RCN1-derived TEIPP neoantigen and its use

Abstract

This specification provides novel nucleic acid sequences, vectors, modified cells, binders, peptides, and pharmaceutical compositions useful for, for example, the prevention or treatment of cancer or viral infections associated with HLA class I antigen presentation disorders. Corresponding methods and applications are also provided.

Description

[Technical Field]

[0001] For example, the present invention provides novel nucleic acid sequences, vectors, modified cells, binders, peptides, and pharmaceutical compositions useful as pharmaceuticals in the prevention or treatment of cancer or viral infections associated with HLA class I antigen presentation disorders. Corresponding methods and applications are also provided. [Background technology]

[0002] background Many T-cell-based immunotherapies used to treat cancer in humans are based on the recognition of tumor antigens presented by tumor cells on HLA class I (HLA-I) molecules (Robbins et al., 2013; Schumacher et al., 2015). Point mutation peptides constitute potent tumor antigens because their non-self nature makes them available to an unrestricted T-cell repertoire. The absolute requirement for such T cells to exert their effect against cancer is the presentation of HLA-I on the surface of tumor cells. However, downmodulation of HLA-I in cancer cells is observed in many immune-evading cancers and is often caused by epigenetic silencing of antigen-processing components such as the peptide transporter TAP (Ritter et al., 2017; Setiadi et al., 2007; Garrido et al., 2016).

[0003] A novel category of tumor antigens called TEIPPs (T-cell epitopes associated with peptide processing dysfunction) are presented on the surface of tumor cells with defects in antigen processing (Van Hall et al., 2006; Seidel et al., 2012). TEIPPs originate from ubiquitously expressed, non-mutated "self" proteins, but their processed peptides fail to load HLA-I to T-cell detectable levels in healthy cells. Their surface presentation is highly facilitated by defects in the antigen processing mechanism, particularly in the absence of the peptide transporter TAP. TAP dysfunction is frequently found in cancer cells, and therefore TEIPP peptides constitute tumor-specific antigens. In a mouse tumor model in which MHC-I presentation is downregulated due to defects in the peptide transporter TAP, selective presentation of TEIPP peptides and successful targeting of immune-evading tumor mutants by TEIPP-specific T cells have been demonstrated by vaccination or transfer of specific T cells (Doorduijn et al., 2016; Doorduijn et al., 2018). Therefore, targeting TEIPP neoantigens is a powerful strategy for inducing an antitumor response against tumors with reduced TAP expression and / or impaired HLA class I antigen presentation.

[0004] Impaired HLA-I mediated antigen presentation has been observed in infections with certain viruses. Such impairment and / or reduced TAP expression may result in surface presentation of the TEIPP antigen on virus-infected cells. Therefore, the TEIPP peptide may also be considered as a target antigen for treating or preventing viral infections associated with reduced TAP expression and / or impaired antigen presentation in HLA class I molecules.

[0005] US2009 / 0220534 and Weinzierl (Weinzierl et al., 2008) describe a screening method for identifying T cell epitopes presented on the cell surface via TAP-independent mechanisms.

[0006] Human TEIPP neoantigens as potential targets for T-cell-based immunotherapy have been previously described in WO2019231326A1 and WO2021107775A1. However, further potentially improved TEIPP neoantigens are needed for the prevention and / or treatment of cancer. [Overview of the project]

[0007] Summary of the disclosure The inventors have previously developed a hybrid forward-reverse immunological screening to identify novel TAP-independent, non-mutant neoantigens selectively presented by immune-evading cancers (Marijt et al., 2018). Their approaches included in silico prediction of TEIPP neoantigen candidates from the whole human proteome, matching of candidates to cancer-specific peptidomes, and in vitro screening to confirm the presence of the TEIPP T cell repertoire in healthy donors.

[0008] The inventors are currently identifying novel TEIPP neoantigen candidates. Thirty-six peptides were selected for their ability to activate CD8+ T cells present in peripheral blood mononuclear cells (PBMCs) from healthy human donors. Following further in vitro screening using healthy donors, HLA-A peptides were selected, demonstrating particularly strong immunogenicity and general T cell availability to such peptides in the human T cell repertoire. * A specific peptide for 02:01 was identified. This peptide was identified as being derived from reticlocarbine 1 (RCN1) and having the sequence VLAPRVLRA (SEQ ID NO: 2).

[0009] Advantageously, the CD8+ T cells identified in the healthy donor repertoire were primarily naive. As disclosed herein, CD8+ T cells did not recognize the peptides presented herein in cells with normal TAP expression, but did recognize them when faced with cells with impaired TAP expression. Therefore, the peptides presented herein are ideal candidates for inducing a T-cell-based immune response in vivo against cells associated with impaired antigen presentation and / or impaired TAP function in HLA class I presenting molecules. The data presented herein therefore provide evidence that these peptides may be used as novel therapies for inducing tumor-specific T-cell-based immune responses in vivo (by activating naive homogeneous T cells present in the patient's native T-cell repertoire). Furthermore, conjugates such as antibodies, TCRs, or CARs (or modified cells expressing the same) that specifically bind to these peptides may be advantageously used as novel immunotherapies for the prevention or treatment of cancer or viral infections associated with impaired antigen presentation and / or impaired TAP function in HLA class I molecules.

[0010] Advantageously, the peptides described herein are of "self" origin, not derived from cancer mutanomes, and therefore constitute universal neoantigens that may be presented on the cell surface of any cell in which antigen presentation in HLA class I molecules is impaired.

[0011] Furthermore, the inventors have previously developed a synthetic long peptide (SLP) vaccine platform in which peptides consisting of 10-35 amino acids (preferably 20-35 amino acids) have the ability to induce CD4 and CD8 T cell responses, leading to the eradication of pre-malignant lesions (Kenter et al., 2009; Toes et al., 1996; Bijker et al., 2007), and have shown that when vaccinated during chemotherapy, they improve the overall survival of cancer patients. Cross-presentation of such peptides by host dendritic cells involves several sequential steps, including uptake via endocytosis, intracellular cleavage of SLPs into short peptides by the proteasome, a major proteolytic enzyme, transport across the ER membrane by TAP, and loading into MHC-I molecules (Rosalia et al., 2013).

[0012] The data presented herein show that dendritic cells cannot cross-present the long version of the peptide VLAPRVLRA, identified as being presented at HLA-A*02:01, when extended in the native adjacent sequence. Notably, amino acid substitution of the C-terminal anchor residue with valine (V) results in enhanced T cell stimulation. A single specific amino acid change in the sequence of the VLAPRVLRA peptide (VLAPRVLRV; to SEQ ID NO: 3) enhances the HLA-A of the peptide. * This results in improved binding affinity to 02:01 and more efficient cross-presentation by monocyte-derived dendritic cells. Remarkably, this specific amino acid change allows the peptide to be used as a more effective vaccine.

[0013] The data presented herein demonstrate that minor modifications to the signal peptide epitope preserve the immunogenicity of the TEIPP antigen, making it a suitable candidate for an SLP vaccine. Such a vaccine may represent salvage therapy for immune-evading cancer by activating RCN1-specific T cells.

[0014] Accordingly, the present invention provides a functional variant of VLAPRVLRA (SEQ ID NO: 2) for pharmaceutical use. In this context, a functional variant means a variant of SEQ ID NO: 2 that still elicits a desired immune response (i.e., a T-cell response capable of recognizing and binding to the native TEIPP of SEQ ID NO: 2). The second amino acid L in this peptide functions as the primary anchor of the peptide in the HLA groove, and therefore, substituting this amino acid with an alternative amino acid is possible without adversely affecting TCR binding to the peptide:HLA complex. Accordingly, in the context of the present invention, when providing a peptide for a peptide vaccine, this amino acid may be altered, and the mutant peptide should still elicit a desired immune response (capable of recognizing and binding to VLAPRVLRA, which is the native TEIPP). This also applies to the C-terminal amino acid (A), which may be altered without adversely affecting TCR specificity, as shown herein. With this in mind, the present invention relates to VLAPRVLRA (SEQ ID NO: 2) and its functional variants, which are represented by SEQ ID NO: 1 (VX1APRVLRX2, where X1 and X2 are any amino acids (i.e., A or R or N or D or C or Q or E or G or H or I or L or K or M or F or P or O or S or U or T or W or Y or V)). References to "SEQ ID NO: 1" herein therefore encompass the amino acid sequences of SEQ ID NOs: 2-21.

[0015] Therefore, a peptide containing the amino acid sequence of SEQ ID NO: 1 is provided. Preferably, the peptide may consist of the amino acid sequence of SEQ ID NO: 1.

[0016] Furthermore, nucleic acid sequences encoding the peptide of the present invention are also provided herein. Thus, nucleic acid sequences encoding the amino acid sequence of SEQ ID NO: 1 are provided.

[0017] In addition, the following vaccines will be offered: (a) a peptide comprising or composed of the amino acid sequence of SEQ ID NO: 1, or a nucleic acid encoding the peptide; and (b) pharmaceutically acceptable excipients, adjuvants, diluents and / or carriers.

[0018] The vaccine encodes or contains an immunogenic peptide, which is a peptide having the ability to elicit an immune response, which is directed against the peptide or the epitope presented by the peptide, with the help of a suitable vaccine adjuvant, if desired.

[0019] The vaccines described herein are examples of the pharmaceutical compositions described herein. Therefore, the term "pharmaceutical composition" as used herein encompasses the vaccines described herein.

[0020] In one embodiment, the peptide contains the amino acid sequence VLAPRVLRA (SEQ ID NO: 2). Preferably, the peptide may consist of the amino acid sequence VLAPRVLRA (SEQ ID NO: 2). Preferably, the peptide may contain or consist of any one of the amino acid sequences of SEQ ID NOs: 22, 23, or 24.

[0021] In one embodiment, the peptide comprises the amino acid sequence VLAPRVLRV (SEQ ID NO: 3). Alternatively, the peptide may consist of the amino acid sequence VLAPRVLRV (SEQ ID NO: 3). Alternatively, the peptide may contain or consist of any one of the amino acid sequences of SEQ ID NOs: 25, 26, or 27.

[0022] In one embodiment, the peptide comprises the amino acid sequence VLAPRVLRI (SEQ ID NO: 4). Alternatively, the peptide may consist of the amino acid sequence VLAPRVLRI (SEQ ID NO: 4).

[0023] In one embodiment, the peptide comprises the amino acid sequence VLAPRVLRL (SEQ ID NO: 5). Alternatively, the peptide may consist of the amino acid sequence VLAPRVLRL (SEQ ID NO: 5).

[0024] In one embodiment, the peptide comprises one amino acid sequence from SEQ ID NOs. 6 to 21. Alternatively, the peptide may consist of one amino acid sequence from SEQ ID NOs. 6 to 21.

[0025] In one embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 22. Alternatively, the peptide may consist of the amino acid sequence of SEQ ID NO: 22.

[0026] In one embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 23. Alternatively, the peptide may consist of the amino acid sequence of SEQ ID NO: 23.

[0027] In one embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 24. Preferably, the peptide may consist of the amino acid sequence of SEQ ID NO: 24.

[0028] In one embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 25. Alternatively, the peptide may consist of the amino acid sequence of SEQ ID NO: 25.

[0029] In one embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 26. Alternatively, the peptide may consist of the amino acid sequence of SEQ ID NO: 26.

[0030] In one embodiment, the peptide comprises the amino acid sequence of SEQ ID NO: 27. Alternatively, the peptide may consist of the amino acid sequence of SEQ ID NO: 27.

[0031] Suitablely, the peptide may have a length not exceeding 35 amino acids. Suitablely, the peptide may consist of 10 to 35 amino acids. Suitablely, the peptide may consist of 20 to 35 amino acids.

[0032] Optionally, the peptide may be conjugated to an immunostimulatory compound. Optionally, the immunostimulatory compound comprises a TLR, NLR, RLR, CLR, or ALR ligand.

[0033] Also provided is a nucleic acid encoding the peptide of the invention. Optionally, the nucleic acid may be mRNA or DNA. Optionally, the nucleic acid may be an isolated nucleic acid.

[0034] In one aspect, the invention provides a complex comprising: a) a peptide comprising the amino acid sequence of SEQ ID NO: 1; and b) an agent that specifically binds to the peptide comprising the amino acid sequence of SEQ ID NO: 1. Optionally, the complex may be an isolated complex.

[0035] Optionally, the binding agent may be an HLA-A * 02 molecule. Optionally, the complex may be a complex of HLA-A * 02 with any one of SEQ ID NOs: 2-21. Optionally, the complex may be an HLA-A * 02:VLAPRVLRA complex, HLA-A * 02:VLAPRVLRV complex, HLA-A * 02:VLAPRVLRI complex, or HLA-A * 02:VLAPRVLRL complex.

[0036] ]>Optionally, the binding agent may be an HLA-A * 02:01 molecule. Optionally, the complex may be a complex of HLA-A * 02:o1 with any one of SEQ ID NOs: 2-21. Optionally, the complex may be an HLA-A * 02:01:VLAPRVLRA complex, HLA-A * 02:01:VLAPRVLRV complex, HLA-A * 02:01:VLAPRVLRI complex, or HLA-A * 02:01:VLAPRVLRL complex.

[0037] The complex may also be: a.VLAPRVLRA:HLA-A * 02 complex, VLAPRVLRV:HLA-A * 02 complex, VLAPRVLRI:HLA-A * 02 complex, VLAPRVLRL:HLA-A * 02 complex, VLAPRVLRR:HLA-A * 02 complex, VLAPRVLRN:HLA-A * 02 complex, VLAPRVLRD:HLA-A * 02 complex, VLAPRVLRC:HLA-A * 02 complex, VLAPRVLRQ:HLA-A * 02 complex, VLAPRVLRE:HLA-A * 02 complex, VLAPRVLRG:HLA-A * 02 complex, VLAPRVLRH:HLA-A * 02 complex, VLAPRVLRK:HLA-A * 02 complex, VLAPRVLRM:HLA-A * 02 complex, VLAPRVLRF:HLA-A * 02 complex, VLAPRVLRP:HLA-A * 02 complex, VLAPRVLRS:HLA-A * 02 complex, VLAPRVLRT:HLA-A * 02 complex, VLAPRVLRW:HLA-A * 02 complex, or VLAPRVLRY:HLA-A * 02 Complex, if desired, the complex is VLAPRVLRA:HLA-A * 02 complex, VLAPRVLRV:HLA-A * 02 complex, VLAPRVLRI:HLA-A * 02 complex or VLAPRVLRL:HLA-A * It may also be an O2 complex, and further, if desired, the complex may be VLAPRVLRA:HLA-A * 02 complex, VLAPRVLRV:HLA-A *02 complex or VLAPRVLRI:HLA-A * It may be a 02 complex; or b.VLAPRVLRA:HLA-A * 02:01 complex, VLAPRVLRV:HLA-A * 02:01 complex, VLAPRVLRI:HLA-A * 02:01 complex, VLAPRVLRL:HLA-A * 02:01 complex, VLAPRVLRR:HLA-A * 02:01 complex, VLAPRVLRN:HLA-A * 02:01 complex, VLAPRVLRD:HLA-A * 02:01 complex, VLAPRVLRC:HLA-A * 02:01 complex, VLAPRVLRQ:HLA-A * 02:01 complex, VLAPRVLRE:HLA-A * 02:01 complex, VLAPRVLRG:HLA-A * 02:01 complex, VLAPRVLRH:HLA-A * 02:01 complex, VLAPRVLRK:HLA-A * 02:01 complex, VLAPRVLRM:HLA-A * 02:01 complex, VLAPRVLRF:HLA-A * 02:01 complex, VLAPRVLRP:HLA-A * 02:01 complex, VLAPRVLRS:HLA-A * 02:01 complex, VLAPRVLRT:HLA-A * 02:01 complex, VLAPRVLRW:HLA-A * 02:01 complex or VLAPRVLRY:HLA-A * 02:01 Complex, if desired, the complex is VLAPRVLRA:HLA-A * 02:01 complex, VLAPRVLRV:HLA-A * 02:01 complex, VLAPRVLRI:HLA-A * The complex may be the 02:01 complex or the VLAPRVLRL:HLA-A*02:01 complex, and further optionally the complex may be the VLAPRVLRA:HLA-A* 02:01 complex, VLAPRVLRV:HLA-A * 02:01 complex or VLAPRVLRI:HLA-A * A 02:01 complex is also acceptable.

[0038] In yet another embodiment, the present invention provides cells loaded with the peptide according to the present invention, or cells loaded with or expressing the complex according to the present invention. The cells may suitably be antigen-presenting cells. The antigen-presenting cells may suitably be selected from macrophages, dendritic cells, monocytes, B cells, or synthetic forms of antigen-presenting cells.

[0039] Appropriately, cells may be loaded with a peptide according to the present invention, comprising an amino acid sequence selected from the group consisting of VLAPRVLRA (SEQ ID NO: 2), VLAPRVLRV (SEQ ID NO: 3), VLAPRVLRI (SEQ ID NO: 4), and VLAPRVLRL (SEQ ID NO: 5). For example, cells may be loaded with a peptide comprising an amino acid sequence selected from any one of SEQ ID NOs. 22 to 27. Appropriately, cells may be loaded with VLAPRVLRA:HLA-A * 02:01 complex, VLAPRVLRV:HLA-A * 02:01 complex, VLAPRVLRI:HLA-A * 02:01 complex, and VLAPRVLRL:HLA-A * A complex according to the present invention, selected from the group consisting of 02:01 complexes, may be loaded or expressed.

[0040] In another embodiment, the present invention provides an isolated nucleic acid composition encoding an RCN1 antigen-specific binding protein having a TCRα chain variable (Vα) domain and a TCRβ chain variable (Vβ) domain, the composition comprising a nucleic acid sequence encoding a TCR Vα domain comprising a CDR3 amino acid sequence; and a nucleic acid sequence encoding a TCR Vβ domain comprising a CDR3 amino acid sequence, wherein the CDR3 sequence specifically binds to a peptide comprising the amino acid sequence of SEQ ID NO: 1 (e.g., when the peptide is complexed with HLA). Preferably, the CDR3 sequence may specifically bind to a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 2 to 21 (e.g., when the peptide is complexed with HLA). Preferably, the CDR3 sequence may specifically bind to a peptide comprising the amino acid sequence of VLAPRVLRA (SEQ ID NO: 2), VLAPRVLRV (SEQ ID NO: 3), VLAPRVLRI (SEQ ID NO: 4), or VLAPRVLRL (SEQ ID NO: 5) (e.g., when the peptide is complexed with HLA). Preferably, the CDR3 sequence may bind to a peptide that is HLA-A * When forming a complex with 02:01, it may specifically bind to the peptide. Preferably, the nucleic acid sequence may encode a T cell receptor.

[0041] In one embodiment, the present invention provides an isolated nucleic acid composition encoding an RCN1 antigen-specific binding protein having a TCRα chain variable (Vα) domain and a TCRβ chain variable (Vβ) domain, the composition comprising: (i) a nucleic acid sequence encoding a TCR Vα domain or a functional fragment thereof containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 42; and a nucleic acid sequence encoding a TCR Vβ domain or a functional fragment thereof containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 45, wherein the CDR3 sequence together specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 1 (for example, wherein the CDR3 sequence together specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 2 (for example, when the peptide forms a complex with HLA)); or (ii) a TCR Vα domain containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 52, or a nucleic acid sequence encoding a functional fragment thereof, or a functional fragment thereof; and a TCR Vβ domain containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 55, or a nucleic acid sequence encoding a functional fragment thereof, wherein the CDR3 sequence together specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 1 (for example, wherein the CDR3 sequence together specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 2 (for example, when the peptide forms a complex with HLA)); or (iii) a nucleic acid sequence encoding a TCR Vα domain or a functional fragment thereof containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 62; and a nucleic acid sequence encoding a TCR Vβ domain or a functional fragment thereof containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 65, wherein the CDR3 sequence together specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 1 (for example, where the CDR3 sequence together specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 2 (for example, when the peptide forms a complex with HLA)); or (iv) a nucleic acid sequence encoding a TCR Vα domain or a functional fragment thereof containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 72; and a nucleic acid sequence encoding a TCR Vβ domain or a functional fragment thereof containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 75, wherein the CDR3 sequence together specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 1 (for example, wherein the CDR3 sequence together specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 2 (for example, when the peptide forms a complex with HLA)); or (v) A nucleic acid sequence encoding a TCR Vα domain or a functional fragment thereof containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 82; and a nucleic acid sequence encoding a TCR Vβ domain or a functional fragment thereof containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 85, wherein the CDR3 sequence together specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 1 (for example, where the CDR3 sequence together specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 2 (for example, when the peptide forms a complex with HLA)); or (vi) a nucleic acid sequence encoding a TCR Vα domain or a functional fragment thereof containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 92; and a nucleic acid sequence encoding a TCR Vβ domain or a functional fragment thereof containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 95, wherein the CDR3 sequence together specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 1 (for example, wherein the CDR3 sequence together specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 2 (for example, when the peptide forms a complex with HLA)); or (vii) A nucleic acid sequence encoding a TCR Vα domain or a functional fragment thereof containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 102; and a nucleic acid sequence encoding a TCR Vβ domain or a functional fragment thereof containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 105, wherein the CDR3 sequence together specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 1 (for example, wherein the CDR3 sequence together specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 2 (for example, when the peptide is complexed with HLA)).

[0042] Preferably, the peptide may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 21. Preferably, the peptide may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 5. Preferably, the peptide may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 4. Preferably, the peptide may contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 5.

[0043] The isolated nucleic acid composition encoding an RCN1 antigen-specific binding protein having a TCRα chain variable (Vα) domain and a TCRβ chain variable (Vβ) domain according to the present invention may also include the following: (i) a Vα domain CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 42, and a Vβ domain CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 45; or (ii) A Vα domain CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 52, and a Vβ domain CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 55; or (iii) A Vα domain CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 62, and a Vβ domain CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 65; or (iv) A Vα domain CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 72, and a Vβ domain CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 75; or (v) A Vα domain CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 82, and a Vβ domain CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 85; or (vi) a Vα domain CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 92, and a Vβ domain CDR3 containing or consisting of the amino acid sequence of SEQ ID NO: 95; or (vii) A Vα domain CDR3 containing or comprising the amino acid sequence of SEQ ID NO: 102, and a Vβ domain CDR3 containing or comprising the amino acid sequence of SEQ ID NO: 105.

