T cell receptors that specifically bind to a her2-derived peptide and uses thereof

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a T cell receptor specifically binding to a HER2-derived peptide segment and application thereof. The T cell receptor specifically binding to the HER2-derived peptide segment comprises an alpha chain variable region and a beta chain variable region, and the sequence of the HER2-derived peptide segment is KIFGSLAFL. The TCR of the present application can specifically recognize and bind to a HER2 peptide-HLA complex presented by tumor cells, thereby mediating precise killing of tumor cells by effector T cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to T-cell receptors that specifically bind to HER2-derived peptides and their uses. Background Technology

[0002] Human epidermal growth factor receptor 2 (HER2) is a transmembrane receptor tyrosine kinase widely expressed in various solid tumors, including breast cancer, gastric cancer, esophageal cancer, and ovarian cancer. Numerous studies have shown that HER2 overexpression or gene amplification is closely related to the high invasiveness, rapid proliferation, and poor prognosis of tumor cells; therefore, HER2 has long been considered an important target for cancer therapy. Current treatments targeting HER2 mainly include monoclonal antibodies, antibody-drug conjugates (ADCs), and tyrosine kinase inhibitors (TKIs). While these strategies have achieved some efficacy in some patients, they still face challenges such as high rates of drug resistance, insufficient duration of efficacy, limited applicability to certain populations, and significant toxic side effects.

[0003] With the development of tumor immunotherapy, T-cell-based targeted therapies have become an important research direction. Compared with antibody therapies, T-cell receptors (TCRs) can recognize intracellular antigenic peptides presented by the major histocompatibility complex (MHC) molecule, theoretically covering a wider range of tumor-associated antigens. However, most naturally occurring TCRs targeting tumor antigens have limited affinity, making it difficult to generate sufficient anti-tumor activity in treatment. Furthermore, the regulation of antigen presentation by tumor cells is complex and variable, and the presentation frequency and stability of specific HER2-derived peptides vary across different tumor types and stages of progression. These factors limit the clinical application of anti-HER2 TCR therapies.

[0004] In recent years, the discovery and engineering of high-affinity, specific TCRs targeting specific HER2-derived peptides has become a research hotspot. Through screening, in vitro affinity optimization, and structure-guided modification, TCR molecules capable of stably recognizing HER2 peptide-MHC complexes can be obtained. Modifying autologous or allogeneic T cells with such TCRs holds promise for achieving precise recognition and killing of HER2-positive tumor cells, overcoming the limitations of traditional antibody therapies in covering intracellular antigens.

[0005] However, the number of publicly available TCRs targeting specific peptides of HER2 is limited. Most have insufficient affinity, unclear cross-reactivity risks, or unstable in vivo effects, failing to meet clinical application needs. Therefore, there is still a need to develop new TCR molecules with high specificity and affinity for HER2 peptides, and which can maintain robust antigen recognition capabilities in the complex tumor microenvironment. These molecules can then be used to construct TCR-T cell or other immune cell therapy products to improve the treatment outcomes for patients with HER2-positive malignancies. Summary of the Invention

[0006] The technical problem that this invention aims to solve is that there are currently few cell therapy products targeting HER2 and their effects are unsatisfactory. Patients need more and more effective products to meet their diverse clinical needs.

[0007] In a first aspect, the present invention provides a T-cell receptor (TCR) that specifically binds to a HER2-derived peptide, the sequence of which is KIFGSLAFL (SEQ ID NO:25), and the T-cell receptor comprises a TCRα chain variable region and a TCRβ chain variable region, wherein the TCRα chain variable region comprises:

[0008] The sequence is CDR1α of DSAIYN (SEQ ID NO:1);

[0009] The sequence is CDR2α of IQSSQRE (SEQ ID NO:2); and

[0010] The sequence is CDR3α, which is APDNDYKLS (SEQ ID NO:3).

[0011] The variable region of the TCRβ chain contains:

[0012] The sequence is DFQATT (SEQ ID NO:4) CDR1β;

[0013] The sequence is CDR2β of SNEGSKA (SEQ ID NO:5); and

[0014] The sequence is CDR3β of SASLLGVSGRASNEQF (SEQ ID NO:6).

[0015] In some implementations, the T-cell receptor is soluble.

[0016] In some implementations, the T-cell receptor binds to a detectable marker or therapeutic agent.

[0017] In some implementations, the T-cell receptor is PEGylated.

[0018] In some implementations, engineered disulfide bonds are provided between the α-chain constant region and the β-chain constant region of the T-cell receptor.

