Separated tumor specific TCR specifically binding to MHC / Survivin epitope complex and application thereof

Abstract

The invention discloses a separated tumor specific TCR specifically combined with an MHC / Survivin epitope complex and application thereof, and belongs to the field of cancer immunotherapy, the separated tumor specific TCR specifically combined with the MHC / Survivin epitope complex comprises a TCR alpha chain, the TCR alpha chain comprises a CDR1 with an amino acid sequence of SEQ ID NO: 1, a CDR2 with an amino acid sequence of SEQ ID NO: 2 and a CDR3 with an amino acid sequence of SEQ ID NO: 3; a TCR [beta] chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO: 6. The TCR can accurately distinguish tumor cells from normal cells, the off-target effect risk is greatly reduced, and immune damage to normal tissue is avoided.

Description

Technical Field

[0001] The present invention belongs to the field of cancer immunotherapy, and in particular relates to a separated tumor-specific TCR specifically binding to an MHC / Survivin epitope complex and its use. Background Art

[0002] Cancer poses a serious threat to human health. Although the human immune system has the ability to recognize and eliminate tumor cells, tumor cells often evade immune surveillance through various mechanisms. Tumor antigens, including mutant proteins specifically expressed by cancer cells and tumor-associated antigens (TAAs), are key agents in triggering immune responses. These antigens are overexpressed in the tissue of origin of cancer and, due to mechanisms such as immune tolerance, are not fully recognized by the immune system as "non-self." Antigen-presenting cells (APCs), particularly dendritic cells (DCs), take up tumor antigens, process them, and present the antigenic peptides bound to the major histocompatibility complex (MHC) on their cell surface. Mature, antigen-loaded DCs activate T cells, triggering an immune response involving a variety of effector T cells, including cytotoxic T cells and helper T cells, as well as memory T cells. Cytotoxic T cells, through the T cell receptor (TCR) on their surface, specifically recognize the tumor antigen-MHC complex and kill tumor cells by releasing cytokines, cytotoxic substances, or inducing apoptosis.

[0003] Adoptive lymphocyte therapy (ACT) is an emerging strategy for cancer treatment, in which patients are infused with ex vivo expanded lymphocytes. Taking tumor-infiltrating lymphocyte (TIL) therapy as an example, TILs are isolated from the patient's tumor tissue, expanded in vitro, and then infused back into the patient's body after the removal of natural non-myeloid lymphocytes. However, this therapy faces many challenges in clinical application: on the one hand, TILs from different patients or donors have significant differences in their affinity for tumor antigens, resulting in unstable anti-tumor efficacy; on the other hand, most antigen-specific mutations that trigger immune responses are individually unique, making standardized treatment difficult to achieve, greatly limiting the widespread application of this therapy and the improvement of therapeutic effects.

[0004] As an important member of the inhibitor of apoptosis protein family (IAPs), Survivin has a unique molecular structure. The baculovirus IAP repeat sequence (BIR) domain it contains is a key functional region for inhibiting cell apoptosis, and the α-helical structure at the C-terminus is involved in protein-protein interactions. In cell biology, Survivin blocks the process of cell apoptosis by inhibiting the activity of caspase, and at the same time, it synergizes with cyclin-dependent kinases (CDKs) in the G2 / M phase of the cell cycle to drive cell proliferation. Studies have shown that Survivin is highly expressed in a variety of malignant tumor tissues such as lung cancer, breast cancer, and colon cancer, while its expression level in normal adult tissues is extremely low or even absent. This tumor-specific expression pattern enables Survivin to not only help tumor cells escape apoptosis and accelerate proliferation, but also promote tumor angiogenesis by regulating vascular endothelial growth factor (VEGF), creating favorable conditions for tumor growth, invasion, and metastasis.

