucleotide may further include the sequence shown in SEQ ID NO:39, the sequence shown in SEQ ID NO:40, the sequence shown in SEQ ID NO:42, the sequence shown in SEQ ID NO:43 Sequence, the sequence shown in SEQ ID NO:45, the sequence shown in SEQ ID NO:46, the sequence shown in SEQ ID NO:47, the sequence shown in SEQ ID NO:48, the sequence shown in SEQ ID NO:49 , one or more of the sequence shown in SEQ ID NO:50, the sequence shown in SEQ ID NO:51 and the sequence shown in SEQ ID NO:52.

The fourth polynucleotide:

The fourth polynucleotide may comprise the sequence set forth in SEQ ID NO:15, SEQ ID NO:36 or SEQ ID NO:53.

Specifically, the fourth polynucleotide includes the sequence of the first polynucleotide. For example, the nucleic acid sequence shown in SEQ ID NO:15 includes the sequences shown in SEQ ID NO:3 and SEQ ID NO:6; such as The nucleic acid sequence shown in SEQ ID NO:36 includes the sequence shown in SEQ ID NO:24 and SEQ ID NO:27; the nucleic acid sequence shown in SEQ ID NO:53 includes the sequence shown in SEQ ID NO:41 and SEQ ID NO: The sequence shown in 44.

Specifically, the above-mentioned polynucleotides may also include other (in addition to the above-mentioned polynucleotides) any nucleotide encoding any of the above-mentioned TCRs, such as a codon-optimized sequence including the sequence shown in SEQ ID NO: 3, Contains the codon-optimized sequence of the sequence shown in SEQ ID NO:6, the codon-optimized sequence of the sequence shown in SEQ ID NO:15; for example, the codon-optimized sequence of the sequence shown in SEQ ID NO:24 , including the codon-optimized sequence of the sequence shown in SEQ ID NO:27, the codon-optimized sequence of the sequence shown in SEQ ID NO:36; for example, the codon-optimized sequence of the sequence shown in SEQ ID NO:41 The sequence includes the codon-optimized sequence of the sequence shown in SEQ ID NO:44, and the codon-optimized sequence of the sequence shown in SEQ ID NO:53.

Another aspect of the present invention provides an expression vector, which may contain any one of the above four polynucleotides.

Furthermore, the expression vector can be selected from lentiviral expression vector, retroviral expression vector, adenovirus expression vector, adeno-associated virus expression vector, DNA vector, RNA vector and 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).

Another aspect of the present invention provides an engineered cell, which can contain the above-mentioned expression vector. Specifically, the engineered cell can be a host cell, and the above-mentioned expression vector is introduced into the host cell to encode the TCR polypeptide; it can also be a T cell, and the above-mentioned expression vector (loaded with the target gene) is transfected into the T cell to obtain TCR-T cells. Recognition and killing of KRAS-G12V and/or KRAS-G12C mutant peptides.

Another aspect of the present invention provides a pharmaceutical composition, which contains 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 thinner.

Specifically, the pharmaceutically acceptable carrier and/or diluent means that 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 can be prepared into various required forms. dosage form. Examples of oral preparations include tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film coatings, pellets, sublingual tablets, ointments, etc., and non-oral preparations, Examples include injections, suppositories, transdermal preparations, ointments, plasters, external solutions, etc. Those skilled in the art can select an appropriate dosage form according to the administration route, administration target, etc.

Another aspect of the present invention provides a method for preparing 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 for improving the secretion of IFN-γ and/or Granzyme-B by T cells. Cytokine levels in pharmaceutical applications.

Further, drugs used to increase the level of IFN-γ and/or Granzyme-B cytokines secreted by T cells include cell drugs, protein drugs, ADC drugs or TCR and antigen combination drugs.

Another aspect of the present invention provides 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 for preparation and detection of expression of KRAS-G12V mutation and/or KRAS-G12C. Mutated tumor cells for use in reagents or kits.

Specifically, the kit involved can be divided into various small boxes, and then contain various detection reagents. Detection reagents and detection kits can be used indirectly or directly in various malignant tumors expressing KRAS-G12C mutations and/or KRAS-G12V mutations, such as pancreatic tumors, colorectal malignant tumors, endometrial malignant tumors, and lung malignancies. Tumors, bile duct malignant tumors, cervical malignant tumors, etc.

Another aspect of the present invention provides 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 for preparation and use in treating patients carrying KRAS-G12C mutation and/or KRAS-G12V. Applications in drugs for diseases caused by mutations.

Further, diseases caused by carrying KRAS-G12C mutation and/or KRAS-G12V mutation include pancreatic cancer, colorectal cancer, endometrial cancer, lung cancer, cholangiocarcinoma, cervical cancer or bladder cancer.

It should be noted that in the above-mentioned applications, the first TCR, the second TCR, the fourth TCR including the nucleic acid sequence shown in SEQ ID NO:15, and the fourth TCR including the nucleic acid sequence shown in SEQ ID NO. : The TCR of the nucleic acid sequence shown in 36 and the expression vector loaded with these TCR, and the engineered cells transfected with these expression vectors can recognize and kill the KRAS-G12V and KRAS-G12C mutant polypeptides; while the third TCR and the third TCR mentioned above can Four types of TCRs containing the nucleic acid sequence shown in SEQ ID NO: 53, expression vectors loaded with these TCRs, and engineered cells transfected with these expression vectors can recognize and kill the KRAS-G12C mutant polypeptide.

Specifically, in the above several applications, for polypeptides with KRAS-G12C mutations and/or KRAS-G12V mutations, HLA-restricted T cell epitope polypeptides are selected, and HLA-A1101 with stronger affinity is selected, that is, KRAS-G12V and /or KRAS-G12C mutated polypeptides are presented by HLA-A1101 molecules, but the selection of other HLA molecules, such as other small molecules in the HLA-A11 series, is not excluded.