[0044] The isolated nucleic acid composition encoding an RCN1 antigen-specific binding protein having a TCRα chain variable (Vα) domain and a TCRβ chain variable (Vβ) domain according to the present invention may also include the following: (i) A Vα domain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 46, and comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 46; and a Vβ domain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 48; or (ii) A Vα domain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 56, or comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 56; and a Vβ domain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 58, or (iii) A Vα domain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 66, and comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 66; and a Vβ domain comprising an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 68, or (iv) A Vα domain comprising an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 76, and comprising an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 76; and a Vβ domain comprising an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 78; or (v) A Vα domain comprising an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 86, and comprising an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 86; and a Vβ domain comprising an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 88; or (vi) A Vα domain containing or comprising SEQ ID NO: 96 and having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 96; and a Vβ domain containing or comprising SEQ ID NO: 98 and having an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 98; or (vii) A Vα domain comprising an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 106, and comprising an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 106; and a Vβ domain comprising an amino acid sequence having at least 80% sequence identity with respect to SEQ ID NO: 108.

[0045] Appropriately, the RCN1 antigen-specific binding protein having the encoded TCRα chain variable (Vα) domain and TCRβ chain variable (Vβ) domain may have the ability to specifically bind to a peptide selected from the group consisting of: VX1APRVLRX2:HLA-A * 02 complex (where X1 and X2 are any amino acids), VLAPRVLRA:HLA-A * 02 complex, VLAPRVLRV:HLA-A * 02 complex, VLAPRVLRI:HLA-A * 02 complex, VLAPRVLRL:HLA-A * 02 complex, VLAPRVLRR:HLA-A * 02 complex, VLAPRVLRN:HLA-A * 02 complex, VLAPRVLRD:HLA-A * 02 complex, VLAPRVLRC:HLA-A * 02 complex, VLAPRVLRQ:HLA-A * 02 complex, VLAPRVLRE:HLA-A * 02 complex, VLAPRVLRG:HLA-A * 02 complex, VLAPRVLRH:HLA-A * 02 complex, VLAPRVLRK:HLA-A * 02 complex, VLAPRVLRM:HLA-A * 02 complex, VLAPRVLRF:HLA-A* 02 complex, VLAPRVLRP:HLA-A * 02 complex, VLAPRVLRS:HLA-A * 02 complex, VLAPRVLRT:HLA-A * 02 complex, VLAPRVLRW:HLA-A * 02 complex, and VLAPRVLRY:HLA-A * 02 complex.

[0046] Suitably, the encoded RCN1 antigen-specific binding protein having a TCR α-chain variable (Vα) domain and a TCR β-chain variable (Vβ) domain is a peptide selected from the group consisting of: peptides that can specifically bind to an HLA complex: VX1APRVLRX2:HLA-A * 02:01 complex (where X1 and X2 are any amino acids), VLAPRVLRA:HLA-A * 02:01 complex, VLAPRVLRV:HLA-A * 02:01 complex, VLAPRVLRI:HLA-A * 02:01 complex, VLAPRVLRL:HLA-A*02:01 complex, VLAPRVLRR:HLA-A * 02:01 complex, VLAPRVLRN:HLA-A * 02:01 complex, VLAPRVLRD:HLA-A * 02:01 complex, VLAPRVLRC:HLA-A * 02:01 complex, VLAPRVLRQ:HLA-A * 02:01 complex, VLAPRVLRE:HLA-A * 02:01 complex, VLAPRVLRG:HLA-A * 02:01 complex, VLAPRVLRH:HLA-A * 02:01 complex, VLAPRVLRK:HLA-A * 02:01 complex, VLAPRVLRM:HLA-A * 02:01 complex, VLAPRVLRF:HLA-A * 02:01 complex, VLAPRVLRP:HLA-A * 02:01 complex, VLAPRVLRS:HLA-A *02:01 complex, VLAPRVLRT:HLA-A * 02:01 complex, VLAPRVLRW:HLA-A * 02:01 complex, and VLAPRVLRY:HLA-A * 02:01 Complex.

[0047] Appropriately, encoded RCN1 antigen-specific binding proteins having a TCRα chain variable (Vα) domain and a TCRβ chain variable (Vβ) domain can specifically bind to a peptide selected from the following group: VLAPRVLRA: HLA-A * 02:01 complex, VLAPRVLRV:HLA-A * 02:01 complex, VLAPRVLRI:HLA-A * 02:01 complex, and VLAPRVLRL:HLA-A * 02:01 complex. Appropriately, encoded RCN1 antigen-specific binding proteins, possessing a TCRα chain variable (Vα) domain and a TCRβ chain variable (Vβ) domain, can specifically bind to HLA complexes selected from the following group: LAPRVLRA: HLA-A * 02:01 complex and VLAPRVLRV:HLA-A * 02:01 Complex.

[0048] Suitablely, the CDR3 sequence may together specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (for example, when the peptide is complexed with HLA). Suitablely, the CDR3 sequence may together specifically bind to a peptide containing any one of the amino acid sequences of SEQ ID NOs: 2 to 21 (for example, when the peptide is complexed with HLA). Suitablely, the peptide may contain the amino acid sequences of VLAPRVLRA (SEQ ID NO: 2), VLAPRVLRV (SEQ ID NO: 3), VLAPRVLRI (SEQ ID NO: 4), or VLAPRVLRL (SEQ ID NO: 5).

[0049] Ideally, the CDR3 sequence, together with the peptide, is HLA-A * 02 or HLA-A *When complexed with 02:01, it can specifically bind to the peptide.

[0050] The encoded binding protein may appropriately include a TCR, an antigen-binding fragment of a TCR, a chimeric antigen receptor (CAR), or an ImmTAC.

[0051] In yet another embodiment, the present invention provides an isolated nucleic acid encoding a chimeric antigen receptor protein comprising a target-binding moiety that specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 1. Preferably, the peptide may contain any one of the amino acid sequences of SEQ ID NOs: 2 to 21. Preferably, the peptide may contain the amino acid sequences of VLAPRVLRA (SEQ ID NO: 2), VLAPRVLRV (SEQ ID NO: 3), VLAPRVLRI (SEQ ID NO: 4), or VLAPRVLRL (SEQ ID NO: 5).

[0052] Modified cells are also provided which are transformed, transfected, or transduce with nucleic acids containing nucleic acid sequences encoding the peptides described herein, nucleic acid compositions described herein, nucleic acids described herein, nucleic acid sequences described herein, or vectors described herein. Preferably, the modified cells may be human cells. Preferably, the modified cells may be selected from the group consisting of CD8 T cells, CD4 T cells, NK cells, NKT cells, gamma-delta T cells, innate lymphocytes (ILCs), hematopoietic stem cells, progenitor cells, T cell lines, or NK-92 cell lines.

[0053] In another embodiment, the present invention provides a pharmaceutical composition comprising a) a peptide, b) a nucleic acid, nucleic acid sequence, or nucleic acid composition, c) a complex, or d) a cell according to the present invention, and a pharmaceutically acceptable excipient, adjuvant, diluent, and / or carrier.

[0054] To avoid any doubt, any one of a), b), or c) may be present in the pharmaceutical composition by being encoded or expressed by cells present in the pharmaceutical composition. For example, either b) or c) may be encoded by cells combined with a pharmaceutically acceptable excipient, adjuvant, diluent, and / or carrier to produce the pharmaceutical composition; or either a) or b) may be expressed by cells combined with a pharmaceutically acceptable excipient, adjuvant, diluent, and / or carrier to produce the pharmaceutical composition. Further details relating thereto are provided in the "Detailed Description" below.

[0055] Preferably, the composition may be formulated as a vaccine. Preferably, the composition may be a peptide vaccine. Preferably, the vaccine, peptide vaccine, or composition may contain an immunostimulatory compound.

[0056] The pharmaceutical compositions described herein are provided for pharmaceutical use. The vaccines or peptide vaccines described herein are provided for pharmaceutical use. The pharmaceutical compositions described herein (including nucleic acids, nucleic acid compositions, complexes, or cells according to the present invention) and the vaccines or peptide vaccines described herein are provided for pharmaceutical use. The pharmaceutical compositions described herein and the vaccines or peptide vaccines described herein may be combined for pharmaceutical use. The combination may preferably include administering the vaccine or peptide vaccine before, during, and / or after administration of the pharmaceutical composition.

[0057] Appropriately, the pharmaceutical composition, vaccine and / or peptide vaccine may be used in human subjects for the prevention or treatment of precancerous, cancerous or viral infections associated with impaired HLA class I antigen presentation. Appropriately, the precancerous or cancerous condition may be one in which a peptide processing mechanism is impaired.

[0058] Appropriately, the pharmaceutical composition, vaccine and / or peptide vaccine may be used in human subjects to treat or prevent precancerous, cancerous or viral infections associated with impaired HLA class I antigen presentation, wherein the subject is X1APRVLRX2:HLA-A in a sample isolated from the subject. * 02 complex (where X1 and X2 are any amino acids), VLAPRVLRA:HLA-A * 02 complex, VLAPRVLRV:HLA-A * 02 complex, VLAPRVLRI:HLA-A * 02 complex, VLAPRVLRL:HLA-A * 02 complex, VLAPRVLRR:HLA-A * 02 complex, VLAPRVLRN:HLA-A * 02 complex, VLAPRVLRD:HLA-A * 02 complex, VLAPRVLRC:HLA-A * 02 complex, VLAPRVLRQ:HLA-A * 02 complex, VLAPRVLRE:HLA-A * 02 complex, VLAPRVLRG:HLA-A * 02 complex, VLAPRVLRH:HLA-A * 02 complex, VLAPRVLRK:HLA-A * 02 complex, VLAPRVLRM:HLA-A * 02 complex, VLAPRVLRF:HLA-A * 02 complex, VLAPRVLRP:HLA-A * 02 complex, VLAPRVLRS:HLA-A * 02 complex, VLAPRVLRT:HLA-A * 02 complex, VLAPRVLRW:HLA-A * 02 complex or VLAPRVLRY:HLA-A * The presence of the O2 complex has been identified as indicating precancerous, cancerous, or viral infections associated with impaired HLA class I antigen presentation.

[0059] Appropriately, the pharmaceutical composition, vaccine and / or peptide vaccine may be used in human subjects to treat or prevent precancerous, cancerous or viral infections associated with impaired HLA class I antigen presentation, wherein the subject is X1APRVLRX2:HLA-A in a sample isolated from the subject. * 02:01 complex (where X1 and X2 are any amino acids), VLAPRVLRA:HLA-A * 02:01 complex, VLAPRVLRV:HLA-A*02:01 complex, VLAPRVLRI:HLA-A * 02:01 complex, VLAPRVLRL:HLA-A * 02:01 complex, VLAPRVLRR:HLA-A * 02:01 complex, VLAPRVLRN:HLA-A * 02:01 complex, VLAPRVLRD:HLA-A * 02:01 complex, VLAPRVLRC:HLA-A * 02:01 complex, VLAPRVLRQ:HLA-A * 02:01 complex, VLAPRVLRE:HLA-A * 02:01 complex, VLAPRVLRG:HLA-A * 02:01 complex, VLAPRVLRH:HLA-A * 02:01 complex, VLAPRVLRK:HLA-A * 02:01 complex, VLAPRVLRM:HLA-A * 02:01 complex, VLAPRVLRF:HLA-A*02:01 complex, VLAPRVLRP:HLA-A * 02:01 complex, VLAPRVLRS:HLA-A * 02:01 complex, VLAPRVLRT:HLA-A * 02:01 complex, VLAPRVLRW:HLA-A * 02:01 complex or VLAPRVLRY:HLA-A * The presence of the 02:01 complex has been identified as being associated with precancerous, cancerous, or viral infections related to impaired HLA class I antigen presentation.

[0060] In another embodiment, the present invention provides a method for treating a condition in a human subject in need, the method comprising administering a therapeutically effective amount of a pharmaceutical composition, vaccine and / or peptide vaccine according to the present invention to the subject. Preferably, the treatment method is for the prevention or treatment of precancerous, cancerous, or viral infections associated with impaired HLA class I antigen presentation.

[0061] A method for generating a T cell receptor is provided, comprising contacting a nucleic acid sequence or nucleic acid composition according to the present invention with cells under conditions in which the nucleic acid sequence is taken up by and expressed in cells, to generate a T cell receptor that specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 1 (or the peptide when it is complexed with HLA). Preferably, the peptide may contain any one of the amino acid sequences of SEQ ID NOs: 2 to 21. Preferably, the method is performed in vitro.

[0062] In another embodiment, the present invention provides a use for identifying a therapeutic binding protein of a peptide having the amino acid sequence of SEQ ID NO: 1, or a complex according to the present invention. The peptide may optionally contain or consist of any one of the amino acid sequences of SEQ ID NOs: 2 to 27. The therapeutic binding protein may optionally be capable of preventing or treating precancerous, cancerous, or viral infections associated with impaired HLA class I antigen presentation. The therapeutic binding protein may optionally contain a TCR, an antigen-binding fragment of a TCR, a chimeric antigen receptor (CAR), or an ImmTAC; or the therapeutic binding protein may be an antibody.

[0063] Throughout this description and claims, “including” and “containing,” and their variations, mean “including but not limited to,” and are not intended to exclude, nor shall they exclude, other parts, additions, components, elements, or processes.

[0064] Throughout the description and claims of this specification, unless the context requires otherwise, the singular form includes the plural. In particular, when an indefinite article is used, the specification is to be understood as contemplating both the singular and the plural as well, unless the context requires otherwise.

[0065] Features, elements, properties, compounds, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the present invention are applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

[0066] Various aspects of the present invention are described in further detail below.

Brief Description of the Drawings

[0067] Embodiments of the present invention are further described below with reference to the accompanying drawings.

[0068] In the present specification, the peptide VLAPRVLRA (SEQ ID NO: 2) is also referred to as "peptide 113" or "p113".

[0069] [Figure 1] Figure 1 shows the specificity of two T cell cultures against the peptide VLAPRVLRA. The T cell cultures of donors 2028 and 4409 were stimulated with the peptide VLAPRVLRA (SEQ ID NO: 2), and analyzed by flow cytometry for the percentage of CD8+ T cells and the percentage of CD8+ T cells that were positively stained with HLA-A*02:01 tetramers presenting the peptide VLAPRVLRA. Both VLAPRVLRA-specific T cell cultures contained more than 85% CD8+ T cells, and more than 98% of them were positively stained with HLA-A*02:01 tetramers loaded with VLAPRVLRA (shown as HLA-A*02:01:VLAPRVLRA TM in Figure 1). These VLAPRVLRA-specific T cells were used in the subsequent experiments described below. In the present application and the drawings, the peptide VLAPRVLRA is also referred to as "peptide 113" or "p113".

[0070] [Figure 2-1] Figure 2 shows the preferential recognition of TAP-deficient tumor cells by VLAPRVLRA-specific T cells. VLAPRVLRA-specific T cells preferentially recognized 518A2 melanoma cells and 08.11 melanoma cells when TAP was knocked out, but did not recognize non-tumor cells such as fibroblasts, immortalized human fetal kidney cells 293 (HEK-293T) cells, or normal adult male human kidney-derived proximal tubular cells (HK2) unless TAP was knocked out (HK2 TAP KO). Figures 2A and 2B show the following: T cell cultures from donors 2028 and 4409 stimulated with the peptide VLAPRVLRA (shown as 2028 p113 and 4409 p113, respectively) were stimulated with wild-type (WT) melanoma cells (518A2) with matching HLA-A*02:01, its mutant with TAP knocked out (518A2 TAP KO), and the corresponding peptide at 25 μg / ml. Additionally, culture 4409 was stimulated with HK2 cells, fibroblasts, and HEK-293T cells with matching HLA-A*02:01. Interferon-gamma (IFNγ) production was evaluated for the cultures, and the results are shown on the vertical axis in units of pg / ml. The dashed line indicates the maximum value of the IFNγ dose titration curve. Figure 2C shows the following: T cell culture 4409 stimulated with the peptide VLAPRVLRA (shown as 4409 p113) was stimulated with wild-type (WT) melanoma cells (518A2, 08.11), their TAP knockout mutants (518A2 TAP KO, 8.11 TAP KO), as well as wild-type HK2 cells and the TAP knockout mutant (HK2 TAP KO). Interferon-gamma (IFNγ) production was evaluated for the cultures, and the results are shown on the vertical axis of Figure 2 in pg / ml. The dashed line in Figure 2 indicates the maximum value of the IFNγ dose titration curve. [Figure 2-2] The same as above.

[0071] [Figure 3-1]Figure 3 shows that recognition of Epstein-Barr virus (EBV) transformed B cells, monocytes, or monocyte-derived dendritic cells (DCs) by VLAPRVLRA-specific T cells was not observed. In Figure 3A, p113 VLAPRVLRA-specific T cell cultures from donor 4409 were stimulated with HLA-A*02:01-matched EBV-transformed B cell lines (EBVs) (shown as "T cells + EBV"). Negative controls are shown as "T cells" and "EBVs". Peptide p113 VLAPRVLRA was added as a control for T cell reactivity (shown as "T cells + EBVs + p113"). In Figure 3B, p113 VLAPRVLRA-specific T cell cultures from donor 4409 were stimulated with monocytes (shown as "Mono's 7110"). As a control for T cell reactivity, the peptide p113 VLAPRVLRA was added (shown as "Loaded and Washed Mono's 7110 p113"). Figure 3C shows the following: p113 VLAPRVLRA-specific T cell cultures from donor 4409 were stimulated with monocyte-derived DCs from two healthy donors 0559 and 4553 (shown as "T cells + moDCs 0559" and "T cells + moDCs 4553," respectively). T cells treated with 25 ug / ml of peptide were used as a control for reactivity (shown as "T cells + p113"). T cells only were used as a negative control (shown as "T cells only"). The amount of GM-CSF produced is shown on the y-axis of Figure 3. [Figure 3-2] Same as above.

[0072] [Figure 4-1]Figure 4 shows the expression levels of RCN1 and TAP1, genes encoding peptide 113. RCN1 and TAP1 expression levels were determined in monocytes from two different donors, 2752 and 4409 (indicated as "2752 mono's" and "4409 mono's"), monocyte-derived dendritic cells from two different donors, 2752 and 4409 (indicated as "2752 moDCs" and "4409 moDCs"), 518A2 melanoma cells (indicated as "518A2 WT") and TAP knockout derivatives (indicated as "518A2 TAP KO"), and HK2 cells (indicated as "HK2 WT") and TAP knockout derivatives (indicated as "HK2 TAP KO"). The expression of the indicated genes was examined by qPCR, and measurements were performed using a triple assay method. Ct values ​​were normalized to the expression of housekeeping genes and CPSF6. Non-tumor cell line HK2 expressed the target genes RCN1 and TAP much more strongly than 518A2 melanoma cells, but recognition of HK2 cells was not observed. However, 518A2 melanoma cells that were TAP1 positive but had lower levels compared to HK2, monocytes, or DCs were recognized to some extent (Figure 2). These data clearly demonstrate that peptide 113 VLAPRVLRA-specific CD8+ T cells selectively recognize tumor cells with low TAP levels, and therefore peptide 113 VLAPRVLRA can be classified as a true TEIPP. [Figure 4-2] Same as above.

[0073] [Figure 5]Figure 5 shows the expression levels of RCN1 and TAP1 in 518A2 wild-type cells, 518A2 TAP knockout cells, and 518A2 TAP KO RCN1 knockout cells. RCN1 and TAP1 expression levels were determined in 518A2 wild-type melanoma cells (518A2 WT), 518A2 cells with TAP knocked out (518A2 TAP KO), and 518A2 cells with both TAP and RCN1 knocked out (518A2 TAP / RCN1 KO clone 2A4). The expression of the indicated genes was examined by qPCR, and measurements were performed using a triple assay method. Ct values ​​were normalized to the expression of housekeeping genes and CPSF6. A key feature of TEIPP-specific T cells in clinical applications is the absence of cross-reactivity to peptides derived from other proteins present within the cell. Therefore, the gene encoding peptide 113(RCN1)VLAPRVLRA was knocked out in 518A2 TAP KO melanoma cells.

[0074] [Figure 6-1] Figure 6 shows that peptide 113 (VLAPRVLRA)-specific T cell cultures exhibit single RCN1 protein specificity. Peptide 113 (VLAPRVLRA)-specific T cell cultures from two different donors (indicated as "4409 p113" and "7151 p113") were stimulated with 518A2 wild-type melanoma cells (518A2 WT), 518A2 cells with TAP knockout (518A2 TAP KO), and 518A2 cells with both TAP and RCN1 knockout (518A2 TAP / RCN1 KO clone 2A4). The amounts of interferon-γ (IFNy) and GM-CSF produced are shown on the y axis in pg / ml. When stimulated with 518A2 TAP / RCN1 KO cells, interferon-γ production was not observed at all, while interferon-γ production was observed in 518A2 cells, particularly when TAP was knocked out. This indicates that peptide 113-specific T cells are truly specific to the RCN1-derived peptide VLAPRVLRA. [Figure 6-2] Same as above.

[0075] [Figure 7-1] Figure 7 shows that peptide 113 VLAPRVLRA is not cross-presented by monocyte-derived dendritic cells from a longer synthetic peptide containing native adjacent amino acids. Peptide 113-specific T cell cultures from donor 4409 were stimulated with monocyte-derived dendritic cells from three different donors (MoDCs 0559, MoDC 4553, MoDC 7401) and loaded with specified synthetic long peptides (10 μM, 20-24 hours) ("p113 long 1" (SEQ ID NO: 22), "p113 long 2" (SEQ ID NO: 23), "p113 long 3" (SEQ ID NO: 24)) or stimulated without peptides as a background control (indicated as "no peptide"). Cells with 25 μg / ml of the short p113 peptide VLAPRVLRA (indicated as "T cells + p113") were used as a positive control for reactivity. The amounts of interferon-γ (IFNy) and GM-CSF produced are shown in pg / ml on the y axis. None of the SLP variants were cross-presented to T cells, and only exogenous pulsing of the short VLAPRVLRA peptide stimulated T cells. These results indicate that cross-presentation from the long peptide region of the VLAPRVLRA epitope is inefficient and requires optimization for vaccine applications. [Figure 7-2] Same as above.