[0005] Given the key role of Survivin in the occurrence and development of tumors, it has become a hot target that has attracted much attention in the field of tumor treatment. Currently, the treatment strategies developed around Survivin include antisense oligonucleotide technology, small molecule inhibitors, and immunotherapy. Among them, TCR-T cell therapy based on Survivin-specific TCR is expected to achieve efficient anti-tumor effects by virtue of its ability to accurately recognize tumor antigens, while reducing off-target effects, opening up new directions for cancer treatment. However, in existing technologies, there is still room for optimization in the research on Survivin-specific TCR, especially how to obtain TCR with high affinity and specificity, which has become the key to improving the clinical efficacy of this therapy. Summary of the Invention

[0006] The purpose of the present invention is to provide an isolated tumor-specific TCR that specifically binds to the MHC / Survivin epitope complex and its use, so as to overcome at least one of the above-mentioned defects in the prior art.

[0007] To achieve this object, the present invention adopts the following technical solutions:

[0008] The present invention provides an isolated tumor-specific TCR that specifically binds to an MHC / Survivin epitope complex, comprising: a TCRα chain, wherein the TCRα chain comprises a CDR1 having an amino acid sequence of SEQ ID NO: 1, a CDR2 having an amino acid sequence of SEQ ID NO: 2, and a CDR3 having an amino acid sequence of SEQ ID NO: 3; and a TCRβ chain, wherein the TCRβ chain comprises a CDR1 having an amino acid sequence of SEQ ID NO: 4, a CDR2 having an amino acid sequence of SEQ ID NO: 5, and a CDR3 having an amino acid sequence of SEQ ID NO: 6.

[0009] Preferably, the Survivin epitope is the amino acid sequence shown in SEQ ID NO:16.

[0010] Preferably, the MHC is HLA-DPAl * 01:03 / HLA-DPB1 * 04:01 or HLA-DPA1 * 02:02 / HLA-DPB1 * 04:01.

[0011] Preferably, it comprises: a TCR α chain having a variable region comprising the amino acid sequence of SEQ ID NO: 7, and a TCR β chain having a variable region comprising the amino acid sequence of SEQ ID NO: 9.

[0012] Preferably, it comprises: a TCR α chain comprising the amino acid sequences of SEQ ID NOs: 7 and 8, and a TCR β chain comprising the amino acid sequences of SEQ ID NOs: 9 and 10.

[0013] Preferably, the TCR alpha chain further comprises an alpha constant region and / or the TCR beta chain further comprises a beta constant region.

[0014] Preferably, the α constant region and the β constant region are mouse constant regions.

[0015] Preferably, the α constant region comprises the amino acid sequence of SEQ ID No: 13 and / or the β constant region comprises the amino acid sequence of SEQ ID NO: 15.

[0016] Preferably, it comprises: a TCR α chain comprising the amino acid sequences of SEQ ID NOs: 12 and 13, and a TCR β chain comprising the amino acid sequences of SEQ ID NOs: 14 and 15.

[0017] Preferably, it further comprises an antigen binding domain.

[0018] Preferably, the TCR or its antigen binding domain is a single chain.

[0019] The present invention provides an isolated nucleic acid molecule encoding the above-mentioned isolated tumor-specific TCR that specifically binds to the MHC / Survivin epitope complex, namely, the TCR α chain and the TCR β chain.

[0020] Preferably, the nucleotide sequence of the nucleic acid molecule is codon-optimized.

[0021] Preferably, the nucleotide sequence encoding the TCR alpha chain and the nucleotide sequence encoding the TCR beta chain are separated by a self-cleaving peptide sequence.

[0022] Preferably, the self-cleaving peptide is P2A, comprising the amino acid sequence shown in SEQ ID NO:11.

[0023] The present invention also provides an engineered immune cell comprising the above-mentioned isolated tumor-specific TCR specifically binding to the MHC / Survivin epitope complex or the above-mentioned isolated nucleic acid molecule, wherein the immune cell is a cell line or a primary cell obtained from a subject or an allogeneic cell.

[0024] The present invention also provides a pharmaceutical composition comprising the engineered immune cells and a pharmaceutically acceptable carrier.

