RAS mutant epitope peptides and T cell receptors that recognize RAS mutants

Abstract

The present invention relates to the fields of immunology and tumor therapy. Specifically, the present invention relates to a Ras G12V mutant epitope peptide, an antigen-presenting cell expressing the epitope peptide, a tumor vaccine containing the same, and the use of the tumor vaccine in preventing or treating tumors with the RAS G12V mutation. The present invention also relates to a T cell receptor (TCR) that specifically recognizes the Ras G12V mutant, a conjugate containing the TCR, a fusion protein containing the TCR, an immune cell expressing the TCR, a T cell drug containing the same, and the use of the T cell drug in preventing or treating tumors with the RAS G12V mutation.

Description

Background of the Invention

[0001] Technical field The present invention relates to the fields of immunology and oncology. Specifically, the present invention relates to RAS G12V mutant epitope peptides, antigen-presenting cells expressing the epitope peptide, tumor vaccines containing the same, and their use for the prevention or treatment of tumors having the RAS G12V mutation. The present invention also relates to T cell receptors (TCRs) that specifically recognize the RAS G12V mutant, conjugates and fusion proteins comprising the TCR, immune cells expressing the TCR, and T cell drugs comprising the same, and their use for the prevention or treatment of tumors having the RAS G12V mutation.

[0002] Background technology RAS is a proto-oncogene with GTPase activity that is involved in many signaling pathways that regulate cell proliferation, differentiation, and apoptosis, including MAPK, PI3K, and STAT signaling pathways. In 1982, Der CJ et al. first confirmed that mutations in the RAS gene are a cancer promoter (Der CJ et al., 1982). There are three RAS-coding genes in humans: HRAS (gene ID: 3265), NRAS (gene ID: 4893), and KRAS (gene ID: 3845). These three RAS genes have high sequence homology (>90%). Approximately 33% of human tumors have RAS gene mutations, making RAS gene mutations the most frequent proto-oncogene mutations (Karnoub AE, 2008). The most common RAS gene mutation in human tumors is KRAS, accounting for approximately 22%, followed by NRAS at approximately 8.0%, and HRAS at approximately 3.3%.

[0003] KRAS gene mutations are most common in solid tumors (accounting for approximately 86% of the three RAS mutations), for example, in colorectal cancer (30%–50%), pancreatic cancer (approximately 85%), and non-small cell lung cancer (15%–25%); over 97% of KRAS mutations are found primarily in exons 2 and 3, with exon 2 having the highest frequency (e.g., G12C, G12V, G12D, G13D, etc.), while G12D and G12V mutations account for approximately 20%–30% of colorectal cancer, 60%–70% of pancreatic cancer, and approximately 38% of non-small cell lung cancer. KRAS mutations are the driving genes for oncological drug resistance. For example, when EGFR TKIs and EGFR monoclonal antibody drugs (such as cetuximab) are administered, the KRAS mutation status of tumor patients should be detected, as patients with KRAS mutations have a very low response to EGFR inhibitors (approximately 0-5%) (Jackman DM et al., 2009). Tumor patients with KRAS mutations have shorter progression-free survival and overall survival compared to tumor patients with wild-type KRAS, and patients with KRAS mutations also have a higher likelihood of postoperative recurrence and metastasis.

[0004] Over the past few decades, scientific research on the structure and biology of KRAS has revealed that GTP binds to the KRAS protein with extremely high affinity (at the pM level), making it difficult for small molecule inhibitors to compete with GTP and inhibit KRAS activity. It has also become clear that other proteins that interact with KRAS within cells are involved in signal transduction, and that inhibiting downstream KRAS signaling through competitive inhibition of protein-protein interactions is also difficult for small molecule compounds. At the same time, the structure of the KRAS protein has a relatively smooth structure and lacks a "pocket" for small molecule inhibitors to bind to, indicating that developing small molecule inhibitors that target the KRAS protein itself and related proteins is extremely difficult. The development of KRAS inhibitors has mainly focused on interfering with KRAS modification and inhibiting its synthesis for the treatment of KRAS-mutated tumors, such as the development of farnesyltransferase inhibitors, but all have ended in failure (Heidi Ledford, 2015). In recent years, drug development against KRAS mutants has mainly focused on the KRAS G12C mutant. By designing compounds that can irreversibly and covalently bind to the cysteine ​​residue of the G12C mutation, the KRAS G12C mutant is blocked in an inactive state, thereby inhibiting the activity of the KRAS mutant G12C. However, there are currently no drugs under development for other KRAS mutants (e.g., G12V and G13D).

[0005] According to World Health Organization (WHO) statistics, in China, the annual incidence of cancers with high RAS mutations (e.g., pancreatic cancer, colorectal cancer, lung cancer, endometrial cancer, ovarian cancer, and prostate cancer) reaches 1.6 million people. Of these, 449,000 have RAS mutations (including G12, G13, Q61, etc.), and 108,000 have the RAS G12V gene mutation. Patients with RAS mutations have a high rate of tumor recurrence, drug resistance, poor prognosis, and short overall survival, making new treatments for these patient groups an urgent need.

[0006] The T cell receptor (TCR) recognizes the peptide sequences of viral proteins and mutant gene transcripts presented by HLA, and since the TCR can specifically recognize peptides containing mutations, mutations in the KRAS gene are ideal TCR targets. Qiong JW et al. used the KRAS G12V p7-16 and KRAS G12D p7-16 peptides to study HLA-A * Transgenic mice expressing 11:01 were immunized and screened for HLA-A * 11:01 Restricted G12V and G12D-recognizing mouse TCRs was obtained (Qiong JW, 2015); Steven A. Rosenberg et al. isolated tumor-infiltrating T cells from patients with advanced metastatic colorectal disease, thereby obtaining HLA-C * Steven A. Rosenberg, 2016 discovered a TCR that recognizes the 08:02 restraining KRAS G12D, which was enlarged in vitro and reinjected into patients, resulting in a favorable clinical response. * Using the 11:01 tetramer staining method, T cell receptors that recognize the KRAS G12V mutant were isolated and obtained from T cells in the peripheral blood of patients with advanced metastatic endometrial cancer (Steven A. Rosenberg, 2017).

[0007] T cell receptors are generated from rearrangements of the VDJ gene, and the naturally occurring T cell receptor library is approximately 10 16-20 It has a capacity of (Harlan S. Robins, 2009); the T cell receptor library capacity is about 1,000 to 10,000 times that of the B cell receptor, and such a vast library capacity corresponds to the human leukocyte antigen system. HLA is divided into type I (A, B, C, etc.) and type II (DP, DR, DQ, etc.), each presenting peptides of different lengths (8 to 16 mers). The discovery of anti-KRAS mutant TCRs is mainly carried out by the National Cancer Center in the United States. Currently, the discovered RAS mutant TCRs are HLA-A * Recognition of KRAS G12V being apprehended at 11:01 and HLA-C *This is the recognition of KRAS G12D being apprehended at 08:02.

[0008] However, HLA alleles are semi-genetic and exhibit strong regional genetic distribution. The HLA distribution in Caucasian populations differs significantly from that of populations in the Asia-Pacific region. Therefore, the discovery of novel T cell receptor pairs for KRAS mutants applicable to Asia-Pacific populations is urgently needed. [Disclosure of the Invention]

[0009] The present invention provides a RAS G12V mutant epitope peptide, a T cell receptor (TCR) that specifically recognizes the epitope peptide, cells and pharmaceutical compositions comprising the epitope peptide or TCR, nucleic acids encoding the epitope peptide or TCR, vectors and host cells for producing the epitope peptide or TCR, and methods for treating a subject by using the epitope peptide or TCR. The epitope peptide and TCR provided by the present invention induce an immune response against tumors having the RAS G12V mutation and can therefore be used to treat tumors in a subject. Furthermore, the epitope peptide and TCR provided by the present invention are MHC-II restricted, and since this MHC-II restriction is an allele that is significantly more frequent in the Asia-Pacific population, this is particularly suitable for patients in the Asia-Pacific region.

[0010] Epitope peptide Therefore, in a first aspect, the present invention provides an isolated epitope peptide or a variant thereof, wherein the epitope peptide consists of consecutive amino acid residues 11 to 30 (e.g., 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11) of the RAS G12V mutant, and comprises amino acid residues 3 to 13 of the RAS G12V mutant.

[0011] This variant differs from the epitope peptide from which it is derived only by substitution of one or several (e.g., 1, 2, or 3) amino acid residues, does not contain amino acid substitutions at positions corresponding to the amino acids at positions 4, 5, 6, 9, and 12 of the RAS G12V mutant, and retains the biological function of the epitope peptide from which it is derived. This biological function includes the ability to be presented by MHC-II molecules and then recognized by T cells, e.g., by an antigen-specific T cell receptor on the T cell.

[0012] In certain embodiments, the epitope peptide or variant thereof of the present invention is an MHC-II restricted antigen, i.e., the epitope peptide or variant thereof of the present invention can present MHC-II molecules expressed on the surface of cells, or exist together with its background, or form a complex with its background. In certain embodiments, the epitope peptide or variant thereof of the present invention has the ability to be presented by MHC-II molecules, and the epitope peptide or variant thereof that associates with MHC-II molecules has the ability to be recognized by T cells, e.g., by an antigen-specific T cell receptor on the T cell.

[0013] In certain embodiments, the MHC-II molecule is HLA-DP. In certain embodiments, HLA-DP comprises HLA-DPB1 * 03:01 or HLA-DPB1 * 14:01. In certain embodiments, HLA-DP further comprises one selected from the group consisting of HLA-DPA1 * 02:02, HLA-DPA1 * 02:01, and HLA-DPA1 * 01:03. In certain embodiments, the MHC-II molecule is HLA-DPB1 * 03:01 or HLA-DPB1 * 14:01, and HLA-DPA1 * 02:02. In certain embodiments, the MHC-II molecule is HLA-DPB1 * 03:01 or HLA-DPB1 *14:01, and HLA-DPA1 * 02:01. In certain embodiments, the MHC-II molecule is HLA-DPB1 * 03:01 or HLA-DPB1 * 14:01, and HLA-DPA1 * It is 01:03.

[0014] In certain embodiments, the epitope peptide consists of consecutive amino acid residues 11-25 (e.g., 11-23, 11-13) of the RAS G12V mutant.

[0015] In certain embodiments, the RAS G12V mutant is a KRAS G12V mutant. In certain embodiments, the RAS G12V mutant has a sequence that has 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% sequence identity with the sequence shown in Sequence ID No. 1.

[0016] In certain embodiments, amino acid residues 3-13 of the RAS G12V mutant have the sequence shown in Sequence ID No. 2.

[0017] In certain embodiments, the epitope peptide comprises or consists of amino acid residues 3 to 13 of the RAS G12V mutant (e.g., shown in SEQ ID NO: 2). In certain embodiments, the epitope peptide comprises or consists of amino acid residues 2 to 13 of the RAS G12V mutant (e.g., shown in SEQ ID NO: 3). In certain embodiments, the epitope peptide comprises or consists of amino acid residues 3 to 14 of the RAS G12V mutant (e.g., shown in SEQ ID NO: 4). In certain embodiments, the epitope peptide comprises or consists of amino acid residues 2 to 14 of the RAS G12V mutant (e.g., shown in SEQ ID NO: 5).

[0018] In certain embodiments, the epitope peptide consists of amino acid residues 2-14 of the RAS G12V mutant (for example, shown in SEQ ID NO: 5).

[0019] In certain embodiments, the epitope peptide comprises or consists of the sequence shown in any one of SEQ ID NOs: 2-5. In certain embodiments, the variant comprises or consists of a sequence selected from the group consisting of (i) a sequence having one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions or additions) compared to the sequence shown in any one of SEQ ID NOs: 2-5; and (ii) a sequence having 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% sequence identity when compared to the sequence shown in any one of SEQ ID NOs: 2-5.

[0020] In another aspect, the present invention provides an MHC-peptide complex comprising the epitope peptide of the present invention or a variant thereof and an MHC-II molecule associated therewith. In a particular embodiment, the MHC-II molecule is HLA-DP. In a particular embodiment, HLA-DP is HLA-DPB1 * 03:01 or HLA-DPB1 * It includes 14:01. In some embodiments, HLA-DP is HLA-DPA1 * 02:02, HLA-DPA1 * 02:01, and HLA-DPA1 * It further comprises one selected from the group consisting of 01:03. In certain embodiments, the MHC-II molecule is HLA-DPB1 * 03:01 or HLA-DPB1 * 14:01, and HLA-DPA1 * 02:02. In certain embodiments, the MHC-II molecule is HLA-DPB1 * 03:01 or HLA-DPB1 *14:01, and HLA-DPA1 * 02:01. In certain embodiments, the MHC-II molecule is HLA-DPB1 * 03:01 or HLA-DPB1 * 14:01, and HLA-DPA1 * It is 01:03.

[0021] In certain embodiments, the MHC-peptide complex is present on the surface of the cell. Therefore, the present invention also encompasses cells that express the MHC-peptide complex.

[0022] T cell receptor In a second aspect, the present invention provides an isolated T cell receptor or an antigen-binding fragment thereof that can specifically recognize the epitope peptide or a variant thereof. In certain embodiments, the epitope peptide or a variant thereof is presented by an MHC-II molecule. In certain embodiments, the MHC-II molecule is HLA-DP. In certain embodiments, HLA-DP is HLA-DPB1 * 03:01 or HLA-DPB1 * It includes 14:01. In some embodiments, HLA-DP is HLA-DPA1 * 02:02, HLA-DPA1 * 02:01, and HLA-DPA1 * It further includes one selected from the group consisting of 01:03.

[0023] In a third aspect, the present invention is capable of specifically recognizing the RAS G12V mutant and comprises an α-chain variable region (Vα) and / or a β-chain variable region (Vβ), (a) Vα comprises CDR1α, CDR2α, and CDR3α, wherein CDR3α comprises the sequence shown in AVRDX1X2X3GGNKLT (SEQ ID NO: 25); X1 is selected from the group consisting of G, A, D, L, M, Q, R, S, V, W, Y; preferably from the group consisting of G, A, D, R; X2 is selected from the group consisting of R, A, D, G, H, I, K, L, M, N, P, Q, S, T, V, W, Y; preferably R or T; X3 is selected from the group consisting of G, A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; preferably from the group consisting of G, A, H, N, S, or W; and / or (b) Vβ comprises CDR1β, CDR2β and CDR3β, wherein CDR3β comprises the sequence shown in ASSX4GX5RDNSPLH (Sequence ID 26); X4 is selected from the group consisting of P, A, H, M, S, T, V, Y; preferably P, A, or T; X5 is selected from the group consisting of Q or S. This provides isolated T cell receptors or their antigen-binding fragments.

[0024] The TCR on the second or third side may use either TCR structure.

[0025] In certain embodiments, the TCR may be a full-length TCR comprising a full-length α chain and a full-length β chain.

[0026] In certain embodiments, the TCR is a soluble TCR lacking one or more transmembrane and / or cytoplasmic domains. In certain embodiments, the soluble TCR is prepared by fusing the extracellular domain of the TCR of the present invention with another protein domain (e.g., a maltose-binding protein, thioredoxin, a human constant κ domain, or a leucine zipper), see, for example, Front Oncol., 2014;4:378, which is incorporated herein by reference in its entirety.

[0027] In certain embodiments, the TCR of the present invention may also be a single-chain TCR (scTCR) comprising Vα and Vβ linked by a peptide linker. Such an scTCR may comprise Vα and Vβ, each linked to a TCR constant region. Alternatively, the scTCR may comprise Vα and Vβ, where Vα, Vβ, or both Vα and Vβ are not linked to a TCR constant region. Exemplary scTCRs are described in PCT Publications: WO2003 / 020763, WO2004 / 033685, and WO2011 / 044186, each of which is by reference incorporated in whole as herein.