[0076] [Figure 8-1]Figure 8 shows that anchor substitution is required for cross-presentation of the RCN1-derived peptide VLAPRVLRA from its long peptide sequence. Peptide 113-specific T cell cultures from donors 4409 and 2028 were stimulated with monocyte-derived dendritic cells from two different HLA-A*02:01 matched donors (indicated as "MoDC donor 0559", "MoDC donor 7401", and "MoDC donor 4553"). They were either loaded with specified synthetic long peptides (10 μM, 20-24 hours) ("p113 long V1" (SEQ ID NO: 25), "p113 long V2" (SEQ ID NO: 26), "p113 long V3" (SEQ ID NO: 27)) or stimulated without peptides as a background control (indicated as "no peptides"). Cells treated with 25 μg / ml of wild-type p113 peptide VLAPRVLRA (indicated as "T cells + p113" or "p113") or the V variant VLAPRVLRV (p113V: SEQ ID NO: 3) (indicated as "T cells + p113V" or "p113V") were used as a positive control for reactivity, while T cells without the peptide were used as a negative control (indicated as "T cells"). The amounts of interferon-γ (IFNy) and GM-CSF generated are shown in pg / ml on the y axis. None of the native A-SLP mutants were able to activate T cell culture (see Figure 7), except for the N-terminal extended V-SLP peptide (p113 long V1), which was presented after being treated with monocyte-derived dendritic cells. [Figure 8-2] Same as above.

[0077] [Figure 9]Figure 9 shows that T cells transduced to express different unique HLA-A*02:01-restricted RCN121-29 CD8 T cell receptors, identified by the inventors, allow the mouse TCRb antibody and HLA-A*02:01 molecule to bind to an MHC multimer displaying the peptide RCN121-29 VLAPRVLRA. The TCRα and TCRβ chains of each TCR were cloned into retroviral expression vectors and transduced into T cells to generate T cells expressing T cell receptors for the RCN121-29 VLAPRVLRA peptide, respectively. These T cells were analyzed by flow cytometry. The transgenes contain a mouse-derived TCR-Cβ domain, which facilitates the correct pairing of the transgenic α and β chains. Expression of this domain was confirmed after incubation of TCR-transduced T cells with the mouse TCRb antibody. T cell specificity was evaluated by positive staining analysis of the MHC multimer of the HLA-A*02:01 molecule that presents the peptide RCN121-29 VLAPRVLRA.

[0078] [Figure 10] Figure 10 shows the identification of HLA-presenting peptides FLGPWPAAS, FLGPWPAAV, VLAPRVLRA, and SLGDWGAEA in different isolated human primary tumor samples. After obtaining various human primary tumor samples, their HLA ligandomes were measured by mass spectrometry using standard techniques in the art. The primary tumor samples included four melanoma samples (Mel0401, Mel1207, Mel1213, and Mel1502), three ovarian cancer samples (OVAL1, OVAL10, and OVAL23), and two cervical cancer samples (COV362-4 and COV413b). The table in Figure 10 shows whether the four possible HLA-presenting peptides (FLGPWPAAS, FLGPWPAAV, VLAPRVLRA, and SLGDWGAEA) were present (+) or absent (none) in each sample after HLA ligandome analysis. VLAPRVLRA has been observed to be present in many samples, suggesting that it may be an attractive target for therapeutic intervention.

[0079] [Figure 11] Figure 11 shows that T cells introduced to express different intrinsic HLA-A*02:01 restriction RCN121-29 CD8 T cell receptors identified by the inventors may be activated by TAP-deficient tumor cells. Figure 11A: TCRs generated by TCR gene transduction and enriched after tetramer-induced MACS fractionation were cultured overnight with HLA-A*02:01-compatible wild-type (WT), TAP-deficient (TAP KO), and TAP and RCN1-deficient (TAP / Ag KO) 518A2 tumor cells, shown from left to right in the figure, and cultured either alone (T cells only) or with 25 μg / ml homologous peptide (T cells + peptide). The amounts of GM-CSF, IFNy, and CCL4 produced are shown for TCR1, 2, 4, 5, 6, 9, and 13. Figure 11B: TCRs generated by TCR gene transfection and enriched after tetramer-induced MACS fractionation were cultured overnight with HLA-A*02:01-compatible wild-type (WT) or TAP-deficient (TAP KO) 08.11 tumor cells, shown from left to right in the figure. The amounts of CCL4 produced are shown for TCR4 and 9. The experiment in Figure 11 was performed using the materials and methods described in the Examples section below. The TCRs used in Figure 11 are the same as those in the Examples, and their sequences are listed in Tables 7-13. It was observed that the best TAP-deficient tumor recognition was achieved by RCN121-29(p113)-specific TCR4, TCR5, and TCR9.

[0080] Detailed explanation Immunogenic peptides The present invention provides a peptide containing the amino acid sequence of SEQ ID NO: 1. The present invention provides a peptide containing the amino acid sequence of SEQ ID NO: 2 (VLAPRVLRA). The present invention provides a peptide containing the amino acid sequence of SEQ ID NO: 3 (VLAPRVLRV). The present invention provides a peptide containing the amino acid sequence of SEQ ID NO: 4 (VLAPRVLRI). The present invention provides a peptide containing the amino acid sequence of SEQ ID NO: 5 (VLAPRVLRL). The present invention provides a peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 21. Preferably, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 2 to 5. More preferably, the amino acid sequence is selected from the group consisting of SEQ ID NOs: 2 to 4, or the group consisting of SEQ ID NOs: 3 to 5. The peptide containing the amino acid sequence of SEQ ID NO: 2 contains the peptides of SEQ ID NOs: 22 to 24. Therefore, a reference to a peptide containing SEQ ID NO: 2 is to be construed as explicitly including the peptides of SEQ ID NOs: 22 to 24. Similarly, the peptide containing the amino acid sequence of SEQ ID NO: 3 contains the peptides of SEQ ID NOs: 25 to 27. Therefore, a reference to a peptide containing SEQ ID NO: 3 is to be construed as explicitly including the peptides of SEQ ID NOs: 25 to 27.

[0081] Preferably, the peptide is an isolated peptide.

[0082] This specification also provides a nucleic acid molecule encoding the above peptide. A nucleic acid encoding the amino acid sequence of SEQ ID NO: 1 is provided. This specification provides a nucleic acid encoding any one of the amino acid sequences of SEQ ID NOs: 1 to 27. Any suitable nucleic acid sequence encoding any one of the amino acid sequences of SEQ ID NOs: 1 to 27 can be used. These can be appropriately selected by those skilled in the art.

[0083] In this specification, "isolated peptide" means a peptide that is not found in its natural environment. Therefore, such peptides may be of synthetic origin (or they may be of natural origin and isolated from their natural environment), and in the context of this specification, the natural environment for these peptides is the human body. Therefore, even if a peptide is present in a pharmaceutical composition (including adjuvants, etc.), it is considered to be in an isolated form because it is not found in its natural environment.

[0084] The peptide described in the present invention may be part of a pharmaceutical composition, which may contain the following: (a) the peptide; and (b) pharmaceutically acceptable excipients, adjuvants, diluents and / or carriers. Such compositions may be referred to herein as vaccines (i.e., peptide vaccines).

[0085] Furthermore, the nucleic acids described in the present invention may be part of a pharmaceutical composition, and such composition may contain the following: (a) Nucleic acids encoding the peptide of the present invention; and (b) pharmaceutically acceptable excipients, adjuvants, diluents and / or carriers. Such compositions may be referred to herein as vaccines (i.e., nucleic acid vaccines).

[0086] An example of such a vaccine containing a nucleic acid sequence encoding the aforementioned peptide, and optionally containing the peptide, is an mRNA vaccine. Preferably, the vaccine according to the present invention may be an mRNA vaccine.

[0087] The peptide of the present invention may consist of only one amino acid sequence from SEQ ID NOs: 1 to 27. Alternatively, the peptide may contain additional amino acids and therefore may contain one amino acid sequence from SEQ ID NOs: 1 to 27.

[0088] In one example, a peptide containing the amino acid sequence VLAPRVLRV (SEQ ID NO: 3) (e.g., any one of SEQ ID NOs. 25-27) may have a higher binding affinity to HLA-A*02 than an equivalent peptide in which the VLAPRVLRV (SEQ ID NO: 3) sequence is substituted with VLAPRVLRA (SEQ ID NO: 2). In a specific example, a peptide containing the amino acid sequence VLAPRVLRV (SEQ ID NO: 3) (e.g., any one of SEQ ID NOs. 25-27) may have a higher binding affinity to HLA-A*02:01 than an equivalent peptide in which the VLAPRVLRV (SEQ ID NO: 3) sequence is substituted with VLAPRVLRA (SEQ ID NO: 2). Methods for determining the binding affinity of a peptide to an HLA molecule are well known in the art (see, for example, the experiments included in the "Examples" section below). Methods for determining the binding affinity of a peptide to an HLA molecule are also well known in the art, particularly as described in Van der Burg et al. 1995 and Van der Burg et al. 1996.

[0089] In a further example, cross-presentation of a peptide containing the amino acid sequence VLAPRVLRV (SEQ ID NO: 3) (e.g., any one of the peptides SEQ ID NOs: 25-27) by monocyte-derived dendritic cells may be more efficient (improved / higher) than when the VLAPRVLRV (SEQ ID NO: 3) sequence is substituted with VLAPRVLRA (SEQ ID NO: 2) in an equivalent peptide. In other words, a peptide containing the amino acid sequence VLAPRVLRV (SEQ ID NO: 3) (e.g., any one of the peptides SEQ ID NOs: 25-27) may be presented more efficiently by monocyte-derived dendritic cells than when the VLAPRVLRV (SEQ ID NO: 3) sequence is substituted with VLAPRVLRA (SEQ ID NO: 2) in an equivalent peptide.

[0090] Methods for determining the effectiveness of cross-presentation of specific peptides by monocyte-derived dendritic cells are well known in the art. For example, the cross-presentation of long peptides of 10-35 amino acids containing equivalent epitope peptides can be evaluated using monocyte-derived dendritic cells (DCs) and CD8+ T cell clones that recognize the peptide VLAPRVLRA in an HLA class I context. Monocyte-derived DCs are obtained by incubating peripheral blood mononuclear cells with anti-CD14 magnetic beads at 4°C for 20 minutes, and then separating CD14-positive monocytes using a magnetic separation column. The obtained CD14+ monocytes are cultured for 6 days in RPMI medium supplemented with 10% FCS, GM-CSF (800 units / ml), and IL-4 (500 units / ml) to produce immature monocyte-derived dendritic cells. On day 6, immature monocyte-derived DCs are incubated with synthetic long-chain peptides at different concentrations (e.g., 20 μg / ml, 10 μg / ml, 5 μg / ml) for 24 hours, and matured on day 7 with LPS (20 ng / ml) stimulation. The cross-presentation of peptides by these monocyte-derived DCs is monitored by the reactivity of peptide-loaded monocyte-derived DCs and CD8+ T cell clones co-cultured at different ratios (e.g., T cells 10:DC1, T cells 5:DC1, T cells 1:DC1). The reactivity of CD8+ T cell clones can be evaluated by measuring cytokine production (e.g., GM-CSF or interferon-γ) in the co-culture supernatant and compared with control co-cultures of DCs loaded with unrelated HLA class I binding peptides or DCs without peptides. As a positive control, monocyte-derived DCs may be loaded with the natural short-chain epitope VLAPRVLRA.

[0091] In a further example, the peptides described herein may have equivalent or higher binding affinity to HLA-A*02 (e.g., HLA-A*02:01) and / or may be presented at an equivalent or more efficient (improved / higher) level in monocyte-derived dendritic cells compared to equivalent peptides containing VLAPRVLRA.

[0092] As will be apparent to those skilled in the art, the term "equivalent peptide" as used above means a peptide having the same amino acid sequence except for the differences described.

[0093] In some embodiments, the peptide may be up to 35 amino acids long; for example, it may be up to 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 9 amino acids long. In one example, the peptide may be up to 30 amino acids long. In another example, the peptide may be up to 29 amino acids long. In one example, the peptide may be up to 28 amino acids long. In another example, the peptide may be up to 27 amino acids long. In yet another example, the peptide may be up to 26 amino acids long. In yet another example, the peptide may be up to 25 amino acids long. In yet another example, the peptide may be up to 24 amino acids long. In yet another example, the peptide may be up to 23 amino acids long.

[0094] Peptides of different lengths have been shown to be particularly effective as peptide vaccines. For example, Ossendorp (Ossendorp et al., (1998)) described that peptides with a length of 9 to 19 amino acids can induce a CD4+ helper T cell response. Therefore, the peptides of the present invention may be 9 to 19 amino acids in length.

[0095] Peptides longer than the conventional 9-residue sequences presented by HLA may be more efficient in inducing immune responses. Therefore, following the teachings of Ossendorp (Ossendorp et al., (1998)), the peptides described herein may be 10 to 19 amino acids in length.

[0096] Bijker (Bijker et al., (2007)) showed that a 9-residue HPV CTL epitope can induce a CD8+ response to RAHYNIVTF, but that a 35-residue peptide containing this epitope is more efficient. Furthermore, Beyranvand-Nejad (Beyranvand-Nejad et al., (2016)) showed that a 35-residue HPV peptide is effective in inducing RAHYNIVTF-specific CD8+ T cells. Furthermore, Rahimian et al. (2015) showed that a 27-residue HPV peptide is effective in inducing a response to RAHYNIVTF. Therefore, in accordance with these teachings, the peptides described herein may be 9 to 35 amino acids in length. To avoid misunderstanding, in this context, a peptide has a total of 9 to 35 amino acids, which includes any of the sequences of SEQ ID NOs. 1 to 21. In other words, a peptide may have any of the sequences of SEQ ID NOs. 1 to 21 and 0 to 26 additional amino acids. The 0 to 26 additional amino acids in the peptide may be located at the N-terminus or C-terminus of any of the sequences of SEQ ID NOs. 1 to 21. Alternatively, if 0 to 26 additional amino acids are present, they may be adjacent to any of the sequences of SEQ ID NOs. 1 to 21 (i.e., additional amino acids may be present at both the N-terminus and C-terminus of the sequences of SEQ ID NOs. 1 to 21). Additional amino acids located at the N-terminus, C-terminus, or both sides of any of the sequences of SEQ ID NOs. 1 to 21 are collectively referred to herein as "additional amino acids."

[0097] Alternatively, to avoid misunderstanding, in this context, a peptide may have a total of 10 to 35 amino acids, which may include any of the sequences of SEQ ID NOs. 1 to 21. In other words, a peptide may have any of the sequences of SEQ ID NOs. 1 to 26 and 1 to 26 additional amino acids. The 1 to 26 additional amino acids of the peptide may be located at the N-terminus or C-terminus of any of the sequences of SEQ ID NOs. 1 to 21. Alternatively, if 1 to 26 additional amino acids are present, they may be located on both sides of any of the sequences of SEQ ID NOs. 1 to 21 (i.e., the additional amino acids may be present at both the N-terminus and C-terminus of the sequences of SEQ ID NOs. 1 to 21).

[0098] Furthermore, in one example, the peptides described herein may be 15 to 30 amino acids long and may contain 6 to 21 additional amino acids. The additional amino acids may be located at the N-terminus or C-terminus of any of the sequences of SEQ ID NOs. 1 to 21. Alternatively, the additional amino acids may be located on both sides of any of the sequences of SEQ ID NOs. 1 to 21 (i.e., additional amino acids may be present at both the N-terminus and C-terminus of the sequences of SEQ ID NOs. 1 to 21).

[0099] Furthermore, in one example, the peptides described herein may be 18 to 27 amino acids long and may contain 9 to 18 additional amino acids. The additional amino acids may be located at the N-terminus or C-terminus of any of the sequences of SEQ ID NOs. 1 to 21. Alternatively, the additional amino acids may be located on both sides of any of the sequences of SEQ ID NOs. 1 to 21 (i.e., additional amino acids may be present at both the N-terminus and C-terminus of the sequences of SEQ ID NOs. 1 to 21).

[0100] In another example, the peptides of this disclosure are 21 to 24 amino acids in total length and may have 12 to 15 additional amino acids. The additional amino acids may be located at the N-terminus or C-terminus of any of the sequences of SEQ ID NOs. 1 to 21. Alternatively, the additional amino acids may be adjacent to any of the sequences of SEQ ID NOs. 1 to 21 (i.e., the additional amino acids may be present at both the N-terminus and C-terminus of any of the sequences of SEQ ID NOs. 1 to 21).

[0101] Examples of peptides containing the amino acid sequence of SEQ ID NO: 2 and having additional amino acids are shown in SEQ ID NOs: 22-24. Examples of peptides containing the amino acid sequence of SEQ ID NO: 3 and having additional amino acids are shown in SEQ ID NOs: 25-27.

[0102] In another example, the peptide of the Disclosure may have 27 amino acids. In yet another example, the peptide of the Disclosure may have 24 amino acids. In yet another example, the peptide of the Disclosure may have 21 amino acids. In a particular example, the peptide of the Disclosure may have 18 amino acids. Preferably, the peptide of the Disclosure is 24 amino acids. In this context, a peptide consists of, for example, 27, 24, 21, or 18 amino acids and includes any of the sequences of SEQ ID NOs: 1 to 21. In other words, a peptide has any of the sequences of SEQ ID NOs: 1 to 21 and a suitable number of additional amino acids (i.e., to produce a peptide of 27, 24, 21, or 18 amino acids in total length).

[0103] The N-terminus of a peptide (also called the amino terminus, NH2 terminus, N-terminal terminus, or amine terminus) is the starting end of the peptide, terminated with an amino acid that has a free amino group (-NH2). By convention, peptide sequences are written from the N-terminus to the C-terminus (from left to right). The C-terminus (also called the carboxyl terminus, C-terminal tail, C-terminal end, or COOH terminus) is the end of an amino acid chain (protein or polypeptide) terminated with a free carboxyl group (-COOH).

[0104] As used herein, the terms “N-terminus” and “C-terminus” are used to indicate, for example, the relative position of a sequence within a peptide. Therefore, a sequence designated as “N-terminus” is located relatively close to the N-terminus of the peptide. Conversely, a domain designated as “C-terminus” is located relatively close to the C-terminus of the peptide. As used herein, the term “located” refers to the position of a sequence within the linear amino acid sequence of the peptide.

[0105] Peptides containing an N-terminal amino acid sequence (A) and a C-terminal amino acid sequence (B) are conventionally described as AB, i.e., from the N-terminus to the C-terminus (left to right).

[0106] If the peptides of this disclosure contain additional amino acids located at the N-terminus, C-terminus, or both sides thereof of any of the sequences of SEQ ID NOs: 1 to 21, they may contain appropriate additional amino acid sequences. For example, the additional amino acids may be amino acid sequences that naturally occur at the N-terminus, C-terminus, or both sides thereof of the VLAPRVLRA sequence in RCN1. In a particular example, all additional amino acids may be located at the N-terminus of any of the sequences of SEQ ID NOs: 1 to 21 and are sequences that naturally occur at the N-terminus of the VLAPRVLRA sequence in RCN1. In another example, all additional amino acids may be located at the C-terminus of any of the sequences of SEQ ID NOs: 1 to 21 and are sequences that naturally occur at the C-terminus of the VLAPRVLRA sequence in RCN1. Alternatively, the additional amino acids may be positioned adjacent to any of the sequences of SEQ ID NOs: 1 to 21 (i.e., additional amino acids are present at the N-terminus and C-terminus of SEQ ID NOs: 1 to 21) and may be natural sequences adjacent to the VLAPRVLRA sequence in RCN1. Preferably, all additional amino acids are located at the N-terminus of the VLAPRVLRV sequence, and optionally, the additional amino acid sequence may be a sequence naturally occurring at the N-terminus of the VLAPRVLRA sequence in RCN1. In this case, the target peptide sequence (e.g., VLAPRVLRV) is located at the C-terminus of the peptide of the present invention.

[0107] A peptide comprising any of the sequences of Sequence IDs 1 to 21 and consisting of 10 to 35 amino acids in total length may contain an appropriate additional amino acid sequence. For example, the additional amino acids may be amino acid sequences naturally occurring at the N-terminus, C-terminus, or both sides of the VLAPRVLRA sequence in RCN1.

[0108] In another example, the additional 1 to 26 amino acids may all be located at the N-terminus of any sequence of SEQ ID NOs: 1 to 21, preferably VLAPRVLRA, VLAPRVLRV, VLAPRVLRI, or VLAPRVLRL, and may be sequences naturally present at the N-terminus of the VLAPRVLRA sequence in RCN1. In yet another example, the additional 1 to 26 amino acids may all be located at the C-terminus of any one of SEQ ID NOs: 1 to 21, preferably VLAPRVLRA, VLAPRVLRV, VLAPRVLRI, or VLAPRVLRL, and may be sequences naturally present at the C-terminus of the VLAPRVLRA sequence in RCN1. Alternatively, if an additional 1 to 26 amino acids are present, the additional amino acids may be adjacent to any one of sequence numbers 1 to 21, preferably VLAPRVLRA, VLAPRVLRV, VLAPRVLRI, or VLAPRVLRL (i.e., the additional amino acids are present at the N-terminus and C-terminus of any one of sequence numbers 1 to 21, preferably VLAPRVLRA, VLAPRVLRV, VLAPRVLRI, or VLAPRVLRL), or they may be natural sequences adjacent to the VLAPRVLRA sequence in RCN1.