Specifically, in the above several applications, the TCR can be any one of the four TCRs mentioned above; the polynucleotide can be any one of the four polynucleotides mentioned above. In the above applications, TCR or antigen-binding fragments or polynucleotides or expression vectors or engineered cells can be added with excipients to form drugs, or can be combined with other active drugs or detection reagents, such as some existing chemotherapy drugs, such as Alkylating agents, anti-metabolites, anti-tumor antibiotics, plant-based anti-cancer drugs, hormones, immune preparations, etc.; can also be combined with surgery. In specific cases, the drug or combination of drugs is used according to the tumor condition.

Specifically, in the above several applications, the polypeptides with KRAS-G12C mutation and KRAS-G12V mutation can be 9 polypeptides, or 10 polypeptides or other numbers of polypeptides, and the KRAS-G12C mutation can be 10 polypeptides. The amino acid sequence is shown in SEQ ID NO: 19, and the amino acid sequence of 10 KRAS-G12V mutated polypeptides is shown in SEQ ID NO: 21.

In order to better understand the present invention, the content of the present invention is further clarified below with reference to specific examples, but the content of the present invention is not limited only to the following examples.

In the following examples, the KRAS-G12 wild-type polypeptide involved in the verification (KRAS-G12 for short), the sequence is as shown in SEQ ID NO: 18; the KRAS-G12C mutant polypeptide (KRAS-G12V for short) involved in the verification, the sequence is as SEQ ID NO. :19; the KRAS-G12D mutant polypeptide involved in the verification (KRAS-G12C for short), the sequence is shown in SEQ ID NO:20; the KRAS-G12V mutant polypeptide involved in the verification (KRAS-G12D for short), the sequence is shown in SEQ ID NO :21.

In the following examples, the gene amplification, sequencing and analysis methods involved are performed according to the following steps:

(1) RT-PCR: The downstream primers used are primers specific to the constant region of the TCR gene (Ca_RV1primer, Cb_RV1primer), and the upstream primers include the outer linker and the initial 20 bp sequence of the TCR signal peptide (AL primers, BLprimers) (specifically according to the literature "Hamana H, Shitaoka K, Kishi H, Ozawa T, Muraguchi A. Anovel, rapid and efficient method of cloning functional antigen-specific T-cell receptors from single human and mouse T-cells. Biochem Biophys ResCommun. 2016 Jun 10; 474( 4):709-714.doi:10.1016/j.bbrc.2016.05.015.Epub 2016May4.PMID:27155153》Examples of the contents recorded);

(2) Second round of PCR: Use the TCR PCR product obtained in (1) above as a template, the upstream primer is the outer adapter primer (P2A-Cprimer), and the downstream primer is a section of the TCR constant region upstream of the α chain constant region. The specific primer (Ca_RV2primer) is used to obtain the second round PCR product of the TCRα chain; the upstream primer is the outer adapter primer (BES-AP primer), and the downstream primer is a specific primer for a section of the TCR constant region upstream of the β chain constant region (Cb_RV2 primer ), the second round PCR product of the TCRβ chain was obtained (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 ResCommun.2016Jun 10;474(4):709-714.doi:10.1016/j.bbrc.2016.05.015.Epub 2016May4.PMID:27155153");

(3) Conduct agarose gel electrophoresis on the amplified second-round PCR amplification product containing the TCR α chain and β chain variable region genes, and obtain the target gene of the TCR α chain or β chain variable region at the 500bp-750bp position. ;

(4) The target band was subjected to first-generation sequencing (Beijing Qingke Company). After TCR sequence analysis, TCR clones that appeared with high frequency were selected and named KT2, KT3 and KT8 respectively.

The amplification system used in the above-mentioned implementation steps involving gene amplification, sequencing and analysis methods is shown in Table 1 below (specifically in accordance with 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.2016Jun 10;474(4):709-714.doi:10.1016/j.bbrc.2016.05.015.Epub 2016May 4. The contents recorded in PMID:27155153 will be implemented).

Table 1 Amplification system

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

In the following examples, the nucleic acid sequence or amino acid sequence of the materials involved is shown in Table 2 below:

Among them, the KT2TCR alpha chain variable region refers to the above-mentioned first TCR consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO :9 and SEQ ID NO:10 The fragment obtained by encoding the alpha chain variable region according to the sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the KT2TCR beta chain variable region refers to the above The sequences shown in the first TCR by SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14 are in accordance with

The fragment encoded by the β-chain variable region composed of the sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; the full-length KT2TCR sequence refers to the KT2α chain variable region and β chain variable region connected to each other respectively. The TCR of the complete structure after the structure is one of the above-mentioned fourth TCRs (obtained from the sequence code shown in SEQ ID NO: 15).

The KT3TCRα chain variable region refers to the sequence shown by SEQIDNO:22, SEQIDNO:23, SEQIDNO:24, SEQIDNO:28, SEQIDNO:29, SEQIDNO:30 and SEQIDNO:31 in the above second TCR according to FR1-CDR1- The fragment encoded by the genome of the alpha chain variable region composed of the sequence of FR2-CDR2-FR3-CDR3-FR4; the KT3TCR beta chain variable region refers to the above-mentioned second TCR consisting of SEQIDNO:25, SEQIDNO:26, and SEQIDNO: 27. Fragments encoded by the β chain variable region genome consisting of the sequences shown in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35 in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; The full-length KT3TCR sequence refers to the complete structure of the TCR after the KT3 alpha chain variable region and beta chain variable region are connected to other structures respectively. It is one of the fourth TCRs mentioned above (encoded by the sequence shown in SEQ ID NO: 36) .

The KT8TCRα chain variable region refers to the sequence shown by SEQIDNO:39, SEQIDNO:40, SEQIDNO:41, SEQIDNO:45, SEQIDNO:46, SEQIDNO:47 and SEQIDNO:48 in the above third TCR according to FR1-CDR1- The fragment encoded by the genome of the alpha chain variable region consisting of the sequence of FR2-CDR2-FR3-CDR3-FR4; the KT8TCR beta chain variable region refers to the above-mentioned first TCR consisting of SEQIDNO: 42, SEQIDNO: 43, and SEQIDNO: 44. Fragments encoded by the β chain variable region genome consisting of the sequences shown in SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51 and SEQ ID NO: 52 in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4; The full-length KT8TCR sequence refers to the complete structure of the TCR after the KT8 alpha chain variable region and beta chain variable region are connected to other structures respectively. It is one of the fourth TCRs mentioned above (encoded by the sequence shown in SEQ ID NO: 53) .