[0028] In certain embodiments, the TCR of the present invention may comprise two polypeptide chains (e.g., an α-chain and a β-chain) which are manipulated to comprise cysteine ​​residues capable of forming intrachain disulfide bonds. Thus, the TCR of the present invention may comprise two polypeptide chains linked by an manipulated disulfide bond. Exemplary TCRs having an manipulated disulfide bond are described in U.S. Patents 8,361,794 and 8,906,383, which are, by reference, whole parts of this specification.

[0029] In certain embodiments, the T cell receptor on the second or third side is a membrane-bound or soluble T cell receptor. In certain embodiments, the T cell receptor on the second or third side is a full-length TCR, a soluble TCR, or a single-chain TCR.

[0030] In certain embodiments, the TCR or its antigen-binding fragment has one or more of the following characteristics: (i) CDR1α comprises the sequence shown in VSGX6PY (Sequence ID 27); X6 is selected from the group consisting of N, A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y (preferably N, E, P, Q, R, or S); (ii) CDR2α comprises the sequence shown in YX7X8GDNLV (Sequence ID 28); X7 is selected from the group consisting of I, A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, Y (preferably I, D, E, F, G, H, N, P, R, W, or Y); X8 is selected from the group consisting of T, A, D, E, H, I, L, N, Q, R, S, W, Y (preferably T, A, D, H, I, Q, or R); (iii) CDR1β comprises the sequence shown in SEQ ID NO: 11; (iv) CDR2β is SX9X 10 VNX 11 X9 comprises the sequence shown in (Sequence ID 29); X9 is selected from the group consisting of Q, A, F, G, H, I, K, L, M, N, P, R, S, T, V, W, Y (preferably Q, A, I, M, N, S, T, or Y); X 10 is selected from the group consisting of I, F, H, T, V (preferably I or T); X 11 The element is selected from the group consisting of D, A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y (preferably D, G, L, M, N, T, or Y).

[0031] In certain embodiments, the TCR or its antigen-binding fragment is (1) CDR1α shown in SEQ ID NO: 8, CDR2α shown in SEQ ID NO: 9, CDR3α shown in SEQ ID NO: 25; and / or CDR1β shown in SEQ ID NO: 11, CDR2β shown in SEQ ID NO: 12, CDR3β shown in SEQ ID NO: 13; (2) CDR1α shown in SEQ ID NO: 8, CDR2α shown in SEQ ID NO: 9, CDR3α shown in SEQ ID NO: 10; and / or CDR1β shown in SEQ ID NO: 11, CDR2β shown in SEQ ID NO: 12, CDR3β shown in SEQ ID NO: 26; (3) CDR1α shown in SEQ ID NO: 27, CDR2α shown in SEQ ID NO: 9, CDR3α shown in SEQ ID NO: 10; and / or CDR1β shown in SEQ ID NO: 11, CDR2β shown in SEQ ID NO: 12, CDR3β shown in SEQ ID NO: 13; (4) CDR1α shown in SEQ ID NO: 8, CDR2α shown in SEQ ID NO: 28, CDR3α shown in SEQ ID NO: 10; and / or CDR1β shown in SEQ ID NO: 11, CDR2β shown in SEQ ID NO: 12, CDR3β shown in SEQ ID NO: 13; (5) CDR1α shown in SEQ ID NO: 8, CDR2α shown in SEQ ID NO: 9, CDR3α shown in SEQ ID NO: 10; and / or CDR1β shown in SEQ ID NO: 11, CDR2β shown in SEQ ID NO: 29, CDR3β shown in SEQ ID NO: 13; or (6) CDR1α shown in SEQ ID NO: 8, CDR2α shown in SEQ ID NO: 9, CDR3α shown in SEQ ID NO: 10; and / or CDR1β shown in SEQ ID NO: 11, CDR2β shown in SEQ ID NO: 12, CDR3β shown in SEQ ID NO: 13 It consists of including.

[0032] In a particular embodiment, Vα comprises FR1α, FR2α, FR3α, and FR4α. FR1α comprises the sequence shown in sequence number 30; FR2α is LFWYVQYPNRGLQFLLX 12 (Sequence ID 38) comprises the sequence shown; X 12 is selected from the group consisting of K, A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y (preferably K, G, H, N, P, R, S, V, W, or Y); FR3α comprises the sequence shown in Sequence ID No. 32; FR4α comprises the sequence shown in Sequence ID No. 33.

[0033] In a particular embodiment, Vβ comprises FR1β, FR2β, FR3β, and FR4β. FR1β comprises the sequence shown in Sequence ID No. 34; FR2β is MYWYRQDPGQGLRLIYX 13 (Sequence ID 39) includes the sequence shown; X 13is selected from the group consisting of Y, A, E, F, I, K, L, M, N, P, Q, R, S, T, V, W (preferably Y, A, E, F, or K); FR3β is FX 14 It comprises the sequence shown in KGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLC (Sequence ID 40); X 14 is selected from the group consisting of Q, A, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, Y (preferably Q, A, D, E, F, H, L, R, T, or W); FR4β comprises the sequence shown in Sequence ID No. 37.

[0034] In certain embodiments, the TCR or its antigen-binding fragment is (1) FR1α shown in SEQ ID NO: 30, FR2α shown in SEQ ID NO: 31, FR3α shown in SEQ ID NO: 32, FR4α shown in SEQ ID NO: 33; and / or FR1β shown in SEQ ID NO: 34, FR2β shown in SEQ ID NO: 35, FR3β shown in SEQ ID NO: 36, FR4β shown in SEQ ID NO: 37; (2) FR1α shown in SEQ ID NO: 30, FR2α shown in SEQ ID NO: 38, FR3α shown in SEQ ID NO: 32, FR4α shown in SEQ ID NO: 33; and / or FR1β shown in SEQ ID NO: 34, FR2β shown in SEQ ID NO: 35, FR3β shown in SEQ ID NO: 36, FR4β shown in SEQ ID NO: 37; (3) FR1α shown in SEQ ID NO: 30, FR2α shown in SEQ ID NO: 31, FR3α shown in SEQ ID NO: 32, FR4α shown in SEQ ID NO: 33; and / or FR1β shown in SEQ ID NO: 34, FR2β shown in SEQ ID NO: 39, FR3β shown in SEQ ID NO: 36, FR4β shown in SEQ ID NO: 37; or (4) FR1α shown in SEQ ID NO: 30, FR2α shown in SEQ ID NO: 31, FR3α shown in SEQ ID NO: 32, FR4α shown in SEQ ID NO: 33; and / or FR1β shown in SEQ ID NO: 34, FR2β shown in SEQ ID NO: 35, FR3β shown in SEQ ID NO: 40, FR4β shown in SEQ ID NO: 37 It consists of including.

[0035] In certain embodiments, Vα comprises the sequence shown in SEQ ID NO: 6 or a variant thereof, and Vβ comprises the sequence shown in SEQ ID NO: 7 or a variant thereof, the variant having one or more amino acid substitutions, deletions or additions (e.g., one, two, three, four or five amino acid substitutions, deletions or additions) compared to the sequence from which it is derived.

[0036] In certain embodiments, Vα comprises the sequence shown in Sequence ID No. 6 or a variant thereof, the variant having amino acid substitutions at the positions of one or more (e.g., 1, 2, 3, or 4) amino acids selected from the group consisting of 30, 49, 51, 52, 96, 97, and 98, as determined according to the IMGT TCR numbering system.

[0037] In certain embodiments, Vβ comprises the sequence shown in Sequence ID No. 7 or a variant thereof, the variant having one or more (e.g., 1, 2, 3, or 4) amino acid substitutions at amino acid positions selected from the group consisting of 48, 50, 51, 54, 56, 95, and 97 when determined according to the IMGT TCR numbering system.

[0038] In certain embodiments, Vα comprises a variant of the sequence shown in Sequence ID No. 6, wherein the amino acid positions are determined by the IMGT TCR numbering system as follows: substitution of the amino acid at position 30 with A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y (preferably E, P, Q, R or S); substitution of the amino acid at position 49 with A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y (preferably G, H, N, P, R, S, V, W or Y); substitution of the amino acid at position 51 with A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W or Y (preferably D, E, F, G, H, N, P, R, W or Y); substitution of the amino acid at position 52 The sequence comprises one or more (e.g., 1, 2, 3, or 4) amino acid substitutions selected from the following: substitution of A, D, E, H, I, L, N, Q, R, S, V, W, or Y (preferably A, D, H, I, Q, or R); substitution of the amino acid at position 96 with A, D, L, M, Q, R, S, V, W, or Y (preferably A, D, or R); substitution of the amino acid at position 97 with A, D, G, H, I, K, L, M, N, P, Q, S, T, V, W, or Y (preferably T); and substitution of the amino acid at position 98 with A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y (preferably A, H, N, S, or W). In such embodiments, Vβ preferably comprises the sequence shown in SEQ ID NO: 7.

[0039] In certain embodiments, Vβ comprises a variant of the sequence shown in Sequence ID No. 7, wherein the amino acid positions are determined by the IMGT TCR numbering system as follows: substitution of the amino acid at position 48 with A, E, F, I, K, L, M, N, P, Q, R, S, T, V, or W (preferably A, E, F, or K); substitution of the amino acid at position 50 with A, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y (preferably A, I, M, N, S, T, or Y); substitution of the amino acid at position 51 with F, H, T, or V (preferably T); substitution of the amino acid at position 54 with A, E, F, G, H, I, K, L, M, The sequence comprises one or more (e.g., 1, 2, 3, or 4) amino acid substitutions selected from: substitutions of N, P, Q, R, S, T, V, W, or Y (preferably G, L, M, N, T, or Y); substitutions of the amino acid at position 56 with A, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, or Y (preferably A, D, E, F, H, L, R, T, or W); substitutions of the amino acid at position 95 with A, H, M, S, T, V, or Y (preferably A or T); and substitutions of the amino acid at position 97 with S. In such embodiments, Vα preferably comprises the sequence shown in Sequence ID No. 6.

[0040] In certain embodiments, the TCR or its antigen-binding fragment of the present invention comprises Vα, represented by SEQ ID NO: 6, and / or Vβ, represented by SEQ ID NO: 7.

[0041] In certain embodiments, the TCR or its antigen-binding fragment can specifically recognize the epitope peptide or its variant (for example, the sequence shown in any one of SEQ ID NOs: 2-5). In certain embodiments, the epitope peptide or its variant is presented by an MHC-II molecule. In certain embodiments, the MHC-II molecule is HLA-DP. In certain embodiments, HLA-DP is HLA-DPB1 * 03:01 or HLA-DPB1 * In some embodiments, including 14:01, HLA-DP is HLA-DPA1 *02:02, HLA-DPA1 * 02:01, and HLA-DPA1 * It further includes one selected from the group consisting of 01:03.

[0042] In certain embodiments, T cells expressing a TCR or its antigen-binding fragment on their surface are activated in co-culture with a second cell presenting the epitope peptide of the present invention or a variant thereof (e.g., presented with respect to MHC-II). In certain embodiments, T cell activation can be measured using any suitable indicator known in the art. Not limited examples of such suitable indicators include increased secretion levels of cytokines (e.g., IL-2, IFN-γ, etc.), proliferative activity, and / or increased expression levels of activation markers (e.g., CD25, CD69, CD107a, etc.). In certain embodiments, T cell activation also includes apoptosis or cell death induced by T cells in a second cell presenting the epitope peptide of the present invention or a variant thereof (e.g., presented with respect to MHC-II).

[0043] Conjugates and Fusion Proteins In a fourth aspect, the present invention provides a conjugate comprising a TCR or its antigen-binding fragment as described in the second or third aspect and an effector portion conjugated thereto.

[0044] In this context, the term “effector moiety” refers to a component or functional group that can modulate (e.g., enhance or reduce) the intrinsic activity of the molecule to which it is linked, or confer novel activity to the molecule. In some embodiments, the effector moiety is a bioactive compound or polypeptide (e.g., a compound or polypeptide that has an effect on cells targeted by the TCR), or a compound or polypeptide having a detectable label.

[0045] In this context, the term “conjugation” refers, but is not limited to, any method known in the art for functionally linking protein domains, including linker-assisted or linker-assisted recombinant fusion, intein-mediated fusion, non-covalent and covalent bonding, such as disulfide bonds, peptide bonds, hydrogen bonds, electrostatic bonds, and conformational bonding, such as biotin-avidin interactions. In certain embodiments, conjugation with an effector moiety may be carried out by chemical or recombinant methods, the chemical method comprising forming a covalent bond between two molecules to form a single molecule.

[0046] In certain embodiments, the effector portion may be a therapeutic component. A therapeutic component refers to a compound or polypeptide useful as a therapeutic agent. The conjugate utilizes the targeting ability of the TCR, enabling the therapeutic component to exert a therapeutic effect on the cells targeted by the TCR.

[0047] In certain embodiments, the TCR or its antigen-binding fragment of the present invention may be conjugated to an effector portion via a linker (e.g., a peptide linker). In certain embodiments, the effector portion is ligated to the N-terminus or C-terminus of the TCR or its antigen-binding fragment of the present invention.

[0048] In certain embodiments, when the effector portion is a peptide or protein, the conjugate is preferably a fusion protein.

[0049] Therefore, in a fifth aspect, the present invention also provides a fusion protein comprising a TCR or its antigen-binding fragment and an additional peptide or protein, as described in the second or third aspect.

[0050] In certain embodiments, the TCR or its antigen-binding fragment of the present invention is optionally fused to an additional peptide or protein via a peptide linker. In certain embodiments, the additional peptide or protein is ligated to the N-terminus or C-terminus of the TCR or its antigen-binding fragment of the present invention.

[0051] In certain embodiments, the additional peptide or protein can be selected from the effector portion, i.e., the peptide or protein, described in the fourth aspect.

[0052] Preparation of epitope peptides, TCRs, and fusion proteins The epitope peptides, TCRs, or fusion proteins containing the TCR of the present invention can be prepared by various methods known in the art, such as genetic engineering and recombination techniques. For example, the DNA molecule encoding it can be obtained by chemical synthesis or PCR amplification, and the obtained DNA molecule can be inserted into an expression vector and then transfected into host cells, and the transfected host cells can be cultured under specific conditions for expressing the epitope peptides, TCRs, or fusion proteins containing the TCR of the present invention.

[0053] Therefore, in the sixth aspect, the present invention is (i) A nucleotide sequence encoding the epitope peptide of the first side or a variant thereof; (ii) Nucleotide sequences encoding the TCR or its antigen-binding fragment or its α-chain variable region and / or β-chain variable region as described in the second or third aspect; (iii) Nucleotide sequence encoding the fusion protein described in the fifth aspect The present invention provides an isolated nucleic acid molecule comprising [the specified substance].

[0054] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the α-chain variable region of the TCR or its antigen-binding fragment described in the second or third aspect, and a second nucleotide sequence encoding the β-chain. In certain embodiments, the first and second nucleotide sequences are optionally linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, or T2A). In certain embodiments, the self-cleaving peptide is P2A.

[0055] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the TCR α chain variable region and a second nucleotide sequence encoding the TCR β chain variable region.

[0056] In certain embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the α chain of the TCR or its antigen-binding fragment and a second nucleotide sequence encoding the β chain of the TCR or its antigen-binding fragment.

[0057] In certain embodiments, the first and second nucleotide sequences are present in different isolated nucleic acid molecules.

[0058] In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are present in any order on the same isolated nucleic acid molecule; in certain embodiments, the first nucleotide sequence and the second nucleotide sequence are linked in any order by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A).