[0025] The present invention also provides use of the engineered immune cells in preparing drugs for treating diseases or conditions expressing Survivin.

[0026] Preferably, the disease or disorder is cancer.

[0027] Preferably, the cancer is oral cancer, lung cancer, breast cancer, colon cancer or ovarian cancer.

[0028] Beneficial effects:

[0029] The unique amino acid sequence confers high specificity to the TCR, effectively avoiding nonspecific binding to low levels of survivin expressed in normal tissues or other homologous antigens. The TCR of this invention can accurately distinguish tumor cells from normal cells, significantly reducing the risk of off-target effects and avoiding immune damage to normal tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a diagram of the Survivin-specific TCR002 construction of the present invention (wild type).

[0031] Figure 2 This is a diagram of the construction of the Survivin-specific TCR002 of the present invention (murinized).

[0032] Figure 3 This is a diagram of the construction of the Survivin-specific TCR002 of the present invention (murinized codon optimized).

[0033] Figure 4 This is a diagram showing the results of TCR002 expression detected by FACS in the present invention.

[0034] Figure 5 This is a diagram showing the results of FACS detection of TCR002 specifically recognizing antigen peptides according to the present invention.

[0035] Figure 6 This is a graph showing the results of the ELISA test of the present invention for TCR002 specifically recognizing antigen peptides.

[0036] Figure 7 This is a diagram showing the results of TCR002 specifically recognizing antigen epitopes of the present invention.

[0037] Figure 8 This figure shows the results of determining HLA restriction of T cells expressing Survivin antigen-specific T cell receptor TCR002 of the present invention through an HLA antibody blocking test.

[0038] Figure 9 This is a diagram showing the results of determining the HLA subtype of T cells expressing the Survivin antigen-specific T cell receptor TCR002 of the present invention through an LCL recognition test.

[0039] Figure 10 This figure shows the results of T cells expressing Survivin antigen-specific T cell receptor TCR002 of the present invention specifically killing oral cancer cells in vitro. DETAILED DESCRIPTION

[0040] The present invention will now be further described with reference to the accompanying drawings and specific embodiments.

[0041] I. Definition

[0042] Before describing the present invention in detail with respect to some preferred embodiments thereof, the following general definitions are provided.Unless otherwise defined below, terms are used herein as commonly used in the art.

[0043] The present invention will be described with respect to specific embodiments and with reference to certain drawings, but the invention is not limited thereto but only by the claims. When the term "comprising" is used in this specification and the claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of" is considered to be a preferred embodiment of the term "comprising". If a group is defined hereinafter as comprising at least a certain number of embodiments, it should also be understood to disclose a group that preferably consists only of these embodiments.

[0044] As used herein, "T cell receptor" or "TCR" refers to an endogenous or engineered T cell receptor that includes an extracellular antigen binding domain that binds to a specific antigen epitope in an MHC molecule. A TCR may include a TCRα polypeptide chain and a TCRβ polypeptide chain. A "tumor-specific TCR" refers to a TCR that specifically recognizes a tumor antigen expressed by a tumor cell. "TCR-T" refers to a T cell that expresses a recombinant TCR. The "antigen binding domain" of a TCR refers to a polypeptide that binds to an antigen with a higher degree of specificity due to its primary, secondary or tertiary sequence and / or post-translational modification and / or charge. The antigen binding domain may be derived from any part or fragment of a TCR that retains the antigen binding activity of the TCR as part of a TCR. The antigen binding portion encompasses, for example, a portion of a TCR that retains specific binding to the amino acid sequence of Survivin or a fragment thereof, or a portion of the ability to detect, treat or prevent cancer to a similar degree, the same degree or a higher degree than a wild-type TCR. The TCR antigen binding domain may comprise, for example, about 10%, about 25%, about 30%, about 50%, about 68%, about 80%, about 90%, about 95% or more of the full-length TCR. The antigen binding domain may comprise the antigen recognition portion of either or both of the α and β chains of the TCR, such as one or more of the complementary determining regions CDR1, CDR2, and CDR3 of the variable regions of the α and / or β chains of the TCR. The antigen binding domain may comprise the following amino acid sequence: CDR1 of the α chain, CDR2 of the α chain, CDR3 of the α chain, CDR1 of the β chain, CDR2 of the β chain, CDR3 of the β chain, or any combination thereof. Preferably, the antigen binding domain comprises the amino acid sequence of CDR1, CDR2, and CDR3 of the α chain of the TCR or the amino acid sequence of CDR1, CDR2, and CDR3 of the β chain; or the amino acid sequence of CDR1, CDR2, and CDR3 of all α and β chains. In one embodiment of the invention, the antigen binding portion may include, for example, the variable region of TCR, which includes a combination of the above-mentioned CDR regions. In this regard, the antigen binding portion may include the amino acid sequence of the variable region (Vα) of the α chain of TCR, the amino acid sequence (Vβ) of the variable region of the β chain, or the amino acid sequence of both Vα and Vβ. In one embodiment of the invention, the antigen binding portion may include a combination of a variable region and a constant region. In this regard, the antigen binding portion may include the full length α or β chain of TCR, or both α and β chains.