Table 2 Nucleic acid sequence or amino acid sequence of materials involved in the examples

In the following examples, KRAS-G12, KRAS-G12V, KRAS-G12C, and KRAS-G12D polypeptides were synthesized by Kingris Biotechnology Co., Ltd.

In the following examples, the tetramer of KRAS-G12/HLA-A1101, the tetramer of KRAS-G12C/HLA-A1101, the tetramer of KRAS-G12D/HLA-A1101 and the tetramer of KRAS-G12V/HLA-A1101 The polymers were all prepared using the QuickSwitch ^TM QuantTetramer Kit from Japan MBL Company, which allows for simple preparation of tetramer reagents in the research laboratory. The specific preparation process is as follows. 1ulKRAS-G12 polypeptide and 1ulKRAS-G12V polypeptide displacer, 1ulKRAS-G12 polypeptide and 1ulKRAS-G12C polypeptide displacer, 1ulKRAS-G12 polypeptide and 1ulKRAS-G12D polypeptide displacer were added to 50ul of QuickSwitchTetramer to obtain KRAS- G12/HLA-A1101 tetramer, KRAS-G12C/HLA-A1101 tetramer, KRAS-G12D/HLA-A1101 tetramer and KRAS-G12V/HLA-A1101 tetramer, place at room temperature for 4 hours at 4℃ Save for later use.

Example 1 Specific TCR screening

(1) Use prediction software (HLAthena) to predict the immunogenicity of KRAS-G12V and KRAS-G12C mutant proteins, and analyze and obtain the immunogenic antigen peptide sequence. The sequence of KRAS-G12V mutation is shown in SEQ ID NO: 21, KRAS- The sequence of the G12C mutation is shown in SEQ ID NO: 19;

(2) Synthesize relevant short peptides with a purity of 85%. The synthesized peptides are dissolved in DMSO into a 10mM solution;

(3) In vitro stimulation and expansion of KRAS-specific T cells: Collect peripheral blood from healthy people, detect their HLA genotypes, select HLA-A1101-positive peripheral blood samples, and isolate mononuclear nuclei from HLA-A1101-positive peripheral blood. For cells and initial T cells, add 800U/ml IL-4 and 800U/ml GM-CSF to the monocyte culture medium and culture for 4 days to differentiate into DC cells. Then add the above-mentioned KRAS-G12C to the DC cell culture medium respectively. The mutant polypeptide (concentration is 10uM) was placed in an incubator and cultured for 16 hours; then, the DC cells loaded with the above mutant peptide were mixed with the initial T cells and cultured for another 3 weeks; the cultured T cell products were analyzed by flow cytometry Technique to detect the content of specific T cells;

(4) Take 1×10 ⁷ amplified T cells, centrifuge at 250g for 10 minutes, discard the supernatant, and add 100ul of antibody staining solution (containing 1ug/ml KRAS-G12C/HLA-A1101 tetramer, 1ug/ml APC-CD3 Antibody PBS), incubate at room temperature for 30 minutes; wash three times with 5 ml of PBS containing 0.5% BSA, and centrifuge at 250g for 10 minutes; resuspend the cells in 1 ml of PBS containing 0.5% BSA, and use a flow cell sorting instrument to sort individual specificities T cells;

(5) Extract total RNA from the sorted specific T cells, and then perform RT-PCR amplification to obtain the TCR gene sequence. Analyze the sequence structure of the TCR through first-generation sequencing and IMGT database comparison to obtain the KT2 variable region, such as SEQ The α chain variable region encoded by the sequence shown in ID NO: 16 and the β chain variable region encoded by the sequence shown in SEQ ID NO: 17 are composed of the KT3 variable region encoded by the sequence shown in SEQ ID NO: 37. The α-chain variable region is composed of the β-chain variable region encoded by the sequence shown in SEQ ID NO:38; the obtained KT8 variable region is composed of the α-chain variable region encoded by the sequence shown in SEQ ID NO:54 and the β-chain variable region encoded by the sequence shown in SEQ ID NO:54. The sequence shown in NO:55 encodes the β-chain variable region.

Example 2 Verification of binding specificity

The KT2 α chain and β chain variable region (V region) are genetically connected to the TCR α chain and β chain constant region (C region), and then the α chain and β chain are connected to obtain the full-length KT2TCR sequence, full-length KT2TCR The sequence gene is shown in SEQ ID NO: 15;

The KT3 α chain and β chain variable region (V region) are genetically connected to the TCR α chain and β chain constant region (C region), and then the α chain and β chain are connected to obtain the full-length KT3TCR sequence, full-length KT3TCR The sequence gene is shown in SEQ ID NO: 36;

The KT8 α chain and β chain variable region (V region) are genetically connected to the TCR α chain and β chain constant region (C region), and then the α chain and β chain are connected to obtain the full-length KT8TCR sequence, full-length KT8TCR The sequence gene is shown in SEQ ID NO: 53;

Using lentiviral expression plasmid pWPXL (purchased from Addegene Company) as the backbone vector, KT2, KT3 and KT8 lentiviral expression vectors were constructed respectively, namely lentiviral expression vectors pWPXL-KT2TCR, pWPXL-KT3TCR and pWPXL-KT8TCR; pWPXL-KT2TCR, The pWPXL-KT3TCR and pWPXL-KT8TCR lentiviral expression vectors were co-transfected into 293T cells with the lentiviral packaging plasmid pMD2.G and psPAX2 plasmid respectively at a mass ratio of 1:0.5:1. The viral supernatant was collected two days later; the viral supernatant was used for infection. For Jurkat cells, the medium was changed 24 hours after infection. After culturing for 2-3 days, Jurkat cells expressing KT2TCR, KT3TCR and KT8TCR were obtained for downstream experiments.