[0059] In a seventh aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of the sixth aspect. In certain embodiments, the vector of the present invention is, for example, a plasmid, cosmid, phage, etc. In certain embodiments, the vector comprises a TCR or its antigen-binding fragment, or its α-chain variable region and / or β-chain variable region, or a nucleotide sequence encoding its α-chain and / or β-chain.

[0060] In certain embodiments, the vector comprises a first nucleotide sequence encoding the TCR α chain variable region and a second nucleotide sequence encoding the TCR β chain variable region.

[0061] In certain embodiments, the vector comprises a first nucleotide sequence encoding the α chain of the TCR or its antigen-binding fragment and a second nucleotide sequence encoding the β chain of the TCR or its antigen-binding fragment.

[0062] In certain embodiments, the first nucleotide sequence and the second nucleotide sequence are present in different vectors.

[0063] In certain embodiments, the first and second nucleotide sequences are present in the same vector in any order. In certain embodiments, the first and second nucleotide sequences are linked in any order by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A).

[0064] In certain embodiments, the vector comprises a nucleotide sequence encoding the fusion protein described above.

[0065] In certain embodiments, the vector is a viral vector such as a lentiviral vector, retroviral vector, adenovirus vector, adeno-associated virus vector, or baculovirus vector.

[0066] In the eighth aspect, the present invention provides a host cell comprising an isolated nucleic acid molecule of the sixth aspect or a vector of the seventh aspect. Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, as well as eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells such as mouse cells and human cells).

[0067] In another aspect, a method is also provided for producing an epitope peptide, TCR, or fusion protein comprising the TCR of the present invention, the method comprising culturing the host cells described in the eighth aspect under conditions that enable protein expression, and recovering an epitope peptide, TCR, or fusion protein comprising the TCR from the culture of the cultured host cells.

[0068] Manipulated antigen-presenting cells (APCs) The epitope peptides and their variants of the present invention can be used in T cell-based immunotherapy. In some cases, T cells can recognize MHC-peptide complexes presented on the surface of APCs via their TCRs to induce an MHC-restricted immune response against RAS mutants.

[0069] Therefore, in the ninth aspect, the present invention provides engineered antigen-presenting cells (APCs) that present the epitope peptide of the first aspect or a variant thereof on their surface.

[0070] In certain embodiments, the epitope peptide or its variant is presented by an MHC-II molecule. In certain embodiments, the MHC-II molecule is HLA-DP. In certain embodiments, HLA-DP is HLA-DPB1 * 03:01 or HLA-DPB1 * It includes 14:01. In some embodiments, HLA-DP is HLA-DPA1 * 02:02, HLA-DPA1 * 02:01, and HLA-DPA1 *It further includes one selected from the group consisting of 01:03.

[0071] In certain embodiments, the APC is selected from the group consisting of dendritic cells, monocytes, macrophages, B lymphocytes (e.g., B-lymphoblastoid cells B-LCL), or any combination thereof.

[0072] In a particular embodiment, the APC is HLA-DPB1 * 03:01 Positive or HLA-DPB1 * 14:01 Positive. In certain embodiments, APC further includes HLA-DPA1 * 02:02 Positive, HLA-DPA1 * 02:01 Positive, or HLA-DPA1 * 01:03 Positive. In certain embodiments, APC is HLA-DPB1 * 03:01 / HLA-DPA1 * 02:02, HLA-DPB1 * 03:01 / HLA-DPA1 * 02:01, or HLA-DPB1 * 03:01 / HLA-DPA1 * 01:03, HLA-DPB1 * 14:01 / HLA-DPA1 * 02:02, HLA-DPB1 * 14:01 / HLA-DPA1 * 02:01, or HLA-DPB1 * 14:01 / HLA-DPA1 * It has the HLA genotype 01:03.

[0073] In a particular embodiment, the APC is HLA-DPB1 * 03:01 Positive subjects or HLA-DPB1 * 14:01 Isolated from positive subjects. In certain embodiments, subjects are further HLA-DPA1 * 02:02 Positive, HLA-DPA1 * 02:01 Positive, or HLA-DPA1 * 01:03 Positive. In certain embodiments, the subject is HLA-DPB1* 03:01 / HLA-DPA1 * 02:02, HLA-DPB1 * 03:01 / HLA-DPA1 * 02:01, or HLA-DPB1 * 03:01 / HLA-DPA1 * 01:03, HLA-DPB1 * 14:01 / HLA-DPA1 * 02:02, HLA-DPB1 * 14:01 / HLA-DPA1 * 02:01, or HLA-DPB1 * 14:01 / HLA-DPA1 * Has the HLA genotype of 01:03.

[0074] In certain embodiments, the engineered APC can be obtained by contacting the APC in vitro with the epitope peptide or its variant described in the first aspect (i.e., exposing the APC to a sufficient amount of the epitope peptide or its variant). In some embodiments, the engineered APC is obtained by introducing in vitro into the APC an expression vector comprising a nucleotide sequence encoding the epitope peptide or its variant described in the first aspect.

[0075] The APC of the ninth aspect can be autologous / self ("self") or allogeneic ("non-self", e.g., allogeneic) cells. "Self" cells refer to cells derived from the same subject; "allogeneic" cells refer to cells of the same species that are genetically different from the cells being compared.

[0076] The APC of the ninth aspect may be isolated or obtained from any tissue in which such cells are found or, alternatively, can be cultured and provided. For example, the APC can be found in mammalian bone marrow or peripheral blood mononuclear cells (PBMCs), mammalian spleen, or mammalian skin (i.e., Langerhans cells having some characteristics similar to those of DCs found in the skin), and then sorted after culturing in a medium containing appropriate cytokines to obtain the APC.

[0077] In another aspect, the present invention provides a method for producing the above-described manipulated APC, the method comprising (1) preparing an APC from a subject; and (2) contacting the APC in vitro with the epitope peptide or a variant thereof described in the first aspect, or introducing an expression vector comprising a nucleotide sequence encoding the epitope peptide or a variant thereof described in the first aspect into the APC to obtain an APC capable of displaying the epitope peptide or a variant thereof on its surface.

[0078] Manipulated immune cells The TCR or its antigen-binding fragment of the present invention can be used in T cell-based immunotherapy. In some cases, T cells expressing the TCR of the present invention can induce an MHC-restricted immune response against RAS mutants by recognizing an MHC-peptide complex.

[0079] Therefore, in the tenth aspect, the present invention provides engineered immune cells expressing the TCR or its antigen-binding fragment described in the second or third aspect on their surface. The engineered immune cells of the present invention have antigen specificity for the RAS G12V mutant. In a particular embodiment, the engineered immune cells of the present invention have one or more features selected from the following: (i) It specifically binds to the RAS G12V mutant, but does not bind to other RAS proteins (including wild-type RAS protein or other mutants), or has low binding affinity to them; (ii) Specifically binding to the epitope peptide or its variant as described in the first aspect (e.g., the sequence shown in any one of SEQ ID NOs. 2-5, particularly SEQ ID NO. 5); (iii) Activation of APCs exhibiting the epitope peptide or a variant of the present invention (e.g., exhibited with respect to MHC-II) when co-cultured, including, but not limited to, increased secretion levels of cytokines (e.g., IL-2, IFN-γ, etc.), increased proliferative activity, and / or increased expression levels of activation markers (e.g., CD25, CD69, CD107a, etc.), and increased killing activity against a second cell exhibiting the epitope peptide or a variant of the present invention (e.g., exhibited with respect to MHC-II).

[0080] In certain embodiments, the manipulated immune cells comprise a nucleotide sequence encoding a TCR or its antigen-binding fragment, as described in the second or third aspect.

[0081] The immune cells of the tenth aspect may be isolated or obtained from any tissue in which such cells are found. For example, APCs may be found in mammalian peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, ascites, pleural fluid, splenic tissue, or tumors, and then, optionally, cultured in a culture medium containing appropriate cytokines, and sorted to obtain the desired immune cells. Alternatively, immune cells may also be cultured and provided in other ways, for example, obtained through induction and differentiation from immune cell progenitor cells (e.g., T lymphocyte progenitor), for example, isolated and / or enriched from bone marrow, umbilical cord blood, or peripheral blood, for example, pluripotent stem cells (e.g., embryonic stem cells, induced pluripotent stem cells), hematopoietic stem cells, or lymphocyte progenitor cells.

[0082] In certain embodiments, the immune cells are lymphocytes. In certain embodiments, the immune cells are selected from the group consisting of T cells, tumor infiltrating lymphocytes (TILs), natural killer (NK) cells, natural killer T (NKT) cells, or any combination thereof. Exemplary immune cells that can be used to express the TCRs of the present invention include PBMCs, TILs, and / or T cells. In certain embodiments, the T cells are selected from the group consisting of αβ T cells, γδ T cells, iPSC-derived T cells, CD8+ cytotoxic T cells, CD4+ cytotoxic T cells, CD4+ helper T cells (e.g., Th1 or Th2 cells), CD4 / CD8 double positive T cells, tumor infiltrating T cells, thymocytes, memory T cells, natural killer T cells, e.g., non-mutant natural killer T cells. In certain embodiments, the immune cells comprise CD4+ T cells. One of ordinary skill in the art will understand that the immune cells can also include progenitor cells (precursor cells) of the immune cells, and that the progenitor cells can be induced to differentiate into immune cells in vivo or in vitro. Thus, in certain embodiments, the immune cells comprise progenitor cells of immune cells, such as hematopoietic stem cells (HSCs) contained within a CD34+ cell population derived from umbilical cord blood, bone marrow, or mobilized peripheral blood, which can be differentiated into mature immune cells when administered to a subject or induced to differentiate into mature immune cells in vitro.

[0083] In a tenth aspect, the immune cells can be autologous / self ( "self") or allogeneic ( "non-self", e.g., allogeneic) cells. "Self" cells refer to cells from the same subject; "allogeneic" cells refer to cells of the same species that are genetically different from the cells being compared.

[0084] In certain embodiments, the immune cells are isolated from HLA-DPB1 * 03:01 positive subjects or HLA-DPB1 * 14:01 positive subjects. In certain embodiments, the subject is further HLA-DPA1 * 02:02 positive, HLA-DPA1 * 02:01 positive, or HLA-DPA1 *01:03 Positive. In certain embodiments, the subject is HLA-DPB1 * 03:01 / HLA-DPA1 * 02:02, HLA-DPB1 * 03:01 / HLA-DPA1 * 02:01, or HLA-DPB1 * 03:01 / HLA-DPA1 * 01:03, HLA-DPB1 * 14:01 / HLA-DPA1 * 02:02, HLA-DPB1 * 14:01 / HLA-DPA1 * 02:01, or HLA-DPB1 * 14:01 / HLA-DPA1 * It has the HLA genotype 01:03.

[0085] It is understood that the manipulated immune cells of the present invention may be contained within an isolated cell population. The cell population may be heterogeneous, for example, comprising, in addition to the manipulated immune cells of the present invention, at least one other type of cell that is not antigen-specific to the RAS G12V mutant; or, for example, the cell population may comprise two or more types of immune cells, all of which express the TCR of the present invention to be antigen-specific to the RAS G12V mutant. Furthermore, the cell population may also be substantially homogeneous, for example, comprising primarily T cells that are antigen-specific to the RAS G12V mutant (e.g., essentially comprising).

[0086] In another aspect, the present invention provides a method for producing the above-described engineered immune cells, the method comprising (1) preparing immune cells from a subject; and (2) introducing a nucleic acid molecule or vector comprising a nucleotide sequence encoding the TCR or its antigen-binding fragment into the immune cells described in step (1) to obtain immune cells expressing the TCR or its antigen-binding fragment.

[0087] In some embodiments, in step (1), immune cells undergo pretreatment; this pretreatment comprises sorting, activating, and / or proliferating the immune cells. In certain embodiments, the pretreatment comprises contacting the immune cells with one or more selected from the group consisting of anti-CD3 antibody, anti-CD28 antibody, IL-2, and IL-15, thereby stimulating the immune cells and inducing their proliferation, and generating pretreated immune cells.

[0088] In some embodiments, in step (2), the nucleic acid molecule or vector can be introduced into immune cells by physical, chemical or biological means for introduction into host cells, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, electroporation, TALEN method, ZFN method, non-viral vector-mediated transfection (e.g., liposome) or viral vector-mediated transfection (e.g., lentivirus infection, retrovirus infection, adenovirus infection), as well as transposon technology, CRISPR-Cas9 and other technologies.

[0089] In some embodiments, after step (2), the method further includes expanding the culture of the immune cells obtained in step (2).

[0090] Therapies based on epitope peptides The epitope peptide of the present invention or the APC presenting the epitope peptide can be used to induce an antitumor immune response in T cell-based immunotherapy.

[0091] Therefore, in the eleventh aspect, the present invention provides a pharmaceutical composition comprising an epitope peptide or a variant thereof described in the first aspect, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the epitope peptide or a variant thereof, or an engineered antigen-presenting cell (APC) described in the ninth aspect; and a pharmaceutically acceptable carrier and / or excipient.

[0092] In certain embodiments, the pharmaceutical composition is a tumor vaccine.

[0093] In certain embodiments, the pharmaceutical composition comprises an adjuvant. The adjuvant is a substance that can nonspecifically enhance the immune response, and examples include Freund's complete adjuvant, Freund's incomplete adjuvant, Toll receptor ligands, immunostimulatory antibodies (e.g., anti-CD3 antibody, anti-CD28 antibody, anti-CD40L (CD154) antibody, anti-41BB (CD137) antibody, anti-OX40 antibody, anti-GITR antibody, or any combination thereof), or immunostimulatory cytokines (e.g., IL-2, IL-3, IL-12, IL-15, IL-18, IFN-γ, IL-10, TGF-β, GM-CSF, or any combination thereof).

[0094] In certain embodiments, the pharmaceutical composition further comprises an additional therapeutic agent, such as an antitumor agent or an immunostimulant.

[0095] In certain embodiments, the antitumor agent is selected from the group consisting of alkylating agents, mitotic inhibitors, anti-cancer antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, radiosensitizers (e.g., gemcitabine, 5-fluorouracil, taxanes, cisplatin, etc.), anti-angiogenic agents, cytokines (e.g., GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-21, etc.), antibodies that specifically target tumor cells (e.g., CD20 antibodies such as rituximab, Her2 antibodies such as trastuzumab, VEGF antibodies such as bevacizumab, EGFR antibodies such as cetuximab, etc.), and immune checkpoint inhibitors (e.g., PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG-3 antibodies, or TIM3 antibodies).

[0096] In certain embodiments, the immunoenhancing agent is selected from the group consisting of immunostimulatory antibodies (e.g., anti-CD3 antibody, anti-CD28 antibody, anti-CD40L (CD154) antibody, anti-41BB (CD137) antibody, anti-OX40 antibody, anti-GITR antibody, or any combination thereof) or immunostimulatory cytokines (e.g., IL-2, IL-3, IL-12, IL-15, IL-18, IFN-γ, IL-10, TGF-β, GM-CSF, or any combination thereof).

[0097] In certain embodiments, the epitope peptide or its variants, the engineered APC, and the additional therapeutic agent of the present invention may be provided as separate components or as a mixture in a pharmaceutical composition.

[0098] In a twelfth aspect, the present invention provides a method for inducing an immune response against a tumor having a RAS G12V mutation in a subject, and / or for preventing or treating a tumor having a RAS G12V mutation in a subject, the method comprising administering to a subject in need an effective amount of the epitope peptide or a variant thereof described in the first aspect, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the epitope peptide or a variant thereof, or an engineered antigen-presenting cell (APC) described in the ninth aspect, or a pharmaceutical composition described in the eleventh aspect.

