A nucleic acid molecule encoding a tumor-specific tcr that specifically binds to a mhc / ccnd1 epitope complex and uses thereof

Abstract

This invention discloses a nucleic acid molecule encoding a tumor-specific TCR that specifically binds to the MHC / CCND1 epitope complex and its application, belonging to the field of cancer immunotherapy. The nucleic acid molecule encodes a tumor-specific TCR that specifically binds to the MHC / CCND1 epitope complex. The TCR comprises a TCRα chain and a TCRβ chain. The TCRα chain comprises CDR1 having the amino acid sequence of SEQ ID NO:1, CDR2 having the amino acid sequence of SEQ ID NO:2, and CDR3 having the amino acid sequence of SEQ ID NO:3. The TCRβ chain comprises CDR1 having the amino acid sequence of SEQ ID NO:4, CDR2 having the amino acid sequence of SEQ ID NO:5, and CDR3 having the amino acid sequence of SEQ ID NO:6. This nucleic acid molecule can encode a tumor-specific TCR that specifically recognizes the MHC / CCND1 epitope complex. By introducing this nucleic acid molecule into recipient cells, engineered immune cells can stably express a TCR with antigen recognition function, thereby endowing them with the ability to specifically recognize and kill CCND1-positive tumor cells.

Description

Technical Field

[0001] This invention belongs to the field of cancer immunotherapy, and particularly relates to a nucleic acid molecule encoding a tumor-specific TCR that specifically binds to the MHC / CCND1 epitope complex and its application. Background Technology

[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, as key substances that trigger immune responses, include mutant proteins specifically expressed by cancer cells and tumor-associated antigens (TAAs). Tumor-associated antigens are overexpressed in cancer-originating tissues and cannot be fully recognized as "non-self" components by the immune system due to mechanisms such as immune tolerance. Antigen-presenting cells (APCs), especially dendritic cells (DCs), after taking up tumor antigens, process them and bind antigen peptides to the major histocompatibility complex (MHC), presenting them to the cell surface. Mature antigen-loaded DCs can activate T cells, triggering an immune response involving various effector T cells, including cytotoxic T cells, helper T cells, and memory T cells. Cytotoxic T cells, through their surface T cell receptors (TCRs), specifically recognize the tumor antigen-MHC complex and kill tumor cells by releasing cytokines, cytotoxic substances, or inducing cancer cell apoptosis.

[0003] Adoptive lymphocyte therapy (ACT), as an emerging strategy in cancer treatment, offers new hope for conquering cancer by infusing patients 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 reinfused into the patient after the removal of natural non-myeloid lymphocytes. However, this therapy faces many challenges in clinical application: on the one hand, the affinity of TILs from different patients or donors for tumor antigens varies significantly, leading to unstable anti-tumor efficacy; on the other hand, most antigen-specific mutations that trigger immune responses are individual-specific, making standardized treatment difficult and greatly limiting the widespread application and improvement of treatment efficacy of this therapy.

[0004] Cyclin D1, encoded by the human CCND1 gene, is a key regulator of the cell cycle, and its abnormal expression is closely related to the development of various cancers. Cyclin D1, encoded by the CCND1 gene, contains five exons, from which two transcripts are derived. Cyclin D1a is transcribed from a five-exon mRNA. In the cell cycle-dependent CDK mechanism, CCND1a binds to and activates G1-phase-specific CDK4 / 6, forming an active CCND1a-CDK4 / 6 complex, leading to phosphorylation of the G1-phase cell cycle repressor protein (RB). The phosphorylated RB protein dissociates from the bound transcription factor E2F1, initiating transcription and driving the cell from G1 phase to S phase, accelerating cell proliferation. Cyclin D1b is encoded by mRNA from an unspliced ​​intron 4, resulting in a unique C-terminal structure. This alternative splicing causes Cyclin D1b to lose a key regulatory motif encoded by exon 5, responsible for its ubiquitin-dependent degradation. Consequently, Cyclin D1b accumulates in the cell nucleus, ultimately leading to tumorigenesis. Studies have shown that CCND1 is highly expressed in various malignant tumors, including breast cancer, colon cancer, and bladder cancer, while its expression is extremely low or absent in normal adult tissues. CCND1b can synergistically regulate αvβ3 expression with HoxD3, thereby enhancing cell invasion and metastasis. The CCND1a-CDK4 / 6 complex activates the small GTPase RAC1 by regulating Pxn phosphorylation, thereby inducing cell invasion and metastasis.