[0109] Furthermore, in certain examples, a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the amino acid sequences of VLAPRVLRA in SEQ ID NO: 2, VLAPRVLRV in SEQ ID NO: 3, VLAPRVLRI in SEQ ID NO: 4, or VLAPRVLRL in SEQ ID NO: 5) and consisting of 15 to 30 amino acids may also contain any appropriate additional amino acid sequences. For example, the additional amino acids may be naturally occurring amino acid sequences located at the N-terminus, C-terminus, or adjacent position of the VLAPRVLRA sequence in RCN1. For example, the additional 6 to 21 amino acids may all be located at the N-terminus of the sequence of SEQ ID NO: 1 (e.g., the VLAPRVLRA, VLAPRVLRV, VLAPRVLRI, or VLAPRVLRL sequence) and may be naturally occurring sequences present at the N-terminus of the VLAPRVLRA sequence in RCN1. In another example, the additional 6–21 amino acids may all be located at the C-terminus of the sequence of SEQ ID NO: 1 (e.g., the VLAPRVLRA, VLAPRVLRV, VLAPRVLRI, or VLAPRVLRL sequence) and may be native sequences present at the C-terminus of the VLAPRVLRA sequence in RCN1. Alternatively, if the additional 6–21 amino acids are present, the additional amino acids may be adjacent to any one of SEQ ID NOs: 1–21, preferably the VLAPRVLRA, VLAPRVLRV, VLAPRVLRI, or VLAPRVLRL sequence (i.e., the additional amino acids may be present at the N-terminus and C-terminus of any one of SEQ ID NOs: 1–21, preferably the VLAPRVLRA, VLAPRVLRV, VLAPRVLRI, or VLAPRVLRL sequence) and may be native sequences adjacent to the VLAPRVLRA sequence in RCN1.

[0110] In another example, a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the amino acid sequences of VLAPRVLRA in SEQ ID NO: 2, VLAPRVLRV in SEQ ID NO: 3, VLAPRVLRI in SEQ ID NO: 4, or VLAPRVLRL in SEQ ID NO: 5) and consisting of 18 to 27 amino acids may also contain any suitable additional amino acid sequences. For example, the additional amino acids may be naturally occurring amino acid sequences located at the N-terminus, C-terminus, or adjacent position of the VLAPRVLRA sequence in RCN1. For example, the additional 9 to 18 amino acids may all be located at the N-terminus of the sequence of SEQ ID NO: 1 (e.g., the VLAPRVLRA, VLAPRVLRV, VLAPRVLRI, or VLAPRVLRL sequence) and may be naturally occurring sequences present at the N-terminus of the VLAPRVLRA sequence in RCN1. In another example, the additional 9–18 amino acids may all be located at the C-terminus of the sequence of Sequence ID No. 1 (e.g., the VLAPRVLRA, VLAPRVLRV, VLAPRVLRI, or VLAPRVLRL sequence) and may be native sequences present at the C-terminus of the VLAPRVLRA sequence in RCN1. Alternatively, if the additional 9–18 amino acids are present, they may be adjacent to the sequence of Sequence ID No. 1 (e.g., the VLAPRVLRA, VLAPRVLRV, VLAPRVLRI, or VLAPRVLRL sequence) (i.e., the additional amino acids may be present at the N-terminus and C-terminus of the sequence) and may be native sequences adjacent to the VLAPRVLRA sequence in RCN1.

[0111] In yet another example, a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the amino acid sequences of SEQ ID NO: VLAPRVLRA, SEQ ID NO: 3, VLAPRVLRV, SEQ ID NO: 4, or VLAPRVLRL) and consisting of 21 to 24 amino acids may also contain any suitable additional amino acid sequences. For example, the additional amino acids may be naturally occurring amino acid sequences located at the N-terminus, C-terminus, or adjacent position of the VLAPRVLRA sequence in RCN1. For example, all 12 to 15 additional amino acids may be naturally occurring sequences located at the N-terminus of the SEQ ID NO: 1 sequence (e.g., the VLAPRVLRA, VLAPRVLRV, VLAPRVLRI, or VLAPRVLRL sequence) and located at the N-terminus of the VLAPRVLRA sequence in RCN1. In another example, the additional 12–15 amino acids may all be located at the C-terminus of the sequence of Sequence ID No. 1 (e.g., the VLAPRVLRA, VLAPRVLRV, VLAPRVLRI, or VLAPRVLRL sequence) and may be native sequences present at the C-terminus of the VLAPRVLRA sequence in RCN1. Alternatively, if the additional 12–15 amino acids are present, they may be adjacent to the sequence of Sequence ID No. 1 (e.g., the VLAPRVLRA, VLAPRVLRV, VLAPRVLRI, or VLAPRVLRL sequence) (i.e., the additional amino acids may be present at the N-terminus and C-terminus of the sequence) and may be native sequences flanking the VLAPRVLRA sequence in RCN1.

[0112] A suitable natural sequence derived from RCN1 is provided in the Examples section below.

[0113] For example, a peptide may contain an additional amino acid at its N-terminus, and may contain, for example, the following sequence: GRGRRLGLALGLLLALVLAPRVLRA (SEQ ID NO: 22). This is an example of a 25-residue sequence with an additional N-terminal amino acid, but other lengths, such as 18, 21, 24, 27 residues, etc., are also acceptable.

[0114] In another example, the peptide may contain additional amino acids at the N-terminus and C-terminus, for example, the sequence:ALGLLLALVLAPRVLRAKPTVRKER (SEQ ID NO: 23). This is an example of a 25-residue peptide with additional N-terminus and C-terminus amino acids, but other lengths, such as 18, 21, 24, 27 residues, etc., are also acceptable.

[0115] In another example, the peptide may contain an additional amino acid at the C-terminus, for example, the sequence: VLAPRVLRAKPTVRKERVVRPDSEL (SEQ ID NO: 24). This is an example of a 25-residue peptide with an additional C-terminal amino acid, but other lengths, such as 18, 21, 24, or 27 residues, are also acceptable.

[0116] For example, a peptide may contain an additional amino acid at its N-terminus, such as the sequence:GRGRRLGLALGLLLALVLAPRVLRV (SEQ ID NO: 25). This is an example of a 25-residue peptide with an additional N-terminal amino acid, but other lengths, such as 18, 21, 24, or 27 residues, are also acceptable.

[0117] In another example, the peptide may contain additional amino acids at its N-terminus and C-terminus, for example, the sequence of SEQ ID NO: 26: ALGLLLALVLAPRVLRVKPTVRKER. This example is a 25-amino acid peptide having additional amino acids at its N-terminus and C-terminus, but other lengths, such as 18 residues, 21 residues, 24 residues, 27 residues, etc., are also acceptable.

[0118] In another example, the peptide may contain a C-terminal additional amino acid, for example, the sequence of SEQ ID NO: 27: VLAPRVLRVKPTVRKERVVRPDSEL. This example is a 25-amino acid peptide with a C-terminal additional amino acid, but other lengths, such as 18 residues, 21 residues, 24 residues, 27 residues, etc., are also acceptable.

[0119] In one example, the peptide may contain the amino acid sequence of SEQ ID NO: 22. In another example, the peptide consists of the amino acid sequence of SEQ ID NO: 22. In one example, the peptide may contain the amino acid sequence of SEQ ID NO: 23. In another example, the peptide consists of the amino acid sequence of SEQ ID NO: 23. In yet another example, the peptide may contain the amino acid sequence of SEQ ID NO: 24. In yet another example, the peptide consists of the amino acid sequence of SEQ ID NO: 24. In yet another example, the peptide may contain the amino acid sequence of SEQ ID NO: 25. In yet another example, the peptide consists of the amino acid sequence of SEQ ID NO: 25. In yet another example, the peptide may contain the amino acid sequence of SEQ ID NO: 26. In yet another example, the peptide consists of the amino acid sequence of SEQ ID NO: 26. In yet another example, the peptide may contain the amino acid sequence of SEQ ID NO: 27. In yet another example, the peptide consists of the amino acid sequence of SEQ ID NO: 27.

[0120] Other suitable natural sequences derived from RCN1 may be identified by those skilled in the art. For example, they may be identified using the full-length RCN1 sequence shown in Sequence ID No. 28: MARGGRGRRLGLALGLLLALVLAPRVLRAKPTVRKERVVRPDSELGERPPEDNQSFQYDHEAFLGKEDSKTFDQLTPDESKERLGKIVDRIDNDGDGFVTTEELKTWIKRVQKRYIFDNVAKVWKDYDRDKDDKISWEEYKQATYGYYLGNPAEFHDSSDHHTFK KMLPRDERRFKAADLNGDLTATREEFTAFLHPEEFEHMKEIVVLETLEDIDKNGDGFVDQDEYIADMFSHEENGPEPDWVLSEREQFNEFRDLNKDGKLDKDEIRHWILPQDYDHAQAEARHLVYESDKNKDEKLTKEEILENWNMFVGSQATNYGEDLTKNHDEL

[0121] In alternative examples, the additional amino acids may be the N-terminus, C-terminus, or adjacent non-naturally located amino acid sequence of the VLAPRVLRA sequence in RCN1. Both natural and non-natural adjacent sequences have been shown to be useful in peptide vaccines and can therefore be used in the peptides described herein. For example, the SIINFEKL epitope of the OVA antigen has been successfully used as a peptide vaccine when it is adjacent to its natural sequence (Bijker et al., (2007)), a non-natural C-terminal adjacent sequence (Varypataki et al., (2015)), or when it lacks an N-terminal adjacent sequence and is conjugated with a glycine linker to which a helper epitope is attached at the C-terminus (i.e., completely outside the context of its own natural adjacent sequence) (Masuko et al., (2015)). Furthermore, Chen (Chen et al., (2016)) has described that 15 CTL epitope vaccines conjugated to the next epitope by a small non-natural linker induce priming to the epitope. Therefore, non-natural additional amino acid sequences at the N-terminus and / or C-terminus may be acceptable in peptide vaccine form.

[0122] The peptides may be “natural peptides,” that is, peptides composed of natural amino acids. Such peptides are composed of conventional amino acids defined by the genetic code and linked to each other by ordinary peptide bonds. Natural peptides may be produced, for example, by cells (via protein expression, for example, using nucleic acids or vectors as described herein), or synthesized extracellularly by chemical synthesis (i.e., extracellularly, using chemical synthesis).

[0123] Alternatively, the peptide may be a "synthetic peptide." A synthetic peptide may contain a mixture of natural amino acids and amino acids other than conventional amino acids defined by the genetic code ("synthetic amino acids"). Alternatively, it may consist solely of synthetic amino acids. Examples of synthetic amino acids are well known in the literature.

[0124] Natural and synthetic peptides may be modified. That is, peptides may contain amino acids modified by natural processes (such as post-translational maturation) or chemical processes. These modifications are well known to those skilled in the art. These modifications can appear at any position within the peptide: within the peptide backbone, within the amino acid chain, or at the carboxyl or amino terminus. Examples of non-limiting peptide modifications include amino acid additions such as acetylation, acylation, ADP-ribosylation, amidation, covalent immobilization of nucleotides or nucleotide derivatives, covalent immobilization of lipids or lipid derivatives, covalent immobilization of phosphatidylinositol, covalent or non-covalent crosslinking, cyclization, disulfide bond formation, demethylation, glycosylation (including PEGylation), hydroxylation, iodization, methylation, myristoylation, oxidation, protease treatment, phosphorylation, prenylation, racemization, seneloylation, sulfated, arginylation, or ubiquitination. These modifications are described in detail in the literature. Therefore, the terms “peptide,” “polypeptide,” and “protein” may include, for example, lipopeptides, lipoproteins, glycopeptides, glycoproteins, and so on. In more non-limiting examples, peptides may be branched by ubiquitination, or may be cyclic with or without branching. This type of modification may be the result of natural or synthetic post-translational processes well known to those skilled in the art.

[0125] The peptides described herein may be conjugated directly or via a linker to a therapeutic site, polymer, polypeptide, ligand, and / or other site, such as a detectable site. Such peptides are referred to herein as “peptide conjugates.”

[0126] The peptides described herein may be bound to immunostimulatory compounds. The peptides described herein may be bound to nonspecific immunostimulatory compounds. The peptide conjugate may contain a peptide covalently bound to an immunostimulatory compound. The peptide conjugate may contain a peptide covalently bound to a nonspecific immunostimulatory compound. The peptides described herein may be included in compositions or vaccines containing immunostimulatory compounds. Accordingly, this specification provides compositions or vaccines containing any peptide (or a nucleic acid molecule encoding such peptide) and an immunostimulatory compound according to the present invention. This specification also provides compositions or vaccines containing any peptide or nucleic acid sequence encoding such peptide and an immunostimulatory compound according to the present invention.

[0127] Immunostimulatory compounds are compounds that stimulate the immune system by inducing the activation of any component of the immune system or by increasing its activity. Immunostimulatory compounds include specific immunostimulatory compounds and nonspecific immunostimulatory compounds. Specific immunostimulatory compounds provide antigen specificity in the immune response, such as any antigen. Nonspecific immunostimulatory compounds act regardless of antigen specificity to enhance the immune response to other antigens or to stimulate components of the immune system, such as adjuvants, without antigen specificity. For example, immunostimulatory compounds may activate receptors of the innate immune system. For example, immunostimulatory compounds may activate pattern recognition receptors.

[0128] The immunostimulatory compound may contain a damage-associated molecular pattern (DAMP). The immunostimulatory compound may contain a pathogen-associated molecular pattern (PAMP). The immunostimulatory compound may contain a ligand for a nucleotide-bound oligomerized domain-like receptor (NLR), such as NOD1 or NOD2. The peptides described herein may be bound to an NLR ligand. The peptide conjugate may contain a peptide covalently bound to an NLR ligand. The immunostimulatory compound may contain a ligand for a RIG-I-like receptor (RLR), such as RIG-I, MDA5, or LGP2. The peptides described herein may be bound to an RLR ligand. The peptide conjugate may contain a peptide covalently bound to an RLR ligand. The immunostimulatory compound may contain a ligand for a C-type lectin receptor (CLR), such as Dectin-1 or Dectin-2. The peptides described herein may be bound to a CLR ligand. The peptide conjugate may contain a peptide covalently bound to a CLR ligand. The immunostimulatory compound may contain a receptor ligand, such as ALR (absent-in-melanoma-2-like receptor). The peptides described herein may be bound to an ALR ligand. The peptide conjugate may contain a peptide covalently bound to an ALR ligand.

[0129] Specific ligands that bind to each receptor are known in the art (for example, disclosed in “Pattern recognition receptors in health and diseases,” Li & Wu, Signal Transduction and Targeted Therapy, 6;291, 2021), and any such ligand or effective variant thereof can be appropriately selected by those skilled in the art as immunostimulatory compounds according to the present invention.

[0130] The immunostimulatory compound may contain a Toll-like receptor (TLR) ligand. The immunostimulatory compound may also be a TLR ligand. The peptides described herein may be conjugated to a TLR ligand. The peptide conjugate may be formed by covalent attachment of the peptide to the TLR ligand.

[0131] TLR ligands may also be referred to as TLR agonists. As used herein, “TLR agonist” is a TLR agonist that binds to TLRs and activates them, in particular, to produce a biological response. “TLR peptide agonist” as used herein refers to a TLR agonist that is a peptide. The above descriptions of TLR agonists are understood to apply equally to NLRs, RLRs, CLRs, and ALRs.

[0132] Peptide conjugates containing TLR agonists covalently bound to peptides, particularly TLR agonists covalently bound to synthetic peptides, are well known to those skilled in the art. For example, Zom (Zom et al., (2018)) described a conjugate of the TLR2 ligand Pam3CSK4 and a synthetic long-chain peptide (SLP). Furthermore, Zom (Zom et al., (2016)) described the binding of a synthetic long-chain peptide encoded by human papillomavirus type 16 (HPV16) to a Pam3CSK4-based TLR2 agonist.

[0133] Toll-like receptors (TLRs) are transmembrane proteins having extracellular, transmembrane, and cytoplasmic domains. The extracellular domain contains leucine-rich repeats (LRRs) with a horseshoe-shaped structure and is involved in the recognition of common molecular patterns from diverse microorganisms. Toll-like receptors include TLR1-10. Compounds capable of activating TLR receptors, as well as their modifications and derivatives, have been well described in the literature by those skilled in the art. TLR1 may be activated by bacterial lipoproteins and their acetylated derivatives. TLR2 may also be activated by glycolipids, LPS, LPA, LTA, pili, outer membrane proteins, bacterial or host-derived heat shock proteins, and mycobacterial lipoarabinomannan from Gram-positive bacteria. TLR3 may be activated by double-stranded RNA, particularly viral-derived, or the chemical poly(LC). TLR4 may be activated by LPS, LTA from Gram-negative bacteria, host or bacterial-derived heat shock proteins, viral capsule or envelope proteins, taxol or its derivatives, hyaluronic acid-containing oligosaccharides, and fibronectin. TLR5 may be activated by bacterial flagella or flagellin. TLR6 may be activated by mycobacterial lipoproteins and group B streptococcal thermounstable soluble factor (GBS-F) or staphylococcal modulin. TLR7 may be activated by imidazoquinoline. TLR9 may be activated by unmethylated CpG DNA or chromatin-IgG complexes.

[0134] TLRs are expressed on the cell surface (TLR1, 2, 4, 5, 6, and 10) or on intracellular organelle membranes such as endosomes (TLR3, 4, 7, 8, and 9). The native ligands for endosomal receptors are nucleic acid-based molecules (except for TLR4). TLR1, 2, 4, 5, 6, and 10, expressed on the cell surface, recognize molecular patterns of extracellular microorganisms (Monie et al., (2009)). Although TLRs are expressed in multiple cell types, virtually all TLRs are expressed on DCs, and these specialized cells are capable of sensing all pathogens and danger signals.

[0135] TLR2, 4, and 5 are constitutively expressed on the surface of dendritic cells (DCs). TLR2 can detect a wide range of ligands from bacteria, viruses, parasites, and fungi. Ligand specificity is often determined by interactions with other TLRs such as TLR1, 6, and 10, and non-TLR molecules such as dectin-1, CD14, and CD36. By forming heterodimers with TLR1, TLR2 can identify triacyllipoproteins and lipopeptides (such as Pam3CSK4 and peptidoglycan (PGA)) from (myco)bacteria (Gay et al., (2007); Spohn et al., (2004)). Heterodimerization of TLR2 and 6 enables the detection of niacyllipopeptides and dimazanes. Lipopolysaccharides (LPS) and their derivatives are ligands for TLR4, and flagellin is a ligand for TLR5 (Bryant et al., (2010)). TLR2 interacts with a wide range of structurally diverse ligands, including molecules expressed by microorganisms and fungi. Natural and synthetic lipopeptides (e.g., Mycoplasma fermentas macrophage-activating lipopeptide (MALP-2)), peptidoglycans (e.g., PG from S. aureus), lipopolysaccharides (LPS) from various bacterial strains, polysaccharides (e.g., dimazan), and glycosylphosphatidylinositol anchor structures from Gram-positive bacteria (e.g., lipoteichoic acid (LTA), lipoarabinomannan from mycobacteria, lipomannan from M. tuberculosis) have been identified as TLR2 agonists. Certain viral determinants may also be induced via TLR2 (Barbalat et al., (2009)). Bacterial lipopeptides are components of the cell wall. They contain acylated S-glycerylcysteine ​​groups and can bind to peptides via cysteine ​​residues. Examples of TLR2 agonists include the bacterial lipopeptide MALP-2 and its synthetic analogs, dipalmitoyl-S-glycerylcysteine ​​(Pam2Cys) and tripalmitoyl-S-glycerylcysteine ​​(Pam3Cys).

[0136] TLR4 interacts with a diverse range of ligands. Examples include monophosphorylated lipid A (MPLA) from Salmonella Minnesota R595, lipopolysaccharide (LPS), mannan (Candida albicans), glycoinositol phospholipid (Trypanosoma), viral membrane proteins (RSV and MMTV), and endogenous antigens including fibrinogen and heat shock proteins. Such TLR4 agonists are described, for example, by Akira (Akira et al., 2006) and Kumar (Kumar et al., 2009). LPS present on the outer membrane of Gram-negative bacteria is the most widely studied TLR4 ligand. Suitable LPS-derived TLR4 agonist peptides are described, for example, in WO2013 / 120073(A1).

[0137] TLR5 is induced by the region of the flagellin molecule expressed by almost all motile bacteria. Therefore, flagellin or flagellin-derived peptides and proteins, as well as flagellin variants and fragments, are also suitable as TLR peptide agonists included in the peptide conjugate of the present invention.

[0138] In such non-limiting examples, TLR peptide agonists include the TLR2 lipopeptide agonists MALP-2, Pam2Cys and Pam3Cys or their modifications, various forms of TLR4 agonists (e.g., N. meningitidis wild-type L3-LPS and the mutant pentaacylated LpxL1-LPS), and the TLR5 agonist flagellin. Further non-limiting examples of TLR2 peptide agonists include annexin II or its immunomodulatory fragments, which are described in detail in WO2012 / 048190A1 and U.S. Patent Application No. 13 / 033,1546.

[0139] In more non-restrictive examples, human high-mobility box-1 protein (HMGB1) and its peptide fragments are considered to be TLR4 agonists. Such HMGB1-derived peptides are described, for example, in US2011 / 0236406A1.

[0140] The peptide conjugate of the present invention may contain at least one TLR agonist. Preferably, the peptide conjugate may contain multiple TLR agonists, particularly 2, 3, 4, 5, 6, 7, 8, 9, 10 or more TLR agonists.

[0141] At least one TLR, NLR, RLR, CLR, or ALR agonist contained in the peptide conjugate of the present invention may be the same or different. Preferably, the various TLR, NLR, RLR, CLR, and / or ALR agonists contained in the peptide conjugate of the present invention may be different from each other.

[0142] It is understood that multiple different TLR agonists activating the same or different TLR receptors may be advantageously included in the single peptide conjugate of the present invention.

[0143] The immunostimulatory compound may also be an adjuvant. In some cases, the adjuvant may be combined with any of the TLR, NLR, RLR, CLR, or ALR agonists described herein.

[0144] The adjuvant may be selected from mineral salts, emulsifiers, and a group of microparticles. Preferably, the mineral salt may be an aluminum salt. The emulsifier may be an oil-in-water emulsifier or a water-in-oil emulsifier. Examples of suitable emulsifiers are Complete Freund's adjuvant, Incomplete Freund's adjuvant, MF59, AS03, and ISA51. Montanide ISA51 (Seppic, France) is an oil-in-water emulsifier consisting of mineral oil and a surfactant derived from the mannide monooleate family, and is a suitable adjuvant for vaccines as used herein. Examples of microparticles include virus-like particles and viromosomes. Virus-like particles are non-infectious, genetically-non-transparent nanoparticles whose outer layer consists of an immunogenic epitope. They are typically icosahedral or rod-shaped nanoparticles (20-200 nm in diameter) with a capsid protein shell.