The Jurkat cells expressing KT2TCR, KT3TCR and KT8TCR were mixed with the K562 cells expressing the A1101 gene at a cell number ratio of 1:1; the K562 cells expressing the KT2TCR and the A1101 gene were mixed and KRAS- at a final concentration of 10uM was added. G12, KRAS-G12C and KRAS-G12V polypeptides, and collect the cells after 6 hours; mix the K562 cells expressing KT3TCR and KT8TCR with the A1101 gene and add KRAS-G12, KRAS-G12C, KRAS-G12V and KRAS-G12V at a final concentration of 10uM respectively. KRAS-G12D peptide, cells were recovered after 6 hours;

Then add 50ul of cell staining solution containing PE-CD69 antibody to the above recovered cells, and incubate at room temperature for 30 minutes; then wash three times with PBS containing 0.5% BSA, and centrifuge at 200-250g for 10 minutes; then repeat with PBS containing 0.5% BSA Suspension cells were used for downstream detection; Jurkat cell activation analysis was performed using flow cytometry technology to detect Jurkat cell activation and CD69 expression.

The analysis results of Jurkat cells expressing KT2TCR are shown in Figure 1. KT2TCR will be specifically activated after stimulation by KRAS-G12V or KRAS-G12C, so it is a KRAS-G12V and/or KRAS-G12C specific TCR.

The analysis results of Jurkat cells expressing KT3TCR are shown in Figure 2. KT3TCR will be specifically activated after stimulation by KRAS-G12V or KRAS-G12C, so it is a KRAS-G12V and/or KRAS-G12C specific TCR.

The analysis results of Jurkat cells expressing KT8TCR are shown in Figure 3. KT8TCR will be specifically activated after stimulation by KRAS-G12C, so it is a KRAS-G12C-specific TCR.

Example 3 Test of secretion levels of IFN-γ and Granzyme-B

KT2-T cells, KT3-T cells and KT8-T cells prepared using human PBMC T cells were compared with A1101-K562 cells loaded with KRAS-G12, KRAS-G12C, KRAS-G12D and KRAS-G12V polypeptides1. :1 Mix, and add 2×10 ⁵ cells/well into a 96-well plate in a volume of 100 μl. Each sample is repeated three times. In the PMA/ionomycin (ION) group, 1 μl PMA is added to every 250 μl of cell culture medium. /ionomycin mixture (250×) was diluted into working solution in advance and used as a positive stimulation control. T cells (D1mock and D2mock) not infected with KT2, KT3 and KT8 lentivirus were added in parallel as negative controls; after culturing at 37°C for 24 hours, take the supernatant from the 96-well plate culture wells and centrifuge at 500g for 5 minutes. Remove the remaining cells, add the supernatant to ELISA plates coated with anti-IFN-γ antibody or anti-Granzyme-B antibody, and detect the IFN-γ level and Granzyme-B level in the supernatant. The results are as follows:

The level results of KT2-mediated T cells secreting Granzyme-B are shown in Figure 4. KT2-T cells will secrete Granzyme-B after being stimulated by KRAS-G12V and/or KRAS-G12C, so KT2 is KRAS-G12V and/or or KRAS-G12C-specific TCR;

The results of KT2-mediated T cells secreting IFN-γ are shown in Figure 5. KT2-T cells will secrete Granzyme-B after being stimulated by KRAS-G12V and/or KRAS-G12C. Therefore, KT2 is KRAS-G12V and/or KRAS-G12C. or KRAS-G12C-specific TCR;

The level results of KT3 mediated T cells secreting Granzyme-B are shown in Figure 6. KT3-T cells will secrete Granzyme-B after being stimulated by KRAS-G12V and/or KRAS-G12C, so KT3 is KRAS-G12V and/or or KRAS-G12C-specific TCR;

The results of KT3-mediated IFN-γ secretion by T cells are shown in Figure 7. KT3-T cells will secrete Granzyme-B after being stimulated by KRAS-G12V and/or KRAS-G12C. Therefore, KT3 is KRAS-G12V and/or KRAS-G12C. or KRAS-G12C-specific TCR;

The results of the level of Granzyme-B secretion by KT8-mediated T cells are shown in Figure 8. KT8-T cells secrete Granzyme-B after stimulation by KRAS-G12C, so KT8 is a KRAS-G12C-specific TCR;

The results of KT8-mediated IFN-γ secretion by T cells are shown in Figure 9. KT8-T cells secrete Granzyme-B after stimulation by KRAS-G12C, so KT8 is a KRAS-G12C-specific TCR.