[0005] Given CCND1's crucial role in tumorigenesis and development, it has become a highly anticipated target in cancer treatment. Currently, therapeutic strategies developed around CCND1 encompass antisense oligonucleotide technology, small molecule inhibitors, and immunotherapy. Among these, TCR-T cell therapy based on CCND1-specific TCRs holds promise for achieving highly effective anti-tumor effects while reducing off-target effects, opening new avenues for cancer treatment. However, existing technologies for CCND1-specific TCRs still have room for optimization, particularly in obtaining TCRs with high affinity and specificity, which is crucial for improving the clinical efficacy of this therapy. Summary of the Invention

[0006] The purpose of this invention is to propose a nucleic acid molecule that encodes a tumor-specific TCR that specifically binds to the MHC / CCND1 epitope complex and its application, in order to overcome at least one of the above-mentioned defects in the prior art.

[0007] To achieve this objective, the present invention adopts the following technical solution: The present invention provides a nucleic acid molecule encoding a tumor-specific TCR that specifically binds to the MHC / CCND1 epitope complex. The TCR comprises a TCRα chain and a TCRβ chain. The TCRα chain comprises CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO: 3. The TCRβ chain comprises CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0008] Preferably, the nucleotide sequence of the nucleic acid molecule is codon-optimized, and the nucleotide sequence encoding the TCRα chain and the nucleotide sequence encoding the TCRβ chain are separated by a self-splitting peptide sequence, wherein the self-splitting peptide is P2A and contains the amino acid sequence shown in SEQ ID NO: 13.

[0009] Preferably, the CCND1 epitope is the amino acid sequence shown in SEQ ID NO:79, and the MHC is HLA-DPA1. * 01:03 / DPB1 * 03:01.

[0010] Preferably, the TCR comprises: a TCRα chain containing a variable region of the amino acid sequence of SEQ ID NO: 7, and a TCRβ chain containing a variable region of the amino acid sequence of SEQ ID NO: 10; or a TCRα chain containing amino acid sequences of SEQ ID NO: 7 and 8, and a TCRβ chain containing amino acid sequences of SEQ ID NO: 10 and 11; or a TCRα chain containing amino acid sequences of SEQ ID NO: 21 and 22, and a TCRβ chain containing amino acid sequences of SEQ ID NO: 24 and 25.

[0011] Preferably, the TCRα chain further comprises an α constant region and / or the TCRβ chain further comprises a β constant region, wherein 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: 22 and / or the β constant region comprises the amino acid sequence of SEQ ID NO: 25.

[0012] Preferably, the TCR further includes an antigen-binding domain, and the TCR or its antigen-binding domain is single-stranded.

[0013] The present invention also provides an engineered immune cell comprising the above-mentioned nucleic acid molecules, wherein the immune cell is a cell line or a primary cell obtained from a subject or an allogeneic cell.

[0014] The present invention also provides a pharmaceutical composition comprising the above-described engineered immune cells and a pharmaceutically acceptable carrier.

[0015] The present invention also provides the use of the above-described engineered immune cells in the preparation of a medicament for treating diseases or conditions expressing CCND1.

[0016] Preferably, the disease or symptom is cancer.

[0017] Preferably, the cancer is oral cancer, head and neck cancer, breast cancer, colon cancer, or bladder cancer.

[0018] The present invention also provides a tumor-specific TCR that specifically binds to the MHC / CCND1 epitope complex, comprising: a TCRα chain, the TCRα chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO: 3; and a TCRβ chain, the TCRβ chain comprising CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6.

[0019] The beneficial effects of this invention are as follows: 1. Nucleic acid molecules can encode tumor-specific TCRs that specifically recognize the MHC / CCND1 epitope complex. By introducing this nucleic acid molecule into recipient cells, engineered immune cells can stably express TCRs with antigen recognition function, thereby endowing them with the ability to specifically recognize and kill CCND1-positive tumor cells.

[0020] 2. This tumor-specific TCR, with its unique amino acid sequence, can accurately identify the MHC / CCND1 epitope complex and effectively identify CCND1-positive tumor cells with corresponding HLA subtypes.

[0021] 3. Targeting the CCND1 target that is highly expressed in tumors, it can be used to treat various CCND1-positive tumors such as oral cancer, head and neck cancer, breast cancer, colon cancer, and bladder cancer, with a wide range of applications.

[0022] 4. After being activated by antigens, B13-modified T cells can efficiently express and secrete a variety of functional cytokines such as IFN-γ, TNF-α, IL-2, and Granzyme B. They are multifunctional effector T cells with strong anti-tumor immune activity.