[0145] The pharmaceutical compositions (e.g., vaccines) described herein may be administered to human subjects to treat or prevent cancer or viral infection associated with HLA class I antigen presentation disorders (e.g., as peptide vaccines). For example, the pharmaceutical compositions (e.g., vaccines) may be administered to subjects to induce or enhance an immune response (e.g., as peptide vaccines). Thus, the pharmaceutical compositions (e.g., vaccines) may be administered to subjects to induce T cell activation (e.g., in vivo T cell activation), and the activated T cells are specific to the peptides and therefore specifically target cancer cells or virus-infected cells.

[0146] The pharmaceutical composition (e.g., vaccine) according to the present invention may be administered as a peptide vaccine for the treatment or prevention of cancer or viral infection associated with HLA class I antigen presentation disorder. The pharmaceutical composition (e.g., vaccine) may be administered to induce or enhance the activation of T cells specific to cancer cells or virus-infected cells.

[0147] The nucleic acid sequences and vectors encoding the peptides according to the present invention may be administered as nucleic acid vaccines for the treatment or prevention of cancer or viral infections associated with HLA class I antigen presentation disorders. The isolated nucleic acid sequences and vectors may be administered to induce or enhance T cell activation specific to cancer cells or virus-infected cells.

[0148] Peptide vaccines and nucleic acid vaccines are examples of vaccines.

[0149] Cross-presentation of long-chain peptides (e.g., SLPs as described herein) by dendritic cells includes endocytosis, intracytoplasmic cleavage of SLPs by proteasomes, transport across the endoplasmic reticulum membrane by TAP, and loading onto MHC-I molecules (Rosalia et al. 2013).

[0150] The peptide according to the present invention (and the corresponding nucleic acid sequence or vector encoding it) is HLA-A * It may be particularly useful as an immunotherapy for O2-positive human subjects.

[0151] HLA-A02 is a globally common human leukocyte antigen serotype belonging to the HLA-A serotype group. * Within group 02, HLA-A * 02:01, HLA-A0 * 2:02, HLA-A * 02:03, HLA-A * 02:04, HLA-A * 02:05, HLA-A * 02:06, HLA-A * 02:09, HLA-A * 02:11, HLA-A * 02:12, HLA-A * 02:16, HLA-A * 02:19, HLA-A * Multiple subtypes exist, such as 02:50. The data presented herein is HLA-A. * Although the focus is on 02:01, as will be obvious to those skilled in the art, HLA-A * Other subtypes within group 02 (not limited to those described herein) may bind to any of the amino acid sequences of SEQ ID NOs. 1-21 (see, e.g., Ressing et al., 1999, particularly Tables 3 and 2). Therefore, all HLA-A * The 02 subtype is included in the present invention, but HLA-A * 02:01 is preferred (see Table 1 below). In the application of the present invention, HLA-A * 02 subtype HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04 and HLA-A *02:09 is also preferable. This HLA-A2 variant has been shown to exhibit comparable binding characteristics according to the A2 supertype (Ressing et al., 1999 and MF Del Guercio et al., J. Immunol. 1995. 154: 685-693).

[0152] [Table 1]

[0153] nucleic acid sequence The isolated nucleic acid sequences encoding the peptide of the present invention are described herein, as are the nucleic acid sequences encoding the binding factors.

[0154] As used herein, “nucleic acid sequence,” “polynucleotide,” “nucleic acid,” and “nucleic acid molecule” are used interchangeably to refer to an oligonucleotide sequence or a polynucleotide sequence. Therefore, the term “nucleic acid sequence” may be replaced with the term “nucleic acid” herein. A nucleotide sequence may be of genomic, synthetic, or recombinant origin, and may be double-stranded or single-stranded (representing a sense strand or antisense strand). The term “nucleotide sequence” includes genomic DNA, cDNA, synthetic DNA, RNA (e.g., mRNA), and analogs of DNA or RNA (e.g., those produced by the use of nucleotide analogs). In one example, the nucleotide sequence lacks introns. In other words, it is a nucleic acid sequence that does not contain introns. For example, the nucleotide sequence may be a DNA sequence that does not contain intron sequences.

[0155] As used herein, “isolated nucleic acid sequence” or “isolated nucleic acid composition” means that the nucleic acid is not linked to a naturally associated sequence (which is also in its natural environment) in its natural environment. In other words, an isolated nucleic acid sequence / composition is not a native nucleotide sequence / composition, while “native nucleotide sequence / composition” means that the sequence is in its natural environment and is functionally linked to all naturally associated promoters (which are also in their natural environment). Such nucleic acid may be part of a vector, and such nucleic acid or polypeptide may be part of a composition (e.g., a cell lysate), but the vector or composition is isolated because it is not part of the natural environment of the nucleic acid or polypeptide. The term “gene” means a portion of DNA involved in the formation of a polypeptide chain, including the pre- and post-coding regions ("leader and trailer") and intervening sequences (introns) between individual coding segments (exons).

[0156] The nucleic acid sequence of the present invention may be a nucleic acid sequence that does not exist in nature (for example, the entire sequence may not exist in its entirety in nature). For example, the nucleic acid sequence of the present invention may be functionally linked to a promoter, where the promoter is not naturally associated in nature with an equivalent human nucleic acid sequence (e.g., a human TCR sequence or a fragment thereof); that is, the promoter is not one that the nucleic acid as a whole is naturally associated with in its natural environment. In this context, such a promoter may be considered an exogenous promoter. Examples of suitable promoters are described elsewhere.

[0157] Vectors and modified cells In one aspect, the present invention provides a vector comprising the nucleic acid sequence described herein (for example, a nucleic acid sequence encoding a peptide comprising the amino acid sequence of SEQ ID NO: 1).

[0158] A vector system comprising the nucleic acid composition described herein is also provided. The vector system may have one or more vectors. As described above, the binding protein components encoded in the nucleic acid composition may be encoded by one or more nucleic acid sequences in the nucleic acid composition. If all binding protein components are encoded by a single nucleic acid sequence, that nucleic acid sequence may reside in a single vector (and therefore the vector system described herein may contain only a single vector). If the binding protein components are encoded by two or more nucleic acid sequences (where multiple nucleic acid sequences cooperate to encode all components of the binding protein), these two or more nucleic acid sequences may reside in a single vector (e.g., in different open reading frames of the vector) or may be distributed across two or more vectors. In this example, the vector system would include multiple different vectors (i.e., vectors having different nucleotide sequences).

[0159] Therefore, in one example, a vector system comprising the nucleic acid composition described herein is provided.

[0160] Any suitable vector can be used. In the example, the vector may be a plasmid, cosmid, or viral vector such as a retroviral vector or lentiviral vector. Adenoviruses, adeno-associated viruses, vaccinia viruses, canary poxviruses, herpesviruses, minicircle vectors, and naked (synthetic) DNA / RNA can also be used (for details on minicircle vectors, see, for example, the nonviral Sleeping Beauty translocation from minicircle vectors published by Monjezi et al. (Leukemia 2016)). Alternatively, the desired TCR can be introduced into lymphocytes using single-stranded or double-stranded DNA or RNA (see Roth et al 2018 Nature vol 559; page 405).

[0161] In the example, the vector is a plasmid, viral vector, or cosmid, and optionally the vector may be selected from the group consisting of retroviruses, lentiviruses, adeno-associated viruses, adenoviruses, vaccinia viruses, canarypox viruses, herpesviruses, minicircle vectors, and synthetic DNA or RNA.

[0162] Where required in this specification, the term “vector” means a nucleic acid sequence capable of carrying other nucleic acid sequences operatively ligated thereto. The vector may be autonomously replicable and may be incorporated into host DNA. The vector may contain restriction enzyme sites for the insertion of recombinant DNA and may contain one or more selection markers or suicide genes. The vector may be a nucleic acid sequence in the form of a plasmid, bacteriophage, or cosmid. Preferably, the vector is suitable for intracellular expression (i.e., the vector is an “expression vector”). Preferably, the vector is suitable for expression in human antigen-presenting cells. Preferably, the vector is CD8 + T cells, CD4 + It is suitable for expression in human T cells such as T cells, stem cells, iPS cells, or NK cells. In certain embodiments, the vector is a viral vector such as a retroviral vector, lentiviral vector, or adeno-associated vector. Optionally, the vector may be selected from the group consisting of adenovirus, vaccinia virus, canarypox virus, herpesvirus, minicircle vector, synthetic DNA, or synthetic RNA.

[0163] Preferably, the (expression) vector is capable of growing within host cells and is reliably transmitted to future generations. Suitable vectors and expression vectors are well known to those skilled in the art.

[0164] The vector may contain a regulatory sequence. Optionally, the vector may contain a nucleic acid sequence of interest operatively ligated to a promoter. The promoter may not be naturally occurring in the host cell (e.g., an exogenous promoter). "Operationally ligated" means that the regulatory elements, individually or in combination, and the coding sequence have a functional relationship with each other, for example, a relationship that directs the expression of the coding sequence.

[0165] Those skilled in the art are familiar with molecular techniques available for the preparation of (expression) vectors and methods by which (expression) vectors may be introduced or transfected into suitable host cells (thereby producing the modified cells described herein). The (expression) vectors of the present invention can be introduced into cells by prior art such as transformation, transduction, or transtransfer.

[0166] "Transformation," "transfection," and "transduction" generally refer to techniques for introducing exogenous nucleic acid sequences into host cells, and therefore include methods such as electroporation, microinjection, gene-oncogenesis, transduction using retroviruses, lentiviruses, and adeno-associated vectors, lipofection, and superfection. The specific method used usually depends on both the type of vector and the type of cell. Appropriate methods for introducing nucleic acid sequences and vectors into host cells such as human cells are well known to those skilled in the art, for example, see Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Ausubel et al. (1987) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY; Cohen et al. (1972) Proc. Natl. Acad. Sci. USA 69, 2110; Luchansky et al. (1988) Mol. Microbiol. 2, 637-646. Furthermore, prior art for preparing expression vectors and introducing them into suitable host cells is described in detail, for example, WO2016 / 071758.

[0167] It is understood that in some embodiments, host cells are contacted with a vector (e.g., a viral vector) in vitro and in vitro, and in some further embodiments, host cells are contacted with a vector (e.g., a viral vector) in vivo.

[0168] The term “host cell” includes any cell into which the nucleic acid sequence or vector described herein may be introduced (e.g., a transduced cell). When a nucleic acid molecule or vector is introduced into a cell, it may be referred to herein as a “modified cell.” Once a nucleic acid molecule or vector is introduced into a host cell, the resulting modified cell should be capable of expressing the encoded polypeptide (and, for example, appropriately localizing the encoded binding protein for its intended function, e.g., transporting the encoded binding protein to the cell surface).

[0169] Nucleic acid compositions or vector systems may be introduced into cells using any conventional method well known to those skilled in the art. For example, nucleic acid compositions or vector systems may be introduced using CRISPR technology. This includes CRISPR / Cas9 and insertion of nucleic acid sequences into endogenous TCR loci by homologous recombination repair (HDR) or non-homologous end joining (NHEJ). Other conventional methods such as transfection, transduction, and transformation can also be used.

[0170] The term "modified cell" refers to a cell that has been genetically modified (e.g., transformed, transfected, or transfected). A modified cell contains at least one exogenous nucleic acid sequence (i.e., a nucleic acid sequence not naturally present in the host cell). This term refers to a specific target cell, and also applies to its offspring or potential offspring. Subsequent generations may undergo modifications due to mutation or environmental factors, but even if the offspring are not entirely identical to the parent cell, they are still included within the scope of the term as used herein.

[0171] In one example, the modified cell comprises a nucleic acid composition or vector system provided herein. In another example, the modified cell comprises a nucleic acid comprising a nucleic acid sequence encoding a peptide provided herein. Any suitable nucleic acid sequence encoding a peptide is available and may be appropriately selected by those skilled in the art.

[0172] The host cell (and therefore modified cell) may be a bacterial cell, but is usually a eukaryotic cell, and is often a human cell capable of overexpressing antigens for uptake by antigen-presenting cells (APCs). More specifically, it is preferable that the host cell (and therefore modified cell) be an antigen-presenting cell such as a dendritic cell (DC), B cell, monocyte, or macrophage. The host cell (and therefore modified cell) may be an autologous cell, meaning a cell obtained from the same individual to which the treatment will be administered. In other words, the host cell (and therefore modified cell) may be a cell derived from the target of treatment. If appropriate, the host cell (and therefore modified cell) may be isolated from a blood sample by methods such as monocyte removal. The modified cell is usually a human cell. The host cell (and therefore modified cell) may be any cell capable of conferring antitumor immunity after TCR gene introduction. Non-limiting examples of suitable cells include autologous or allogeneic CD8 T cells, CD4 T cells, natural killer (NK) cells, NKT cells, gamma-delta T cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells or other progenitor cells, as well as other autologous or allogeneic cells or cell lines capable of conferring antitumor immunity (e.g., NK-92 or T cell lines).

[0173] Therefore, in one example, the modified cells are selected from the group consisting of CD8 T cells, CD4 T cells, NK cells, NK-T cells, gamma-delta T cells, innate lymphoid cells (ILCs), hematopoietic stem cells, induced pluripotent stem cells, progenitor cells, T cell lines, and NK-92 cell lines.

[0174] In the context of the therapeutic methods described herein, the host cells (and therefore modified cells) to be administered to the subject may be autologous or allogeneic.

[0175] Preferably, the modified cells are capable of expressing polypeptides encoded by the nucleic acid sequences or vectors described herein, thereby enabling the modified cells to provide immunotherapy that specifically targets cancer cells or virus-infected cells with impaired HLA class I antigen presentation, and can be used to treat or prevent cancer or viral infections with impaired HLA class I antigen presentation. Details of this use are given below.

[0176] Method for preparing peptides As described above, the peptide according to the present invention may be a natural peptide or a synthetic peptide. In other words, the peptide according to the present invention may be modified.

[0177] Methods for preparing the peptides of the present invention are also provided herein. In one aspect, the methods for preparing the peptides of the present invention provided herein may be natural methods. In another aspect, the methods for preparing the peptides of the present invention may be synthetic methods. Alternatively, the methods for preparing the peptides of the present invention may include both natural and synthetic methods.

[0178] The methods for preparing the peptides of the present invention provided herein may be natural methods. Such methods include culturing modified cells transformed, transfected, or transfected with a nucleic acid (e.g., a vector) encoding the peptide of interest in a culture medium, and separating the peptide from the medium or modified cell lysate after cell lysis. In this context, the modified cells are used to express the peptide of interest. Examples of such cells include, but are not limited to, bacterial cells (e.g., E. coli) and eukaryotic cells (e.g., yeast cells, animal cells, or plant cells). In one example, the cells are mammalian cells, e.g., human cells, CHO, HEK293T, PER.C6, NS0, myeloma, or hybridoma cells. Dendritic cells and dendritic cell lines are particularly preferred.

[0179] Typically, as described above, the nucleic acid encoding the target peptide resides within a vector, such as an expression vector. In some cases, an appropriate secretion signal can be incorporated into the vector so that the peptide encoded by the target nucleic acid is guided toward, for example, the lumen of the endoplasmic reticulum, the periplasmic space, the membrane, or the extracellular environment. The selection of an appropriate secretion signal facilitates subsequent protein purification. The selection of an appropriate secretion signal is usually possible within the ordinary skill of a person skilled in the art. Typically, the selection of the culture medium depends, in particular, on the selection of the cell type and / or cell line used to express the target peptide. A person skilled in the art is familiar with culture media suitable for the selected cell type and / or cell line.

[0180] The cells are cultured in a suitable culture medium for a period sufficient to induce the expression of the encoded peptide. The appropriate duration and conditions for cell culture are well known to those skilled in the art and depend on the specific cell type and / or cell line used.

[0181] After the peptide has been expressed by cells, it may be purified using standard methods. For example, commercially available kits and / or reagents for protein extraction, such as Novagen's BugBuste®, may be used. Other standard methods that may be used include affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and immunoaffinity chromatography.

[0182] Alternatively, the peptides of the present invention may be prepared by synthetic methods. Such methods are described in detail in the literature. Non-limiting examples include liquid-phase peptide synthesis and solid-phase peptide synthesis, such as the Merrifield method, t-Boc solid-phase peptide synthesis, Fmoc solid-phase peptide synthesis, and solid-phase peptide synthesis using BOP (Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate).

[0183] Peptide-carrying cells Cells carrying the peptides described herein may also be provided. These cells may be advantageously used in the therapeutic methods described below.

[0184] In this specification, a cell “loaded” with a peptide may refer to a cell in which the peptide is bound to the MHC (major histocompatibility complex) on its cell surface. Typically, a peptide-loaded cell does not express the peptide itself, but presents the exogenous peptide in the context of the MHC. The cell may be pulsed with the exogenous peptide to “load” the peptide. Therefore, a peptide-loaded cell may also be called a cell possessing the target peptide (e.g., an exogenous peptide), where the target peptide is part of the MHC complex on the cell surface. In other words, such a cell has MHC present extracellularly (or on the cell surface) that forms a complex with the target peptide. The presence of a peptide within the MHC of an antigen-presenting cell is referred to herein as “antigen presentation.” Antigen presentation means expressing an antigen molecule on the surface of a macrophage or other antigen-presenting cell, meaning that when the antigen is presented to CD4+ helper T cells, it is presented in association with an MHC class II molecule, and when it is presented to CD8+ cytotoxic T cells, it is presented in association with an MHC class I molecule.

[0185] Alternatively, the cells may be modified cells transduced, transformed, or transduced with nucleic acids containing the nucleic acid sequence encoding the peptide described herein. Since such modified cells express the peptide, it is also possible to load the peptide into the same cells or other cells.

[0186] Cells carrying the peptides defined herein may be cells derived from the target being treated. In particular, they may be cells isolated from the target being treated. Alternatively, cell lines such as antigen-presenting cell lines may also be used.

[0187] Preferably, the cells carrying the peptides defined herein may be antigen-presenting cells (APCs). Preferably, the antigen-presenting cells may be selected from dendritic cells (DCs), macrophages, monocytes, B cells, and synthetic forms of antigen-presenting cells. In particular, dendritic cells (conventional and / or plasmacytoid dendritic cells) isolated from the target of treatment are most preferred.

[0188] Methods for isolating antigen-presenting cells, particularly dendritic cells, from a subject are well known to those skilled in the art. These include methods for collecting monocytes or hematopoietic stem cells from bone marrow, umbilical cord blood, or peripheral blood. Furthermore, the use of embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) is also included. Antigen-presenting cells, particularly dendritic cells or their progenitor cells, can be enriched by methods such as elution or separation using magnetic beads, which may include enrichment of CD14+ progenitor cells.

[0189] Methods for loading the complexes defined herein onto the aforementioned antigen-presenting cells, more preferably dendritic cells, and for preparing these cells before administration to a target, are well known to those skilled in the art. For example, the preparation of dendritic cells may include culture or differentiation using cytokines, which may contain GM-CSF and IL-4, etc. Dendritic cell lines may also be used.

[0190] A method for loading peptides into cells, preferably APCs, and more preferably dendritic cells, may include co-culturing the peptides with the cells. Additional culture to efficiently mature the thus loaded cells, such as dendritic cells, may include the addition of cytokines, including IL-1β, IL-6, TNFα, PGE2, IFNα, and adjuvants. Suitable methods and reagents are well known to those skilled in the art.

[0191] Pharmaceutical composition A pharmaceutical composition is provided comprising a peptide (a), a nucleic acid and / or vector (b), a complex or binder (c), or a cell as described herein (d), and a pharmaceutically acceptable excipient, adjuvant, diluent and / or carrier.

[0192] To avoid any doubt, any of a), b), or c) may be present in the pharmaceutical composition by being encoded or expressed by cells present in the pharmaceutical composition. For example, either b) or c) may be encoded by cells combined with pharmaceutically acceptable excipients, adjuvants, diluents, and / or carriers to produce the pharmaceutical composition. Alternatively, either a) or b) may be expressed by cells combined with pharmaceutically acceptable excipients, adjuvants, diluents, and / or carriers to produce the pharmaceutical composition. Further details are provided below.

[0193] Particularly suitable compositions may be selected based on the target HLA serotype. Further details are provided elsewhere in this specification.

[0194] The nucleic acids, vectors, complexes, cells, binders, and / or peptides described herein may be provided as part of a pharmaceutical composition. Preferably, such composition may be administered to a person and used to treat or prevent cancer or viral infection in which HLA class I antigen presentation is impaired (for example, by inducing or enhancing a specific immune response against such cancer cells or virus-infected cells).

[0195] In this specification, the terms "pharmaceutical composition" and "composition" are used synonymously unless otherwise specified in the context.

[0196] The pharmaceutical composition may include nucleic acid sequences, vectors, complexes, binders, cells, and / or peptides as described herein, along with pharmaceutically acceptable excipients, adjuvants, diluents, and / or carriers.

[0197] To avoid any doubt, nucleic acid sequences, vectors, complexes, binders, and / or peptides may be present in a pharmaceutical composition as part of a cell. In other words, nucleic acid sequences or vectors may be incorporated into cells, and binders, complexes, or peptides may be expressed by cells. The cells may be, for example, bacterial cells, or mammalian cells, such as eukaryotic cells, such as dendritic cells (DCs) (in which case the mammalian cells are usually extracorporeal cells). Therefore, pharmaceutical compositions comprising nucleic acid sequences, vectors, complexes, binders, and / or peptides as described herein also include pharmaceutical compositions comprising cells (e.g., bacterial cells, DCs, etc.) that encode the nucleic acid sequence or vector, or that are capable of expressing the peptide, complex, or binder.