[0099] In certain embodiments, tumors having the RAS G12V mutation are selected from the group consisting of colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, endometrial cancer, ovarian cancer, multiple myeloma, melanoma, thyroid cancer, bladder cancer, prostate cancer, breast cancer, head and neck cancer, or acute myeloid leukemia.

[0100] In certain embodiments, the subject is human. In certain embodiments, the subject is HLA-DPB1 * 03:01 Positive. In certain embodiments, the subject is further HLA-DPA1 * 02:02 Positive, HLA-DPA1 * 02:01 Positive, or HLA-DPA1 *01:03 positive, or HLA-DPB1 * 14:01 Positive. In certain embodiments, the subject is HLA-DPB1 * 03:01 / HLA-DPA1 * 02:02, HLA-DPB1 * 03:01 / HLA-DPA1 * 02:01, or HLA-DPB1 * 03:01 / HLA-DPA1 * 01:03, HLA-DPB1 * 14:01 / HLA-DPA1 * 02:02, HLA-DPB1 * 14:01 / HLA-DPA1 * 02:01, or HLA-DPB1 * 14:01 / HLA-DPA1 * It has the HLA genotype 01:03.

[0101] In certain embodiments, the epitope peptide or a variant thereof described in the first aspect, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the epitope peptide or a variant thereof, or the engineered antigen-presenting cell (APC) described in the ninth aspect, or the pharmaceutical composition described in the eleventh aspect, can be administered in combination with an additional therapeutic agent (e.g., an immunostimulant or an antitumor agent). Thus, in certain embodiments, the method further comprises administering the additional therapeutic agent (e.g., an immunostimulant or an antitumor agent) to the subject, for example, simultaneously, separately, or sequentially.

[0102] In certain embodiments, the epitope peptide or a variant thereof described in Aspect 1, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the epitope peptide or a variant thereof, or the engineered antigen-presenting cell (APC) described in Aspect 9, or the pharmaceutical composition described in Aspect 11, may be administered in combination with an additional therapy, for example, simultaneously, separately, or sequentially. The additional therapy may be any therapy known to be used for tumors, such as surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative therapy.

[0103] The epitope peptide or its variant, nucleic acid molecules or vectors comprising a nucleotide sequence encoding the epitope peptide or its variant, host cells, engineered antigen-presenting cells (APCs), or pharmaceutical compositions containing them may be formulated in any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection, and concentrated solutions for injection), inhalants, and sprays. The preferred dosage form depends on the intended mode of administration and therapeutic use. The agents of the present invention must be sterile and stable under manufacturing and storage conditions. The preferred dosage form is an injection. Such an injection may be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: formulating the epitope peptide or its variants, engineered antigen-presenting cells (APCs), or a pharmaceutical composition containing them in the required dose in a suitable solvent, optionally incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. Furthermore, the sterile injectable solution may also be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze-drying) for easier storage and use. Such sterile freeze-dried powders can be dispersed before use in suitable vehicles such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate-buffered saline), lingual solution, and any combination thereof.

[0104] Therefore, in certain exemplary embodiments, the pharmaceutical composition of the eleventh aspect comprises a sterile injectable solution (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such a sterile injectable solution is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate-buffered saline), Ringer's solution, and any combination thereof.

[0105] The epitope peptides or their variants of the present invention, nucleic acid molecules or vectors or host cells comprising nucleotide sequences encoding the epitope peptides or their variants, engineered antigen-presenting cells (APCs), or pharmaceutical compositions comprising them may be administered by any preferred method known in the art, including, but not limited to, oral, sublingual, intraocular, topical, parenteral, rectal, subarachnoid, cisterna magna, inguinal, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or mode of administration will vary depending on the intended purpose. In certain embodiments, the epitope peptide or a variant thereof of the present invention, a nucleic acid molecule or vector comprising a nucleotide sequence encoding the epitope peptide or a variant thereof, a host cell, an engineered antigen-presenting cell (APC), or a pharmaceutical composition containing the same, is administered by intravenous injection or bolus injection.

[0106] The pharmaceutical compositions described in the eleventh aspect may comprise a "therapeutably effective amount" or a "preventively effective amount" of the epitope peptide of the present invention or a variant thereof, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the epitope peptide or variant thereof, or an engineered antigen-presenting cell (APC). In this context, "therapeutably effective amount" means an amount that can generate an immune response in the target to be treated, which can reduce or inhibit the proliferation of tumor cells and / or eliminate them. "Preventively effective amount" means an amount that can generate an immune response against target cells (e.g., tumor cells with RAS mutations) in the target to be treated, which can prevent tumor formation in the target or substantially reduce the chances of tumor development or continued tumor development in the target.

[0107] In another aspect, the present invention provides the use in the manufacture of a drug comprising an epitope peptide or a variant thereof described in the first aspect, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the epitope peptide or a variant thereof, or an engineered antigen-presenting cell (APC) described in the ninth aspect, or a pharmaceutical composition described in the eleventh aspect, the drug being used to induce an immune response against a tumor having the RAS G12V mutation in a subject, and / or to prevent or treat a tumor having the RAS G12V mutation in a subject.

[0108] Therapy based on tCR The TCR or immune cells expressing the TCR of the present invention can be used in T cell-based immunotherapy to kill tumors having the RAS G12V mutation.

[0109] Accordingly, in the thirteenth aspect, the present invention provides a pharmaceutical composition comprising a TCR or its antigen-binding fragment as described in the second or third aspect, a conjugate as described in the fourth aspect, or a fusion protein as described in the fifth aspect, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding a TCR or its antigen-binding fragment or conjugate or fusion protein, or an engineered immune cell as described in the tenth aspect; and a pharmaceutically acceptable carrier and / or excipient.

[0110] In certain embodiments, the pharmaceutical composition further comprises an additional therapeutic agent, such as an antitumor agent or an immunostimulant.

[0111] In certain embodiments, the antitumor agent is selected from the group consisting of alkylating agents, mitotic inhibitors, anti-cancer antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, radiosensitizers (e.g., gemcitabine, 5-fluorouracil, taxanes, cisplatin, etc.), anti-angiogenic agents, cytokines (e.g., GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-21, etc.), antibodies that specifically target tumor cells (e.g., CD20 antibodies such as rituximab, Her2 antibodies such as trastuzumab, VEGF antibodies such as bevacizumab, EGFR antibodies such as cetuximab, etc.), and immune checkpoint inhibitors (e.g., PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG-3 antibodies, or TIM3 antibodies).

[0112] In certain embodiments, the immunostimulator is selected from the group consisting of immunostimulatory antibodies (e.g., anti-CD3 antibody, anti-CD28 antibody, anti-CD40L (CD154) antibody, anti-41BB (CD137) antibody, anti-OX40 antibody, anti-GITR antibody, or any combination thereof) or immunostimulatory cytokines (e.g., IL-2, IL-3, IL-12, IL-15, IL-18, IFN-γ, IL-10, TGF-β, GM-CSF, or any combination thereof).

[0113] In certain embodiments, the TCR or its antigen-binding fragment, conjugate, fusion protein or engineered immune cells and additional therapeutic agents of the present invention may be used as separate components or as mixed components in a pharmaceutical composition.

[0114] In a fourteenth aspect, the present invention provides a method for inducing an immune response against a tumor having a RAS G12V mutation in a subject, and / or for preventing or treating a tumor having a RAS G12V mutation in a subject, the method comprising administering to a subject in need an effective amount of a TCR or its antigen-binding fragment of a second or third aspect, a conjugate of a fourth aspect, or a fusion protein of a fifth aspect, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or its antigen-binding fragment or conjugate or fusion protein, or engineered immune cells of a tenth aspect, or a pharmaceutical composition of an eleventh aspect.

[0115] In certain embodiments, tumors having the RAS G12V mutation are selected from the group consisting of colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, endometrial cancer, ovarian cancer, multiple myeloma, melanoma, thyroid cancer, bladder cancer, prostate cancer, breast cancer, head and neck cancer, or acute myeloid leukemia.

[0116] In certain embodiments, the subject is human. In certain embodiments, the subject is HLA-DPB1 * 0301 positive or HLA-DPB1 * 14:01 Positive subject. In certain embodiments, the subject is further HLA-DPA1 * 02:02 Positive, HLA-DPA1 * 02:01 Positive, or HLA-DPA1 * 01:03 Positive. In certain embodiments, the subject is HLA-DPB1 * 03:01 / HLA-DPA1 * 02:02, HLA-DPB1 * 03:01 / HLA-DPA1 * 02:01, or HLA-DPB1* 03:01 / HLA-DPA1 * 01:03, HLA-DPB1 * 14:01 / HLA-DPA1 * 02:02, HLA-DPB1 * 14:01 / HLA-DPA1 * 02:01, or HLA-DPB1 * 14:01 / HLA-DPA1 * It has the HLA genotype 01:03.

[0117] In certain embodiments, the method comprises (1) preparing immune cells required by a subject; (2) introducing a nucleotide sequence encoding the TCR or its antigen-binding fragment as described in the second or third aspect into the immune cells described in step (1) to obtain immune cells expressing the TCR or its antigen-binding fragment on their surface; and (3) administering the immune cells of step (2) to the subject.

[0118] In certain embodiments, prior to step (1), the method further comprises a step of obtaining immune cells from a subject. Immune cells may be isolated or obtained from any tissue in which such cells are found (e.g., peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, ascites, pleural fluid, spleen tissue, and tumors), or otherwise may be cultured and provided, and can be obtained, for example, by induction and differentiation from immune cell progenitor cells (e.g., T lymphocyte precursors).

[0119] In certain embodiments, immune cells are selected from lymphocytes. In certain embodiments, immune cells are selected from the group consisting of T cells (e.g., αβ T cells, γδ T cells, or iPSC-derived T cells), tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, natural killer T (NKT) cells, or any combination thereof. In certain embodiments, immune cells include CD4+ T cells.

[0120] In certain exemplary embodiments, peripheral blood mononuclear cells (PBMCs) and / or TILs are obtained from a subject and directly genetically modified to express the TCR.

[0121] In certain exemplary embodiments, T cells are obtained from a subject and directly genetically modified to express the TCR. T cells can be obtained from various sources; for example, T cells can be obtained from blood units collected from a subject using various techniques known to those skilled in the art (e.g., sedimentation, e.g., Ficoll® isolation). In one embodiment, cells from individual circulating blood are obtained by apheresis. The product of apheresis is typically lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction, and these cells are placed in a suitable buffer or medium for subsequent processing. As will be understood to those skilled in the art, the washing step can be achieved by methods known to those skilled in the art, such as using semi-automatic flow-through centrifugation. After washing, the cells can be resuspended in various biocompatible buffers or other physiological salines, with or without the buffer. In certain embodiments, unwanted components of the apheresis sample can be removed from the medium in which the cells are directly resuspended. In certain embodiments, T cells are isolated from peripheral blood mononuclear cells (PBMCs) by lysing erythrocytes and depleting monocytes (e.g., by Percoll® gradient centrifugation). A specific T cell subpopulation expressing one or more of the following markers, CD3, CD28, CD4, CD8, CD45RA, and CD45RO, can be further isolated by positive or negative selection techniques. In one embodiment, a specific T cell subpopulation expressing CD3, CD28, CD4, CD8, CD45RA, and CD45RO is further isolated by positive or negative selection techniques. Enrichment of the T cell population by negative selection can be achieved, for example, using a combination of antibodies against surface markers specific to the cells that have been negatively selected. Exemplary methods include cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, which utilize a mixture of monoclonal antibodies against cell surface markers present on the negatively selected cells.For example, to enrich CD4+ cells by negative selection, a mixture of monoclonal antibodies generally includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate the target cell population used in this invention.

[0122] In certain embodiments, immune cells (e.g., T cells) can be activated and cultured in vitro (or differentiated, in the case of progenitor cells) before genetic modification of the immune cells.

[0123] In some embodiments, the TCR or its antigen-binding fragment of the second or third aspect, the conjugate of the fourth aspect, or the fusion protein of the fifth aspect, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or its antigen-binding fragment or conjugate or fusion protein, or manipulated immune cells of the tenth aspect, or the pharmaceutical composition of the thirteenth aspect can be administered in combination with an additional therapeutic agent (e.g., an immunostimulant or an antitumor agent). Thus, in certain embodiments, the method further comprises administering the additional therapeutic agent (e.g., an immunostimulant or an antitumor agent) to the subject, for example, simultaneously, separately, or sequentially.

[0124] In some embodiments, the TCR or its antigen-binding fragment of the second or third aspect, the conjugate of the fourth aspect, or the fusion protein of the fifth aspect, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or its antigen-binding fragment or conjugate or fusion protein, or manipulated immune cells of the tenth aspect, or the pharmaceutical composition of the thirteenth aspect may be administered in combination with an additional therapy, for example, simultaneously, separately, or sequentially. The additional therapy may be any therapy known to be used for tumors, such as surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative therapy.

[0125] The present invention relates to TCRs or their antigen-binding fragments, conjugates, fusion proteins, nucleic acid molecules or vectors comprising nucleotide sequences encoding the TCRs or their antigen-binding fragments, conjugates, or fusion proteins, or to host cells, engineered immune cells, or pharmaceutical compositions comprising these, which may be prepared in any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The agents of the present invention must be sterile and stable under manufacturing and storage conditions. The preferred dosage form is an injection. Such an injection may be a sterile injectable solution. For example, a sterile injectable solution can be prepared by the following method: formulating the TCR or its antigen-binding fragment, conjugate, fusion protein, engineered immune cells, or pharmaceutical composition comprising these in a suitable solvent in the required dose, optionally simultaneously formulating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. Furthermore, the sterile injectable solution may also be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze-drying) for easier storage and use. Such sterile freeze-dried powders can be dispersed before use in suitable vehicles such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate-buffered saline), lingual solution, and any combination thereof.

[0126] Therefore, in certain exemplary embodiments, the pharmaceutical composition of the thirteenth aspect comprises a sterile injectable solution (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such a sterile injectable solution is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate-buffered saline), Ringer's solution, and any combination thereof.

[0127] The TCR or its antigen-binding fragment, conjugate, or fusion protein, nucleic acid molecules or vectors or host cells, engineered immune cells, or pharmaceutical compositions comprising the TCR or its antigen-binding fragment or conjugate or fusion protein may be administered by any preferred method known in the art, including, but not limited to, oral, sublingual, intraocular, topical, parenteral, rectal, subarachnoid, cisterna magna, inguinal, bladder, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route and / or mode of administration will vary depending on the intended purpose. In certain embodiments, the TCR or its antigen-binding fragment, conjugate, or fusion protein of the present invention, a nucleic acid molecule or vector or host cell, engineered immune cells, or a pharmaceutical composition comprising the TCR or its antigen-binding fragment or conjugate or fusion protein comprising a nucleotide sequence encoding the TCR or its antigen-binding fragment or conjugate or fusion protein, is administered by intravenous injection or bolus injection.

[0128] The pharmaceutical compositions described in the 13th aspect may comprise a "therapeutically effective amount" or a "preventively effective amount" of the TCR or its antigen-binding fragment, conjugate, or fusion protein of the present invention, a nucleic acid molecule or vector or host cell, engineered immune cells, or a pharmaceutical composition comprising a nucleotide sequence encoding the TCR or its antigen-binding fragment, conjugate, or fusion protein. In this context, "therapeutably effective amount" means an amount that can generate an immune response in the target to be treated, which can reduce or inhibit the proliferation of tumor cells and / or eliminate them. "Preventively effective amount" means an amount that can generate an immune response against target cells (e.g., tumor cells with RAS mutations) in the target to be treated, which can prevent tumor formation in the target or substantially reduce the chances of tumor development or continued tumor development in the target.