sequence list

<110> Chongqing Medical University, Feng Yulin

<120> TCR or its antigen-binding fragment and its application

<130> 2022-3-29

<160> 55

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 1

aacagtgcttctcagtct 18

<210> 2

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 2

gtatactcca gtggtaat 18

<210> 3

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 3

gtggtgaacg atggaaacaa gctggtc 27

<210> 4

<211> 15

<212> DNA

<213> Artificial Sequence

<400> 4

atgaaccata actcc 15

<210> 5

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 5

tcagcttctg agggtacc 18

<210> 6

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 6

gccagcagtg aagttgtatt gggcattcag tac 33

<210> 7

<211> 78

<212> DNA

<213> Artificial Sequence

<400> 7

cggaaggagg tggagcagga tcctggaccc ttcaatgttc cagagggagc cactgtcgct 60

ttcaactgta cttacagc 78

<210> 8

<211> 51

<212> DNA

<213> Artificial Sequence

<400> 8

ttcttctggt acagacagga ttgcaggaaa gaacctaagt tgctgatgtc c 51

<210> 9

<211> 99

<212> DNA

<213> Artificial Sequence

<400> 9

gaagatggaa ggtttacagc acagctcaat agagccagcc agtatatttc cctgctcatc 60

agagactcca agctcagtga ttcagccacc tacctctgt 99

<210> 10

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 10

tttggcgcag gaaccattct gagagtcaag tcc 33

<210> 11

<211> 72

<212> DNA

<213> Artificial Sequence

<400> 11

ggtgtcactc agacccccaaa attccaggtc ctgaagacag gacagagcat gacactgcag 60

tgtgcccagg at 72

<210> 12

<211> 51

<212> DNA

<213> Artificial Sequence

<400> 12

atgtactggt atcgacaaga cccaggcatg ggactgaggc tgatttatta c 51

<210> 13

<211> 111

<212> DNA

<213> Artificial Sequence

<400> 13

actgacaaag gagaagtccc caatggctac aatgtctcca gattaaacaa acgggagttc 60

tcgctcaggc tggagtcggc tgctccctcc cagacatctg tgtacttctg t 111

<210> 14

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 14

ttcggcgccgggacccggctctcagtgctg 30

<210> 15

<211> 1452

<212> DNA

<213> Artificial Sequence

<400> 15

gtattgggca ttcagtactt cggcgccggg acccggctct cagtgctgga ggacctgcgc 60

aacgtgacac cccctaaggt gtccctgttt gagccttcta aggccgagat cgccaataag 120

cagaaggcca ccctggtgtg cctggcccgc ggcttctttc cagatcacgt ggagctgtcc 180

tggtgggtga acggcaagga ggtgcactcc ggcgtgtcta cagaccccca ggcctacaag 240

gagagcaatt actcctattg cctgagctcc aggctgcgcg tgagcgccac cttttggcac 300

aacccaagga atcacttccg ctgtcaggtg cagtttcacg gcctgtctga ggaggataag 360

tggccagagg gcagcccaaa gcctgtgaca cagaacatct ccgccgaggc ctggggaagg 420

gcagactgtg gcatcaccag cgcctcctat caccagggcg tgctgagcgc cacaatcctg 480

tacgagatcc tgctgggcaa ggccaccctg tatgccgtgc tggtgtctgg cctggtgctg 540

atggctatgg tgaagaagaa gaacagcaga gccaaaagaa gtggttctgg cgcgacgaat 600

tttagtttgc ttaagcaagc cggagatgtg gaggaaaatc ctggaccgat gatatccttg 660

agagttttac tggtgatcct gtggcttcag ttaagctggg tttggagcca acggaaggag 720

gtggagcagg atcctggacc cttcaatgtt ccagaggggag ccactgtcgc tttcaactgt 780

acttacagca acagtgcttc tcagtctttc ttctggtaca gacaggattg caggaaagaa 840

cctaagttgc tgatgtccgt atactccagt ggtaatgaag atggaaggtt tacagcacag 900

ctcaatagag ccagccagta tatttccctg ctcatcagag actccaagct cagtgattca 960

gccacctacc tctgtgtggt gaacgatgga aacaagctgg tctttggcgc aggaaccatt 1020

ctgagagtca agtccgacat ccagaaccca gagcccgccg tgtaccagct gaaggacccc 1080

agaagccagg atagcaccct gtgcctgttc accgactttg attctcagat caatgtgcct 1140

aagacaatgg agagcggcac cttcatcaca gacaagaccg tgctggatat gaaggctatg 1200

gactccaagt ctaacggcgc catcgcctgg tctaatcaga ccagcttcac ctgccaggat 1260

atctttaagg agacaaatgc cacctatcct tcctctgacg tgccatgtga tgccaccctg 1320

acagagaaga gcttcgagac cgacatgaac ctgaattttc agaacctgtc cgtgatgggc 1380

ctgagaatcc tgctgctgaa ggtggccggc ttcaatctgc tgatgacact gaggctgtgg 1440

agctcctgat aa 1452

<210> 16

<211> 324

<212> DNA

<213> Artificial Sequence

<400> 16

cggaaggagg tggagcagga tcctggaccc ttcaatgttc cagagggagc cactgtcgct 60

ttcaactgta cttacagcaa cagtgcttct cagtctttct tctggtacag acaggattgc 120

aggaaagaac ctaagttgct gatgtccgta tactccagtg gtaatgaaga tggaaggttt 180

acagcacagc tcaatagagc cagccagtat atttccctgc tcatcagaga ctccaagctc 240

agtgattcag ccacctacct ctgtgtggtg aacgatggaa acaagctggt ctttggcgca 300

ggaaccattc tgagagtcaa gtcc 324

<210> 17

<211> 330

<212> DNA

<213> Artificial Sequence

<400> 17

ggtgtcactc agacccccaaa attccaggtc ctgaagacag gacagagcat gacactgcag 60

tgtgcccagg atatgaacca taactccatg tactggtatc gacaagaccc aggcatggga 120

ctgaggctga tttattactc agcttctgag ggtaccactg acaaaggaga agtccccaat 180

ggctacaatg tctccagatt aaacaaacgg gagttctcgc tcaggctgga gtcggctgct 240

ccctcccaga catctgtgta cttctgtgcc agcagtgaag ttgtattggg cattcagtac 300

ttcggcgccgggacccggctctcagtgctg 330

<210> 18

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 18

Val Val Val Gly Ala Gly Gly Val Gly Lys

1 5 10

<210> 19

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 19