[0045] II. Survivin Tumor-Specific TCR

[0046] The present application provides TCRs that specifically recognize tumor survivin and / or specifically recognize an MHC / survivin epitope complex. In some embodiments, the tumor-specific TCR specifically recognizes survivin. In some embodiments, the tumor-specific TCR specifically recognizes an MHC / survivin epitope complex. Nucleic acids and vectors encoding tumor-specific TCRs, and engineered immune cells expressing tumor-specific TCRs are also within the scope of this patent application.

[0047] Exemplary TCRs identified using the methods described herein are shown in Table 1 below. The V, J, and C segments are named according to the IMGT database. Other nomenclature and segment delineation algorithms known in the art can be used. See, for example, Lefranc, M.-P., The Immunologist, 7, 132-136 (1999), and worldwideweb.imgt.org / IMGTScientificChart / Nomenclature / IMGT-FRCDRdefinition.html. The CDR1, CDR2, and CDR3 of exemplary TCRs are shown in the "Sequence Listing" section.

[0048] Table 1: Exemplary tumor-specific TCRs

[0049]

[0050] In some embodiments, a Survivin tumor-specific TCR or an antigen-binding domain thereof is provided that specifically binds to an epitope of a Survivin peptide, for example, an epitope of Survivin comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, a Survivin tumor-specific TCR or an antigen-binding domain thereof specifically binds to an epitope of Survivin comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, a Survivin tumor-specific TCR or an antigen-binding domain thereof is provided that specifically binds to a Survivin peptide / MHC complex, wherein Survivin comprises an epitope of Survivin comprising the amino acid sequence of SEQ ID NO: 16.

[0051] In some embodiments, a Survivin tumor-specific TCR or an antigen-binding domain thereof is provided, which comprises the amino acid sequence of any one of SEQ ID NOs: 3, 6, 17, 20, 31, and 34. In some embodiments, a Survivin tumor-specific TCR or antigen-binding domain thereof is provided, comprising: (a) a TCRα CDR3 having at least about 90% sequence identity (e.g., 100% identity) to SEQ ID NO: 3; and a TCRβ CDR3 having at least about 90% sequence identity (e.g., 100% identity) to SEQ ID NO: 6; (b) a TCRα CDR3 having at least about 90% sequence identity (e.g., 100% identity) to SEQ ID NO: 17; and a TCRβ CDR3 having at least about 90% sequence identity (e.g., 100% identity) to SEQ ID NO: 20; (c) a TCRα CDR3 having at least about 90% sequence identity (e.g., 100% identity) to SEQ ID NO: 31; and a TCRβ CDR3 having at least about 90% sequence identity (e.g., 100% identity) to SEQ ID NO: 32. NO:34. A Survivin tumor-specific TCR or its antigen-binding domain having an amino acid sequence with at least about 90% sequence identity (e.g., 100% identity) has higher affinity.