[0019] In some implementations, the sequence of the variable region of the TCRα chain is as follows:

[0020] KQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAPDNDYKLSFGAGTTVTVRAN (SEQ ID NO: 19),

[0021] The sequence of the variable region of the TCRβ chain is:

[0022] GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSASLLGVSGRASNEQFFGPGTRLTVLE (SEQ ID NO: 20).

[0023] In some implementations, the T-cell receptor is a fusion protein.

[0024] Secondly, the present invention provides a synthetic nucleic acid molecule that encodes the T-cell receptor of the first aspect of the present invention.

[0025] Thirdly, the present invention provides a carrier containing the nucleic acid molecule synthesized by the present invention.

[0026] Fourthly, the present invention provides a host cell containing the vector of the present invention.

[0027] In some embodiments, the present invention also provides a host cell in which the synthetic nucleic acid molecules of the present invention are integrated into the chromosome of the host cell.

[0028] Fifthly, the present invention provides a method for preparing a T-cell receptor, comprising:

[0029] (i) Culturing host cells of the present invention to express the T cell receptor of the first aspect of the present invention; and

[0030] (ii) Isolate T cell receptors.

[0031] In a sixth aspect, the present invention provides a companion diagnostic kit for tumor immunotherapy, comprising: a T-cell receptor protein, wherein the T-cell receptor protein includes a TCRα chain variable region and a TCRβ chain variable region, and the TCRα chain variable region includes:

[0032] The sequence is CDR1α of DSAIYN (SEQ ID NO:1);

[0033] The sequence is CDR2α of IQSSQRE (SEQ ID NO:2); and

[0034] The sequence is CDR3α, which is APDNDYKLS (SEQ ID NO:3).

[0035] The variable region of the TCRβ chain contains:

[0036] The sequence is DFQATT (SEQ ID NO:4) CDR1β;

[0037] The sequence is CDR2β of SNEGSKA (SEQ ID NO:5); and

[0038] The sequence is CDR3β of SASLLGVSGRASNEQF (SEQ ID NO:6).

[0039] T-cell receptors target tumor antigen peptides derived from the HER2 gene (e.g., HER2), with the sequence of the tumor antigen peptide being KIFGSLAFL.

[0040] In some implementations, the kit also includes a colorimetric system, a positive control, a negative control, a staining buffer, and a blocking solution.

[0041] In some implementations, the samples for the companion diagnostic kit include: tissue samples and peripheral blood.

[0042] In some implementations, detectable markers for diagnostic purposes include, for example, fluorescent markers, radioactive markers, enzymes, nucleic acid probes, and contrast agents.

[0043] In some implementations, the companion diagnostic kit includes: an immunofluorescence staining (IHC) kit or a flow cytometry detection kit.

[0044] In some implementation schemes, the method of using the companion diagnostic kit includes:

[0045] 1) Use the TCR-Fc protein probe to stain tumor tissue sections;

[0046] 2) Detect the presence of the HER2 / HLA complex;

[0047] 3) Assess HER2 antigen presentation capability based on test results.

[0048] In some implementation schemes, the method of using the companion diagnostic kit includes:

[0049] 1) Use TCR protein probes for flow cytometry staining;

[0050] 2) Detect whether the HER2 / HLA complex is expressed on the cell surface;

[0051] 3) Dynamic immune monitoring is performed by assessing antigen presentation levels or HER2 expression.

[0052] In a seventh aspect, the present invention provides a composition comprising a fusion polypeptide comprising the aforementioned TCRα chain and / or TCRβ chain.

[0053] In some embodiments, cells are genetically modified by introducing isolated nucleic acid molecules encoding polypeptides, wherein the polypeptides contain at least one of the aforementioned TCRα and TCRβ chains.

[0054] In some implementations, the cells are immune cells.

[0055] In some implementation schemes, immune cells are selected from the group consisting of: antigen-presenting cells, B cells, dendritic cells, macrophages, Langerhans cells, T cells, NK cells, and NK T cells.

[0056] Eighthly, the present invention provides the use of the aforementioned T-cell receptor or synthetic nucleic acid molecule or carrier or host cell in the preparation of a medicament for treating cancer.

[0057] In some implementations, the drug may be suitable for administration via any appropriate route, preferably parenteral (including subcutaneous, intramuscular, or preferably intravenous) route.

[0058] In a ninth aspect, the present invention also provides a method for treating cancer in a patient, comprising administering to the patient the T-cell receptor of the present invention, a synthetic nucleic acid molecule, a carrier, a host cell, or a drug.