[0129] In another aspect, the present invention provides the use of a TCR or its antigen-binding fragment according to the second or third aspect, a conjugate according to the fourth aspect, a fusion protein according to the fifth aspect, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or its antigen-binding fragment or conjugate or fusion protein, or an engineered immune cell according to the tenth aspect, or a pharmaceutical composition according to the thirteenth aspect, in the manufacture of a drug for inducing an immune response against a tumor having a RAS G12V mutation in a subject, and / or for preventing or treating a tumor having a RAS G12V mutation in a subject.

[0130] Definition of Terms In this invention, unless otherwise specified, the scientific and technical terms used herein have meanings that are generally understood by those skilled in the art. Furthermore, the immunological, molecular biology, biochemistry, nucleic acid chemistry, and cell culture procedures used herein are all common procedures widely used in their respective fields. On the other hand, to better understand this invention, the definitions and explanations of relevant terms are given below.

[0131] As used herein, the term "RAS" refers to a proto-oncogene, the RAS protein it encodes, which possesses GTPase activity in many signaling pathways, including MAPK, PI3K, and STAT signaling pathways, regulating cell proliferation, differentiation, and apoptosis. Human genes contain three RAS genes: HRAS (gene ID: 3265), NRAS (gene ID: 4893), and KRAS (gene ID: 3845), which share a high degree of sequence homology (>90%). RAS gene mutations are a driving factor in cancer, and are the most frequent proto-oncogene mutations. The sequences of the RAS proteins encoded by these RAS genes are well known to those skilled in the art and can be found in various public databases. For example, the sequence of the KRAS protein can be found at NCBI:NP_001356715.1, the sequence of the NRAS protein can be found at NCBI:NP_002515.1, and the sequence of the HRAS protein can be found at NCBI:NP_001123914.1.

[0132] As used herein, the term “RAS G12V mutant” refers to a RAS mutant in which the amino acid residue Gly at position 12 is mutated to Val. In some embodiments, the RAS G12V mutant refers to the KRAS G12V mutant. Where the amino acid sequence of the RAS G12V mutant is referred to, it is described with reference to the sequence shown in SEQ ID NO: 1. For example, “amino acid residues at positions 3–13 of the RAS G12V mutant protein” refers to the amino acid residues at positions 3–13 of the sequence shown in SEQ ID NO: 1 or the corresponding fragment of another RAS G12V mutant amino acid sequence. “Corresponding fragment” refers to the fragment that is at the same position in the sequence being compared when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage of identity.

[0133] As used herein, the terms “Major Histocompatibility Complex” and “MHC” are used interchangeably and refer to a closely related set of genes, primarily MHC-I and MHC-II molecules, that determine whether transplanted tissues are identical and are closely related to the immune response. “MHC-I molecule” refers to a dimer of MHC type Iα and β2 microglobulin chains, and “MHC-II molecule” refers to a dimer of MHC type IIα and MHC type IIβ chains. Human MHC is called the human leukocyte antigen (HLA) complex.

[0134] As used herein, the term “MHC-peptide complex” refers to an MHC molecule (MHC type I or MHC type II) containing a peptide bound within an MHC peptide-binding pocket, as is well known in the art. In some cases, the MHC molecule may be a membrane-bound protein expressed on the cell surface. In other cases, the MHC molecule may be a soluble protein lacking a transmembrane or cytoplasmic domain.

[0135] As used herein, the term “epitope” with respect to the TCR refers to the localization region of an antigen (e.g., a peptide or peptide-MHC complex) to which the TCR can bind. In certain embodiments, the TCR-binding epitope can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography, ELISA assay, hydrogen / deuterium exchange mass spectrometry (e.g., liquid chromatography-electrospray mass spectrometry), flow cytometry, mutagenesis mapping (e.g., site-directed mutagenesis mapping), and / or structural modeling. In some exemplary embodiments, the antigen epitope is determined using alanine scanning mutagenesis studies. In certain embodiments, the antigen is a peptide presented by a peptide-MHC complex or an MHC molecule.

[0136] As used herein, the terms “T cell receptor” and “TCR” are interchangeable and refer to molecules comprising a CDR or variable region derived from an αβ or γδ T cell receptor. Examples of TCRs include, but are not limited to, full-length TCRs, antigen-binding fragments of TCRs, soluble TCRs lacking transmembrane and cytoplasmic regions, single-chain TCRs containing a TCR variable region attached by a flexible linker, and TCR chains linked by manipulated disulfide bonds.

[0137] As used herein, the term “full-length TCR” refers to a TCR comprising a dimer of a first polypeptide chain and a second polypeptide chain, each of which comprises a TCR variable region and a TCR constant region comprising a TCR transmembrane region and a TCR cytoplasmic region. In certain embodiments, the full-length TCR comprises a mature full-length TCR α chain and a mature full-length TCR β chain. In certain embodiments, the full-length TCR comprises a mature full-length TCR γ chain and a mature full-length TCR δ chain.

[0138] As used herein, the term “TCR variable region” refers to a portion of a mature TCR polypeptide chain (e.g., TCR α or β chain) that is not encoded by the TRAC gene of the TCR α chain, the TRBC1 or TRBC2 gene of the TCR β chain, the TRDC gene of the TCR δ chain, or the TRGC1 or TRGC2 gene of the TCR γ chain. In certain embodiments, the TCR variable region of the TCR α chain encompasses all amino acids of the mature TCR α chain polypeptide encoded by the TRAV gene and / or TRAJ gene, and the TCR variable region of the TCR β chain encompasses all amino acids of the mature TCR β chain polypeptide encoded by the TRBV gene, TRBD gene, and / or TRBJ gene (see, for example, “T cell receptor Facts book”, (2001), LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, the entire contents of which are hereafter referred to herein). The TCR variable region generally comprises framework regions (FR) 1, 2, 3, and 4 and complementarity-determining regions (CDR) 1, 2, and 3. Here, the terms "α-chain variable region" and "Vα" are used interchangeably to refer to the variable region of the TCR α chain. The terms "β-chain variable region" and "Vβ" are used interchangeably to refer to the variable region of the TCR β chain.

[0139] As used herein, the term “CDR” or “complementarity-determining region” with respect to the TCR refers to discontinuous antigen-binding sites found within the variable region of the TCR chain (e.g., the α-chain or β-chain). These regions are described in Lefranc, (1999) “The Immunologist” 7:132-136; Lefranc et al., (1999) “Nucleic Acids Res” 27:209-212; LeFranc (2001) “T cell receptor facts book”, Academic Press, ISBN 0-12-441352-8; Lefranc et al., (2003) Dev Comp Immunol 27(1):55-77; and Kabat et al., (1991) Sequences of proteins of immunological interest, each of which is incorporated herein by reference in its entirety. In certain embodiments, the CDR is defined according to the IMGT numbering system described above in Lefranc (1999). In certain embodiments, the CDR is defined according to the Kabat numbering system described above in Kabat.

[0140] As used herein, the term "FR" or "framework region" with respect to TCRs refers to amino acid residues in the variable region of a TCR chain other than the CDR as defined above (e.g., the α-chain or β-chain).

[0141] As used herein, the term “constant region” with respect to the TCR refers to a portion of the TCR encoded by the TRAC gene (for the TCR α chain), the TRBC1 or TRBC2 gene (for the TCR β chain), the TRDC gene (for the TCR δ chain), or the TRGC1 or TRGC2 gene (for the TCR γ chain), which may, in some cases, lack all or part of the transmembrane region and / or all or part of the cytoplasmic region. In certain embodiments, the TCR constant region lacks both the transmembrane and cytoplasmic regions. The TCR constant region does not contain the amino acids encoded by the TRAV, TRAJ, TRBV, TRBD, TRBJ, TRDV, TRDD, TRDJ, TRGV, or TRGJ genes (for example, the T cell receptor facts book, (2001), LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, which is entirely included herein by reference).

[0142] Regarding TCRs, the term "extracellular" refers to one or more portions of the TCR chain located outside the cell, "transmembrane region" refers to one or more portions of the TCR chain embedded in the cell membrane, and "cytoplasmic region" refers to one or more portions of the TCR chain located in the cytoplasm of the cell.

[0143] As used herein, the term “antigen-binding moiety” with respect to a TCR refers to any portion or fragment of a TCR that possesses the biological activity of the TCR (parent TCR). Biological activity may include the ability to specifically bind to the same antigen (e.g., RAS G12V mutant) or MHC-antigen complex to which the parent TCR binds.

[0144] As used herein, the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets. The strength or affinity of a specific binding interaction is determined by the equilibrium dissociation constant (K) for that interaction. D ) can be expressed in relation to. In the present invention, the term "K DThe "equilibrium dissociation constant" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction and is used to represent the binding affinity between the antibody and the antigen. A smaller equilibrium dissociation constant results in stronger antibody-antigen binding and a stronger affinity between the antibody and the antigen. The specific binding properties between two molecules can be determined using methods well known in the art. One method involves measuring the formation rate and dissociation rate of the antigen-binding site / antigen complex. Both the "association rate constant" (ka or kon) and the "dissociation rate constant" (kdis or koff) can be calculated from the concentration and the actual association and dissociation rates (see Malmqvist M, Nature, 1993, 361:186-187). The kdis / kon ratio is the dissociation constant K D This corresponds to (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). K D The kon and kdis values ​​may be measured by any effective method, for example, by surface plasmon resonance (SPR) with Biacore, or by bioluminescence interference spectroscopy or Kinexa.

[0145] With respect to TCRs, the terms “specific binding” or “specific recognition” refer to the ability of a TCR to preferentially bind to a specific antigen (e.g., a specific peptide or a specific peptide-MHC complex). Generally, a TCR that specifically binds to one antigen will not bind to other antigens, or will bind to them with lower affinity. For example, an antigen-specific TCR will bind to a target antigen with an association constant (Ka) at least 2, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000 times higher than other antigens to which the TCR does not specifically bind. In certain embodiments, the TCR or its antigen-binding fragment disclosed herein specifically binds to the RAS G12V mutant. In certain embodiments, the TCR or its antigen-binding fragment disclosed herein specifically binds to the epitope peptide of the first aspect or its variant. In certain embodiments, the TCR or its antigen-binding fragment disclosed herein specifically binds to the sequence shown in any one of SEQ ID NOs. 2-5 (particularly SEQ ID NO. 5).

[0146] As used herein, the term “antigen-presenting cell” or “APC” refers to any cell capable of presenting peptide fragments of proteins associated with major histocompatibility complex (MHC) molecules on its cell surface. Such cells are well known to those skilled in the art and include, but are not limited to, dendritic cells, monocytes, macrophages, and B lymphocytes (e.g., B-lymphoblastoid cells, B-LCL).

[0147] As used herein, the term “immune cell” refers to any cell of the immune system that has one or more effector functions. Immune cells generally include cells that play a role in the immune response, and they are usually of hematopoietic origin. The term “effector function” refers to a specialized function of an immune cell, such as a function or response that enhances or promotes an immune attack against a target cell (e.g., killing the target cell or inhibiting its growth or proliferation). For example, the effector function of a T cell may be, for example, cytolytic activity or activity that assists or includes the secretion of cytokines. Examples of immune cells include T cells (e.g., α / β T cells and γ / δ T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloid macrophages.

[0148] The immune cells of the present invention may be self / autologous ("self") or non-self ("non-self," e.g., allogeneic, syngeneic, or heteroallelic) cells. As used herein, "self" cells refer to cells originating from the same subject; "allogeneic" cells refer to cells of the same species that are genetically different from the cells being compared; "synogeneic" cells refer to cells that are genetically identical to the cells being compared but originating from different subjects; and "heteroallelic" cells refer to cells that originate from a different species than the cells being compared. In certain embodiments, the immune cells of the present invention are self or allogeneic cells.

[0149] As used herein, the term “cytotoxic agent” includes any agent that is harmful to cells (e.g., kills them), such as chemotherapeutic agents, bacterial toxins, plant toxins, or radioisotopes.

[0150] As used herein, the term “vector” refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. A vector is called an expression vector if it can achieve the expression of a protein encoded by the inserted polynucleotide. A vector can be introduced into a host cell by transformation, transduction, or transfection so that the genetic material elements it contains can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages such as lambda phages or M13 phages; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). A vector can contain, but is not limited to, various elements that control gene expression, including promoter sequences, transcription start sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, a vector may also contain an origin of replication.

[0151] As used herein, the term “host cell” refers to, but is not limited to, cells into which a vector can be introduced, including prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells, and immune cells (e.g., T lymphocytes, NK cells, monocytes, macrophages or dendritic cells). Host cells may consist of a single cell or a population of cells.

[0152] As used herein, the term “isolated” means separated or purified from components that are naturally associated with them (e.g., nucleic acids, proteins, or other naturally occurring biological or organic molecules).

[0153] As used herein, the term “identity” refers to the degree of similarity between two polypeptides or two nucleic acids. Two sequences for comparison are identical at a particular site if they have the same monomer subunit base or amino acid at that site (for example, two DNA molecules each having adenine at a particular site, or two polypeptides each having lysine at a particular site). The percentage of identity between two sequences is a function of the number of identical sites shared by the two sequences divided by the total number of sites being compared, multiplied by 100. For example, if six out of ten positions of two sequences match, these two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Generally, the comparison of two sequences is performed to obtain the greatest possible identity. Such alignments can be performed using computer programs such as the Align program (DNAstar, Inc.), based on the method of Needleman, et al. (J. Mol. Biol. 48:443-453, 1970). The percentage of identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)), which is included in the ALIGN program (version 2.0), using a PAM120 weight reciprocal table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percentage of identity between two amino acid sequences can be determined using the algorithm by Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)), which is included in the GAP program of the GCG software package (available at http: / / www.gcg.com), using either the Blossum 62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0154] As used herein, the terms “conservative substitution” and “conservative amino acid substitution” refer to amino acid substitutions that do not adversely affect or alter the expected properties of a protein / polypeptide comprising its amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain to the corresponding amino acid residue, for example, a physically or functionally similar residue (e.g., similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds). The group of amino acid residues having similar side chains is defined in the art. These groups include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid and glutamic acid), amino acids with non-charged side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (e.g., threonine, valine, and isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, the corresponding amino acid residue is preferably substituted with another amino acid residue from the same side chain group. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).

[0155] The descriptions of the 20 conventional amino acids referenced herein follow conventional usage. See, for example, Immunology-A Synthesis (2nd edition, edited by ES Golub and DR Gren, Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. In this invention, amino acids are generally represented by one- and three-letter abbreviations known in the art. For example, alanine can be represented as A or Ala.

[0156] As used herein, the term “pharmaceutically acceptable carrier and / or excipient” refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and active ingredient, and includes, but is not limited to, those well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th edition. Pennsylvania: Mack Publishing Company, 1995), pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic maintenance agents, absorption delay agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Drugs that maintain osmotic pressure include, but are not limited to, sugars and NaCl. Drugs that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (e.g., buffered saline), alcohols and polyols (e.g., glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, and sorbic acid. Stabilizers have a meaning generally understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the pharmaceutical, and are not limited to, monosodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein) or their degradation products (e.g., lactalbumin hydrolysate).In certain exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable solution (e.g., an aqueous or non-aqueous suspension or solution). In certain exemplary embodiments, such a sterile injectable solution is selected from the group consisting of water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), dextrose solution (e.g., 5% dextrose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate-buffered saline), Ringer's solution, and any combination thereof.

[0157] As used herein, the term “prevention” refers to a method performed to prevent or delay the development of a disease, disorder, or symptom (e.g., tumor) in a subject. As used herein, the term “treatment” refers to a method performed to obtain a beneficial or desired clinical outcome. For the purposes of the present invention, beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of the scope of the disease, stabilization of the disease state (i.e., no further exacerbation), delay or slowing of disease progression, improvement or relief of the disease state, and relief (partial or complete) of detectable or undetectable symptoms. Furthermore, “treatment” may also refer to extending survival time compared to the expected survival time (if no such treatment is received).