[0052] In some embodiments, a Survivin tumor-specific TCR or an antigen-binding domain thereof is provided, which includes a TCR α chain comprising CDR1, CDR2, and CDR3 of any one of the amino acid sequences of SEQ ID NO: 21, 23, or 37.

[0053] In some embodiments, a Survivin tumor-specific TCR or an antigen-binding domain thereof is provided, which includes a TCR β chain comprising CDR1, CDR2, and CDR3 of any one of the amino acid sequences of SEQ ID NO: 22, 26, or 40.

[0054] In some embodiments, a Survivin tumor-specific TCR or its antigen-binding domain is provided, which includes: a TCRα chain, which includes the CDRs of any one of the amino acid sequences of SEQ ID NO: 21, 23, or 37; and a TCRβ chain, which includes the CDRs of any one of the amino acid sequences of SEQ ID NO: 22, 26, or 40.

[0055] The Survivin tumor-specific TCR or its antigen-binding domain described herein also includes a TCR constant domain. In some embodiments, the Survivin tumor-specific TCR or its antigen-binding domain includes: a TCR α chain including a TCR α constant domain (TRAC) of any one of SEQ ID NO: 21, 23, 37, or a variant thereof; and a TCR β chain including a TCR β constant domain (TRBC) of any one of the amino acid sequences of SEQ ID NO: 22, 26, 40, or a variant thereof. In some embodiments, the Survivin tumor-specific TCR or its antigen-binding domain includes: human TRAC and human TRBC, such as human Cα and human Cβ1, or human Cα and human Cβ2. In some embodiments, the Survivin tumor-specific TCR or its antigen-binding domain includes murine TRAC and murine TRBC, such as murine Cα and murine Cβ1, or murine Cα and murine Cβ2.

[0056] In some embodiments, a Survivin tumor-specific TCR or an antigen-binding domain thereof is provided, comprising: a TCR α chain comprising an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 98%, or any one of higher identity, or 100% identity) to any one of the amino acid sequences of SEQ ID NOs: 21, 23, and 37; and a TCR β chain comprising an amino acid sequence having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 98%, or any one of higher identity, or 100% identity) to any one of the amino acid sequences of SEQ ID NOs: 22, 26, and 40.

[0057] Also provided is an isolated nucleic acid encoding the TCR alpha chain and / or the TCR beta chain of a tumor-specific TCR or its antigen-binding domain according to any of the above-mentioned tumor-specific TCRs or their antigen-binding domains; a vector comprising the isolated nucleic acid(s). In some embodiments, the isolated nucleic acid is a murinized nucleic acid sequence. In some embodiments, the isolated nucleic acid is a codon-optimized murinized nucleic acid sequence.

[0058] III. Immune cells expressing survivin tumor-specific TCR or its antigen-binding domain

[0059] Provided is an engineered immune cell comprising a tumor-specific TCR or its antigen-binding domain, an isolated nucleic acid, or a vector according to any one of the above tumor-specific TCR or its antigen-binding domain.

[0060] In some embodiments, an effector cell (e.g., a T cell) expressing a Survivin tumor-specific TCR on its surface is provided, comprising a) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 21 and b) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 22; and / or c) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 23 and d) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 26; and / or e) a first nucleic acid sequence encoding a first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 37 and f) a second nucleic acid sequence encoding a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the effector cell expressing the Survivin tumor-specific TCR or its antigen-binding domain further comprises a P2A nucleic acid sequence encoding SEQ ID NOs: 11, 27, or 41. In some embodiments, an effector cell (eg, a T cell) expressing a Survivin tumor-specific TCR or its antigen-binding domain on its surface is provided, comprising a nucleic acid sequence encoding an amino acid sequence of one or more of SEQ ID NOs: 24, 28, or 42.