[0059] This invention achieves the following beneficial effects: 1. High specificity: It can distinguish between HER2 peptides and wild-type peptides, effectively achieving specific killing of tumor tissues and improving patient survival rate and quality of life. 2. Improved safety: It reduces toxic side effects on normal tissues. 3. Great potential for clinical application: It can be used for various HER2 mutation-related tumors such as breast cancer, ovarian cancer, lung cancer, gastric cancer, prostate cancer, and bladder cancer. Attached Figure Description

[0060] Figure 1 The figure shows the experimental results of the binding affinity of TCR to the HER2 peptide.

[0061] Figure 2 A figure illustrating the experimental results of TCR stability expression on the cell membrane.

[0062] Figure 3 The figure shows the experimental results illustrating the effect of T cells overexpressing TCR on the specific IFN-γ secretion of antigen-positive target cells.

[0063] Figure 4 The figure shows the experimental results of the effect of T cells overexpressing TCR on the specific IL-2 secretion of antigen-positive target cells.

[0064] Figure 5 A figure showing the experimental results of the effect of T cells overexpressing TCR on CD137 expression.

[0065] Figure 6 This figure shows the experimental results of the specific killing activity of T cells overexpressing TCR against antigen-positive tumor cells. Detailed Implementation

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this invention, exemplary methods and materials are described.

[0067] As used in this article, “CDR” is defined as the amino acid sequence of the complementarity-determining region of the TCR or TCR chain.

[0068] In the context of this invention, the following abbreviations for common nucleic acid bases are used: “A” for adenosine, “C” for cytidine, “G” for guanosine, “T” for thymidine, and “U” for uridine.

[0069] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can make up a protein or peptide. A polypeptide includes any peptide or protein containing two or more amino acids linked together by peptide bonds.

[0070] As used in this article, "vector" can refer to a nucleic acid sequence containing an origin of replication. Vectors can be plasmids, bacteriophages, bacterial artificial chromosomes, or yeast artificial chromosomes. Vectors can be DNA or RNA vectors. Vectors can be self-replicating extrachromosomal vectors or vectors integrated into the host genome.

[0071] The TCRs of the present invention may be non-naturally occurring and / or purified and / or engineered. Relative to the parental TCR, the TCRs of the present invention may have more than one mutation present in the α-chain variable region and / or the β-chain variable region. "Engineered TCR" and "mutated TCR" are used synonymously herein and generally refer to a TCR having one or more introduced mutations relative to the parental TCR, particularly in its α-chain variable region and / or β-chain variable region. These mutations can improve binding affinity against KIFGSLAFL (SEQ ID NO:25) complexed with HLA-A*02. In some embodiments, 1, 2, 3, 4, 5, 6, 7, or 8 mutations are present in the α-chain variable region, for example, 4 or 8 mutations, and / or 1, 2, 3, 4, or 5 mutations are present in the β-chain variable region, for example, 5 mutations.

[0072] TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature and are linked to the IMGT public database of TCR sequences. Natural α-β heterodimer TCRs have an α-chain and a β-chain. Broadly, each chain contains a variable region, a linker region, and a constant region. The β-chain typically also contains a short polyvariant region between the variable and linker regions, but this polyvariant region is usually considered part of the linker region. Each variable region contains three CDRs (complementarity-determining regions) embedded in the frame sequence, one of which is a hypervariable region called CDR3. There are several types of α-chain variable (Vα) regions and several types of β-chain variable (Vβ) regions, distinguished by their frame, CDR1 and CDR2 sequences, and partially defined CDR3 sequence.

[0073] In some implementations, the TCR includes a TCR α-chain variable region and a TCR β-chain variable region, and the three complementarity-determining regions (CDRs) of the TCR α-chain variable region are:

[0074] CDR1α-DSAIYN (SEQ ID NO:1);

[0075] CDR2α-IQSSQRE (SEQ ID NO:2); and

[0076] CDR3α-APDNDYKLS (SEQ ID NO:3), and

[0077] The three complementarity-determining regions (CDRs) of the TCRβ chain variable region are:

[0078] CDR1β-DFQATT (SEQ ID NO:4);

[0079] CDR2β-SNEGSKA (SEQ ID NO:5); and

[0080] CDR3β-SASLLGVSSGRASNEQF (SEQ ID NO: 6).