[0023] 5. Engineered immune cells expressing this TCR can specifically kill CCND1-positive tumor cells that are positive for the corresponding HLA subtype. The killing effect is dose-dependent, and the anti-tumor effect is clear.

[0024] 6. We have developed a variety of TCR constructs, including wild-type, murine-derived, and murine-derived with codon optimization, which can be used with P2A self-splitting peptides to achieve double-chain co-expression. The sequence is codon optimized to facilitate cell expression. At the same time, it supports multiple structural forms such as full-length chain, variable region, and single chain to meet different experimental and clinical modification needs.

[0025] 7. Clearly define the identification pin as CCND1 93-110 The restricted HLA subtype is HLA-DPA1. * 01:03 / DPB1 * 03:01, with a clear mechanism of action, facilitating standardized research and development and application.

[0026] 8. It solves the problems of unstable efficacy and difficulty in standardization of traditional TIL therapy, and provides a core component with high affinity and high specificity for CCND1-targeted TCR-T tumor immunotherapy. Attached Figure Description

[0027] Figure 1 This is the construction diagram of CCND1-specific B13 (wild type) of the present invention.

[0028] Figure 2 This is a diagram of the CCND1-specific B13 construction (mouse-derived) of the present invention.

[0029] Figure 3 This is the CCND1-specific B13 construction diagram (mouse-derived codon optimization) of the present invention.

[0030] Figure 4 This is a graph showing the results of FACS detection of B13 expression according to the present invention.

[0031] Figure 5 This is a graph showing the results of FACS detection of B13-specific recognition antigen peptides according to the present invention.

[0032] Figure 6 This is a graph showing the results of ELISA detection of B13-specific antigen peptides according to the present invention.

[0033] Figure 7 This is a diagram showing the results of the B13 specific recognition of antigen epitopes in this invention.

[0034] Figure 8 This is a diagram showing the HLA restriction results of T cells expressing the CCND1 antigen-specific T cell receptor B13 as determined by an HLA antibody blocking assay according to the present invention.

[0035] Figure 9This is a diagram showing the results of the LCL recognition assay used to determine the HLA subtype of T cells expressing the CCND1 antigen-specific T cell receptor B13, as per the present invention.

[0036] Figure 10 This invention relates to T cells expressing the CCND1 antigen-specific T cell receptor B13 recognizing the LCL-loaded antigen peptide, and then IFN... TNF Figure showing the specific expression results of three functional factors: IL-2, IL-3, and IL-4.

[0037] Figure 11 This invention relates to T cells expressing the CCND1 antigen-specific T cell receptor B13 recognizing the LCL-loaded antigen peptide, and then IFN... TNF A graph showing the specific secretion results of four functional factors: IL-2, Granzyme B, etc.

[0038] Figure 12 This is a diagram showing the results of T cells expressing the CCND1 antigen-specific T cell receptor B13 in vitro specifically killing oral cancer cells. Detailed Implementation

[0039] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments.

[0040] I. Definition Before describing the 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 they are generally used in the art to which they pertain.

[0041] The invention will be described with reference to specific embodiments and certain accompanying drawings, but the invention is not limited thereto, but is defined solely by the claims. The use of the term "comprising" in this specification and claims does not exclude other elements. For the purposes of this invention, the term "consisting of" is considered a preferred embodiment of the term "comprising." If a group is defined below as including at least a certain number of embodiments, it should also be understood as disclosing a group preferably consisting only of these embodiments.

[0042] 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 antigenic epitope in an MHC molecule. A TCR may include a TCRα polypeptide chain and a TCRβ polypeptide chain. “Tumor-specific TCR” refers to a TCR that specifically recognizes tumor antigens expressed by tumor cells. “TCR-T” refers to T cells expressing a recombinant TCR. The “antigen-binding domain” of a TCR refers to a polypeptide that binds to an antigen with a high degree of specificity due to its primary, secondary, or tertiary sequence and / or post-translational modifications and / or charge. The antigen-binding domain may be derived from any part or fragment of the TCR that retains the antigen-binding activity of the TCR as part of the TCR. The antigen-binding portion encompasses, for example, a portion of the TCR that retains specific binding to the amino acid sequence of CCND1 or a fragment thereof, or a portion that possesses the ability to detect, treat, or prevent cancer to a similar, equal, or greater degree compared to 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 any one or both of the α and β chains of the TCR, such as portions of one or more of the complementarity-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 sequences: CDR1, CDR2, CDR3 of the α chain, CDR1, CDR2, CDR3 of the β chain, or any combination thereof. Preferably, the antigen-binding domain comprises the amino acid sequences of CDR1, CDR2, and CDR3 of the α chain or the amino acid sequences of CDR1, CDR2, and CDR3 of the β chain; or the amino acid sequences of CDR1, CDR2, and CDR3 of both the α and β chains. In one embodiment of the invention, the antigen-binding portion may include, for example, a variable region of the TCR, which includes a combination of the aforementioned CDR regions. In this regard, the antigen-binding portion may contain the amino acid sequence of the variable region (Vα) of the α chain of the TCR, the amino acid sequence of the variable region (Vβ) 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 contain a combination of a variable region and a constant region. In this regard, the antigen-binding portion may contain the full-length α or β chain of the TCR, or both the α and β chains.