[0061] Example 1: Preparation of Survivin-specific T cells

[0062] A Survivin antigen-specific T cell receptor (named TCR002) was prepared for characterization and function verification experiments. For each tumor antigen-specific T cell receptor, a human (i.e., wild-type) TCR construct and a murinized TCR construct with a constant region (C region) were prepared; codon optimization was performed on the murinized TCR002 with a constant region. The murinized TCR construct has murine constant domains (mCα and mCβ1). Schematic diagrams of the wild-type, murinized, and murinized codon-optimized constructs of the Survivin antigen-specific T cell receptor TCR002 are shown in FIG. Figure 1-3 shown.

[0063] Example 2: Characteristics and Functional Verification of Survivin-Specific T Cell Receptor TCR002

[0064] The purpose of this example is to verify the expression of T cell receptor (TCR) TCR002, recognition of Survivin antigen peptide, determination of recognition epitope, determination of HLA restriction, determination of HLA recognition subtype and in vitro tumor cell killing.

[0065] TCR expression and survivin 96-122Antigenic peptide recognition

[0066] TCR expression was determined by flow cytometry. TCR-transduced T cells were collected, washed with 15 mL of PBS containing 2% FBS, centrifuged at 350 g for 5 minutes, and the supernatant discarded. The cell pellet was resuspended in PBS containing 2% FBS and the density was adjusted to 1 × 10 6 cells / mL; 200 μL / sample cell suspension was added, CD3 antibody and TCRβ chain antibody were added, and incubated on ice for 30 minutes for staining; after staining, 10 mL of PBS containing 2% FBS was added for washing once, and TCR expression was detected by flow cytometry.

[0067] like Figure 3 As shown, TCR002 lentivirus-transduced T cells can express TCR002 at high levels.

[0068] Flow cytometry and IFNγ-ELISA were used to determine the expression of Survivin in TCR002-transduced T cells. 96-122 Antigen peptide recognition. For flow cytometry detection, collect TCR-transduced T cells, add 15 mL of PBS containing 2% FBS to wash, centrifuge at 350 g for 5 minutes, and discard the supernatant. Resuspend TCR002-transduced T cells and adjust the density to 1×10 by adding AIM-V medium. 6 cells / mL, 100 μL / well was transferred to a 96-well plate; 96-122 After 24 hours of co-incubation with the peptides, the LCL cells were washed with 15 mL of PBS containing 2% FBS, centrifuged at 350 g for 5 minutes, and the supernatant was discarded. The LCL cells were resuspended by adding AIM-V medium and adjusted to 1×10 6 Cells / mL, 100 μL / well were transferred to a 96-well plate and mixed with TCR-transduced T cells; brefeldin A was added to the wells and cultured for 4-6 hours; after 4-6 hours, the cells were collected and stained for cell surface CD3 and intracellular IFN-γ and detected by flow cytometry. ELISA for IFN-γ: The resuspension method of LCL cells and transduced T cells was consistent with that of flow cytometry. 100 μL / well of resuspended LCL (1×10 6 cells / mL) and 100 μL / well of TCR002-transduced T cells (1×10 6 Cells were mixed into a 96-well plate at 100 cells / mL and cultured in an incubator for 16-24 hours. After 16-24 hours, 100 μL of supernatant was collected from each well, and the IFN-γ content in the supernatant was detected using an IFN-γ ELISA kit.

[0069] like Figure 4-6As shown, TCR002 can specifically recognize Survivin 96-122 Antigenic peptide.