[0081] In some implementations, the TCR includes a TCR α-chain variable region and a TCR β-chain variable region, and the three complementarity-determining regions (CDRs) of the TCR α-chain variable region are:

[0082] CDR1α-ATGYPS (SEQ ID NO:7);

[0083] CDR2α-ATKADDK (SEQ ID NO:8); and

[0084] CDR3α-ALSDHDNYGQNFV (SEQ ID NO:9), and

[0085] The three complementarity-determining regions (CDRs) of the TCRβ chain variable region are:

[0086] CDR1β-MNHEY (SEQ ID NO:10);

[0087] CDR2β-SVGAGI (SEQ ID NO:11); and

[0088] CDR3β-ASRPHQPTNEKLF (SEQ ID NO: 12).

[0089] In some implementations, the TCR includes a TCR α chain variable region and a TCR β chain variable region, and the three complementarity-determining regions (CDRs) of the TCR α chain variable region are:

[0090] CDR1α-TSDPSYG (SEQ ID NO:13);

[0091] CDR2α-QGSYDQQN (SEQ ID NO:14); and

[0092] CDR3α-AMREGTLNTGFQKLV (SEQ ID NO:15), and

[0093] The three complementarity-determining regions (CDRs) of the TCRβ chain variable region are:

[0094] CDR1β-MNHNS (SEQ ID NO:16);

[0095] CDR2β-SASEGT (SEQ ID NO:17); and

[0096] CDR3β-ASSEGSGYSEAF (SEQ ID NO: 18).

[0097] In some embodiments (Y24347-C1), the TCR includes the α-chain variable region amino acid sequence as shown in SEQ ID NO:19:

[0098] KQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAPDNDYKLSFGAGTTVTVRAN;

[0099] Furthermore, the TCR contains the β-chain variable region amino acid sequence as shown in SEQ ID NO:20:

[0100] GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSASLLGVSGRASNEQFFGPGTRLTVLE.

[0101] In some embodiments (Y24348-C1), the TCR includes the α-chain variable region amino acid sequence as shown in SEQ ID NO:21:

[0102] GNSVTQMEGPVTLSEEAFLTINCTYTATGYPSLFWYVQYPGEGLQLLLKATKADDKGSNKGFEATYRKETTSFHLEKGSVQVSDSAVYFCALSDHDNYGQNFVFGPGTRLSVLPN;

[0103] Furthermore, the TCR contains the β-chain variable region amino acid sequence as shown in SEQ ID NO:22:

[0104] NAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASRPHQPTNEKLFFGSGTQLSVLE.

[0105] In some embodiments (Y24350-C3), the TCR includes the α-chain variable region amino acid sequence as shown in SEQ ID NO:23:

[0106] AQKITQTQPGMFVQEKEAVTLDCTYDTSDPSYGLFWYKQPSSGEMIFLIYQGSYDQQNATEGRYSLNFQKARKSANLVISASQLGDSAMYFCAMREGTLNTGFQKLVFGGTRLLVSP;

[0107] Furthermore, the TCR contains the β-chain variable region amino acid sequence as shown in SEQ ID NO:24:

[0108] NAGVTQTPKFQVLKTGQSMTLQCAQDMNHNSMYWYRQDPMGLRLIYYSASEGTTDKGEVPNGYNVSRLNKREFSLRLESAAPSQTSVYFCASSEGSGYSEAFFGQGTRLTVV.

[0109] In some embodiments, the α-chain variable region of the TCR of the present invention may contain an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid residue sequences shown in sequences such as SEQ ID NO: 19, SEQ ID NO: 21, or SEQ ID NO: 23. In some embodiments, the β-chain variable region of the TCR of the present invention may contain an amino acid sequence that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the amino acid residue sequences shown in sequences such as SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 24.

[0110] In some implementations, the TCR is single-stranded.

[0111] In some implementations, the TCR is formed by linking the α-chain variable region and the β-chain variable region through a peptide linker sequence.

[0112] In some implementations, cysteine ​​residues form artificial disulfide bonds between the α and β chain constant regions of the TCR.

[0113] In some embodiments, the C- or N-terminus of the α-chain and / or β-chain of the TCR is bound with a conjugate, preferably a detectable marker, a therapeutic agent, a PK-modified moiety, or any combination of these substances.

[0114] In some embodiments, therapeutic agents that can bind to the TCR of the present invention include immunomodulators, radioactive compounds, enzymes (e.g., perforin), or chemotherapeutic agents (e.g., cisplatin). To ensure toxicity at the desired site, the therapeutic agent can be contained within a liposome linked to the TCR, allowing for slow release. This prevents damaging effects during in vivo transport and ensures maximum toxicity after the TCR binds to the relevant antigen-presenting cells.