[0158] As used herein, the term “effective dose” means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective dose for preventing a disease (e.g., a tumor) means an amount sufficient to prevent, inhibit, or delay the onset of the disease (e.g., a tumor), and an effective dose for treating a disease means an amount sufficient to cure, or at least partially prevent, the existing disease or its complications in a patient suffering from the disease. Determining such an effective dose is well within the capabilities of a person skilled in the art. For example, an effective dose for therapeutic use will vary depending on the severity of the disease being treated, the general state of the patient’s own immune system, the patient’s general condition such as age, weight, and sex, the mode of drug administration, and any other therapies used in combination.

[0159] As used herein, the term “subject” refers to mammals, e.g., primate mammals, e.g., humans. In certain embodiments, the term “subject” means an organism capable of eliciting an immune response. In certain embodiments, the subject (e.g., human) has a RAS G12V mutation-positive tumor and is at risk of developing the disease described above.

[0160] Beneficial Effects of the Present Invention The present invention provides an epitope peptide of a RAS G12V mutant, a T cell receptor (TCR) capable of specifically recognizing the epitope peptide, cells and pharmaceutical compositions comprising the epitope peptide or TCR, nucleic acids encoding the epitope peptide or TCR, vectors and host cells for producing the epitope peptide or TCR, and methods for using the epitope peptide or TCR to treat a subject. The epitope peptide and TCR provided by the present invention can induce an immune response against tumors having the RAS G12V mutation, thereby treating the aforementioned tumor in a subject. Furthermore, the epitope peptide and TCR provided by the present invention are MHC-II restricted, where MHC-II restriction refers to an allele that is frequently found primarily in populations in the Asia-Pacific region; therefore, it is particularly suitable for patients in the Asia-Pacific region. Thus, the present invention provides a novel T cell-based immunotherapy for the treatment of RAS G12V mutation-positive tumors and therefore has significant clinical value.

[0161] Embodiments of the present invention will be described in detail below with reference to the drawings and examples, but those skilled in the art will understand that the following drawings and examples are merely illustrative and do not limit the scope of the invention. From the accompanying drawings and the detailed description of the following preferred embodiments, various purposes and advantages of the present invention will become apparent to those skilled in the art. [Brief explanation of the drawing]

[0162] [Figure 1]Figure 1 shows the results of IFNγ-specific release for various LCL clones in Example 5. [Figure 2] Figure 2 shows the results of IFNγ release after co-culturing antigen-presenting cells with various peptides attached and B13.14.1 TCR-T cells in Example 6. [Figure 3] Figure 3 shows the results of IFNγ release assays of B13.14.1 TCR-T induced by various G12V peptide segments in Example 7. [Figure 4] Figure 4 shows the results of the IFN release assay of B13.14.1 TCR-T induced by the G12V peptide segment containing the mutation in Example 8. [Figure 5] Figure 5 shows the results of selective killing of SW620-CIITA-DPA02:02 / DPB03:01 cells by B13.14.1 TCR-T in Example 9. [Figure 6A-B] Figures 6A-6B show the results of the B13.14.1 TCR-T affinity assay against the RAS G12V mutant in Example 10. [Figure 7] Figure 7 shows the results of the in vivo efficacy assay of B13.14.1 TCR-T in Example 11. [Figure 8] Figure 8 shows the results of determining the binding activity of the TCR mutant (CDR3 region) to the KRAS-G12V antigen peptide in Example 12. [Figure 9] Figure 9 shows the results of determining the binding activity of the TCR mutant (MHC-II binding domain) to the KRAS-G12V antigen peptide in Example 12.

[0163] Sequence information The table below shows information regarding the partial sequences involved in the present invention.

[0164] [Table 1] TIFF2023550515000003.tif251170TIFF2023550515000004.tif252170TIFF2023550515000005.tif25217 0TIFF2023550515000006.tif250170TIFF2023550515000007.tif252168TIFF2023550515000008.tif28170 [Examples]

[0165] The present invention will be described below with reference to examples, but these are illustrative examples of the present invention and do not limit it.

[0166] Unless otherwise noted, the molecular biological experimental and immunoassay methods used in this invention are based on the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and bF. M. Ausubel et al., Short protocols in Molecular Biology, 3rd edition, John Wiley & Sons, Inc., 1995, with restriction enzymes used according to the conditions recommended by the product manufacturer. Those skilled in the art will understand that these examples illustrate the invention as illustrative examples and do not limit the scope of the claimed invention.

[0167] Example 1: Culture and expansion of tumor-infiltrating T cells Tumor samples obtained from surgical resection of colorectal cancer patients with RAS mutation G12V were cut into 2mm-4mm tumor pieces with a scalpel, washed twice with DPBS, and cultured in 24-well plates in TIL medium containing IL2 (6000 IU / ml), human AB serum (2%), Hepes (25 mM), and Xvivo15. Half of the medium was changed every 2-3 days until tumor-infiltrating T lymphocytes (TILs) reached a concentration density of 60-80% (approximately 0.5-3.0 × 10⁶). 6When the TILs had grown to a certain number (including 1 individual TIL), they were collected and stored in CS10 cryogenic protection solution.

[0168] TIL cells were co-cultured in a ratio of 1:30 to 1:200 with peripheral blood mononuclear cells (pooled PBMCs, donor > 3) from different donors that had been irradiated with gamma rays (4000 cGy), and then placed in a T175 culture flask at a rate of 1 × 10⁶. 8 The cells can be expanded by culturing at a rate of 1.0 × 10⁶ cells per flask (using TIL medium supplemented with 10 ng / ml of OKT3). After 3 days of culture, half of the medium (Xvivo15 medium containing 3000 IU / ml of IL2 and 2% human AB serum) is replaced, and after 7 days of culture, the cells are washed once, the medium is replaced, and the cells are expanded to 1.0 × 10⁶ cells. 6 The cells were subcultured at / ml, harvested after 10-14 days of culture, and cryopreserved.

[0169] Example 2: Preparation of RAS G12V antigen mRNA Vector UTR-LAMP3 Lumenal-KRAS G12V The -LMP3 Sorting-UTR was constructed for a sequence containing the RAS mutation G12V according to the following design, which is shown as Sequence ID No. 14 in Table 1, where KRAS is indicated by a single underline and the G12V mutation by a double underline. The above sequence was synthesized, cloned into a pcDNA3.1 vector, and mRNA transcription was prepared in vitro using the T7 promoter (mMESSAGE mMACHINE T7 Transcription Kit, Thermofisher). The transcribed mRNA was subpackaged and stored at -80°C.

[0170] Example 3: Screening using antigen-presenting cells Maturation of DC cells (dendritic cells): Patient autologous peripheral blood CD14-positive cells were isolated using the MACS CD14 Isolation Kit and cultured in AIM-V medium containing IL4 (1000 IU / ml), GM-CSF (1000 IU / ml), and 1% human AB serum. On day 3, the culture medium was replaced with fresh medium, and after culturing for 5-6 days, the cells were cryopreserved in CS10 cryoprotection solution.

[0171] Induction and generation of LCL cells (lymphoblastoid cell line): 5 × 10⁶ peripheral blood mononuclear cells from the patient. 6 The cells were resuspended in RPMI1640 medium containing 10% fetal bovine serum, and the B95.8 supernatant of a cell culture containing EBV for induction was added. Induction was generally completed in 14 to 30 days, with half of the medium being replaced every 7 days during the induction period. After induction, the established LCL cell line was enlarged and cryopreserved.

[0172] After harvesting, TIL cells were cultured for at least 48 hours for screening. DC cells or LCL cells, called APCs (antigen-presenting cells), were transfected with KRAS G12V mRNA using a Neon electroporator. The APC cells were transfected in an electroporation solution at a rate of 1 × 10⁶. 7 The cells were resuspended to a concentration of 1 / ml, and 5-8 μg of mRNA was added to 100 μl of cells for each electroporation (1500 V, 30 ms, 1 pulse). Transfected APCs were ready for use the day after culturing. 1-2 × 10 5 A single TIL cell and 0.5 × 10⁶ 5 Individual electroporated DC cells or 4 × 10 5 Individual electroporated LCL cells were cultured in Xvivo15 medium in a 96-well U-bottom plate. After 16 hours of incubation, the cell culture supernatant was collected, and the amount of IFNγ released from the supernatant was determined using a human IFNγ Flex Set. Patient number B13 had KRAS G12V The patient had metastatic colorectal cancer with a mutation. The results of the TIL screening were as follows:

[0173] [Table 2]

[0174] Example 4: Selection and expansion of TIL TIL cells stimulated by APC cells were sorted by flow cytometry. 1 × 10 6TIL cells were resuspended in flow buffer (DPBS solution containing 1% human AB serum, 2 mM EDTA), CD3 / CD137 antibody and PI (propidium iodide solution) were added, incubated at 4°C for 1 hour, washed twice with flow buffer, and sorted using a BD FACSAira II flow sorter. The sorted population consisted of PI-negative, CD3-positive, and CD137-positive cell populations. The sorted cells were stored in RPMI1640 medium containing 10% human AB serum and kept on ice. The harvested sorted cells (CD3 and CD137+) were centrifuged at 300 g at 4°C for 10 minutes to remove 80% of the storage solution, washed twice with DPBS solution, resuspended in DPBS, and subjected to 10× Genomics single-cell sequencing.

[0175] Example 5: KRAS G12V Screening for recognizing TCRs TCR clones obtained by single-cell sequencing were subjected to gene synthesis according to the sequence TRAVmCa-P2A-TRBVmCb, where TRAV is the α-chain variable region of the TCR, mCa is the mouse TCRα constant region (its amino acid and nucleotide sequences are shown in SEQ ID NOs. 18 and 19, respectively), TRBV is the β-chain variable region of the TCR, mCb is the mouse TCRβ constant region (its amino acid and nucleotide sequences are shown in SEQ ID NOs. 20 and 21, respectively), and P2A is a self-cleaving peptide (its nucleotide sequence is shown in SEQ ID NOs. 22); the above sequence was cloned into a lentiviral transvector (GV401), and the transvector and package vector were transiently transfected into 293T cells according to a standard lentiviral vector packaging method. The culture supernatant containing the lentiviral vector expressing the TCR was collected. T cells from healthy donors were activated for 24 hours in OKT3 / 15E8 antibody-coated 6-well plates, then transformed with a lentiviral vector containing a TCR, followed by TCR screening (transformed T cells were harvested, washed with FACS buffer solution, and 1 × 10⁶ cells were selected). 6Individual modified T cells were stained with a mouse TCRβ constant region recognition antibody and cultured for 6-8 days to detect recombinant TCR expression, thereby obtaining T cells modified with recombinant TCR.

[0176] Following the electroporation method in "Example 3," autologous LCL cells from patient number B13 were subjected to KRAS. G12V LCL cells were transiently transfected with mRNA, cultured overnight, and then electroporated with recombinant TCR-modified T cells in a 96-well U-bottom plate. (1 × 10⁶ LCL cells) 5 :1×10 5 Inoculation and co-culture were performed in the specified ratio, and IFNγ-specific release in the supernatant was detected. The results are shown in Figure 1. The screening results showed that the TCR of clone number B13.14.1 specifically recognized the KRAS G12V point mutation and did not recognize wild-type RAS.

[0177] B13.14.1 The TCR is shown in the table below.

[0178] [Table 3]

[0179] Example 6: B13.14.1 Determination of HLA constraints in TCR 6.1 HLA Restriction Determination 1 T cells expressing recombinant B13.14.1 TCR (hereinafter referred to as B13.14.1 1 TCR-T) were prepared according to the method described in Example 5, which includes introducing a lentiviral vector containing the nucleotide sequence encoding the recombinant TCR into T cells from a healthy donor, where the nucleotide sequences of the α chain (TRAVmCa) and β chain (TRBVmCb) of the recombinant TCR are shown in SEQ ID NOs. 23 and 24, respectively. HLA restriction of B13.14.1 TCR was determined by the method described below.

[0180] The SW620 cell line (colorectal cancer lymph node metastasis) contains a KRAS G12V homozygous mutation, and the CFPAC-1 cell line (pancreatic cancer) contains a KRAS G12V heterozygous mutation. Their HLA-DP compatibility types are shown in the table below.

[0181] [Table 4]

[0182] SW620-CIITA or CFPAC1-CIITA cells, SW620 / CFPAC1-CIITA-DPB1 * 03:01 cells and SW620 / CFPAC1-CIITA-DPA1 * 02:02~DPB1 * 03:01 Cells were harvested, resuspended in RPMI1640 medium, incubated at 37°C for 2 hours, and washed twice with DPBS solution. Antigen-presenting cells (the modified SW620 or CFPAC-1 cells described above) were mixed with B13.14.1 TCR-T cells in RPMI1640 medium containing 2% fetal bovine serum, 2 × 10⁶ cells. 4 :2×10 4 The cells were co-cultured overnight in the specified ratio, and the IFN release from the supernatant was measured. The results are shown in Figure 2.

[0183] These results indicate that B13.14.1 TCR is HLA-DPB1 * It was confined at 03:01 and was able to recognize the presented G12V mutation, and all three alleles of HLA-DPA1 were DPB1 * It could be used in combination with 03:01 for presentation purposes.

[0184] 6.2 HLA Restriction Determination 2 LCL cell lines prepared from various donor sources were added with KRAS-G12V T15 antigen peptide (10 μg / ml), and then co-incubated with Mock T or B13.14.1 TCR-T in RPMI1640 medium containing 2% FBS. The IFNγ content in the supernatant was determined. The ability of B13.14.1 TCR to recognize KRAS-G12V presented by the human high-frequency HLA-DPB1 gene was determined, and the results are shown in the table below.

[0185] [Table 5]

[0186] The results in Table 4 show that B13.14.1 TCR is HLA-DPB1 * In addition to being able to recognize the KRAS-G12V antigen peptide presented by 03:01, HLA-DPB1 * We demonstrated that the KRAS-G12V antigen peptide presented at 14:01 can also be recognized.

[0187] Example 7: Determination of epitope presentation in RAS G12V Peptides containing the G12V mutation site, with lengths of 9-23 (SEQ ID NOs. 2-5, 18-34), were synthesized according to the table below. These peptides were then subjected to antigen presentation by autologous LCL cells, and the amount of IFN released was determined to screen for RAS G12V epitopes recognized by the B13.14.1 TCR.

[0188] [Table 6]

[0189] After synthesizing the above peptide, DMSO is added to dissolve it, patient B13's autologous LCL cells are resuspended in RPMI1640 medium, the peptide is added to a final concentration of 1 μg / ml, incubated for 2 hours, washed twice with DPBS solution, and 2 × 10⁶ cells are added to RPMI1640 medium containing % fetal bovine serum. 5 The peptide-supported antigen-presenting cells were resuspended to a concentration of / ml. 4 :2×104 They were co-cultured with B13.14.1 TCR-T cells overnight according to the ratio, the IFNγ release in the supernatant was measured, and the results are shown in Figure 3.

[0190] The results showed that all peptides of G12V-T3, G12V-T9, G12V-T15, and G12V-T16 could effectively induce the IFNγ release of B13.14.1 TCR-T, and HLA-DPB1 * 03:01 was shown to be able to present the above peptides. Also, the above results showed that peptides comprising amino acid residues at positions 3 to 13 of the RAS G12V mutant could effectively activate T lymphocytes, thereby inducing an immune response against tumors having the RAS G12V mutant.