Val Val Val Gly Ala Cys Gly Val Gly Lys

1 5 10

<210> 20

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 20

Val Val Val Gly Ala Asp Gly Val Gly Lys

1 5 10

<210> 21

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 21

Val Val Val Gly Ala Val Gly Val Gly Lys

1 5 10

<210> 22

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 22

aacagtgcttctcagtct 18

<210> 23

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 23

gtatactcca gtggtaat 18

<210> 24

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 24

gtggtgaaca ccggtaacca gttctat 27

<210> 25

<211> 15

<212> DNA

<213> Artificial Sequence

<400> 25

atgaaccata actcc 15

<210> 26

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 26

tcagcttctg agggtacc 18

<210> 27

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 27

gccagcagtg aagtcatctt gggaacgcag tat 33

<210> 28

<211> 78

<212> DNA

<213> Artificial Sequence

<400> 28

cggaaggagg tggagcagga tcctggaccc ttcaatgttc cagagggagc cactgtcgct 60

ttcaactgta cttacagc 78

<210> 29

<211> 51

<212> DNA

<213> Artificial Sequence

<400> 29

ttcttctggt acagacagga ttgcaggaaa gaacctaagt tgctgatgtc c 51

<210> 30

<211> 99

<212> DNA

<213> Artificial Sequence

<400> 30

gaagatggaa ggtttacagc acagctcaat agagccagcc agtatatttc cctgctcatc 60

agagactcca agctcagtga ttcagccacc tacctctgt 99

<210> 31

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 31

tttggggacag ggacaagttt gacggtcatt cca 33

<210> 32

<211> 78

<212> DNA

<213> Artificial Sequence

<400> 32

aatgctggtg tcactcagac cccaaaattc caggtcctga agacaggaca gagcatgaca 60

ctgcagtgtgcccaggat 78

<210> 33

<211> 51

<212> DNA

<213> Artificial Sequence

<400> 33

atgtactggt atcgacaaga cccaggcatg ggactgaggc tgatttatta c 51

<210> 34

<211> 111

<212> DNA

<213> Artificial Sequence

<400> 34

actgacaaag gagaagtccc caatggctac aatgtctcca gattaaacaa acgggagttc 60

tcgctcaggc tggagtcggc tgctccctcc cagacatctg tgtacttctg t 111

<210> 35

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 35

tttggcccag gcacccggct gacagtgctc 30

<210> 36

<211> 1383

<212> DNA

<213> Artificial Sequence

<400> 36

ccccctaagg tgtccctgtt tgagccttct aaggccgaga tcgccaataa gcagaaggcc 60

accctggtgt gcctggcccg cggcttcttt ccagatcacg tggagctgtc ctggtgggtg 120

aacggcaagg aggtgcactc cggcgtgtct acagaccccc aggcctacaa ggagagcaat 180

tactcctatt gcctgagctc caggctgcgc gtgagcgcca ccttttggca caacccaagg 240

aatcacttcc gctgtcaggt gcagtttcac ggcctgtctg aggaggataa gtggccagag 300

ggcagcccaa agcctgtgac acagaacatc tccgccgagg cctggggaag ggcagactgt 360

ggcatcacca gcgcctccta tcaccagggc gtgctgagcg ccacaatcct gtacgagatc 420

ctgctgggca aggccaccct gtatgccgtg ctggtgtctg gcctggtgct gatggctatg 480

gtgaagaaga agaacagcag agccaaaaga agtggttctg gcgcgacgaa ttttagtttg 540

cttaagcaag ccggagatgt ggaggaaaat cctggaccga tgatatcctt gagagtttta 600

ctggtgatcc tgtggcttca gttaagctgg gtttggagcc aacggaagga ggtggagcag 660

gatcctggac ccttcaatgt tccagaggga gccactgtcg ctttcaactg tacttacagc 720

aacagtgctt ctcagtcttt cttctggtac agacaggatt gcaggaaaga acctaagttg 780

ctgatgtccg tatactccag tggtaatgaa gatggaaggt ttacagcaca gctcaataga 840

gccagccagt atatttccct gctcatcaga gactccaagc tcagtgattc agccacctac 900

ctctgtgtgg tgaacaccgg taaccagttc tattttggga cagggacaag tttgacggtc 960

attccagaca tccagaaccc agagcccgcc gtgtaccagc tgaaggaccc cagaagccag 1020

gatagcaccc tgtgcctgtt caccgacttt gattctcaga tcaatgtgcc taagacaatg 1080

gagagcggca ccttcatcac agacaagacc gtgctggata tgaaggctat ggactccaag 1140

tctaacggcg ccatcgcctg gtctaatcag accagcttca cctgccagga tatctttaag 1200

gagacaaatg ccacctatcc ttcctctgac gtgccatgtg atgccaccct gacagagaag 1260

agcttcgaga ccgacatgaa cctgaatttt cagaacctgt ccgtgatggg cctgagaatc 1320

ctgctgctga aggtggccgg cttcaatctg ctgatgacac tgaggctgtg gagctcctga 1380

taa 1383

<210> 37

<211> 324

<212> DNA

<213> Artificial Sequence

<400> 37

cggaaggagg tggagcagga tcctggaccc ttcaatgttc cagagggagc cactgtcgct 60

ttcaactgta cttacagcaa cagtgcttct cagtctttct tctggtacag acaggattgc 120

aggaaagaac ctaagttgct gatgtccgta tactccagtg gtaatgaaga tggaaggttt 180

acagcacagc tcaatagagc cagccagtat atttccctgc tcatcagaga ctccaagctc 240

agtgattcag ccacctacct ctgtgtggtg aacaccggta accagttcta ttttgggaca 300

gggacaagtt tgacggtcat tcca 324

<210> 38

<211> 336

<212> DNA

<213> Artificial Sequence

<400> 38

aatgctggtg tcactcagac cccaaaattc caggtcctga agacaggaca gagcatgaca 60

ctgcagtgtg cccaggatat gaaccataac tccatgtact ggtatcgaca agacccaggc 120

atgggactga ggctgattta ttactcagct tctgagggta ccactgacaa aggagaagtc 180

cccaatggct acaatgtctc cagattaaac aaacggggagt tctcgctcag gctggagtcg 240

gctgctccct cccagacatc tgtgtacttc tgtgccagca gtgaagtcat cttgggaacg 300

cagtattttg gcccaggcac ccggctgaca gtgctc 336

<210> 39

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 39

gatagcgcta tttacaac 18

<210> 40