[0043] II. CCND1 Tumor-Specific TCR This application provides a tumor-specific TCR that specifically recognizes tumor-specific CCND1 and / or specifically recognizes the MHC / CCND1 epitope complex. In some embodiments, the tumor-specific TCR specifically recognizes CCND1. In some embodiments, the tumor-specific TCR specifically recognizes the MHC / CCND1 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.

[0044] 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 delineation algorithms known in the art may 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 the exemplary TCRs are shown in the "Sequence Listing" section.

[0045] Table 1: Exemplary Tumor-Specific TCRs

[0046] In some embodiments, a CCND1 tumor-specific TCR or its antigen-binding domain is provided that specifically binds to an epitope of the CCND1 peptide, such as a CCND1 epitope comprising the amino acid sequence of SEQ ID NO: 79. In some embodiments, the CCND1 tumor-specific TCR or its antigen-binding domain specifically binds to a CCND1 epitope comprising the amino acid sequence of SEQ ID NO: 79. In some embodiments, a CCND1 tumor-specific TCR or its antigen-binding domain is provided that specifically binds to a CCND1 peptide / MHC complex, wherein CCND1 comprises a CCND1 epitope comprising the amino acid sequence of SEQ ID NO: 79.

[0047] In some embodiments, a CCND1 tumor-specific TCR or its antigen-binding domain is provided, comprising the amino acid sequence of any one of SEQ ID NO: 3, 6, 17, 20, 31, 34. In some embodiments, a CCND1 tumor-specific TCR or its antigen-binding domain is provided, comprising: (a) TCRα CDR3 having at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO: 3; and TCRβ CDR3 having at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO: 6; (b) TCRα CDR3 having at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO: 17; and TCRβ CDR3 having at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO: 20; (c) TCRα CDR3 having at least about 90% sequence identity (e.g., 100% identity) with SEQ ID NO: 31; and TCRβ ... 34 CCND1 tumor-specific TCRs or their antigen-binding domains having an amino acid sequence with at least about 90% sequence identity (e.g., 100% identity) have higher affinity.

[0048] In some embodiments, a CCND1 tumor-specific TCR or its antigen-binding domain is provided, comprising a TCRα chain comprising CDR1, CDR2, and CDR3 of any of the amino acid sequences of SEQ ID NO: 9, 23, or 37.

[0049] In some embodiments, a CCND1 tumor-specific TCR or its antigen-binding domain is provided, comprising a TCRβ chain comprising CDR1, CDR2, and CDR3 of any of the amino acid sequences of SEQ ID NO: 12, 26, or 40.

[0050] In some embodiments, a CCND1 tumor-specific TCR or its antigen-binding domain is provided, comprising: a TCRα chain comprising a CDR of any one of the amino acid sequences of SEQ ID NO: 9, 23, or 37; and a TCRβ chain comprising a CDR of any one of the amino acid sequences of SEQ ID NO: 12, 26, or 40.

[0051] The CCND1 tumor-specific TCR or its antigen-binding domain described herein also includes a TCR constant domain. In some embodiments, the CCND1 tumor-specific TCR or its antigen-binding domain comprises: a TCRα chain comprising the TCRα constant domain (TRAC) of any one of SEQ ID NO: 9, 23, 37, or a variant thereof; and a TCRβ chain comprising the TCRβ constant domain (TRBC) of any one of the amino acid sequences of SEQ ID NO: 12, 26, 40, or a variant thereof. In some embodiments, the CCND1 tumor-specific TCR or its antigen-binding domain comprises: human TRAC and human TRBC, such as human Cα and human Cβ1, or human Cα and human Cβ2. In some embodiments, the CCND1 tumor-specific TCR or its antigen-binding domain comprises murine TRAC and murine TRBC, such as murine Cα and murine Cβ1, or murine Cα and murine Cβ2.