[0115] In some implementations, the TCR is a mouse-derived TCR, a human-mouse chimeric TCR, or a humanized TCR.

[0116] In some implementations, the vector includes an expression vector, i.e., a construct capable of being expressed in vivo or in vitro. Commonly used vectors include bacterial plasmids, bacteriophages, and viral vectors.

[0117] In some embodiments, the viral vector includes, but is not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, herpesvirus vectors, retrovirus vectors, lentivirus vectors, and baculovirus vectors. Preferably, the vector can transfer the nucleotides of the present invention into cells, such as T cells, causing the cells to express HER2 antigen-specific TCRs. The vector should be able to be expressed at a sustained high level in T cells.

[0118] In some implementations, the lentiviral vector may include: the lentiviral expression vector pLenti (addgene).

[0119] In some embodiments, the host cell is a mammalian cell. For example, the host cell is a human cell. Although the host cell can be any cell type, can originate from any type of tissue, and can be a cell at any developmental stage, the host cell is preferably a peripheral blood lymphocyte (PBL) or peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell.

[0120] In some embodiments, when host cells or related cell populations are administered, the host cells may be allogeneic or autologous to mammals. Preferably, the cells are autologous to mammals.

[0121] In some embodiments, mammal refers to any mammal, including but not limited to: rodent mammals such as mice and hamsters, and lagomorph mammals such as rabbits; preferably, the mammal is from the order Carnivora, including felines (cats) and canids (dogs). More preferably, the mammal is from the order Artiodactyla, including bovines (cattle) and suidae (pigs), or from the order Perissodactyla, including equines (horses); most preferably, the mammal is from the order Primates, apes, or monkeys, or from the suborder Anthropoidea (humans and apes). Particularly preferred is the mammal being human.

[0122] In some embodiments, the TCR, drug, carrier, synthetic nucleic acid molecule and host cell of the present invention can be provided in a substantially pure form, for example at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% purity.

[0123] This document uses specific embodiments to illustrate the principles and implementation methods of the present invention. The descriptions of these embodiments are merely illustrative and are intended to aid in understanding the method and central ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0124] Example 1: Cloning Antigen Short Peptide-Specific T Cells

[0125] Peripheral blood lymphocytes (PBLs) from healthy volunteers with genotype HLA-A*02:01 were stimulated with the synthetic short peptide HER2 (KIFGSLAFL, SEQ ID NO:25). The short peptide was then annealed with biotin-labeled HLA-A*02:01 to prepare pHLA haploids. These haploids were combined with PE-labeled streptavidin (BD Biosciences) to form PE-labeled tetramers. The tetramers and anti-CD8-APC double-positive cells were sorted using a BD Melody flow cytometry system, with one positive cell per well in each 96-well plate.

[0126] Example 2: Construction of TCR gene and vector for antigen-specific T cell clones

[0127] Cells obtained in Example 1 were lysed using 0.1% Triton-X (Sangon Biotech), and amplified using the Clontech SMARTRACE cDNA amplification kit. Primers were designed for the conserved C-terminal region of the human TCR gene. The downstream primer for the conserved C-terminal region of the TCR α chain was tcagctggaccacagc (SEQ ID NO:26); the downstream primer for the conserved C-terminal region of the TCR β chain was tcagaaatcctttctcttgac (SEQ ID NO:27). The full-length TCR α and β chains were cloned into the lentiviral expression vector pCDH (SBI) using overlap PCR. Specifically, the full-length TCR α and β chains were ligated using overlap PCR to obtain the TCRα-2A-TCRβ fragment. The lentiviral expression vector and TCRα-2A-TCRβ restriction enzyme were digested and ligated to obtain the pCDH-TRA-2A-TRB plasmid, which was then sequenced and confirmed (IMGT) to obtain plasmids of three TCR clones: Y24347-C1, Y24348-C1 and Y24350-C3.

[0128] Y24347-C1 The sequencing results of the CDR1α, CDR2α, and CDR3α regions of the TCRα chain variable region are confirmed as SEQ ID NO:1-3, respectively; the sequencing results of the CDR1β, CDR2β, and CDR3β regions of the TCRβ chain variable region are confirmed as SEQ ID NO:4-6, respectively. The sequence of the TCRα chain variable region is SEQ ID NO:19, and the sequence of the TCRβ chain variable region is SEQ ID NO:20.