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 40

attcagtcaa gtcagagaga g 21

<210> 41

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 41

gctgtgaggg actctggggc tggggagttac caactcact 39

<210> 42

<211> 15

<212> DNA

<213> Artificial Sequence

<400> 42

agccaagtca ccatg 15

<210> 43

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 43

gcaaatcagg gctctgaggc c 21

<210> 44

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 44

agcgttgagg aggggagaaa tacgcagtat 30

<210> 45

<211> 78

<212> DNA

<213> Artificial Sequence

<400> 45

aaacaggagg tgacgcagat tcctgcagct ctgagtgtcc cagaaggaga aaacttggtt 60

ctcaactgca gtttcact 78

<210> 46

<211> 51

<212> DNA

<213> Artificial Sequence

<400> 46

ctccagtggt ttaggcagga ccctgggaaa ggtctcacat ctctgttgct t 51

<210> 47

<211> 102

<212> DNA

<213> Artificial Sequence

<400> 47

caaacaagtg gaagacttaa tgcctcgctg gataaatcat caggacgtag tactttatac 60

attgcagctt ctcagcctgg tgactcagcc acctacctct gt 102

<210> 48

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 48

ttcgggaagg ggaccaaact ctcggtcata cca 33

<210> 49

<211> 78

<212> DNA

<213> Artificial Sequence

<400> 49

agtgctgtca tctctcaaaa gccaagcagg gatatctgtc aacgtggaac ctccctgacg 60

atccagtgtc aagtcgat 78

<210> 50

<211> 51

<212> DNA

<213> Artificial Sequence

<400> 50

atgttctggt accgtcagca acctggacag agcctgacac tgatcgcaac t 51

<210> 51

<211> 114

<212> DNA

<213> Artificial Sequence

<400> 51

acatatgaga gtggatttgt cattgacaag tttcccatca gccgcccaaa cctaacattc 60

tcaactctga ctgtgagcaa catgagccct gaagacagca gcatatatct ctgc 114

<210> 52

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 52

tttggcccag gcacccggct gacagtgctc 30

<210> 53

<211> 1755

<212> DNA

<213> Artificial Sequence

<400> 53

agtgctgtca tctctcaaaa gccaagcagg gatatctgtc aacgtggaac ctccctgacg 60

atccagtgtc aagtcgatag ccaagtcacc atgatgttct ggtaccgtca gcaacctgga 120

cagagcctga cactgatcgc aactgcaaat cagggctctg aggccacata tgagagtgga 180

tttgtcattg acaagtttcc catcagccgc ccaaacctaa cattctcaac tctgactgtg 240

agcaacatga gccctgaaga cagcagcata tatctctgca gcgttgagga ggggagaaat 300

acgcagtatt ttggcccagg cacccggctg acagtgctcg aggacctgcg caacgtgaca 360

ccccctaagg tgtccctgtt tgagccttct aaggccgaga tcgccaataa gcagaaggcc 420

accctggtgt gcctggcccg cggcttcttt ccagatcacg tggagctgtc ctggtgggtg 480

aacggcaagg aggtgcactc cggcgtgtct acagaccccc aggcctacaa ggagagcaat 540

tactcctatt gcctgagctc caggctgcgc gtgagcgcca ccttttggca caacccaagg 600

aatcacttcc gctgtcaggt gcagtttcac ggcctgtctg aggaggataa gtggccagag 660

ggcagcccaa agcctgtgac acagaacatc tccgccgagg cctggggaag ggcagactgt 720

ggcatcacca gcgcctccta tcaccagggc gtgctgagcg ccacaatcct gtacgagatc 780

ctgctgggca aggccaccct gtatgccgtg ctggtgtctg gcctggtgct gatggctatg 840

gtgaagaaga agaacagcag agccaaaaga agtggttctg gcgcgacgaa ttttagtttg 900

cttaagcaag ccggagatgt ggaggaaaat cctggaccga tggagaccct cttgggcctg 960

cttatccttt ggctgcagct gcaatgggtg agcagcaaac aggaggtgac gcagattcct 1020

gcagctctga gtgtcccaga aggagaaaac ttggttctca actgcagttt cactgatagc 1080

gctatttaca acctccagtg gtttaggcag gaccctggga aaggtctcac atctctgttg 1140

cttattcagt caagtcagag agagcaaaca agtggaagac ttaatgcctc gctggataaa 1200

tcatcaggac gtagtacttt atacattgca gcttctcagc ctggtgactc agccacctac 1260

ctctgtgctg tgagggactc tggggctggg agttaccaac tcactttcgg gaaggggacc 1320

aaactctcgg tcataccaga catccagaac ccagagcccg ccgtgtacca gctgaaggac 1380

cccagaagcc aggatagcac cctgtgcctg ttcaccgact ttgattctca gatcaatgtg 1440

cctaagacaa tggagagcgg caccttcatc acagacaaga ccgtgctgga tatgaaggct 1500

atggactcca agtctaacgg cgccatcgcc tggtctaatc agaccagctt cacctgccag 1560

gatatcttta aggagacaaa tgccacctat ccttcctctg acgtgccatg tgatgccacc 1620

ctgacagaga agagcttcga gaccgacatg aacctgaatt ttcagaacct gtccgtgatg 1680

ggcctgagaa tcctgctgct gaaggtggcc ggcttcaatc tgctgatgac actgaggctg 1740

tggagctcct gataa 1755

<210> 54

<211> 342

<212> DNA

<213> Artificial Sequence

<400> 54

aaacaggagg tgacgcagat tcctgcagct ctgagtgtcc cagaaggaga aaacttggtt 60

ctcaactgca gtttcactga tagcgctatt tacaacctcc agtggtttag gcaggaccct 120

gggaaaggtc tcacatctct gttgcttatt cagtcaagtc agagagagca aacaagtgga 180

agacttaatg cctcgctgga taaatcatca ggacgtagta ctttatacat tgcagcttct 240

cagcctggtg actcagccac ctacctctgt gctgtgaggg actctggggc tgggagttac 300

caactcactt tcgggaaggg gaccaaactc tcggtcatac ca 342

<210> 55

<211> 339

<212> DNA

<213> Artificial Sequence

<400> 55

agtgctgtca tctctcaaaa gccaagcagg gatatctgtc aacgtggaac ctccctgacg 60

atccagtgtc aagtcgatag ccaagtcacc atgatgttct ggtaccgtca gcaacctgga 120

cagagcctga cactgatcgc aactgcaaat cagggctctg aggccacata tgagagtgga 180

tttgtcattg acaagtttcc catcagccgc ccaaacctaa cattctcaac tctgactgtg 240

agcaacatga gccctgaaga cagcagcata tatctctgca gcgttgagga ggggagaaat 300

acgcagtatt ttggcccagg cacccggctg acagtgctc 339

Claims (17)