[0052] In some embodiments, a CCND1 tumor-specific TCR or its antigen-binding domain is provided, comprising: a TCRα chain comprising an amino acid sequence of any one of SEQ ID NO: 9, 23, and 37 having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 98%, or higher, or 100% identity); and a TCRβ chain comprising an amino acid sequence of any one of SEQ ID NO: 12, 26, and 40 having at least about 80% identity (e.g., at least about 85%, 90%, 95%, 98%, or higher, or 100% identity).

[0053] Also provided is an isolated nucleic acid encoding the TCRα chain and / or the TCRβ 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; and a vector comprising the isolated nucleic acid. In some embodiments, the isolated nucleic acid is a murine nucleic acid sequence. In some embodiments, the isolated nucleic acid is a codon-optimized murine nucleic acid sequence.

[0054] III. Immune cells expressing CCND1 tumor-specific TCR or its antigen-binding domain An engineered immune cell is provided, comprising a tumor-specific TCR or its antigen-binding domain based on any one of the tumor-specific TCRs or their antigen-binding domains, an isolated nucleic acid, or a vector.

[0055] In some embodiments, an effector cell (e.g., a T cell) expressing a CCND1 tumor-specific TCR on its surface is provided, comprising a) a first nucleic acid sequence encoding a first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 9 and b) a second nucleic acid sequence encoding a second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 12; and / or c) a first nucleic acid sequence encoding the first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 23 and d) a second nucleic acid sequence encoding the second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 26; and / or e) a first nucleic acid sequence encoding the first polypeptide chain, the first polypeptide chain comprising the amino acid sequence of SEQ ID NO: 37 and f) a second nucleic acid sequence encoding the second polypeptide chain, the second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 40. In some embodiments, the effector cell expressing a CCND1 tumor-specific TCR or its antigen-binding domain further comprises a P2A nucleic acid sequence encoding SEQ ID NOs: 13, 27, 41. In some embodiments, an effector cell (e.g., a T cell) expressing a CCND1 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: 14, 28, 42.

[0056] Example 1: Preparation of CCND1-specific T cells A CCND1 antigen-specific T-cell receptor (named B13) was prepared for characterization and functional validation experiments. For each tumor antigen-specific T-cell receptor, a corresponding human (i.e., wild-type) TCR construct and a murine TCR construct with the constant region (C region) were prepared; codon optimization was performed on the murine constant region B13. The murine TCR construct possesses murine constant domains (mCα and mCβ1). Schematic diagrams of the wild-type, murine, and murine codon-optimized constructs of the CCND1 antigen-specific T-cell receptor B13 are shown below. Figure 1-3 As shown.

[0057] Example 2: Characterization and functional verification of CCND1-specific T cell receptor B13 The purpose of this embodiment is to verify the expression of T cell receptor (TCR) B13, recognition of CCND1 antigen peptide, identification of recognition epitopes, determination of HLA restriction, determination of HLA recognition subtypes, and in vitro killing of tumor cells.

[0058] TCR expression and CCND1 90-119 Antigen peptide recognition TCR expression was measured using flow cytometry. TCR-transduced T cells were collected, washed with 15 mL of PBS containing 2% FBS, centrifuged at 350g for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in PBS containing 2% FBS and the density was adjusted to 1×10⁻⁶. 6 Cells / mL; Take 200 µL of cell suspension per sample, add CD3 antibody and TCRβ chain antibody, incubate on ice for 30 minutes for staining; After staining, wash once with 10 mL of PBS containing 2% FBS, and then detect TCR expression using flow cytometry.

[0059] like Figure 4 As shown, B13 lentivirus-transduced T cells can express B13 at high levels.

[0060] Using flow cytometry and IFN ELISA technology measures the effect of B13-transduced T cells on CCND1. 90-119 Recognition of antigenic peptides. For flow cytometry detection; collect TCR-transduced T cells, wash with 15 mL of PBS containing 2% FBS, centrifuge at 350g for 5 minutes, discard the supernatant; resuspend B13-transduced T cells by adding AIM-V medium and adjust the density to 1×10⁻⁶. 6 Transfer 100 µL / well to a 96-well plate at 1 cell / mL; collect CCND1. 90-119 LCL cells co-incubated with peptides for 24 hours were washed with 15 mL of PBS containing 2% FBS, centrifuged at 350g for 5 minutes, and the supernatant was discarded. The LCL cells were then resuspended by adding AIM-V medium and adjusted to a concentration of 1×10⁻⁶ cells / mL. 6 Cells / mL, 100 µL / well transferred to 96-well plates and mixed with TCR-transduced T cells; brefeldin A added to wells and cultured for 4-6 hours; after 4-6 hours, cells were collected and used for CD3 staining of the cell surface and IFN-γ staining of the intracellular cells. Staining and detection by flow cytometry. Targeting IFN- ELISA assay: The resuspending method for LCL cells and transduced T cells was the same as that for flow cytometry. 100 µL / well of resuspended LCL (1×10⁻⁶ cells) was used. 6 100 µL / well of B13 transduced T cells (1×10⁻⁶ cells / mL) and 100 µL / well of B13 transduced T cells (1×10⁻⁶ cells / mL). 6 Mix (cells / mL) into 96-well plates and incubate for 16-24 hours; after 16-24 hours, collect 100 µL of supernatant from each well and use IFN-γ. ELISA kit for detecting IFN-α in supernatant The content of.