[0191] Example 8: Determination of important amino acids presented by the RAS G12V epitope by alanine scanning. By performing alanine substitution on the RAS G12V epitope peptide one by one, important amino acids involved in antigen presentation in the RAS G12V epitope could be screened. The peptide segments after alanine mutation (mutated amino acids are underlined) are shown in the following table.

[0192]

Table 7

[0193] After synthesizing the above peptides, IFNγ or IL2 in the supernatant after co-culturing antigen-presenting cells and B13.14.1 TCR-T was measured according to the method of Example 7, and the results are shown in Figure 4. The above results showed that TEYKLVVVGA V In the GV epitope, the important amino acids involved in antigen presentation are p3Y, p4K, p5L, and p8V. After mutating these four amino acids to alanine, the ability of B13.14.1 TCR-T to recognize the RAS G12V epitope decreased, and it was found that the most important among them are p3Y, p4K, and p5L.

[0194] Example 9: B13.14.1 Extent of tumor lysis by TCR-T SW620-CIITA-DPA02:02 / DPB03:01 cells were resuspended in RPMI1640 medium containing 2% FBS, and 10 4 Inoculate into 96-well plates according to the / well ratio, and Mock-T, B13.14.1 TCR-CD4 according to the E:T ratio of 10, 3, 1, 0.3, 0.1. + T and B13.14.1 TCR-CD8 + T cells were added to establish a control without T cells. After 48 hours of co-incubation, the medium was aspirated, and each well was washed once with 200 μl of DPBS solution. Then, 100 μl of RPMI1640 medium containing 2% FBS and 10 μl of CCK8 detection reagent (Cell Counting Kit-8) was added to each well, and the mixture was incubated at 37°C for 1 hour. The absorbance at 450 nm light was read.

[0195] Lethality = 1 - (Absorbance / Absorbance of the control well)

[0196] The results are shown in Figure 5. These results demonstrate that B13.14.1 TCR-T can dose-dependently kill tumor cells and show a CD4 dependency.

[0197] Example 10: B13.14.1 Determination of functional avidity of TCR-T B13 autologous LCL cells were tagged with RAS G12V-T15 peptide (SEQ ID NO: 5) and the corresponding wild-type peptide (SEQ ID NO: 17) at various concentrations (10 μg / ml, 1 μg / ml, 0.1 μg / ml, 0.01 μg / ml), respectively. After culturing at 37°C for 2 hours, the cells were washed twice with DPBS solution, and the antigen-presenting cells were placed in RPMI1640 medium containing 2% fetal bovine serum (2 × 10⁶ cells). 4 :2×10 4The B13.14.1 TCR-T cells were co-cultured overnight with the TCR-T cells according to the specified ratio, and the release of IL2 and IFNγ in the supernatant was measured. The results are shown in Figures 6A-6B. These results indicate that the B13.14.1 TCR-T cells can specifically recognize the RAS G12V mutant peptide within the tested concentration range, release IFNγ and IL2, and still efficiently recognize the RAS G12V mutant peptide even at an antigen peptide concentration of 0.1 μg / ml, demonstrating that T cells expressing this TCR have high affinity and high specificity for the RAS G12V mutant.

[0198] Example 11: B13.14.1 Determination of in vivo efficacy of TCR-T SW620-DPAB tumor cell line (HLA-DPB1 * (overexpressing 03:01) was subcutaneously inoculated into NSG mice (1 × 10 7 (in mice), tumor volume approximately 50 mm 3 When the patient reaches [a certain level], administer the following via tail vein: blank preparation, MockT (non-transduced T cell control), B13.14.1CD4TCRT (CD4 T cells genetically modified with B13.14.1TCR), B13.14.1CD8TCRT (CD8 T cells genetically modified with B13.14.1TCR), or B13.14.1 MixTCRT (a 1:1 mixture of CD4 T cells and CD8 T cells genetically modified with B13.14.1TCR). The dose is 1 × 10⁻¹⁶. 7 Tumor growth was measured using mTCRβ+ cells / animals. The results are shown in Figure 7.

[0199] These results demonstrate that B13.14.1TCRT can eliminate tumors in the SW620-DPAB cell line and show dependence on the CD4 coreceptor.

[0200] Example 12: B13.14.1 Affinity maturation of TCR As shown in the table below, point mutations occurred in Vα and Vβ (including the CDR3 region and MHC II binding region) of the B13.14.1 TCR. Lentiviral vector shuttle plasmids encoding these TCR mutants were constructed, packaged with standard lentiviral vectors in 293T cells, and functional screening was performed on the B13.14.1 TCR mutants.

[0201] [Table 8]

[0202] 12.1 TCR CDR3 Mutant Screening A lentiviral vector containing a TCR mutant (CDR3 region) was transduced into the Jurkat-NFAT-Luc cell line, and B13 LCL cells to which the KRAS-G12V antigen (RAS G12V-T15 peptide, SEQ ID NO: 5) was added were used as antigen-presenting cells. 2 × 10 4 TCR-T cells and 2 × 10 4 Antigen-added B13 LCL cells were co-cultured for 16-24 hours, and ONE Glo luciferase was added to detect the expression of a fluorescent signal. The TCR Mut RLU / WT RLU (ratio of the RLU signal value of the TCR mutant to the RLU signal value of the wild-type B13.14.1 TCR) was calculated. The results were as follows:

[0203] [Table 9] TIFF2023550515000017.tif251170TIFF2023550515000018.tif252170TIFF2023550515000019.tif254170TIFF2023550515000020.tif111170

[0204] The above results showed the following: 1) The results of detecting mTCRβ expression showed that it was expressible on the surface of Jurkat-NFAT-Luc cells; 2) In the mutations in the CDRα3 and CDRβ3 regions of the B13.14.1 TCR, the amino acid positions that had an important impact on TCR specificity were CDRα3~95D, CDRβ3~96G, CDRβ3~98R, and CDRβ3~99D. After the amino acids mutated at these positions, all mutants were unable to effectively recognize the KRAS G12V antigen peptide presented by antigen-presenting cells;

[0205] TCR CDRα3 and CDRβ3 mutants with activity (maintaining more than 30% of the wild-type B13.14.1 TCR RLU signal value) are shown in the following table.

[0206]

Table 10

[0207] In the above table, those with a higher TCR Mut RLU / WT RLU ratio are underlined, and these were the preferred TCR mutants. The Emax and EC50 (functional avidity) of these TCR mutants in the recognition of the KRAS-G12V antigen peptide were further determined. Various concentrations of the G12V antigen peptide (10 μg / ml, 10-fold dilution, 5 concentrations) were added to B13 LCL cells and co-incubated with Jurkat-NFAT-Luc cells expressing the TCR mutant for 16 - 24 hours. Fluorescent signal expression was detected by adding ONE Glo luciferase, and the results are shown in Figure 8 and the following table. These results indicated that the preferred TCR mutants could recognize the KRAS-G12V mutant antigen peptide in a concentration-dependent manner and had a stronger antigen recognition ability than the wild-type B13.14.1WT.

[0208]

Table 11

[0209] 12.2 Screening of TCR MHC-II binding region mutants A lentiviral vector containing a TCR mutant (MHC-II binding domain) was transduced into the Jurkat-NFAT-Luc cell line, and B13 LCL cells to which the KRAS-G12V antigen (RAS G12V-T15 peptide, SEQ ID NO: 5) was added were used as antigen-presenting cells. 2 × 10 4 TCR-T and 2x10 4 Antigen-added B13 LCL cells were co-cultured for 16-24 hours, and ONE Glo-luciferase was added to detect the expression of a fluorescent signal. The TCR Mut RLU / WT RLU (ratio of the RLU signal value of the TCR mutant to the RLU signal value of the wild-type B13.14.1 TCR) was calculated. The results are shown in the table below.

[0210] [Table 12] TIFF2023550515000024.tif249170TIFF2023550515000025.tif250170TIFF2023550515000026.tif249170TIFF2023550515000027.tif49170

[0211] The above results showed the following: 1) The results of mTCRβ expression detection showed that all TCR mutants can be expressed on the surface of Jurkat-NFAT-Luc cells; 2) B13.14.1 In mutations in the MHCII binding region of the TCR, the amino acid position that had a significant impact on TCR specificity was Vα-32Y. After the amino acid mutation at the above position, the mutant was present in the KRAS presented by antigen-presenting cells. G12V The antigen peptide could not be effectively recognized; 3) TCR mutants that exhibit activity (maintaining 30% or more of the wild-type B13.14.1 TCR RLU signal value) are shown in the table below.

[0212] [Table 13]

[0213] In the table above, TCR mutants with a TCR Mut RLU / WT RLU ratio of 80% or higher are underlined, and these were considered desirable TCR mutants. The Emax and EC50 (functional avidity) of the TCR mutants recognizing the KRAS-G12V antigen peptide were further determined. SW620-DPB cells (HLA-DPB1 * SW620 cells expressing 03:01 were treated with various concentrations of the G12V antigen peptide (10 μg / ml, 10-fold dilution, 5 concentrations), co-incubated with Jurkat-NFAT-Luc cells expressing TCR mutants for 16-24 hours, and ONE Glo luciferase was added to detect the expression of a fluorescent signal. The results are shown in Figure 9 and the table below. These results indicate that the preferred TCR mutant could recognize the KRAS-G12V mutant antigen peptide in a concentration-dependent manner and had stronger antigen recognition ability than wild-type B13.14.1WT.

[0214] [Table 14]

[0215] Although specific models for carrying out the present invention have been described in detail, those skilled in the art will know that various modifications and changes can be made to the details according to all the disclosed teachings, and all such changes are within the scope of the protection of the present invention. The entire scope of the present invention is given by the claims and equivalents appended herein.

Claims

1. An isolated T cell receptor (TCR) or antigen-binding fragment thereof capable of specifically recognizing a RAS G12V mutant, the TCR or antigen-binding fragment thereof comprising an α chain variable region (Vα) and / or a β chain variable region (Vβ); (a) Vα comprises CDR1α, CDR2α and CDR3α, and CDR3α is AVRDX １ X ２ X ３ comprising the sequence shown in GGNKLT (SEQ ID NO:25); X １ is selected from the group consisting of G, A, D, L, M, Q, R, S, V, W, Y; preferably, G, A, D or R; X ２ is selected from the group consisting of R, A, D, G, H, I, K, L, M, N, P, Q, S, T, V, W, Y; preferably, R or T; X ３ is selected from the group consisting of G, A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y; preferably, G, A, H, N, S or W; and / or (b) Vβ comprises CDR1β, CDR2β and CDR3β, and CDR3β is ASSX ４ GX ５ Comprising the sequence shown in RDNSPLH (SEQ ID NO:26); X ４ is selected from the group consisting of P, A, H, M, S, T, V, Y; preferably, P, A or T; X ５ is selected from the group consisting of Q or S; Preferably, the TCR is soluble or membrane bound; Preferably, the TCR is a full length TCR, a soluble TCR or a single chain TCR. TCR or an antigen-binding fragment thereof.

2. Features: (i) CDR1α comprises the sequence set forth in VSGX6PY (SEQ ID NO:27); X6 is selected from the group consisting of N, A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W, Y (preferably, N, E, P, Q, R or S); (ii) CDR2α is YX ７ X ８ GDNLV (SEQ ID NO: 28); ７ is selected from the group consisting of I, A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W, Y (preferably I, D, E, F, G, H, N, P, R, W or Y); X ８ is selected from the group consisting of T, A, D, E, H, I, L, N, Q, R, S, W, Y (preferably, T, A, D, H, I, Q or R); (iii) CDR1β comprises the sequence set forth in SEQ ID NO:11; (iv) CDR2β is SX ９ X １０ VNX １１ (SEQ ID NO: 29); ９ is selected from the group consisting of Q, A, F, G, H, I, K, L, M, N, P, R, S, T, V, W, Y (preferably Q, A, I, M, N, S, T or Y); X １０ is selected from the group consisting of I, F, H, T, V (preferably I or T); １１ is selected from the group consisting of D, A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, Y (preferably D, G, L, M, N, T or Y). The TCR or antigen-binding fragment thereof of claim 1, comprising one or more of the following:

3. (a) Vα comprises FR1α, FR2α, FR3α and FR4α; FR1α comprises the sequence shown in SEQ ID NO:30; FR2α is LFWYVQYPNRGLQFLLX １２ (SEQ ID NO: 38); １２ is selected from the group consisting of K, A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W, Y (preferably K, G, H, N, P, R, S, V, W or Y); FR3α comprises the sequence shown in SEQ ID NO:32; FR4α comprises the sequence set forth in SEQ ID NO:33; and / or (b) Vβ comprises FR1β, FR2β, FR3β and FR4β; FR1β comprises the sequence set forth in SEQ ID NO:34; FR2β is MYWYRQDPGQGLRLIYX １３ (SEQ ID NO: 39); １３ is selected from the group consisting of Y, A, E, F, I, K, L, M, N, P, Q, R, S, T, V, W (preferably, Y, A, E, F or K); FR3β is FX １４ KGDIAEGYSVSREKKESSFPLTVTSAQKNPTAFYLC (SEQ ID NO: 40); １４ is selected from the group consisting of Q, A, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W, Y (preferably Q, A, D, E, F, H, L, R, T or W); FR4β comprises the sequence set forth in SEQ ID NO:37; A TCR or an antigen-binding fragment thereof according to claim 1 or 2.

4. (a) Vα comprises the sequence set forth in SEQ ID NO:6 or a variant thereof, wherein the variant comprises an amino acid substitution at one or more (e.g., 1, 2, 3, or 4) amino acid positions selected from the group consisting of amino acid positions 30, 49, 51, 52, 96, 97, 98 as determined according to the IMGT TCR numbering system; and / or (b) the Vβ comprises a sequence set forth in SEQ ID NO: 7 or a variant thereof, wherein the variant comprises an amino acid substitution at one or more (e.g., one, two, three, or four) amino acid positions selected from the group consisting of amino acid positions 48, 50, 51, 54, 56, 95, and 97, as determined according to the IMGT TCR numbering system; A TCR or an antigen-binding fragment thereof according to any one of claims 1 to 3.

5. Vα comprises a variant of the sequence shown in SEQ ID NO:6, the variant comprising the following amino acid positions as determined by the IMGT TCR numbering system: substitution of the amino acid at position 30 with A, D, E, F, G, H, I, K, L, M, P, Q, R, S, T, V, W or Y (preferably E, P, Q, R or S); substitution of the amino acid at position 49 with A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y (preferably G, H, N, P, R, S, V, W or Y); substitution of the amino acid at position 51 with A, D, E, F, G, H, K, L, M, N, P, Q, R, S, T, V, W or Y (preferably D, E, F, G, H, N, P, R, W or Y); substitution of the amino acid at position 52 with A, D, E, F, G, H, I, L, M, N, P, Q, R, S, T, V, W or Y (preferably D, E, F, G, H, N, P, R, W or Y); a substitution of the amino acid at position 97 with A, D, G, H, I, K, L, M, N, P, Q, S, T, V, W or Y (preferably A, D or R); a substitution of the amino acid at position 98 with A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, W or Y (preferably A, H, N, S or W); Preferably, the Vβ comprises the sequence shown in SEQ ID NO:

7. The TCR or antigen-binding fragment thereof described in claim 4.

6. Vβ comprises a variant of the sequence set forth in SEQ ID NO:7, where the amino acid positions are determined by the IMGT TCR numbering system: a substitution of the amino acid at position 48 with A, E, F, I, K, L, M, N, P, Q, R, S, T, V or W (preferably A, E, F or K); a substitution of the amino acid at position 50 with A, F, G, H, I, K, L, M, N, P, R, S, T, V, W or Y (preferably A, I, M, N, S, T or Y); a substitution of the amino acid at position 51 with F, H, T or V (preferably T); a substitution of the amino acid at position 54 with A, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V or W (preferably A, I, M, N, S, T or Y); , N, P, Q, R, S, T, V, W or Y (preferably G, L, M, N, T or Y); substitution of the amino acid at position 56 with A, D, E, F, G, H, I, K, L, M, N, P, R, S, T, V, W or Y (preferably A, D, E, F, H, L, R, T or W); substitution of the amino acid at position 95 with A, H, M, S, T, V or Y (preferably A or T); substitution of the amino acid at position 97 with S; Preferably, Vα comprises the sequence shown in SEQ ID NO:

6. A TCR or an antigen-binding fragment thereof according to claim 4 or 5.

7. A TCR or antigen-binding fragment thereof according to any one of claims 1 to 6 and an effector moiety conjugated thereto, Preferably, the TCR or antigen-binding fragment thereof is in a soluble form. Conjugate.