[0198] Preferably, the cells (e.g., bacterial cells, DCs, etc.) may be modified to introduce a suitable nucleic acid sequence / vector into the cell (e.g., by transduction, transfection, or transformation), so that the modified cells encode the nucleic acid sequence / vector and are capable of expressing the nucleic acid sequence, vector, complex, peptide, or binder. These cells may be combined with pharmaceutically acceptable excipients, adjuvants, diluents, and / or carriers to produce the pharmaceutical compositions of the present invention. The cells may be modified in vitro. For example, they may be autologous cells obtained from a subject for a pharmaceutical composition to treat a subject (or to be administered to treat or prevent cancer or viral infection with impaired HLA class I antigen presentation). The cells may be modified in vitro, for example by introducing a nucleic acid sequence or vector into the cells, so that the modified cells encode the nucleic acid sequence / vector and express the nucleic acid sequence or vector to produce the desired peptide, complex, or binder. The thus modified cells may be administered to a subject as a pharmaceutical composition.

[0199] The composition may typically contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, adjuvants and cytokines, and, if necessary, other therapeutic agents or compounds.

[0200] As used herein, “pharmaceutically acceptable” means a material that does not have any undesirable biological or other effects. That is, a material that, when administered to an organism together with selected nucleic acid sequences, vectors, cells, binders, or peptides, does not cause any undesirable biological effects without adverse interactions with other components in the pharmaceutical composition.

[0201] Excipients are natural or synthetic substances prepared together with the active ingredient (e.g., nucleic acid sequences, nucleic acid compositions, vectors or vector systems, modified cells, or isolated nucleic acids as described herein) and may be included to increase the volume of the composition or to enhance the therapeutic effect of the active ingredient in the final dosage form (e.g., improved drug absorption or solubility). Excipients are also useful in the manufacturing process, assisting in the handling of the active ingredient, such as improving powder flowability and anti-adhesion properties, and aiding in vitro stability, such as preventing denaturation during the expected storage period. Medicinally acceptable excipients are widely known in the art; therefore, suitable excipients can be easily identified by those skilled in the art. For example, suitable pharmaceutically acceptable excipients include saline, water, glucose aqueous solution, glycerol, and ethanol. Adjuvants are pharmacological and / or immunological substances that modify the effects of other components in the composition. Medicinally acceptable adjuvants are widely known in the art, and suitable adjuvants can be easily identified by those skilled in the art. In another embodiment of the present invention, the excipients do not include water.

[0202] The pharmaceutical composition may include immunostimulatory compounds as disclosed elsewhere in this specification.

[0203] Diluents are diluents. Medicinally acceptable diluents are widely known in the art, and suitable diluents can be easily identified by those skilled in the art.

[0204] The carrier is nontoxic to the receptor at the dose and concentration used and is compatible with other components in the composition. “Carrier” means a natural or synthetic organic or inorganic component that facilitates the combination and application of the active ingredient. Medicinally acceptable carriers are widely known in the art, and suitable carriers can be easily identified by those skilled in the art.

[0205] The pharmaceutical compositions described herein may be administered to a subject as monotherapy or as part of a combination therapy. For example, the combination of the vaccine described herein with an immune checkpoint inhibitor or other immunomodulatory compound is particularly useful for targeting immune-evading TAP-deficient cancers, as demonstrated by the combination of a cancer virus vaccine with a PD-1 inhibitor.

[0206] Therefore, the pharmaceutical compositions provided herein may be used in combination with immune checkpoint inhibitors that inhibit PD-1, CTLA-4, PD-L1, TIM3, TIGIT, VISTA, NKG2A, or LAG-3.

[0207] Therefore, the pharmaceutical compositions provided herein may be used in combination with immune checkpoint inhibitors that inhibit PD-1, CTLA-4, PD-L1, TIM3, TIGIT, VISTA, NKG2A, or LAG-3.

[0208] In specific examples, an immune checkpoint inhibitor may be an inhibitor of PD-1 and / or PD-L1 activity. In other words, an immune checkpoint inhibitor may result in a block of PD-1 or PD-L1. An inhibitor of PD-1 and / or PD-L1 activity may be, for example, an antibody that inhibits PD-L1 from binding to PD-1 (or vice versa).

[0209] The pharmaceutical composition and the immune checkpoint inhibitor may be administered in any order. Preferably, the pharmaceutical composition may be administered simultaneously with or after the immune checkpoint inhibitor. Alternatively, the pharmaceutical composition may be administered simultaneously with or before the immune checkpoint inhibitor.

[0210] The pharmaceutical compositions described herein (including nucleic acids, nucleic acid compositions, complexes, or cells according to the present invention) and the vaccines or peptide vaccines described herein may be provided for use as pharmaceuticals. The pharmaceutical compositions described herein and the vaccines or peptide vaccines described herein may be used in combination for pharmaceutical purposes. This combination may include administering the vaccine or peptide vaccine before, during, and / or after administration of the pharmaceutical composition.

[0211] Target treatment The pharmaceutical compositions or vaccines described herein may be used advantageously as pharmaceuticals. These compositions or vaccines may be used in human subjects to treat or prevent precancerous, cancerous, or viral infections in which HLA class I antigen presentation is impaired. Preferably, the human subjects are HLA-A * HLA-A such as 02:01 * It is acceptable for the O2 test to be positive.

[0212] "Precancerous" or "precancerous" usually refers to a condition or growth that precedes or develops into cancer. Precancerous growth or precancerous growth is characterized by cells with abnormalities in cell cycle control, cell proliferation, or differentiation, which can be determined by markers of cell cycle control, cell proliferation, or differentiation.

[0213] Methods for treating or prophylactically treating precancerous, cancerous, or viral infections with impaired HLA class I antigen presentation described herein may result in an induced or enhanced immune response (e.g., a cellular immune response) in the subject (e.g., a targeted immune response against cancer cells or virus-infected cells presenting an HLA-A restriction peptide).

[0214] "Induced or enhanced immune response" refers to an increase in the immune response of the target (e.g., a T-cell mediated cellular immune response) during or after treatment compared to before treatment. An "induced or enhanced" immune response includes any measurable increase in an immune response directly or indirectly targeted by the precancerous, cancerous, or viral infection being treated (or prevented).

[0215] The compositions of the present invention may be used to treat or prevent cancers in which HLA class I antigen presentation is impaired. Those skilled in the art have a good understanding of cancers in which HLA class I antigen presentation is impaired and therefore recognize that they can be treated according to the present invention.

[0216] In another example, the pharmaceutical composition or vaccine may be used to stimulate a cellular immune response against a target cell population or tissue in a human subject. The pharmaceutical composition or vaccine may be used to provide antitumor immunity to a human subject.

[0217] In some cases, the cancer may be a cancer in which the peptide processing mechanism is impaired. In one example, the cancer may be melanoma. In another example, the cancer may be lung cancer.

[0218] The compositions or vaccines of the present invention may be used to treat or prevent precancerous conditions in which HLA class I antigen presentation is impaired. Those skilled in the art have a good understanding of precancerous conditions in which HLA class I antigen presentation is impaired and therefore recognize that they can be treated according to the present invention.

[0219] Depending on the circumstances, the precancerous cells may be those in which the peptide processing mechanism is impaired.

[0220] The compositions or vaccines of the present invention may also be used to treat or prevent viral infections in which HLA class I antigen presentation is impaired. Those skilled in the art are well aware of viral infections in which HLA class I antigen presentation is impaired and therefore may treat them according to the present invention.

[0221] As used herein, “precancerous, cancerous, or viral infections with impaired HLA class I antigen presentation” refers to precancerous, cancerous, or viral infections in which alterations occur in the HLA class I antigen presentation pathway in precancerous cells, cancerous cells, or virus-infected cells, resulting in reduced HLA class I antigen presentation in these cells. In this context, reduced presentation of non-TEIPP HLA class I restriction antigens on the cell surface is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, etc., compared to control cells (e.g., cells from the same subject that are not precancerous, cancerous, or viral-infected).

[0222] Multiple molecular pathways that impair HLA class I antigen presentation may be altered in precancerous cells, cancer cells, or virus-infected cells. For example, 1-2% of melanomas have harmful mutations in TAP1 or TAP2, and it is known that TAP1 expression is reduced by epigenetic silencing in high-frequency metastatic melanomas (Ritter et al., 2017; Setiadi et al., 2007; Garrido et al., 2016). Downregulation of TAP has also been observed in lung cancer specimens ("Different Expression Levels of the TAP Peptide Transporter Lead to Recognition of Different Antigenic Peptides by Tumor-Specific CTL", A. Durgeau et al., J Immunol 2011, 187 (11) 5532-5539; "Loss of antigen-presenting molecules (MHC class I and TAP-1) in lung cancer", Korkolopoulou P. et al., 1996. Br. J. Cancer 73: 148-153; "Restoration of the expression of transporters associated with antigen processing in lung carcinoma increases tumor-specific immune responses and survival", Lou Y. et al., 2005. Cancer Res. 65: 7926-7933).

[0223] Therefore, precancerous, cancerous, or viral infections with impaired HLA class I antigen presentation may also be precancerous, cancerous, or viral infections in which tumor cells or infected cells have mutations in the TAP1 or TAP2 gene. In one example, the mutation reduces the expression of TAP1 or TAP2 (so that pretumor cells, tumor cells, or virus-infected cells have low TAP1 or TAP2 expression); in another example, the mutation reduces intracellular TAP1 or TAP2 activity (so that pretumor cells, tumor cells, or virus-infected cells have reduced / low TAP1 or TAP2 activity); in yet another example, the mutation reduces the level of TAP1 or TAP2 protein in cells (e.g., pretumor cells, tumor cells, or virus-infected cells have reduced / low TAP1 or TAP2 protein expression and / or reduced / low TAP1 or TAP2 protein stability).

[0224] TAP1 or TAP2 expression may be reduced or low due to epigenetic silencing in precancerous cells, cancer cells, or virus-infected cells. Methods for detecting epigenetic silencing of TAP1 or TAP2 are well known in the art. TAP1 or TAP2 expression, activity, protein levels, and / or protein stability may also be reduced or low in precancerous cells, cancer cells, or virus-infected cells due to reasons other than mutations in the TAP1 or TAP2 gene (e.g., by precancerous / cancer / viruses altering the molecular mechanisms and pathways of cells).

[0225] Therefore, precancerous, cancerous, or viral infections may be associated with reduced (or low) TAP1 or TAP2 protein expression, activity, quantity, or stability.

[0226] Methods for determining the presence of mutations in TAP1 or TAP2 are well known in the art. Furthermore, methods for determining the expression level of TAP1 or TAP2, the activity level of TAP1 or TAP2, the protein quantity of TAP1 or TAP2, and the protein stability of TAP1 or TAP2 are also well known in the art.

[0227] For example, expression levels may be detected by measuring mRNA (e.g., Northern blot analysis, RNA probes (e.g., spatial transcriptomics (provided by NanoString)), or in situ hybridization (e.g., RNAscope®)), and protein levels may be measured using TAP1 or TAP2-specific antibodies (e.g., with detectable labels). Methods such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunofluorescence, enzyme immunoassay (EIA), radioimmunoassay (RIA), spatial proteomics (provided by NanoString), and Western blot analysis may be used. Other standard methods for determining these parameters are also well known in the art.

[0228] As described above, precancerous, cancerous, or viral infections may be associated with reduced (or low) TAP1 or TAP2 protein expression, activity, quantity, or stability.

[0229] In this specification, “reduced (or lower) TAP1 or TAP2 protein expression, activity, quantity, or stability” means a decrease in protein expression, activity, quantity, or stability compared to a control or reference level (e.g., a decrease of at least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). As used herein, “reference level” or “control” refers to a cell sample having normal TAP1 or TAP2 protein expression, activity, quantity, or stability, e.g., a sample from a healthy control subject that does not have or is not suspected of having precancerous, cancerous, or viral infection, or a cell sample from the same control subject under test, where the reference level or control cell sample is not (and is not suspected of having) precancerous, cancerous, or viral infection. Alternatively, the reference level may be a value of TAP1 or TAP2 protein expression, activity, quantity, or stability from a reference database which may be used to generate a predetermined cutoff value. That is, it may be a diagnostic score that statistically predicts the presence or absence of symptoms or disease. Furthermore, the reference level may be a predetermined reference level based on a standard population sample, or a predetermined reference level based on the subject's baseline expression level, i.e., the level before or suspected of developing precancerous, cancerous, or viral infection. For example, decreased or low expression may be determined using immunohistochemistry with an anti-TAP1 or anti-TAP2 antibody, such as anti-TAP1 antibody, clone mAb 148.3 (MABF125 EMD Millipore). In one example, the evaluation of normal levels of TAP1 or TAP2 protein expression in a sample is determined by the "De Ruiter" evaluation method compared to decreased or low expression levels. For example, in such a method, the sample is a precancerous, pre-tumorous, cancerous, or tumorous sample. Alternatively, if the sample is a viral sample, the presence of immunomodulatory viral gene products such as CMV, HSV, or BVS may decrease the expression and / or activity of TAP function, and the presence of these gene products may be used as an indicator of decreased or low TAP1 or TAP2 expression and / or activity.

[0230] Other molecular pathways that may be altered to impair HLA class I antigen presentation in precancerous cells, cancer cells, or virus-infected cells include, for example, the deficiency of tapasin (a chaperone protein involved in peptide loading onto MHC class I molecules via TAP) and the inhibition of proteasome-mediated degradation that breaks down proteins for MHC class I presentation into peptides (see, for example, US2009 / 0220534 for details).

[0231] In this specification, the terms “to treat,” “to treat,” and “treatment” are understood to include interventions performed with the intention of preventing the development of a particular condition, disorder, or symptom (i.e., in this case, precancerous, cancerous, or viral infection with impaired HLA class I antigen presentation) or altering its pathology. Accordingly, “treatment” refers to both therapeutic treatments and prophylactic or preventive measures, the purpose of which is to prevent or delay (mitigate) the condition, disorder, or symptom of the subject. Accordingly, “treatment” includes, for example, a reduction, delay, or suppression of the amount or concentration of precancerous, cancerous, or virally infected cells by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the amount or concentration of such cells (precancerous, cancerous, or virally infected cells) before treatment, as measured in a sample obtained from the subject. Methods for measuring the amount or concentration of precancerous, cancerous, or virally infected cells include, for example, qRT-PCR and the quantification of specific biomarkers in a sample obtained from the subject.

[0232] As used herein, “Subject” refers to an individual (e.g., a human) who has, or is at risk of having, a particular condition, disease, or symptom. The Subject may be a patient requiring treatment according to the present invention. The Subject may have previously received treatment for the condition, disease, or symptom. Alternatively, the Subject may not have received treatment prior to receiving treatment according to the present invention. Preferably, the Subject is human, and more preferably HLA * O2-positive human, more preferably HLA * 02:01 Tested positive.

[0233] The compositions or vaccines described herein may be administered to subjects by any conventional route of administration, including injection or sustained-release administration over time. Administration may be, for example, by intravenous infusion, intramuscular, intravascular, intracavitary, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, spinal, or transdermal administration.

[0234] The compositions or vaccines described herein may be in any form suitable for the method of administration. For example, a composition containing cells may be in any form suitable for intravenous administration. Further examples include sterile solutions, suspensions, or emulsions suitable for injection (including subcutaneous, intramuscular, intravascular, or intravenous administration), ointments or creams suitable for topical administration, and suppositories suitable for rectal administration. Alternatively, the route of administration may be direct injection to the target site, regional administration, or topical administration. Identifying appropriate doses of the compositions of the present invention is readily apparent within the scope of the routine skills of those skilled in the art.

[0235] Preferably, the compositions of the present invention may be prepared for use as a vaccine (for example, a composition containing a peptide, wherein the peptide contains the amino acid sequence of SEQ ID NO: 1 (or the corresponding nucleic acid sequence or vector), can be prepared as a pharmaceutical composition usable as a (peptide) vaccine). Alternatively, a composition containing cells may also be prepared as a pharmaceutical composition usable as a vaccine. Methods for preparing suitable cells, binders (e.g., antibodies), peptides, and nucleic acid vaccines are well known to those skilled in the art.

[0236] Preferably, the compositions described herein may be prepared for use in T cell receptor (TCR) gene transfer. TCR gene transfer is rapid, reliable, independent of the patient's existing immune repertoire, and capable of generating large quantities of T cells specific to the RCN1 antigen peptide (e.g., any of the peptides in SEQ ID NOs: 1-27, particularly SEQ ID NO: 2). Using TCR gene transfer, modified cells suitable for intravenous administration may be generated within a few days.

[0237] This pharmaceutical composition or vaccine is preferably intended for administration to a subject and is therefore prepared to be suitable for administration to a subject, preferably a human or animal subject. Preferably, administration is other than percutaneous, for example by infusion, subcutaneous, intramuscular, intradermal, subcutaneous and / or intratumoral administration, i.e., by injection.

[0238] Preferably, the pharmaceutical composition or vaccine contains, or consists solely of, an amount of active ingredients (e.g., nucleic acid sequences, peptides, vectors, binders, or cells) constituting a pharmaceutical dose unit. Here, a pharmaceutical dose unit refers to the amount of active ingredients applied to a subject at a specific time (i.e., the total amount of peptides in a peptide vaccine). A pharmaceutical dose unit may be applied to a subject as a single dose, i.e., a single administration, or as two, three, four, five or more separate doses or injections. These are preferably administered to different parts of the body, for example, the right arm and the left arm. Here, the separate doses of the pharmaceutical dose may have different compositions, i.e., the type and composition of the active ingredients and / or adjuvants may differ.

[0239] The single injection volume or single dose (i.e., the amount applied to one site at a particular time) may be in the range of 100 μL to 2 mL, or 100 μL to 1 mL, as part of the total medicinal dose, or as part of multiple doses administered approximately simultaneously at the same time. The single injection volume may be 100 μL, 200 μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2 mL, 3 mL, or any value in between.

[0240] The pharmaceutical dosage unit, or the total amount of active ingredient administered to a subject at a specific time, depends on the type of vaccine (e.g., peptide, cell, nucleic acid, etc.). For example, the pharmaceutical dosage unit, or total peptide amount administered to a subject in the case of a single injection or multiple doses at specific time points, may include peptide amounts ranging from 0.1 μg to 20 mg. In specific examples, approximately 0.1 μg, 0.5 μg, 1 μg, 5 μg, 10 μg, 15 μg, 20 μg, 30 μg, 40 μg, 50 μg, 60 μg, 70 μg, 80 μg, 90 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 650 μg, 700 μg, 75 μg The dosage may be 0 μg, 800 μg, 850 μg, 900 μg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg, 3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 15 mg, or approximately 20 mg, or any value in between. The preferred range for the pharmaceutical dosage unit is 0.1 μg to 20 mg, 1 μg to 10 mg, 10 μg to 5 mg, 0.5 mg to 2 mg, 0.5 mg to 10 mg, 1 mg to 5 mg, or 2 to 4 mg.

[0241] The compositions or vaccines described herein are intended for administration in an effective dose. “Effective dose” means the amount, alone or in combination with additional doses, that can produce the desired (therapeutic or non-therapeutic) response. The effective dose depends, for example, on the therapeutic (or non-therapeutic) purpose, the route of administration, the condition of the subject, etc. For example, the appropriate dose of the composition of the present invention for a particular subject is determined by the attending physician (or the person administering the composition), taking into account various factors known to affect the action of the composition, such as the severity and type of hematological malignancy, body weight, sex, diet, time and route of administration, other drugs, and other relevant clinical factors. Dosage and administration schedule may be varied depending on the specific condition, disorder or symptom and the overall condition of the subject. The effective dose may be determined in vitro or in vivo. For convenience, the pharmaceutical compositions of the present invention are preferably presented in the form of pharmaceutical dosage units.

[0242] Binder The binders described herein specifically bind to peptides containing (or composed of) the amino acid sequence of SEQ ID NO: 1. These binders are useful in human subjects for the prevention or treatment of precancerous, cancerous, or viral infections in which HLA class I antigen presentation is impaired. The specification also describes binders that specifically bind to peptides containing (or composed of) any of the amino acid sequences SEQ ID NOs: 2 to 21, preferably any of SEQ ID NOs: 2 to 5, more preferably any of SEQ ID NOs: 2 to 4, or any of SEQ ID NOs: 3 to 5.

[0243] The binder may specifically bind to an epitope in the amino acid sequence provided by SEQ ID NO: 1. The binder may also specifically bind to an epitope in the amino acid sequence provided by any of SEQ ID NOs: 2 to 21, preferably any of SEQ ID NOs: 2 to 5, more preferably any of SEQ ID NOs: 2 to 4, or any of SEQ ID NOs: 3 to 5. As used herein, "epitope" refers to the site on the target molecule (the peptide in this example) to which the binder binds. An epitope is an aggregate of molecules such as amino acids or sugar side chains, and usually has specific stereochemical and charge properties. A single peptide (antigen) may have multiple epitopes. Epitopes may be formed from continuous or discontinuous residues (e.g., amino acid residues) of the target molecule. Epitopes formed from continuous residues (e.g., amino acid residues) are usually also called linear epitopes. Epitopes usually contain at least 5 residues and up to about 12 residues, mainly 6 to 10 residues (e.g., amino acid residues). Epitopes may be steric (nonlinear). In one example, the binder specifically binds to the epitope produced by the peptide itself. In another example, the binder (e.g., an antibody) binds to the epitope produced by the combination of the peptide and the HLA molecule that presents it (i.e., the peptide presents HLA class I, e.g., HLA * (An epitope generated when presented on the cell surface by 02:01).

[0244] The binder of the present invention may be any suitable binder that specifically binds to a peptide containing (or composed of) the amino acid sequence of SEQ ID NO: 1. The binder of the present invention may also be any suitable binder that specifically binds to a peptide containing (or composed of) any of the amino acid sequences of SEQ ID NOs: 2 to 21, preferably binding to any of SEQ ID NOs: 2 to 5, more preferably to any of SEQ ID NOs: 2 to 4, or any of SEQ ID NOs: 3 to 5.

[0245] One example of a suitable binder of the present invention is the HLA-A02 molecule, which specifically binds to a peptide containing (or composed of) the amino acid sequence of SEQ ID NO: 1. Another example is the HLA-A02 molecule, which specifically binds to a peptide containing (or composed of) the amino acid sequence of SEQ ID NO: 1. * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * One example is the 02:01 molecule. Another example is the HLA-A*02 molecule, which specifically binds to peptides containing (or composed of) any of the amino acid sequences of SEQ ID NOs. 2-21. Yet another example is the HLA-A*02 molecule, which specifically binds to peptides containing (or composed of) any of the amino acid sequences of SEQ ID NOs. 2-21. * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 molecules are listed. These HLA-A * 02, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, HLA-A * 02:09, or HLA-A *02:01 molecules are useful, for example, as part of a multimeric structure when administered to a target to stimulate target T cells (e.g., in the form of synthetic dendritic cells (synthetic DCs)).