[0129] Y24348-C1 The sequencing results of the CDR1α, CDR2α, and CDR3α regions of the TCRα chain variable region are confirmed as SEQ ID NO:7-9, respectively; the sequencing results of the CDR1β, CDR2β, and CDR3β regions of the TCRβ chain variable region are confirmed as SEQ ID NO:10-12, respectively. The sequence of the TCRα chain variable region is SEQ ID NO:21, and the sequencing result of the TCRβ chain variable region is SEQ ID NO:22.

[0130] Y24350-C3 The sequencing results of the CDR1α, CDR2α, and CDR3α regions of the TCRα chain variable region are confirmed as SEQ ID NO:13-15, respectively; the sequencing results of the CDR1β, CDR2β, and CDR3β regions of the TCRβ chain variable region are confirmed as SEQ ID NO:16-18, respectively. The sequencing result of the TCRα chain variable region is SEQ ID NO:23, and the sequencing result of the TCRβ chain variable region is SEQ ID NO:24;

[0131] The pseudoviruses were then packaged using 293T cells. Specifically, the plasmids were mixed with VSVG, RRE, and Rev (Addgene) plasmids in a 4:5:4:10 ratio, and 20 μL of each mixture was diluted in 1.25 mL of DMEM medium to prepare the DNA solution. 20 μL of polyetherimide (PEI 1 µg / μL) was added to 1.25 mL of DMEM, and the PEI / DMEM mixture was then added to the prepared DNA solution. After incubation at room temperature for 20 minutes, the mixture was added to 293T cells cultured in 15 cm dishes and mixed thoroughly. After 6 hours, the DMEM medium was replaced with fresh medium. After 72 hours, the supernatant containing the lentivirus was collected; this was the lentivirus supernatant for each TCR.

[0132] Example 3: Construction of a cell line overexpressing antigen short peptide-specific TCR

[0133] The NFAT-GFP element (Addgene) was synthesized and inserted into the expression vector pCDH(SBI) using the standard methods described in *Molecular Cloning Laboratory Manual*. The fragment was confirmed to be correct by sequencing. Then, pseudoviruses were packaged using 293T (Pronosai CL-0130). Jurkat cell lines were infected with pseudoviruses containing the NFAT-GFP element and pseudoviruses containing the TCR element. Through limiting dilution and monoclonal amplification, Jurkat-NFAT-GFP-TCR overexpressing cell lines were obtained, namely: Jurkat-NFAT-GFP-Y24347-C1-TCR, Jurkat-NFAT-GFP-Y24348-C1-TCR, and Jurkat-NFAT-GFP-Y24350-C3-TCR.

[0134] Example 4: Binding Affinity Experiment

[0135] (1) Construction of K562-CD80-HLA-A*02:01 and 293T-CD80-HLA-A*02:01

[0136] The HLA-A*02:01 element (IMGT / HLA Acc No: HLA00043) and CD80 (NP_005182.1) were synthesized and inserted into the expression vector pCDH(SBI). The fragment was confirmed to be correct after sequencing. Lentiviral virus was then packaged using the 293T cell line. Specifically, the plasmid containing the CD80-HLA-A*02:01 element was mixed with VSVG plasmid, RRE plasmid, and Rev plasmid (purchased from Addgene) in a ratio of 4:5:4:10, and 20 μL was diluted in DMEM medium (1.25 mL) to prepare the DNA solution. 20 μL of polyetherimide (PEI 1 µg / µL) was added to DMEM (1.25 mL), and the PEI / DMEM mixture was added to the prepared DNA solution. After incubation at room temperature for 20 minutes, the mixture was added to 293T cells cultured in 15 cm plates and mixed thoroughly. Six hours later, the medium was replaced with fresh DMEM. After 72 hours, the supernatant containing the lentivirus was collected, which was the lentivirus supernatant containing CD80-HLA-A*02:01. K562 or 293T cell lines were infected with pseudoviruses containing the CD80-HLA-A*02:01 element. Through limiting dilution and monoclonal amplification, K562-CD80-HLA-A*02:01 overexpressing cell lines and 293T-CD80-HLA-A*02:01 were obtained.