1. The TCR targeting the polypeptide-HLA-A1101 complex is characterized in that it includes an α chain variable region and a β chain variable region; the α chain variable region includes the α chain CDR1 encoded by the sequence shown in SEQ ID NO: 1, The α-chain CDR2 encoded by the sequence shown in SEQIDNO:2 and the α-chain CDR3 encoded by the sequence shown in SEQIDNO:3, and the β-chain variable region includes the β-chain CDR1 encoded by the sequence shown in SEQIDNO:4 and the sequence encoded by SEQIDNO:5. The β chain CDR2 and the β chain CDR3 encoded by the sequence shown in SEQ ID NO: 6; the polypeptide sequence is shown in SEQ ID NO: 19 or SEQ ID NO: 21. 2. The TCR according to claim 1, wherein the α chain variable region further includes: α chain FR1 encoded by the sequence shown in SEQ ID NO: 7, α chain FR2 encoded by the sequence shown in SEQ ID NO: 8, such as One or more of the α-chain FR3 encoded by the sequence shown in SEQIDNO:9 and the α-chain FR4 encoded by the sequence shown in SEQIDNO:10; the β-chain variable region also includes: the β-chain encoded by the sequence shown in SEQIDNO:11 One or more of FR1, β chain FR2 encoded by the sequence shown in SEQ ID NO: 12, β chain FR3 encoded by the sequence shown in SEQ ID NO: 13, and β chain FR4 encoded by the sequence shown in SEQ ID NO: 14. 3. TCR targeting the polypeptide-HLA-A1101 complex, characterized in that it includes an α chain variable region and a β chain variable region; the α chain variable region includes the α chain CDR3 encoded by the sequence shown in SEQ ID NO: 22, The α chain CDR3 encoded by the sequence shown in SEQ ID NO: 23 and the α chain CDR3 encoded by the sequence shown in SEQ ID NO: 24. The β chain variable region includes the β chain CDR3 encoded by the sequence shown in SEQ ID NO: 25 and the sequence encoded by SEQ ID NO: 26. The β chain CDR3 and the β chain CDR3 encoded by the sequence shown in SEQ ID NO: 27; the polypeptide sequence is shown in SEQ ID NO: 19 or SEQ ID NO: 21. 4. The TCR according to claim 3, wherein the α chain variable region further includes: α chain FR1 encoded by the sequence shown in SEQ ID NO: 28, α chain FR2 encoded by the sequence shown in SEQ ID NO: 29, such as One or more of the α-chain FR3 encoded by the sequence shown in SEQIDNO:30 and the α-chain FR4 encoded by the sequence shown in SEQIDNO:31; the β-chain variable region also includes: the β-chain encoded by the sequence shown in SEQIDNO:32 One or more of FR1, β chain FR2 encoded by the sequence shown in SEQ ID NO: 33, β chain FR3 encoded by the sequence shown in SEQ ID NO: 34, and β chain FR4 encoded by the sequence shown in SEQ ID NO: 35. 5. TCR, characterized in that it contains a fragment encoded by the sequence shown in SEQ ID NO: 15 or SEQ ID NO: 36. 6. Antigen-binding fragment, characterized by comprising the TCR according to any one of claims 1 to 5. 7. The polynucleotide encoding the TCR of claim 1, characterized by comprising the sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. 8. The polynucleotide according to claim 7, further comprising a sequence shown in SEQ ID NO: 8, a sequence shown in SEQ ID NO: 9, a sequence shown in SEQ ID NO: 10, a sequence shown in SEQ ID NO: 11 , one or more of the sequence shown in SEQ ID NO: 12, the sequence shown in SEQ ID NO: 13, and the sequence shown in SEQ ID NO: 14. 9. The polynucleotide encoding the TCR of claim 3, characterized by comprising the sequences shown in SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27. 10. The polynucleotide according to claim 9, further comprising a sequence shown in SEQ ID NO: 28, a sequence shown in SEQ ID NO: 29, a sequence shown in SEQ ID NO: 30, a sequence shown in SEQ ID NO: 31 , one or more of the sequence shown in SEQ ID NO: 32, the sequence shown in SEQ ID NO: 33, the sequence shown in SEQ ID NO: 34 and the sequence shown in SEQ ID NO: 35. 11. A polynucleotide encoding TCR, characterized in that it contains the sequence shown in SEQ ID NO: 15 or SEQ ID NO: 36. 12. Expression vector, characterized in that it contains the polynucleotide according to any one of claims 7 to 11. 13. Engineered cells, characterized by comprising the expression vector of claim 12. 14. Pharmaceutical composition, characterized by comprising the TCR according to any one of claims 1 to 5, the antigen-binding fragment according to claim 6, or the multinucleus according to any one of claims 7 to 11. or the expression vector of claim 12 or the engineered cell of claim 13, and a pharmaceutically acceptable carrier and/or diluent. 15. The TCR according to any one of claims 1 to 5, the antigen-binding fragment according to claim 6, the polynucleotide according to any one of claims 7 to 11, or the polynucleotide according to claim 12. The expression vector or the engineered cell according to claim 13 is used in the preparation of drugs for treating pancreatic cancer, colon cancer, endometrial cancer, lung cancer, cholangiocarcinoma, cervical cancer or bladder cancer with positive polypeptide-HLA-A1101 expression. Application in; the polypeptide sequence is shown in SEQ ID NO: 19 or SEQ ID NO: 21. 16. The application according to claim 15, characterized in that the drugs include cell drugs, protein drugs, ADC drugs or TCR and antigen combination drugs. 17. The TCR according to any one of claims 1 to 5, the antigen-binding fragment according to claim 6, the polynucleotide according to any one of claims 7 to 11, or the polynucleotide according to claim 12. The expression vector or the application of the engineered cells according to claim 13 in the preparation of tumor cell reagents or kits for detecting positive expression of polypeptide-HLA-A1101; the polypeptide sequence is shown in SEQ ID NO: 19 or SEQ ID NO: 21.