[0061] like Figure 5-6 As shown, B13 can specifically recognize CCND1. 90-119 Antigenic peptides.

[0062] Identification of epitopes For CCND1 90-119 The antigenic peptide was truncated and a truncated polypeptide was synthesized. The truncated antigenic peptide was incubated with LCL overnight to a final concentration of 10 µg / mL. After incubation, the LCL was collected in 15 mL test tubes, washed with 10 mL of PBS containing 2% FBS, centrifuged at 350g 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. 6 Cells / mL; collect TCR-transduced T cells, wash with 15 mL of PBS containing 2% FBS, centrifuge at 350g for 5 minutes, and discard the supernatant; resuspend B13-transduced T cells by adding AIM-V medium and adjust the density to 1×10⁶ cells / mL. 6 Cells / mL; 100 µL / well of resuspended LCL (1×10⁻⁶ cells / mL); 6 100 µL / well of B13 transduced T cells (1×10⁻⁶ cells / mL) and 100 µL / well of B13 transduced T cells (1×10⁻⁶ cells / mL). 6 Mix (cells / mL) into 96-well plates and incubate for 16-24 hours; after 16-24 hours, collect 100 µL of supernatant from each well and use IFN-γ. ELISA kit for detecting IFN-α in supernatant The content of.

[0063] like Figure 7 As shown, B13 specifically recognizes the antigen epitope CCND1. 93-110 Its amino acid sequence is PVKKSRLQLLGATCMFVA.

[0064] HLA-restricted determination HLA antibody blocking assays were performed using ELISA to detect 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). LCL loaded with CCND1 was used. 90-119 After overnight incubation of the antigenic peptide, LCL was collected in a 15 mL test tube, washed with 10 mL of PBS containing 2% FBS, centrifuged at 350g 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. 6Cells / mL; 100 µL / well of 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 the plates were incubated for 2 hours; TCR-transduced T cells were collected, washed with 15 mL of PBS containing 2% FBS, centrifuged at 350g for 5 minutes, and the supernatant was discarded; B13-transduced T cells were resuspended by adding AIM-V medium and the density was adjusted to 1×10⁶ cells / mL. 6 100 µL / well of TCR-transduced cell suspension was added to each well, mixed with LCL, and incubated for 16–24 hours. After 16–24 hours, 100 µL of supernatant was collected from each well and incubated with IFN-γ. ELISA kit for detecting IFN-α in supernatant The content of.

[0065] The results are as follows Figure 8 As shown, the HLA restriction of B13 is HLA-DP.

[0066] HLA identification and subtype determination HLA-DP subtype assays were performed using ELISA. Multiple LCLs of different HLA-DP subtypes were loaded with CCND1. 93-110 After overnight incubation of the antigenic peptide, LCL was collected in a 15 mL test tube, washed with 10 mL of PBS containing 2% FBS, centrifuged at 350g 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. 6 Cells / mL; Add 100 µL / well of cell suspension to a 96-well plate; Collect TCR-transduced T cells, wash with 15 mL of PBS containing 2% FBS, centrifuge at 350g for 5 minutes, and discard the supernatant; Resuspend B13-transduced T cells by adding AIM-V medium and adjust the density to 1×10⁶ cells / mL. 6 100 µL / well of TCR-transduced cell suspension was added to each well, mixed with LCL, and incubated for 16–24 hours. After 16–24 hours, 100 µL of supernatant was collected from each well and incubated with IFN-γ. ELISA kit for detecting IFN-α in supernatant The content of.

[0067] like Figure 9 As shown, T cells transfected with B13 can recognize HLA-DPA1. * 01:03 / DPB1 * 03:01 Subtype presentation of CCND1 93-110 Antigenic peptides.