8. A TCR or antigen-binding fragment thereof according to any one of claims 1 to 6 and an additional peptide or protein, Preferably, the TCR or antigen-binding fragment thereof is in a soluble form. Fusion proteins.

9. An isolated epitope peptide or variant thereof, wherein the epitope peptide consists of 11 to 30 (e.g., 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, or 11) consecutive amino acid residues of a RAS G12V mutant, comprising amino acid residues at positions 3 to 13 of the RAS G12V mutant; The variant differs from the epitope peptide from which it is derived only by the substitution of one or a few (e.g., 1, 2 or 3) amino acid residues, does not contain amino acid substitutions at positions corresponding to amino acids at positions 4, 5, 6, 9 and 12 of the RAS G12V mutant, and retains the biological function of the epitope peptide from which it is derived; Preferably, the epitope peptide consists of consecutive amino acid residues 11 to 25 (e.g., 11 to 23, 11 to 13) of the RAS G12V mutant. Epitope peptide or variant thereof.

10. The epitope peptide or variant thereof is capable of presentation by an MHC-II molecule, and the epitope peptide or variant thereof associated with the MHC-II molecule is capable of recognition by a T cell, e.g., recognition by an antigen-specific T cell receptor on a T cell; Preferably, the MHC-II molecule is HLA-DP; Preferably, the HLA-DP comprises HLA-DPB1*03:01 and / or HLA-DPB1*14:01; preferably, the HLA-DP further comprises one selected from the group consisting of HLA-DPA1*02:02, HLA-DPA1*02:01, HLA-DPA1*01:03; The epitope peptide according to claim 9 or a variant thereof.

11. The epitope peptide or a variant thereof according to claim 9 or 10, wherein the amino acid residues at positions 3 to 13 of the RAS G12V mutant have the sequence shown in SEQ ID NO:

2.

12. The epitope peptide comprises amino acid residues at positions 3 to 13, amino acid residues at positions 2 to 13, amino acid residues at positions 3 to 14, or amino acid residues at positions 2 to 14 of the RAS G12V mutant; Preferably, the amino acid residues at positions 3 to 13, the amino acid residues at positions 2 to 13, the amino acid residues at positions 3 to 14 or the amino acid residues at positions 2 to 14 of the RAS G12V mutant have the sequences shown in SEQ ID NOs: 2 to 5, respectively. The epitope peptide according to any one of claims 9 to 11, or a variant thereof.

13. The epitope peptide or its variant according to any one of claims 9 to 12, wherein the RAS G12V mutant has the sequence shown in SEQ ID NO:

1.

14. The epitope peptide or variant thereof according to any one of claims 9 to 13, wherein the epitope peptide comprises a sequence as set forth in any one of SEQ ID NOs: 2 to 5; and the variant comprises a sequence selected from the group consisting of: (i) a sequence having one or several amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence as set forth in any one of SEQ ID NOs: 2 to 5; or (ii) a sequence having 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% sequence identity when compared to the sequence as set forth in any one of SEQ ID NOs: 2 to 5.

15. An isolated T cell receptor or antigen-binding fragment thereof, capable of specifically recognizing an epitope peptide or variant thereof according to any one of claims 9 to 14; Preferably, the epitope peptide or variant thereof is presented by an MHC-II molecule; Preferably, the MHC-II molecule is HLA-DP; Preferably, the HLA-DP comprises HLA-DPB1*03:01 and / or HLA-DPB1*14:01; preferably, the HLA-DP further comprises one selected from the group consisting of HLA-DPA1*02:02, HLA-DPA1*02:01, HLA-DPA1*01:03; Preferably, the TCR is soluble or membrane bound; Preferably, the TCR is a full length TCR, a soluble TCR or a single chain TCR. A T cell receptor or an antigen-binding fragment thereof.

16. The TCR or antigen-binding fragment thereof, capable of specifically recognizing an epitope peptide or a variant thereof according to any one of claims 9 to 14; Preferably, the epitope peptide or variant thereof is presented by an MHC-II molecule; Preferably, the MHC-II molecule is HLA-DP; Preferably, the HLA-DP comprises HLA-DPB1*03:01 and / or HLA-DPB1*14:01; preferably, the HLA-DP further comprises one selected from the group consisting of HLA-DPA1*02:02, HLA-DPA1*02:01, HLA-DPA1*01:03; Preferably, T cells expressing said TCR or an antigen-binding fragment thereof on their surface are activated in co-culture with a second cell (e.g., APC) presenting an epitope peptide or a variant thereof according to any one of claims 9 to 14. A TCR or an antigen-binding fragment thereof according to any one of claims 1 to 6.

17. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the epitope peptide or a variant thereof according to any one of claims 9 to 14, or comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof according to any one of claims 6 to 16, or an α chain variable region and / or a β chain variable region thereof, or comprising a nucleotide sequence encoding the fusion protein according to claim 8.

18. 18. The isolated nucleic acid molecule of claim 17. Preferably, it comprises a nucleotide sequence encoding the epitope peptide or a variant thereof according to any one of claims 9 to 14; Preferably, it comprises a nucleotide sequence encoding a TCR or an antigen-binding fragment thereof according to any one of claims 1 to 6 and 16, or an α chain variable region and / or a β chain variable region thereof; Preferably, it comprises a nucleotide sequence encoding a fusion protein according to claim 8; Preferably, it is a viral vector such as a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector or a baculoviral vector. vector.

19. Comprising an isolated nucleic acid molecule according to claim 17, or a vector according to claim 18; Preferably, it comprises a nucleotide sequence encoding the epitope peptide or a variant thereof according to any one of claims 9 to 14; Preferably, the TCR or antigen-binding fragment thereof comprises a nucleotide sequence encoding the α chain variable region and / or the β chain variable region thereof according to any one of claims 1 to 6 and 16; Preferably, it comprises a nucleotide sequence encoding a fusion protein according to claim 8; Preferably, it comprises E. coli, yeast, insect cells, or mammalian cells. host cell.

20. A method for producing an epitope peptide or variant thereof according to any one of claims 9 to 14, or a TCR or antigen-binding fragment thereof according to any one of claims 1 to 6 and 16, or a fusion protein according to claims, comprising culturing a host cell according to claim 19 under conditions allowing protein expression, and recovering the epitope peptide or variant thereof, or the TCR or antigen-binding fragment thereof, or the fusion protein from a culture of the cultured host cell.

21. An engineered antigen-presenting cell (APC) that presents on its surface the epitope peptide according to any one of claims 9 to 14 or a variant thereof, Preferably, the epitope peptide or variant thereof is presented by an MHC-II molecule; Preferably, the MHC-II molecule is HLA-DP; Preferably, the HLA-DP comprises HLA-DPB1*03:01 and / or HLA-DPB1*14:01; preferably, the HLA-DP further comprises one selected from the group consisting of HLA-DPA1*02:02, HLA-DPA1*02:01, HLA-DPA1*01:03; Preferably, the APC is selected from the group consisting of dendritic cells, monocytes, macrophages, B-lymphocytes (e.g., B-lymphoblastoid cells, B-LCL), or any combination thereof; Preferably, the APCs are HLA-DPB1*03:01 positive or HLA-DPB1*14:01 positive; preferably, the APCs are further HLA-DPA1*02:02 positive, HLA-DPA1*02:01 positive or HLA-DPA1*01:03 positive; Preferably, the APCs are isolated from an HLA-DPB1*03:01 positive subject or an HLA-DPB1*14:01 positive subject; preferably, the subject is further HLA-DPA1*02:02 positive, HLA-DPA1*02:01 positive, or HLA-DPA1*01:03 positive. APC.

22. A method for producing an engineered APC according to claim 21, comprising: (1) preparing an APC from a subject; (2) contacting the APC in vitro with an epitope peptide or variant thereof according to any one of claims 9 to 14, or introducing an expression vector comprising a nucleotide sequence encoding the epitope peptide or variant thereof according to any one of claims 9 to 14 into an APC, thereby obtaining an APC that presents the epitope peptide or variant thereof on its surface.

23. 17. An engineered immune cell expressing on its surface a TCR or an antigen-binding fragment thereof according to any one of claims 1 to 6 and 16, Preferably, the engineered immune cell comprises a nucleotide sequence encoding a TCR or an antigen-binding fragment thereof according to any one of claims 1 to 6 and 16; Preferably, the immune cell is a lymphocyte; Preferably, the immune cells are engineered immune cells selected from the group consisting of T cells (e.g., αβ T cells, γδ T cells, or iPSC-derived T cells), tumor infiltrating lymphocytes (TIL), natural killer (NK) cells, natural killer T (NKT) cells, or any combination thereof.

24. A method for producing an engineered immune cell according to claim 23, comprising: (1) providing an immune cell from a subject; (2) introducing the isolated nucleic acid molecule according to claim 17 or the vector according to claim 18 into the immune cell of step (1), the nucleic acid molecule or vector comprising a nucleotide sequence encoding the TCR or an antigen-binding fragment thereof according to any one of claims 1 to 6 and 16, thereby obtaining an immune cell expressing the TCR or an antigen-binding fragment thereof; Preferably, in step (1), the immune cells are subjected to a pretreatment; said pretreatment comprises the selection, activation and / or proliferation of the immune cells; Preferably, the pretreatment comprises contacting the immune cells with one or more selected from the group consisting of an anti-CD3 antibody, an anti-CD28 antibody, IL-2 and IL-15 to stimulate the immune cells and induce their proliferation, thereby generating pretreated immune cells. method.

25. The epitope peptide or variant thereof according to any one of claims 1 to 6, a nucleic acid molecule or carrier or host cell comprising a nucleotide sequence encoding said epitope peptide or variant thereof, or an engineered antigen-presenting cell (APC) according to claim 21; and a pharma- ceutically acceptable carrier and / or excipient. For inducing an immune response against a tumor having a RAS G12V mutation in a subject and / or for preventing or treating a tumor having a RAS G12V mutation in a subject; 1. A pharmaceutical composition comprising: Preferably, the pharmaceutical composition is a tumor vaccine; Preferably, the pharmaceutical composition comprises an adjuvant; Preferably, the pharmaceutical composition further comprises an additional therapeutic agent, such as an anti-tumor agent or an immune enhancing agent; Preferably, the anti-tumor drug is selected from the group consisting of alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, radiosensitizers, anti-angiogenic agents, cytokines, immune checkpoint inhibitors (e.g., PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG-3 antibodies, or TIM3 antibodies); Preferably, the immune enhancing agent is selected from the group consisting of an immunostimulatory antibody (e.g., an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD40L (CD154) antibody, an anti-41BB (CD137) antibody, an anti-OX40 antibody, an anti-GITR antibody, or any combination thereof), or an immunostimulatory cytokine (e.g., IL-2, IL-3, IL-12, IL-15, IL-18, IFN-γ, IL-10, TGF-β, GM-CSF, or any combination thereof); Preferably, the tumor harboring the RAS G12V mutation is selected from the group consisting of colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, endometrial cancer, ovarian cancer, multiple myeloma, melanoma, thyroid cancer, bladder cancer, prostate cancer, breast cancer, head and neck cancer, or acute myeloid leukemia; Preferably, the subject is a human; Preferably, the subject is HLA-DPB1*03:01 positive or HLA-DPB1*14:01 positive; Preferably, the subject is further HLA-DPA1*02:02 positive, HLA-DPA1*02:01 positive, or HLA-DPA1*01:03 positive; Preferably, said epitopic peptide or variant thereof, nucleic acid molecule or vector or host cell, engineered antigen-presenting cell (APC), or pharmaceutical composition is administered in combination with an additional therapeutic agent, e.g., administered simultaneously, separately or sequentially; preferably, the additional therapeutic agent is an immunostimulant or an antitumor drug. Pharmaceutical compositions.

26. A pharmaceutical composition comprising a TCR or antigen-binding fragment thereof according to any one of claims 1 to 6 and 16, a conjugate according to claim 7, a fusion protein according to claim 8, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding said TCR or antigen-binding fragment thereof or fusion protein, or an engineered immune cell according to claim 23; and a pharma- ceutically acceptable carrier and / or excipient for inducing an immune response against a tumor carrying a RAS G12V mutation in a subject and / or for preventing or treating a tumor carrying a RAS G12V mutation in a subject; Preferably, the pharmaceutical composition further comprises an additional therapeutic agent, such as an anti-tumor agent or an immune enhancing agent; Preferably, the anti-tumor drug is selected from the group consisting of alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, radiosensitizers, anti-angiogenic agents, cytokines, immune checkpoint inhibitors (e.g., PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG-3 antibodies, or TIM3 antibodies); Preferably, the immune enhancing agent is selected from the group consisting of an immunostimulatory antibody (e.g., an anti-CD3 antibody, an anti-CD28 antibody, an anti-CD40L (CD154) antibody, an anti-41BB (CD137) antibody, an anti-OX40 antibody, an anti-GITR antibody, or any combination thereof) or an immunostimulatory cytokine (e.g., IL-2, IL-3, IL-12, IL-15, IL-18, IFN-γ, IL-10, TGF-β, GM-CSF, or any combination thereof); Preferably, the tumor harboring the RAS G12V mutation is selected from the group consisting of colorectal cancer, pancreatic cancer, gastric cancer, lung cancer, endometrial cancer, ovarian cancer, multiple myeloma, melanoma, thyroid cancer, bladder cancer, prostate cancer, breast cancer, head and neck cancer, or acute myeloid leukemia; Preferably, the subject is a human; Preferably, the subject is HLA-DPB1*03:01 positive or HLA-DPB1*14:01 positive; preferably, the subject is further HLA-DPA1*02:02 positive, HLA-DPA1*02:01 positive, or HLA-DPA1*01:03 positive; Preferably, said TCR or antigen-binding fragment thereof, conjugate, fusion protein, nucleic acid molecule or vector or host cell, engineered immune cell, or pharmaceutical composition is administered in combination with an additional therapeutic agent, e.g., administered simultaneously, separately or sequentially; preferably, the additional therapeutic agent is an immunostimulant or an anti-tumor agent; Preferably, inducing an immune response against a tumor carrying a RAS G12V mutation in a subject and / or preventing or treating a tumor carrying a RAS G12V mutation in a subject comprises: (1) providing immune cells required by the subject; (2) introducing a nucleotide sequence encoding a TCR or an antigen-binding fragment thereof according to any one of claims 1 to 6 and 16 into the immune cells of step (1), thereby obtaining immune cells expressing said TCR or an antigen-binding fragment thereof on their surface; (3) administering the immune cells obtained in step (2) to the subject; Preferably, the immune cell is a lymphocyte; Preferably, the immune cells are selected from the group consisting of T cells (e.g., αβ T cells, γδ T cells, or iPSC-derived T cells), tumor infiltrating lymphocytes (TIL), natural killer (NK) cells, natural killer T (NKT) cells, or any combination thereof.

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