[0070] Determination of recognition epitopes

[0071] Survivin 96-122 The antigenic peptide was truncated and a truncated peptide was synthesized; the truncated antigenic peptide was incubated with LCL overnight, with a final concentration of 10 μg / mL; after incubation, the LCL was collected in a 15 mL test tube, washed with 10 mL of PBS containing 2% FBS, centrifuged at 350 g for 5 minutes, and the supernatant was discarded; the cell pellet was resuspended in AIM-V medium and the density was adjusted to 1×10 6 TCR002 transduced T cells were collected, washed with 15 mL of PBS containing 2% FBS, centrifuged at 350 g for 5 minutes, and the supernatant was discarded. TCR002 transduced T cells were resuspended and adjusted to a density of 1 × 10 cells / mL by adding AIM-V medium. 6 cells / mL; 100 μL / well of resuspended LCL (1×10 6 cells / mL) and 100 μL / well of TCR002-transduced T cells (1×10 6 Cells were mixed into a 96-well plate at 100 cells / mL and cultured in an incubator for 16-24 hours. After 16-24 hours, 100 μL of supernatant was collected from each well, and the IFN-γ content in the supernatant was detected using an IFN-γ ELISA kit.

[0072] like Figure 7 As shown, TCR002 specifically recognizes the antigen epitope Survivin 100-118 , whose amino acid sequence is KKQFEELTLGEFLKLDRER.

[0073] HLA restriction determination

[0074] HLA antibody blocking test was carried out by ELISA assay, and different HLA antibody blocking groups (including no blocking, isotype control group, HLA-DR antibody blocking group, HLA-DP antibody blocking group, and HLA-DQ antibody blocking group) were tested. 100-118 After overnight exposure to antigenic peptides, LCLs were collected in 15 mL tubes, washed with 10 mL of PBS containing 2% FBS, centrifuged at 350 g for 5 minutes, and the supernatant discarded. The cell pellet was resuspended in AIM-V medium and the density was adjusted to 1 × 10 6cells / mL; 100 μL / well of the cell suspension was added to a 96-well plate, and HLA blocking antibody (final concentration 25 μg / mL) was added to the corresponding wells and cultured in an incubator for 2 hours; TCR-transduced T cells were collected, washed with 15 mL of PBS containing 2% FBS, centrifuged at 350 g for 5 minutes, and the supernatant was discarded; TCR002-transduced T cells were resuspended and adjusted to a density of 1×10 by adding AIM-V medium. 6 cells / mL, add 100 μL / well of TCR-transduced cell suspension to the corresponding wells, mix with LCL, and culture in an incubator for 16-24 hours; after 16-24 hours, collect 100 μL of supernatant from each well and use an IFN-γ ELISA kit to detect the IFN-γ content in the supernatant.

[0075] The results are as follows Figure 8 As shown, the HLA restriction of TCR002 is HLA-DP.

[0076] HLA subtype determination

[0077] HLA-DP subtype test was carried out by ELISA method, and multiple LCLs with different HLA-DP subtypes were loaded with Survivin 100-118 After overnight exposure to antigenic peptides, LCLs were collected in 15 mL tubes, washed with 10 mL of PBS containing 2% FBS, centrifuged at 350 g for 5 minutes, and the supernatant discarded. The cell pellet was resuspended in AIM-V medium and the density was adjusted to 1 × 10 6 cells / mL; 100 μL / well of the cell suspension was added to a 96-well plate; TCR-transduced T cells were collected, washed with 15 mL of PBS containing 2% FBS, centrifuged at 350 g for 5 minutes, and the supernatant was discarded; TCR002-transduced T cells were resuspended and adjusted to a density of 1 × 10 by adding AIM-V medium. 6 cells / mL, add 100 μL / well of TCR-transduced cell suspension to the corresponding wells, mix with LCL, and culture in an incubator for 16-24 hours; after 16-24 hours, collect 100 μL of supernatant from each well and use an IFN-γ ELISA kit to detect the IFN-γ content in the supernatant.

[0078] like Figure 9 As shown, T cells transfected with TCR002 can recognize HLA-DPA1 * 01:03 / HLA-DPB1 * 04:01 or HLA-DPA1 * 02:02 / HLA-DPB1 * 04:01 Two isoforms of Survivin 100-118 Antigenic peptide.

[0079] Killing tumor cells in vitro

[0080] TCR002-transduced T cells were used as effector cells using HLA-DPA1 * 01:03 / HLA-DPB1 * Oral cancer cell line TCA8113, which is positive for 04:01 and survivin protein, was used as target cells and mixed with T cells at a ratio of 4:1 as the experimental group. T cells not transfected with the T cell receptor were mixed with the same target cells at the same ratio as the control group. The killing rate was measured over 48 hours.