[0246] Therefore, in one example, the binder that specifically binds to a peptide containing (or composed of) the amino acid sequence of SEQ ID NO: 1 is HLA-A * It contains the HLA-A02 molecule. Typically, in this case, the HLA-A02 molecule specifically binds to peptides containing (or composed of) the amino acid sequence of SEQ ID NO: 1. In another example, the binder that specifically binds to peptides containing (or composed of) the amino acid sequence of SEQ ID NO: 1 is HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 contains molecules. Typically, in this case, HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * The 02:01 molecule specifically binds to peptides containing (or composed of) the amino acid sequence of SEQ ID NO: 1.

[0247] In another example, the binder that specifically binds to a peptide containing (or constituting) any of the amino acid sequences of SEQ ID NOs: 2-21 is HLA-A * Contains 02 molecules. Typically, in this case, HLA-A * The 02 molecule specifically binds to peptides containing (or constituting) any of the amino acid sequences of SEQ ID NOs. 2-21. In another example, the binder that specifically binds to peptides containing (or constituting) any of the amino acid sequences of SEQ ID NOs. 2-21 is HLA-A. * 02:01, HLA-A *02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, more preferably HLA-A * 02:01 contains molecules. Typically, in this case, HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, more preferably HLA-A * The 02:01 molecule specifically binds to peptides containing (or constituting) any of the amino acid sequences of SEQ ID NOs: 2 to 21. More preferably, it specifically binds to any of the amino acid sequences of SEQ ID NOs: 2 to 5, even more preferably, any of the amino acid sequences of SEQ ID NOs: 2 to 4, or any of the amino acid sequences of SEQ ID NOs: 3 to 5.

[0248] Such binders are useful as pharmaceutical compositions, as described elsewhere here.

[0249] Provided herein are a complex comprising a) a peptide containing the amino acid sequence of SEQ ID NO: 1, and b) a binder that specifically binds to the peptide containing the amino acid sequence of SEQ ID NO: 1; optionally, the binder is HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, more preferably HLA-A * 02:01 molecules are also acceptable.

[0250] Provided herein is a complex comprising a) a peptide containing the amino acid sequence VLAPRVLRA of SEQ ID NO: 2, and b) a binder that specifically binds to the peptide containing the amino acid sequence VLAPRVLRA of SEQ ID NO: 2; optionally, the binder is HLA-A02:01, HLA-A * 02:02, HLA-A *02:03, HLA-A * 02:04, or HLA-A * 02:09, more preferably HLA-A * 02:01 molecules are also acceptable.

[0251] Provided herein are a complex comprising a) a peptide containing the amino acid sequence VLAPRVLRV of SEQ ID NO: 3, and b) a binder that specifically binds to the peptide containing the amino acid sequence VLAPRVLRV of SEQ ID NO: 3; optionally, the binder is HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, more preferably HLA-A * 02:01 molecules are also acceptable.

[0252] Provided herein are a complex comprising a) a peptide containing the amino acid sequence VLAPRVLRI of SEQ ID NO: 4, and b) a binder that specifically binds to the peptide containing the amino acid sequence VLAPRVLRI of SEQ ID NO: 4; optionally, the binder is HLA-A02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, more preferably HLA-A * 02:01 molecules are also acceptable.

[0253] Provided herein is a complex comprising: a) a peptide containing the amino acid sequence VLAPRVLRL of SEQ ID NO: 5; and b) a binder that specifically binds to the peptide containing the amino acid sequence VLAPRVLRL of SEQ ID NO: 5; optionally, the binder is HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, more preferably HLA-A* 02:01 molecules are also acceptable.

[0254] Provided herein is a complex comprising: a) a peptide containing the amino acid sequence VLAPRVLRR of SEQ ID NO: 6; and b) a binder that specifically binds to the peptide containing the amino acid sequence VLAPRVLRR of SEQ ID NO: 6; optionally, the binder is HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, more preferably HLA-A * 02:01 molecules are also acceptable.

[0255] Provided herein is a complex comprising a) a peptide containing the amino acid sequence VLAPRVLRN of SEQ ID NO: 7, and b) a binder that specifically binds to the peptide containing the amino acid sequence VLAPRVLRN of SEQ ID NO: 7; optionally, the binder is HLA-A * 02:01, HLA-A * 02:02, HLA-A 02:03, HLA-A * 02:04, or HLA-A * 02:09, more preferably HLA-A * 02:01 molecules are also acceptable.

[0256] The complex described herein may also include a) a peptide comprising the amino acid sequence VLAPRVLRD (SEQ ID NO: 8), and b) a binder that specifically binds to the peptide comprising the amino acid sequence VLAPRVLRD (SEQ ID NO: 8); optionally, the binder may be HLA-A02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 molecules are also acceptable.

[0257] The complex described herein may also include a) a peptide comprising the amino acid sequence VLAPRVLRC (SEQ ID NO: 9), and b) a binder that specifically binds to the peptide comprising the amino acid sequence VLAPRVLRC (SEQ ID NO: 9); optionally, the binder may be HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 molecules are also acceptable.

[0258] The complex described herein may also comprise a) a peptide comprising the amino acid sequence VLAPRVLRE (SEQ ID NO: 10), and b) a binder that specifically binds to the peptide comprising the amino acid sequence VLAPRVLRE (SEQ ID NO: 10); optionally, the binder may be HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 molecules are also acceptable.

[0259] The complex described herein may also comprise a) a peptide comprising the amino acid sequence VLAPRVLRQ (SEQ ID NO: 11), and b) a binder that specifically binds to the peptide comprising the amino acid sequence VLAPRVLRQ (SEQ ID NO: 11); optionally, the binder may be HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 molecules are also acceptable.

[0260] The complex described herein may also include a) a peptide comprising the amino acid sequence VLAPRVLRG (SEQ ID NO: 12), and b) a binder that specifically binds to the peptide comprising the amino acid sequence VLAPRVLRG (SEQ ID NO: 12); optionally, the binder may be HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 molecules are also acceptable.

[0261] The complex described herein may also comprise a) a peptide comprising the amino acid sequence VLAPRVLRH (SEQ ID NO: 13), and b) a binder that specifically binds to the peptide comprising the amino acid sequence VLAPRVLRH (SEQ ID NO: 13); optionally, the binder may be HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 molecules are also acceptable.

[0262] The complex described herein may also comprise a) a peptide containing the amino acid sequence VLAPRVLRK (SEQ ID NO: 14), and b) a binder that specifically binds to the peptide containing the amino acid sequence VLAPRVLRK (SEQ ID NO: 14); optionally, the binder may be HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 molecules are also acceptable.

[0263] The complex described herein may also comprise a) a peptide containing the amino acid sequence VLAPRVLRM (SEQ ID NO: 15), and b) a binder that specifically binds to the peptide containing the amino acid sequence VLAPRVLRM (SEQ ID NO: 15); optionally, the binder may be HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 molecules are also acceptable.

[0264] The complex described herein may also comprise a) a peptide containing the amino acid sequence VLAPRVLRF (SEQ ID NO: 16), and b) a binder that specifically binds to the peptide containing the amino acid sequence VLAPRVLRF (SEQ ID NO: 16); optionally, the binder may be HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 molecules are also acceptable.

[0265] The complex described herein may also comprise a) a peptide containing the amino acid sequence VLAPRVLRP (SEQ ID NO: 17), and b) a binder that specifically binds to the peptide containing the amino acid sequence VLAPRVLRP (SEQ ID NO: 17); optionally, the binder may be HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 molecules are also acceptable.

[0266] The complex described herein may also comprise a) a peptide containing the amino acid sequence VLAPRVLRS (SEQ ID NO: 18), and b) a binder that specifically binds to the peptide containing the amino acid sequence VLAPRVLRS (SEQ ID NO: 18); optionally, the binder may be HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 molecules are also acceptable.

[0267] The complex described herein may also comprise a) a peptide comprising the amino acid sequence VLAPRVLRT (SEQ ID NO: 19), and b) a binder that specifically binds to the peptide comprising the amino acid sequence VLAPRVLRT (SEQ ID NO: 19); optionally, the binder may be HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 molecules are also acceptable.

[0268] The complex described herein may also comprise a) a peptide comprising the amino acid sequence VLAPRVLRW (SEQ ID NO: 20), and b) a binder that specifically binds to the peptide comprising the amino acid sequence VLAPRVLRW (SEQ ID NO: 20); optionally, the binder may be HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 molecules are also acceptable.

[0269] The complex described herein may also comprise a) a peptide containing the amino acid sequence VLAPRVLRY (SEQ ID NO: 21), and b) a binder that specifically binds to the peptide containing the amino acid sequence VLAPRVLRY (SEQ ID NO: 21); optionally, the binder may be HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 molecules are also acceptable.

[0270] Preferably, the binder forms a complex with the peptide or is bound to the peptide.

[0271] HLA-A as described in this specification * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, preferably HLA-A * 02:01 The molecule may be useful, for example, as part of a multimeric structure used to stimulate the target's T cells when administered to a target. For example, it may be used in the form of an artificial antigen-presenting cell (aAPC). aAPC is a synthetic structure that carries a tumor antigen. aAPC is designed to mimic dendritic cells (DCs) and is also called a synthetic DC. The aim is to induce an efficient and specific T cell response against tumors in cancer treatment.

[0272] In one example, the complex of the present invention is present in a cell, and the binder may carry a peptide according to the present invention. In another example, a cell expresses the complex according to the present invention. The cell may be an antigen-presenting cell. The antigen-presenting cell may be selected from macrophages, dendritic cells, monocytes, B cells, or synthetic forms of antigen-presenting cells.

[0273] The binders described above may be useful as pharmaceutical compositions as described elsewhere in this specification.

[0274] In one example, the binder may be an isolated binder. In this specification, “isolated binder” means a binder that is not found in its natural environment. Therefore, the binder may be a recombinant binder, or a synthetically derived binder (or it may be naturally derived or isolated from its natural environment). In the context of this disclosure, HLA-A2 * 02 and HLA-A2 * 02:01 The natural environment for binders such as molecules is the human body. Therefore, the binder (e.g., HLA-A2) * 02 and HLA-A2 * 02:01 When molecules are present in a pharmaceutical composition (including adjuvants, etc.), they are considered isolated forms because they do not exist in the natural environment.

[0275] As used herein, the terms “specific binding” and “specifically binding” (or equivalent terms) are used interchangeably to indicate that other biomolecules do not significantly bind to the region of interest (e.g., the peptide containing SEQ ID NO: 1). In some embodiments, the binding levels to biomolecules other than the target peptide are negligible and cannot be measured by ELISA or affinity assays.

[0276] "Negligible binding" means binding that is at least approximately 85%, particularly at least approximately 90%, more particularly at least approximately 95%, even more particularly at least approximately 98%, and particularly at least 99% to 100% compared to binding to the target peptide (e.g., the peptide containing SEQ ID NO: 1).

[0277] In the present invention, the binding affinity of a binder to the target peptide (for example, a peptide containing the amino acid sequence described in SEQ ID NO: 1) may be measured using a standard binding assay such as surface plasmon resonance (BIAcore®, GE-Healthcare Uppsala, Sweden). As used herein, "surface plasmon resonance" refers to an optical phenomenon that enables the real-time analysis of specific biological interactions by detecting changes in protein concentration within a biosensor substrate, and is performed, for example, using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For further details, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51: 19-26; Jonsson, U., et al. (1991) Biotechniques 11: 620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8: 125-131; and Johnsson, B., et al. (1991) Anal. Biochem. 198: 268-277.

[0278] Nucleic acid compositions encoding binding protein components The present invention provides an isolated nucleic acid composition encoding a reticulocalbin 1 (RCN1) antigen-specific binding protein. The binding protein has a TCR α-chain variable (Vα) domain and a TCR β-chain variable (Vβ) domain, and the composition comprises the following: (a) A nucleic acid sequence encoding a TCR Vα domain having a CDR3 amino acid sequence; and (b) A nucleic acid sequence encoding a TCR Vβ domain having a CDR3 amino acid sequence, Here, these CDR3 sequences specifically bind to peptides containing the target peptide (for example, when combined with HLA).

[0279] The RCN1 antigen according to the present invention has a peptide comprising an amino acid sequence selected from the group consisting of: a peptide selected from SEQ ID NOs: 1 to 21, preferably a peptide selected from SEQ ID NOs: 1 to 5 or SEQ ID NOs: 2 to 5, and more preferably a peptide selected from any one of SEQ ID NOs: 2 to 4 or SEQ ID NOs: 3 to 5. Therefore, the CDR3 sequence according to the present invention preferably specifically binds to a peptide having an amino acid sequence of any one of these SEQ ID NOs: 1 to 21, more preferably SEQ ID NOs: 1 to 5 or SEQ ID NOs: 2 to 5, and even more preferably SEQ ID NOs: 2 to 4 or SEQ ID NOs: 3 to 5, in order to specifically bind to the target peptide (for example, when compounded with HLA).

[0280] As will be apparent to those skilled in the art, the CDR3 amino acid sequences described herein specifically bind to a target (in this case, a peptide containing the amino acid sequence of SEQ ID NO: 1, e.g., VLAPRVLRA peptide, VLAPRVLRV peptide, VLAPRVLRI peptide, or VLAPRVLRL peptide) when the target (i.e., the appropriate peptide) is presented in the context of HLA. The binding proteins (and CDR3 sequences) specifically described herein are therefore capable of specifically binding to appropriate peptide:HLA complexes. These complexes are described in further detail elsewhere in this specification.

[0281] The present invention provides an isolated nucleic acid composition encoding a binding protein containing a T cell receptor (TCR) component that specifically binds to the RCN1 antigen (for example, to a peptide containing the sequence of SEQ ID NO: 1, e.g., VLAPRVLRA peptide, VLAPRVLRV peptide, VLAPRVLRI peptide, or VLAPRVLRL peptide). Thus, the encoded binding protein can specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (for example, a peptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 21, preferably selected from the group consisting of SEQ ID NOs: 2 to 5, more preferably selected from the group consisting of SEQ ID NOs: 2 to 4, or selected from the group consisting of SEQ ID NOs: 3 to 5), but will not bind to a peptide that does not contain the amino acid sequence of SEQ ID NO: 1 (for example, a peptide that does not contain an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 21, preferably selected from the group consisting of SEQ ID NOs: 2 to 5, more preferably selected from the group consisting of SEQ ID NOs: 2 to 4, or selected from the group consisting of SEQ ID NOs: 3 to 5).

[0282] The nucleic acid composition comprises (a) a nucleic acid sequence encoding a TCR Vα domain having predetermined characteristics as described herein, and (b) a nucleic acid sequence encoding a TCR Vβ domain having predetermined characteristics as described herein. The encoded TCR component forms an RCN1 antigen-specific binding protein.

[0283] The nucleic acid sequences in (a) and (b) above may be different nucleic acid sequences within the nucleic acid composition. Therefore, the TCR components of the binding protein may be encoded by two or more nucleic acid sequences (having different nucleotide sequences) that encode all of the TCR components of the binding protein. In other words, some of the TCR components may be encoded by one nucleic acid sequence in the nucleic acid composition, and other components may be encoded by another (different) nucleic acid sequence in the nucleic acid composition.

[0284] Alternatively, the nucleic acid sequences in (a) and (b) may be part of a single nucleic acid sequence. Therefore, all TCR components of the binding protein may be encoded by a single nucleic acid sequence (for example, having a single open reading frame, or having multiple open reading frames (e.g., two or more, three or more, etc.)).

[0285] The nucleic acid sequences described herein may form part of a larger nucleic acid sequence encoding a larger component of a functional binding protein. For example, a nucleic acid sequence encoding a TCR Vα domain having certain characteristics described herein may be part of a larger nucleic acid sequence encoding a functional TCRα chain (including the constant domain). In another example, a nucleic acid sequence encoding a TCR Vβ domain having certain characteristics described herein may be part of a larger nucleic acid sequence encoding a functional TCRβ chain (including the constant domain). In yet another example, both nucleic acid sequences in (a) and (b) above may be part of a larger nucleic acid sequence encoding a combination of a functional TCRα chain (including the constant domain) and a functional TCRβ chain (including the constant domain), and optionally, the sequence encoding the functional TCRα chain may be separated from the sequence encoding the functional TCRβ chain by a linker sequence that coordinately expresses two proteins or polypeptides within the same nucleic acid sequence. Further details are provided below.

[0286] The nucleic acid sequences described herein may encode only small components of the T cell receptor, such as the TCR Vα domain or the TCR Vβ domain. These nucleic acid sequences can be considered “constituent blocks” that provide the necessary components for peptide bond specificity. The nucleic acid sequences described herein may be incorporated into another nucleic acid sequence (e.g., a vector) that encodes other elements of a functionally binding protein such as the TCR, and this incorporation generates a novel nucleic acid sequence encoding the TCRα and / or TCRβ chains that specifically bind to the RCN1 antigen (for example, if the RCN1 antigen contains an amino acid sequence selected from the group of SEQ ID NOs: 1-21, preferably selected from the group of SEQ ID NOs: 1-5, or from the group of SEQ ID NOs: 2-5, more preferably selected from the group of SEQ ID NOs: 2-4, or from the group of SEQ ID NOs: 3-5). Thus, the nucleic acid sequences described herein have utility as essential components that confer binding specificity to the RCN1 antigen and can be used to generate a larger nucleic acid sequence that encodes a binding protein with the required antigen-binding activity and specificity.

[0287] The nucleic acid sequences described herein may be codon-optimized for expression in host cells. For example, they may be codon-optimized for expression in human cells such as immune system cells, inducible pluripotent stem cells (iPSCs), hematopoietic stem cells, T cells, primary T cells, T cell lines, NK cells, innate lymphoid cells (ILCs), or natural killer T cells (Scholten et al, Clin. Immunol. 119: 135, 2006). The T cells may be CD4+ or CD8+ T cells. Codon optimization is a well-known method in the art for maximizing the expression of nucleic acid sequences in specific host cells. For example, it is also possible to introduce one or more cysteine ​​residues into the components of the encoded TCRα and TCRβ chains to reduce the risk of mispairing with the endogenous TCR chain.

[0288] In one example, the nucleic acid sequences described herein may be codon-optimized for expression in a suitable host cell and modified to introduce codons encoding one or more cysteine ​​amino acids into the constant domains of the encoding TCRα and / or TCRβ chains in order to reduce the risk of mispairing with endogenous TCR chains. In one example, the nucleic acid sequences described herein may be codon-optimized for expression in a suitable host cell, and optionally, the host cell may be a human cell.

[0289] In certain cases, the TCR constant domain is modified to enhance the pairing of desired TCR chains. For example, if the modification enhances the pairing between heterolog TCRα and heterolog TCRβ chains, a TCR containing both heterolog chains may be preferentially assembled over undesirable mispairings between heterolog TCR chains and endogenous TCR chains (see, e.g., Govers et al, Trends Mol. Med. 16(2):11 (2010)). Typical modifications to enhance heterolog TCR chain pairing include introducing complementary cysteine ​​residues to each heterolog TCRα and β chain.

[0290] The binding proteins encoded by the nucleic acid compositions described herein are specific to the RCN1 antigen and include an RCN1 antigen-specific TCR component. However, the encoded binding proteins are not limited to TCRs. Other suitable binding proteins containing the specified RCN1 antigen-specific TCR component are also included. For example, the encoded binding proteins may include TCRs, antigen-binding fragments of TCRs, chimeric antigen receptors (CARs), or ImmTACs. TCRs, antigen-binding fragments of TCRs, CARs, and ImmTACs are well-defined in the art. Non-limiting examples of antigen-binding fragments of TCRs include single-chain TCRs (scTCRs) or chimeric dimers in which single-chain TCRs (scTCRs) or antigen-binding fragments of TCRα and TCRβ chains are linked to the transmembrane and intracellular domains of a dimeric complex to form a chimeric dimeric TCR (cdTCR). ImmTACs are TCRs linked to anti-CD3 antibodies and are bispecific molecules combining an RCN1-recognizing TCR component with an immunoactivating complex.

[0291] In certain examples, the antigen-binding fragment of the TCR may include a single-chain TCR (scTCR), which contains both the TCR Vα and TCR Vβ domains but only one constant domain. In another example, the antigen-binding fragment of the TCR may include a chimeric TCR dimer in which the antigen-binding fragment of the TCR is linked to alternative transmembrane and intracellular signaling domains that are not naturally present in the TCR. In yet another example, the antigen-binding fragment of the TCR or a chimeric antigen receptor may be chimeric (e.g., containing amino acid residues or motifs from multiple donors or species), humanized (e.g., modified or substituted with non-human residues to reduce the risk of immunogenicity), or human-derived.

[0292] A "chimeric antigen receptor (CAR)" refers to a fusion protein designed to contain two or more naturally occurring amino acid sequences that are not naturally linked in nature or do not naturally exist in host cells, and which may function as a receptor when present on the cell surface. The CARs described herein have an extracellular domain containing an antigen-binding domain (i.e., an immunoglobulin or immunoglobulin-like molecule such as scFv derived from an antibody or TCR, e.g., an antigen-binding domain derived from a cancer antigen-specific antibody or a killer immune receptor derived from NK cells), which is linked to a transmembrane domain and one or more intracellular signaling domains (including optionally a costimulatory domain) (see, for example, Sadelain et al, Cancer Discov., 3(4):388 (2013); Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2016); Stone et al, Cancer Immunol. Immunother., 63(11):1163 (2014)).

[0293] Methods for artificially producing TCRs are described, for example, in Bowerman et al, Mol. Immunol, 5(15):3000 (2009). Methods for producing CARs are well known in the art and are described, for example, in US Patent Nos. 6,410,319 and 7,446,191, US Patent Publication 2010 / 065818, US Patent No. 8,822,647, PCT Publication WO 2014 / 031687, US Patent No. 7,514,537, and Brentjens et al, 2007, Clin. Cancer Res. 73:5426.