[0068] Determination of expression of multiple functional factors Flow cytometry was used to determine the recognition of CCND1 by B13-transduced T cells. 90-119 Following the antigen peptide, multiple functional factors are expressed. For flow cytometry detection: TCR-transduced T cells are collected, washed with 15 mL of PBS containing 2% FBS, centrifuged at 350g for 5 minutes, and the supernatant is discarded; B13-transduced T cells are resuspended in AIM-V medium and the density is adjusted to 1×10⁻⁶. 6 Transfer 100 µL / well to a 96-well plate at 1 cell / mL; collect CCND1. 90-119 LCL cells co-incubated with peptides for 24 hours were washed with 15 mL of PBS containing 2% FBS, centrifuged at 350g for 5 minutes, and the supernatant was discarded. The LCL cells were then resuspended by adding AIM-V medium and adjusted to a concentration of 1×10⁻⁶ cells / mL. 6 100 µL / well was 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 used for CD3 staining on the cell surface and staining for IFN-γ, TNF-α, and IL-2 intracellularly, and the results were analyzed by flow cytometry.

[0069] like Figure 10 As shown, engineered T cells transfected with B13 TCR were processed via CCND1. 90-119 After stimulation with antigenic peptides (right column) or control peptides (left column), CD3 was detected by intracellular staining (ICS). + IFN-γ in T cells The expression of TNF-α and IL-2 was observed. Results showed that under control peptide stimulation, the positive rates of all three cytokines were at extremely low background levels (1.21%-5.10%); while under CCND1... 90-119 Upon stimulation by the antigen peptide, B13 TCR-T cells are specifically activated and can simultaneously express IFN-γ. The cell population exhibited the characteristics of multifunctional effector T cells, showing positive rates of TNF-α (9.50%), IL-α (74.6%), and IL-2 (28.3%). The positive rates of TNF-α and IL-2 were particularly significant, indicating that this cell population possesses highly efficient tumor-killing potential and good in vivo survival and expansion capabilities. The control peptide is a 30-amino acid polypeptide derived from amino acids 108-137 of the HIV-1 gp120 protein, as shown in SEQ ID NO: 80, with the amino acid sequence LWDQSLKPCVKLTPLCVTLKCTNVNTTNLN.

[0070] Multiple functional factors secretion determined The recognition of CCND1 by B13-transduced T cells was measured using an ELISA method.90-119 Following the antigenic peptide, multiple functional factors are secreted. LCLs are loaded with CCND1. 90-119 After overnight incubation of the antigen peptide or control peptide, LCL was collected in 15 mL test tubes, washed with 10 mL of PBS containing 2% FBS, centrifuged at 350g 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. 6 Cells / mL; Add 100 µL / well of cell suspension to a 96-well plate; Collect TCR-transduced T cells, wash with 15 mL of PBS containing 2% FBS, centrifuge at 350g for 5 minutes, and discard the supernatant; Resuspend B13-transduced T cells by adding AIM-V medium and adjust the density to 1×10⁶ cells / mL, add 100 µL / well of TCR-transduced cell suspension to the corresponding wells, mix with LCL, and incubate for 16-24 hours; After 16-24 hours, collect 100 µL of supernatant from each well and use IFN-γ to treat the cells. -ELISA, TNFα-ELISA, IL-2-ELISA, and Granzyme B-ELISA kits are used to detect the content of factors in the supernatant.

[0071] like Figure 11 As shown, after T cells transfected with B13 recognized the antigenic peptide, ELISA results confirmed that the engineered T cells expressing the B13 TCR of this invention, upon stimulation with specific antigens, could efficiently secrete multiple functional cytokines such as IFN-γ, TNF-α, IL-2, and Granzyme B. Among these, the secretion level of Granzyme B was particularly prominent (approximately 90,000 pg / mL), exhibiting extremely strong direct cytotoxic killing potential; simultaneously, high concentrations of IFN-γ (approximately 45,000 pg / mL) and TNF-α (approximately 22,000 pg / mL) were secreted, demonstrating significant immunomodulatory and microenvironment remodeling capabilities; the effective secretion of IL-2 (approximately 12,000 pg / mL) ensured the in vivo expansion and sustained activity of the T cells. These results indicate that the T cells obtained in this invention are typical highly active, multifunctional effector T cells.

[0072] Killing tumor cells in vitro Transduced B13 T cells were used as effector cells, using HLA-DPA1. * 01:03 / DPB1 * Oral cancer cell line SAS, which is positive for 03:01 and CCND1 protein expression, was used as the target cell line and mixed with other cells at ratios of 1:1 and 4:1 as the experimental group. Untransfected T cell receptor T cells were mixed with the same target cells at the same ratio as the control group. The killing rate was measured after 48 hours.