[0081] like Figure 10 As shown, T cells transfected with TCR002 had a significant killing effect on TCA8113 oral cancer cells.

[0082] The CDR sequences of the TCR of the present invention are shown in Table 2:

[0083] Table 2: CDR sequence list of TCR of the present invention

[0084]

[0085] The sequence involved in the present invention is as follows:

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, it should be understood by those skilled in the art that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. An isolated tumor-specific TCR that specifically binds to an MHC / Survivin epitope complex, characterized in that: include: a TCR alpha chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 1, a CDR2 having the amino acid sequence of SEQ ID NO: 2, and a CDR3 having the amino acid sequence of SEQ ID NO: 3; A TCR β chain comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4, a CDR2 having the amino acid sequence of SEQ ID NO: 5, and a CDR3 having the amino acid sequence of SEQ ID NO:

6.

2. The isolated tumor-specific TCR that specifically binds to the MHC / Survivin epitope complex according to claim 1, characterized in that: The Survivin epitope is the amino acid sequence shown in SEQ ID NO: 16; The MHC is HLA-DPAl * 01:03 / HLA-DPB1 * 04:01 or HLA-DPA1 * 02:02 / HLA-DPB1 * 04:

01.

3. The isolated tumor-specific TCR that specifically binds to the MHC / Survivin epitope complex according to claim 1, characterized in that include: a TCR alpha chain comprising the variable region of the amino acid sequence of SEQ ID NO: 7, and a TCR beta chain comprising the variable region of the amino acid sequence of SEQ ID NO: 9; or a TCR alpha chain comprising the amino acid sequences of SEQ ID NOs: 7 and 8, and a TCR beta chain comprising the amino acid sequences of SEQ ID NOs: 9 and 10; or A TCR alpha chain comprising the amino acid sequence of SEQ ID NOs: 12 and 13, and a TCR beta chain comprising the amino acid sequence of SEQ ID NOs: 14 and 15.

4. The isolated tumor-specific TCR that specifically binds to the MHC / Survivin epitope complex according to claim 1, characterized in that: The TCR alpha chain further comprises an alpha constant region and / or the TCR beta chain further comprises a beta constant region; The α constant region and the β constant region are mouse constant regions; The α constant region comprises the amino acid sequence of SEQ ID No: 13 and / or the β constant region comprises the amino acid sequence of SEQ ID NO:

15.

5. The isolated tumor-specific TCR that specifically binds to the MHC / Survivin epitope complex according to any one of claims 1 to 4, characterized in that: It also contains an antigen binding domain; The TCR or its antigen binding domain is a single chain.

6. An isolated nucleic acid molecule encoding the TCR alpha chain and TCR beta chain of the isolated tumor-specific TCR that specifically binds to the MHC / Survivin epitope complex according to any one of claims 1 to 5.

7. The isolated nucleic acid molecule according to claim 6, characterized in that: The nucleotide sequence of the nucleic acid molecule is codon-optimized; The nucleotide sequence encoding the TCRα chain and the nucleotide sequence encoding the TCRβ chain are separated by a self-cleaving peptide sequence; The self-cleaving peptide is P2A, comprising the amino acid sequence shown in SEQ ID NO:

11.

8. An engineered immune cell characterized by: A method comprising the isolated tumor-specific TCR that specifically binds to the MHC / Survivin epitope complex according to any one of claims 1 to 5 or the isolated nucleic acid molecule according to any one of claims 6 to 7; The immune cells are cell lines, primary cells obtained from a subject, or allogeneic cells.

9. A pharmaceutical composition comprising the engineered immune cell of claim 13 and a pharmaceutically acceptable carrier.

10. Use of the engineered immune cell according to claim 13 in the preparation of a medicament for treating a disease or condition expressing Survivin.

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