[0137] (2) Jurkat-NFAT-GFP-TCR overexpressing cell lines were co-cultured with K562-CD80-HLA-A*02:01

[0138] Cell lines (Jurkat-NFAT-GFP-Y24347-C1-TCR, Jurkat-NFAT-GFP-Y24348-C1-TCR, and Jurkat-NFAT-GFP-Y24350-C3-TCR) were co-incubated with K562-CD80-HLA-A*02:01 cells loaded with different concentrations of the target antigen peptide HER2 (KIFGSLAFL, SEQ ID NO:25). Specifically, K562 cells were incubated with different concentrations of the target antigen peptide at 37°C for 1 h. After centrifugation, the cells were resuspended in culture medium. Jurkat and K562 cells were counted separately, and 2 × 10⁶ cells each of Jurkat and peptide-loaded K562 cells were aspirated. 4 Cells were mixed and co-cultured in 96-well plates. After 24 hours of co-culture, flow cytometry was used to detect the activation levels of Jurkat cell reporter genes and CD69 cell activation levels.

[0139] The results are as follows Figure 1The results show that TCR-T cells expressing Y24347-C1, Y24348-C1, and Y24350-C3 exhibit strong reactivity and specificity to the target antigen peptide HER2, but no response to unrelated peptides (another target, the PRAME epitope peptide: VLDGLDVL); therefore, they are well-suited for the use of related T cell receptor proteins for diagnostic and therapeutic purposes.

[0140] Example 5: Stability Experiment

[0141] TCR-T cells expressing TCR (Y25084 and Y25085) were constructed using TCR elements (TCR α-2A-TCR β fragments) from Y24347-C1 and Y24348-C1, respectively. The specific steps are as follows:

[0142] (1) Preparation of TCR lentivirus: Each TCR element and GFP (Addgene) were synthesized and inserted into the expression vector pCDH (SBI) using the standard method described in Molecular Cloning Laboratory Manual. The fragments were confirmed to be correct after sequencing. Then, pseudoviruses were packaged using 293T (Pronosai CL-0130) according to the specific operation steps in Example 2.

[0143] (2) Construction of TCR-T cells expressing TCR: After thawing PBMCs, they were cultured in an appropriate amount of X-VIVO 15 medium containing 100 IU / mL rhIL-2, and the density was adjusted to 1×10⁻⁶ cells / mL. 6 / mL. Every 2×10 6 Cells were added to 10 μL of MACS CD3 / CD28 T cellTransact beads in X-VIVO15 medium containing 100 IU / mL rhIL-2, gently mixed, and cultured in a cell culture incubator. After 24 hours, the cells were centrifuged and the supernatant was discarded to remove the magnetic beads. The cells were resuspended in 1 mL of X-VIVO 15 medium containing 100 IU / mL rhIL-2. Target lentivirus (with an MOI of 10) was added based on the total cell count and viral titer. A control group without lentivirus was also included. Culture medium was added to each control group to a final volume of 2 mL of X-VIVO 15 medium containing 100 IU / mL IL-2, and polybrene was added to a final concentration of 10 μg / mL. The cells were centrifuged at 37°C and 2000g for 60 minutes. The infected cells were then cultured in a CO2 incubator for 24 hours. The medium was changed periodically and the cell density was adjusted until day 14.

[0144] GFP was used to detect the expression rate of TCR-T. The results showed (see...). Figure 2 Each TCR can be stably expressed in the cell membrane.

[0145] Example 6: Specific IFN-γ and IL-2 secretion assays of TCR-T cells overexpressing antigen-positive target cells

[0146] 1. T cells expressing TCR (same as in Example 5) were used as effector cells, and PBMCs that were not transduced with TCR were expanded and cultured in parallel as control effector cells.

[0147] 2. Using a load of 10 -7 The short peptide of HER2 (KIFGSLAFL) or unrelated peptide (PRAME epitope peptide: VLDGLDVL) K562-CD80-HLA-A*02:01 was used as a positive target cell (same as in Example 4); the effector-to-target ratio (ratio of effector cells to target cells) was 1:1, and the expression of IL-2 and / or IFN-γ in the cells was detected after 24 h of incubation.

[0148] 3. Using a load of 10 -11 M to 10 -5 M. Different concentrations of HER2 (KIFGSLAFL) short peptide K562-CD80-HLA-A*02:01 were used as positive target cells (same as in Example 4); E:T effector-to-target ratio (ratio of effector cells to target cells) was 1:1, and the expression of CD137 in cells was detected after 24 h of incubation.