[0073] like Figure 12 As shown, T cells transfected with B13 have a significant killing effect on SAS oral cancer cells in a dose-dependent manner, as detailed below: Specific killing: The NCI of control T cells (effect-to-target ratio 4:1) increased slowly over time (no effective killing); while the NCI of B13TCR-T cells (effect-to-target ratio 4:1 or 1:1) decreased continuously over time, indicating that B13 TCR-T cells can initiate a highly efficient killing effect by specifically recognizing SAS cell surface antigens.

[0074] Dose-dependent: The NCI decrease in the B13 TCR-T group (green line) with an effector-to-target ratio of 4:1 was significantly greater than that in the 1:1 group (red line), demonstrating that the killing efficiency increases with the number of effector cells (dose-dependent).

[0075] Time-dependent: The NCI of the B13 TCR-T group continued to decrease over time, indicating that the killing effect was persistent (the longer the treatment time, the more target cells died).

[0076] In summary, B13 TCR-T exhibits specific, dose-dependent, and time-dependent potent killing ability against SAS oral cancer cells, providing key functional evidence for its clinical anti-tumor application.

[0077] The CDR sequences of the TCR of this invention are shown in Table 2: Table 2: CDR sequence list of the TCR of the present invention

[0078] The sequences involved in this invention are shown in the table below:

[0079] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nucleic acid molecule encoding a tumor-specific TCR that specifically binds to the MHC / CCND1 epitope complex, said TCR comprising a TCRα chain and a TCRβ chain; The TCRα chain comprises CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO: 3; The TCRβ chain comprises CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO:

6.

2. The nucleic acid molecule according to claim 1, characterized in that: The nucleotide sequence of nucleic acid molecules is optimized using codons; The nucleotide sequences encoding the TCRα chain and the nucleotide sequences encoding the TCRβ chain are separated by self-splitting peptide sequences; The self-splitting peptide is P2A, which contains the amino acid sequence shown in SEQ ID NO:

13.

3. The nucleic acid molecule according to claim 1, characterized in that: The CCND1 epitope is the amino acid sequence shown in SEQ ID NO:79; The MHC is HLA-DPA1 * 01:03 / DPB1 * 03:

01.

4. The nucleic acid molecule according to claim 3, characterized in that, The TCR includes: A TCRα chain containing the variable region of the amino acid sequence of SEQ ID NO: 7, and a TCRβ chain containing the variable region of the amino acid sequence of SEQ ID NO: 10; or A TCRα chain containing the amino acid sequences of SEQ ID NO: 7 and 8, and a TCRβ chain containing the amino acid sequences of SEQ ID NO: 10 and 11; or The TCRα chain containing the amino acid sequences of SEQ ID NO: 21 and 22, and the TCRβ chain containing the amino acid sequences of SEQ ID NO: 24 and 25.

5. The nucleic acid molecule according to claim 3, characterized in that: The TCRα chain also contains an α constant region and / or the TCRβ chain also contains a β constant region; The α-constant region and β-constant region are mouse constant regions; The α constant region contains the amino acid sequence of SEQ ID No: 22 and / or the β constant region contains the amino acid sequence of SEQ ID NO:

25.

6. The nucleic acid molecule according to any one of claims 1-5, characterized in that: The TCR also includes an antigen-binding domain; The TCR or its antigen-binding domain is single-stranded.

7. Engineered immune cells, characterized in that: It includes the nucleic acid molecule according to any one of claims 1-6; The immune cells are cell lines or primary cells obtained from the subject or allogeneic cells.

8. A pharmaceutical composition comprising the engineered immune cells of claim 7 and a pharmaceutically acceptable carrier.

9. Use of the engineered immune cells of claim 7 in the preparation of a medicament for treating oral cancer expressing CCND1.

10. A tumor-specific TCR that specifically binds to the MHC / CCND1 epitope complex, characterized in that, include: The TCRα chain comprises CDR1 having the amino acid sequence of SEQ ID NO: 1, CDR2 having the amino acid sequence of SEQ ID NO: 2, and CDR3 having the amino acid sequence of SEQ ID NO: 3; The TCRβ chain comprises CDR1 having the amino acid sequence of SEQ ID NO: 4, CDR2 having the amino acid sequence of SEQ ID NO: 5, and CDR3 having the amino acid sequence of SEQ ID NO: 6.

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