[0149] The results showed that in the presence of positive target cells, the TCR-T overexpression group produced IL-2 and IFN-γ, and CD137 expression was upregulated, while the TCR-T overexpression group did not produce IFN-γ or IL-2 in the presence of negative target cells. Partial results of IL-2 and IFN-γ expression and secretion are shown in [the table below]. Figure 3 and Figure 4 The results of CD137 upregulation are shown in the table below. Figure 5 The above data indicate that T cells overexpressing TCR have a specific activation effect on antigen-positive target cells, and the TCR protein obtained in this invention is suitable for use for diagnostic and therapeutic purposes.

[0150] Example 7: Specific killing activity of TCR-T cells overexpressing against antigen-positive tumor cells

[0151] 1. T cells expressing TCR (same as in Example 5) were used as effector cells, and PBMCs that were not transduced with TCR were expanded and cultured in parallel as a control (blank).

[0152] 2. Using a load of 10 -9 M to 10 -7M's HER2 (KIFGSLAFL) short peptide 293T-CD80-HLA-A*02:01 (same as Example 5) was used as a positive target cell (+); the E:T effector-target ratio (effector cell:target cell ratio) was 10:1. The adhesion ability of the target cells was detected in real time using an RTCA (Real-Time Label-Free Cell Analysis System, which integrates a microelectronic cell sensor chip into the bottom of the cell detection plate and obtains biological information related to cell physiological functions, including cell growth, extension, morphological changes, death, and adhesion, through real-time dynamic electrode impedance detection) instrument. Specifically, the instrument collected cell adhesion ability values ​​(Cell Index) for each well every 15 minutes. In subsequent data processing, the data from the last time point before the addition of T cells was used as the normalized value to calculate the normalized cell adhesion ability value (Normalized Cell Index) for each group at each time point. The results showed (see Figure 6 The TCR-T overexpression group showed significant killing activity only against 293T-CD80-HLA-A*02:01 loaded with tumor-associated antigen peptides, and had no killing effect on 293T-CD80-HLA-A*02:01 loaded with unrelated peptides or unrelated peptides. Among them, Y25085 showed better killing effect.

Claims

1. A T cell receptor that specifically binds to a HER2-derived peptide segment, characterized in that, the sequence of the HER2-derived peptide segment is KIFGSLAFL, and the T cell receptor comprises an alpha chain variable region and a beta chain variable region, wherein the alpha chain variable region comprises: a CDR1a of the sequence ATGYPS; a CDR2a of the sequence ATKADDK; and a CDR3a of the sequence ALSDHDNYGQNFV, the beta chain variable region comprises: a CDR1b of the sequence MNHEY; a CDR2b of the sequence SVGAGI; and a CDR3b of the sequence ASRPHQPTNEKLF.

2. The T cell receptor of claim 1, wherein, the T cell receptor is conjugated to a detectable label, a therapeutic agent, or the T cell receptor is PEGylated.

3. The T cell receptor of claim 1, wherein, an engineered disulfide bond is disposed between the alpha chain constant region and the beta chain constant region of the T cell receptor.

4. The T cell receptor according to any one of the preceding claims, characterized in that, the amino acid sequence of the alpha chain variable region is: GNSVTQMEGPVTLSEEAFLTINCTYTATGYPSLFWYVQYPGEGLQLLLKATKADDKGSNKGFEATYRKETTSFHLEKGSVQVSDSAVYFCALSDHDNYGQNFVFGPGTRLSVLPN; the amino acid sequence of the beta chain variable region is: NAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASRPHQPTNEKLFFGSGTQLSVLE.

5. A synthetic nucleic acid molecule, characterized in that, the synthetic nucleic acid molecule encodes the T cell receptor of any one of claims 1 to 4.

6. A vector, characterized in that, the vector comprises the synthetic nucleic acid molecule of claim 5.

7. A host cell, characterized in that, the host cell comprises the synthetic nucleic acid molecule of claim 5 or the vector of claim 6.

8. Use of the T cell receptor of any one of claims 1 to 4 or the synthetic nucleic acid molecule of claim 5 or the vector of claim 6 or the host cell of claim 7 in the manufacture of a medicament for the treatment of cancer.

9. A method of making a T cell receptor, the method comprising, comprises: (i) culturing the host cell of claim 7 to express the T cell receptor of any one of claims 1 to 4; and (ii) isolating the T cell receptor from the host cell.

10. A companion diagnostic kit for tumor immunotherapy, the kit comprising: a T cell receptor protein capable of recognizing a HER2 protein-derived peptide segment, wherein the sequence of the HER2 protein-derived peptide segment is KIFGSLAFL, and the T cell receptor protein comprises the alpha chain variable region and the beta chain variable region of claim 1.

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