[0294] The binding proteins described herein may be expressed as part of a transgene configuration encoding additional accessory proteins such as safety switch proteins, tags, selection markers, CD8 coreceptor β-chain, α-chain, or both, or a combination thereof.

[0295] T cell receptors (TCRs) are molecules present on the surface of T cells (T lymphocytes) that recognize peptides bound to (presented) major histocompatibility complex (MHC) molecules on target cells. This invention relates to nucleic acid compositions encoding binding proteins containing TCR components that interact in the context of specific peptides and appropriate MHC serotypes. Specifically, HLA-A * 02 or HLA-A * 02:01, HLA-A * 02:02, HLA-A * 02:03, HLA-A * 02:04, or HLA-A * 02:09, HLA-A as requested * This corresponds to the RCN1 antigen in the context of 02:01 (in other words, the encoded binding protein is specifically capable of binding to the RCN1 antigen-specific HLA complex).

[0296] In one example, the present invention relates to a nucleic acid composition encoding a binding protein comprising a TCR component that interacts with a specific peptide in the context of an appropriate MHC serotype. That is, the peptide of SEQ ID NO: 1 is HLA-A * In the context of 02:01, the peptide VLAPRVLRA (SEQ ID NO: 2) is HLA-A * In the context of 02:01, the peptide VLAPRVLRV (SEQ ID NO: 3) is HLA-A * In the context of 02:01, the peptide VLAPRVLRI (SEQ ID NO: 4) is HLA-A * In the context of 02:01, the peptide VLAPRVLRL (SEQ ID NO: 5) is HLA-A * In the context of 02:01, the peptide VLAPRVLRR (sequence number 6) is HLA-A * In the context of 02:01, the peptide VLAPRVLRN (sequence number 7) is HLA-A * In the context of 02:01, the peptide VLAPRVLRD (SEQ ID NO: 8) is HLA-A * In the context of 02:01, the peptide VLAPRVLRC (SEQ ID NO: 9) is HLA-A * In the context of 02:01, the peptide VLAPRVLRE (SEQ ID NO: 10) is HLA-A *In the context of 02:01, the peptide VLAPRVLRQ (SEQ ID NO: 11) is HLA-A * In the context of 02:01, the peptide VLAPRVLRG (SEQ ID NO: 12) is HLA-A * In the context of 02:01, the peptide VLAPRVLRH (SEQ ID NO: 13) is HLA-A * In the context of 02:01, the peptide VLAPRVLRK (SEQ ID NO: 14) is HLA-A * In the context of 02:01, the peptide VLAPRVLRM (SEQ ID NO: 15) is HLA-A * In the context of 02:01, the peptide VLAPRVLRF (SEQ ID NO: 16) is HLA-A * In the context of 02:01, the peptide VLAPRVLRP (SEQ ID NO: 17) is HLA-A * In the context of 02:01, the peptide VLAPRVLRS (SEQ ID NO: 18) is HLA-A * In the context of 02:01, the peptide VLAPRVLRT (SEQ ID NO: 19) is HLA-A * In the context of 02:01, the peptide VLAPRVLRW (SEQ ID NO: 20) is HLA-A * In the context of 02:01, and / or the peptide VLAPRVLRY (SEQ ID NO: 21) is HLA-A * They interact in the context of 02:01.

[0297] HLA-A * 02:01 is a globally common human leukocyte antigen (HLA) serotype within the HLA-A serotype group. * The peptide presented to the TCR by 02:01 is "HLA-A * It is described as "02:01 restrictive." Other serotypes are described similarly.

[0298] As described herein, the inventors have developed HLA-A * Multiple RCN1-derived peptides presented to cells were identified on 02:01. Specifically, the inventors identified the RCN1-derived peptide SEQ ID NO: 2 (VLAPRVLRA).

[0299] Therefore, the RCN1 antigen to which the binding protein described herein specifically binds may include an amino acid sequence selected from SEQ ID NOs: 1 to 21. More preferably, it may include an amino acid sequence selected from SEQ ID NOs: 1 to 5 or SEQ ID NOs: 2 to 5. The antigen may be an antigenic fragment (i.e., a portion) of an amino acid sequence selected from SEQ ID NOs: 1 to 21. More preferably, it may be a fragment selected from SEQ ID NOs: 1 to 5 or SEQ ID NOs: 2 to 5, and even more preferably, a fragment of any of SEQ ID NOs: 2 to 4 or SEQ ID NOs: 3 to 5. Alternatively, the antigen may be a complete sequence containing an amino acid sequence selected from SEQ ID NOs: 1 to 21, more preferably a sequence selected from SEQ ID NOs: 1 to 5 or SEQ ID NOs: 2 to 5, and even more preferably a sequence selected from SEQ ID NOs: 2 to 4 or SEQ ID NOs: 3 to 5. Or, it may be a longer sequence containing (i.e., encapsulating) an amino acid sequence selected from SEQ ID NOs: 1 to 21.

[0300] The inventors have found that the RCN1-derived peptide VLAPRVLRA (SEQ ID NO: 2) is HLA-A * It was identified that it may be presented by 02:01. As stated here, the second amino acid L of this peptide can be modified without affecting the TCR's ability to bind to the peptide:HLA complex. The same applies to the C-terminal amino acid A, which may also be modified without adversely affecting TCR specificity. The present invention relates to this VLAPRVLRA (SEQ ID NO: 2) and its functional variant, represented by SEQ ID NO: 1 (VX1APRVLRX2, where X1 and X2 are any amino acids).

[0301] Therefore, in one example, the binding protein described herein may specifically bind to a peptide selected from the following group: HLA complex: VX1APRVLRX2: HLA-A * 02:01 complex (where X1 and X2 are any amino acids), VLAPRVLRA:HLA-A02:01 complex, VLAPRVLRV:HLA-A * 02:01 complex, VLAPRVLRI:HLA-A *02:01 complex, VLAPRVLRL:HLA-A * 02:01 complex, VLAPRVLRR:HLA-A02:01 complex, VLAPRVLRN:HLA-A02:01 complex, VLAPRVLRD:HLA-A02:01 complex, VLAPRVLRC:HLA-A * 02:01 complex, VLAPRVLRQ:HLA-A02:01 complex, VLAPRVLRE:HLA-A * 02:01 complex, VLAPRVLRG:HLA-A02:01 complex, VLAPRVLRH:HLA-A02:01 complex, VLAPRVLRK:HLA-A * 02:01 complex, VLAPRVLRM:HLA-A02:01 complex, VLAPRVLRF:HLA-A02:01 complex, VLAPRVLRP:HLA-A * 02:01 complex, VLAPRVLRS:HLA-A * 02:01 complex, VLAPRVLRT:HLA-A * 02:01 complex, VLAPRVLRW:HLA-A02:01 complex, and VLAPRVLRY:HLA-A * 02:01 complex, more preferably selected from the following group: VX1APRVLRX2:HLA-A02:01 complex (where X1 and X2 are any amino acids), VLAPRVLRA:HLA-A * 02:01 complex, VLAPRVLRV:HLA-A02:01 complex, VLAPRVLRI:HLA-A * 02:01 complex, and VLAPRVLRL:HLA-A * 02:01 complex, more preferably selected from the following group: VX1APRVLRX2:HLA-A * 02:01 complex (where X1 and X2 are any amino acids), VLAPRVLRA:HLA-A02:01 complex, VLAPRVLRV:HLA-A * 02:01 complex, and VLAPRVLRI:HLA-A * 02:01 complex. In other examples, the above complex contains HLA-A * 02:01 is HLA-A * 02:02, HLA-A * 02:03, HLA-A* 02:04, or HLA-A * It may be replaced with 02:09.

[0302] In yet another example, the encoded binding protein may specifically bind to a peptide selected from the following group: HLA complex: VX1APRVLRX2: HLA-A * 02 complex (where X1 and X2 are any amino acids), VLAPRVLRA:HLA-A * 02 complex, VLAPRVLRV:HLA-A * 02 complex, VLAPRVLRI:HLA-A * 02 complex, VLAPRVLRL:HLA-A * 02 complex, VLAPRVLRR:HLA-A * 02 complex, VLAPRVLRN:HLA-A * 02 complex, VLAPRVLRD:HLA-A * 02 complex, VLAPRVLRC:HLA-A * 02 complex, VLAPRVLRQ:HLA-A * 02 complex, VLAPRVLRE:HLA-A02 complex, VLAPRVLRG:HLA-A * 02 complex, VLAPRVLRH:HLA-A02 complex, VLAPRVLRK:HLA-A * 02 complex, VLAPRVLRM:HLA-A02 complex, VLAPRVLRF:HLA-A * 02 complex, VLAPRVLRP:HLA-A02 complex, VLAPRVLRS:HLA-A * 02 complex, VLAPRVLRT:HLA-A02 complex, VLAPRVLRW:HLA-A * 02 complex, and VLAPRVLRY:HLA-A * 02 complex, more preferably selected from the following group: VX1APRVLRX2:HLA-A * 02 complex (where X1 and X2 are any amino acids), VLAPRVLRA:HLA-A * 02 complex, VLAPRVLRV:HLA-A * 02 complex, VLAPRVLRI:HLA-A *02 complex, and VLAPRVLRL:HLA-A * 02 complex, more preferably selected from the following group: VX1APRVLRX2:HLA-A * 02 complex (where X1 and X2 are any amino acids), VLAPRVLRA:HLA-A * 02 complex, VLAPRVLRV:HLA-A * 02 complex, and VLAPRVLRI:HLA-A * 02 complex.

[0303] In one example, the RCN1-derived peptide of the peptide:HLA complex contains an antigenic fragment of an amino acid sequence selected from the following group: SEQ ID NOs: 1-21, more preferably SEQ ID NOs: 1-5 or SEQ ID NOs: 2-5, even more preferably selected from SEQ ID NOs: 2-4 or SEQ ID NOs: 3-5. In yet another example, the RCN1-derived peptide of the peptide:HLA complex contains or constitutes an amino acid sequence selected from the following group: SEQ ID NOs: 1-21, more preferably SEQ ID NOs: 1-5 or SEQ ID NOs: 2-5, even more preferably selected from SEQ ID NOs: 2-4 or SEQ ID NOs: 3-5.

[0304] Advantageously, the encoded binding protein may interact with the native RCN1 amino acid sequence of SEQ ID NO: 2 (but not with peptides that do not contain the amino acid sequence of SEQ ID NO: 1).

[0305] The TCR is composed of two different polypeptide chains. In humans, 95% of the TCR consists of alpha (α) and beta (β) chains (encoded by TRA and TRB, respectively). When the TCR binds to a peptide in the context of HLA (e.g., HLA-A), * (In the context of 02:01) T cells are activated through signal transduction.

[0306] The α and β chains of the TCR are highly variable in sequence. Each chain consists of two extracellular domains: a variable domain (V) and a constant domain (C). The constant domain is adjacent to the T cell membrane, followed by a transmembrane region and a short cytoplasmic tail, while the variable domain binds to the peptide / HLA complex.

[0307] This specification provides isolated nucleic acid compositions encoding RCN1 antigen-specific binding proteins having a TCRα chain variable (Vα) domain and a TCRβ chain variable (Vβ) domain. In one example, the nucleic acid compositions described herein may contain a TCRα chain constant domain and / or a TCRβ chain constant domain.

[0308] Each chain's variable domain has three highly variable regions (also called complementarity-determining regions (CDRs)). Therefore, the TCR alpha variable domain (hereinafter referred to as the TCR Vα domain, TCR V-alpha domain, Vα domain, V-alpha domain, alpha variable domain, etc.) contains CDR1, CDR2, and CDR3 regions. Similarly, the TCR beta variable domain (hereinafter referred to as the TCR Vβ domain, TCR V-beta domain, Vβ domain, V-beta domain, beta variable domain, etc.) also contains (different) CDR1, CDR2, and CDR3 regions. In both the alpha and beta variable domains, CDR3 is primarily responsible for peptide recognition.

[0309] As will be apparent to those skilled in the art, the phrase “TCR α-chain variable domain” refers to the variable (V) domain (extracellular domain) of the TCR α-chain, which includes three highly variable regions (CDR1, CDR2, and the identified CDR3) as well as intervening sequences, but does not include the constant (C) domain of the α-chain, which does not form part of the variable domain.

[0310] As will be apparent to anyone with ordinary knowledge of this art, the phrase “TCRβ chain variable domain” refers to the variable (V) domain (extracellular domain) of the TCRβ chain, which includes three highly variable regions (CDR1, CDR2, and the identified CDR3) as well as intervening sequences, but does not include the constant (C) domain of the β chain, which does not form part of the variable domain.

[0311] TCR component The isolated nucleic acid compositions described herein encode RCN1 antigen-specific binding proteins. As described herein, the inventors have identified HLA-A * 02 (for example, HLA-A * We identified multiple TCRs that interact with the native RCN1 amino acid sequence VLAPRVLRA (SEQ ID NO: 2), presented by 02:01).

[0312] (i) TCR clone TCR-1:VLAPRVLRA (SEQ ID NO: 2) TCR components that interact with TCR clone TCR-1:VLAPRVLRA As provided elsewhere in this specification, the inventors identified a TCR clone TCR-1 that interacts with VLAPRVLRA (SEQ ID NO: 2) in the context of HLA-A*02:01. The sequences corresponding to the TCR clone TCR-1 provided herein are SEQ ID NOs: 40-49.

[0313] In one embodiment, an isolated nucleic acid composition is provided that encodes an RCN1 antigen-specific binding protein having a TCRα chain variable (Vα) domain and a TCRβ chain variable (Vβ) domain. The composition comprises: a nucleic acid sequence encoding a TCR Vα domain or a functional fragment thereof, comprising a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 42; and a nucleic acid sequence encoding a TCR Vβ domain or a functional fragment thereof, comprising a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 45; where both CDR3 sequences specifically bind to a peptide, for example, SEQ ID NO: 1, when compounded with HLA.

[0314] An example of a suitable TCR Vα domain CDR3 amino acid sequence that specifically binds to the RCN1 antigen, particularly peptides containing SEQ ID NO: 1 (e.g., VLAPRVLRA (SEQ ID NO: 2)), is shown in SEQ ID NO: 42.

[0315] As will be apparent to those skilled in the art, variants of the CDR3 amino acid sequence provided herein may also be functional (i.e., if CDR3 is part of the TCR Vα domain, it retains the ability to specifically bind to the peptide containing SEQ ID NO: 1 (e.g., the peptide VLAPRVLRA of SEQ ID NO: 2)). Therefore, such functional variants are also included herein.

[0316] For example, a suitable (functional) Vα-domain CDR3 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 42. That is, it may have at least 80%, at least 81%, at least 90%, or even 100% sequence identity with SEQ ID NO: 42. In other words, a suitable (functional) Vα-domain CDR3 amino acid sequence may differ from the sequence shown in SEQ ID NO: 42 by one or more amino acids (e.g., two).

[0317] Sequence identity is calculated as a percentage of the total length of the reference sequence (e.g., sequence number 42), depending on the context.

[0318] As described above, the functional variants of the CDR3 amino acid sequence provided herein retain the ability to specifically bind to the peptide containing SEQ ID NO: 1 (e.g., the peptide of SEQ ID NO: 2) when CDR3 is part of the TCR Vα domain. Functional variants may be naturally occurring, artificially created, or artificially modified functional variants of the described CDR3 amino acid sequence. The term “variant” also includes homologs and fragments. Functional variants typically consist only of one, two, or more conserved amino acid substitutions, or substitutions, deletions, or insertions of non-essential amino acids in non-essential regions of CDR3.

[0319] Non-functional mutants are variants of the CDR3 amino acid sequence that do not specifically bind to the peptide containing SEQ ID NO: 1 (e.g., the peptide in SEQ ID NO: 2). Non-functional mutants typically include non-conservative substitutions, deletions, insertions, premature cleavage of the CDR3 amino acid sequence, or substitutions, insertions, or deletions in key amino acids or regions. Methods for identifying functional and non-functional mutants are well known to those skilled in the art.

[0320] In one embodiment, the CDR3 of the Vα domain contains or consists of the amino acid sequence of SEQ ID NO: 42. If the TCR Vα domain CDR3 has the amino acid sequence of SEQ ID NO: 42, CDR3 may be encoded by any suitable nucleic acid sequence.

[0321] The encoded TCR Vα domain may include, in addition to the specified CDR3, CDR1 having the amino acid sequence of SEQ ID NO: 40, or a functional variant thereof.

[0322] Functional variants of the CDR1 sequence refer to variants that retain the ability to specifically bind to the peptide containing SEQ ID NO: 1 (e.g., the peptide in SEQ ID NO: 2). Such functional variants may be naturally occurring, artificially created, or artificially modified functional variants of the original CDR1 sequence. The term “variant” also includes homologs and fragments. Functional variants typically consist only of one or more conserved amino acid substitutions, or substitutions, deletions, or insertions of non-essential amino acids in non-essential regions of a protein.

[0323] Non-functional variants are amino acid sequence variants of the original CDR1 sequence that do not specifically bind to the peptide containing SEQ ID NO: 1 (e.g., the peptide in SEQ ID NO: 2). Non-functional variants typically include non-conservative substitutions, deletions, insertions, premature cleavage of the original CDR1 amino acid sequence, or substitutions, insertions, or deletions in key amino acids or regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0324] For example, a suitable functional Vα domain CDR1 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 40, i.e., at least 80%, at least 83%, or even 100% sequence identity with SEQ ID NO: 40. In other words, a suitable functional Vα domain CDR1 amino acid sequence may differ from the sequence shown in SEQ ID NO: 1 by one or more amino acids.

[0325] In one embodiment, the Vα domain CDR1 contains or consists of the amino acid sequence of SEQ ID NO: 40. If the TCR Vα domain CDR1 has the amino acid sequence of SEQ ID NO: 40, CDR1 may be encoded by any suitable nucleic acid sequence.

[0326] The encoded TCR Vα domain may include, in addition to the specified CDR3 (and the specified CDR1 above may be omitted), a CDR2 having the amino acid sequence of SEQ ID NO: 41, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to HLA-A*02, most preferably HLA-A*02:01).

[0327] Functional variants may be naturally occurring, artificially created, or artificially modified functional variants of the original CDR2 sequence. The term “variant” also includes homologs and fragments. Functional variants typically consist only of one or more conserved amino acid substitutions, or substitutions, deletions, or insertions of non-essential amino acids in non-essential regions of a protein.

[0328] Non-functional mutants are amino acid sequence variants of the original CDR2 sequence that do not specifically bind to HLA-A*02, preferably HLA-A*02:01. Non-functional mutants typically include non-conservative substitutions, deletions, insertions, premature cleavage of the amino acid sequence of the original CDR2 sequence, or substitutions, insertions, or deletions in key amino acids or key regions. Methods for identifying functional and non-functional mutants are well known to those skilled in the art.

[0329] For example, a suitable functional Vα domain CDR2 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 41, i.e., at least 80%, at least 85%, or 100% sequence identity with SEQ ID NO: 41. In other words, a suitable (functional) Vα domain CDR2 amino acid sequence may differ by one or more amino acids from the sequence shown in SEQ ID NO: 41. Suitable % identity is calculated as the percentage of identity with respect to the full length of the reference sequence (e.g., SEQ ID NO: 41). As mentioned above, the variant may contain conserved amino acid substitutions compared to the original CDR2 sequence. As described above, the functional variant of CDR2 retains the ability to specifically bind to HLA-A*02, preferably HLA-A*02:01.

[0330] In one example, the CDR2 of the Vα domain contains or consists of the amino acid sequence of SEQ ID NO: 41. If the TCR Vα domain CDR2 has the amino acid sequence of SEQ ID NO: 41, the CDR2 may be encoded by any suitable nucleic acid sequence. Thus, the encoded TCR Vα domain may contain the CDRs described in detail above (specifically, SEQ ID NO: 42, SEQ ID NO: 40, SEQ ID NO: 41 or their functional variants), with appropriate spacing sequences placed between the CDRs.

[0331] The encoded TCR Vα domain may include the amino acid sequence of SEQ ID NO: 46, or a functional variant thereof (i.e., the mutant TCR Vα domain retains the ability to specifically bind to the peptide containing SEQ ID NO: 1 (e.g., the peptide of SEQ ID NO: 2) as part of the binding protein described in this invention). Such functional variants may be naturally occurring, artificially created, or artificially modified functional variants of SEQ ID NO: 46. Functional variants typically consist only of one or more conserved amino acid substitutions, or substitutions, deletions, or insertions of non-essential amino acids in non-essential regions of the protein.

[0332] Non-functional variants are amino acid sequence variants that do not specifically bind to the peptide containing SEQ ID NO: 1 (e.g., the peptide in SEQ ID NO: 2). Non-functional variants typically involve non-conservative substitutions, deletions, insertions, or premature cleavage of the amino acid sequence of the original TCR Vα domain, or substitutions, insertions, or deletions in key amino acids or regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0333] In one example, the encoded TCR Vα domain has at least 75%, at least 80%, at least 85%, or at least 90% (or 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 amino acid sequence of SEQ ID NO: 46 and retains the ability to specifically bind to the peptide containing SEQ ID NO: 1 (e.g., the peptide of SEQ ID NO: 2). In other words, this also includes functional TCR Vα domains that contain one or more amino acid substitutions compared to the sequence of SEQ ID NO: 46. As mentioned above, the amino acid substitutions may be conserved amino acid substitutions. Sequence variations compared to SEQ ID NO: 46 may be present only in regions of the TCR Vα domain that do not form a CDR (i.e., the variant may have the CDRs of SEQ ID NO: 42, SEQ ID NO: 40, and / or SEQ ID NO: 41 and have 25% (or less) sequence variation compared to SEQ ID NO: 46). In other words, the sequence of CDR of sequence number 46 is preserved, and the sequence of the remaining part may change appropriately within the parameter of "at least 75% identity" mentioned above. Appropriate % identity is calculated as the percentage of identity relative to the total length of the reference sequence (e.g., sequence number 46).

[0334] In one example, the encoded TCR Vα domain has at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) sequence identity with the amino acid sequence of SEQ ID NO: 46, and the TCR Vα domain includes CDR3 having the amino acid sequence of SEQ ID NO: 42. In this example, CDR1 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 40, and CDR2 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 41.

[0335] In another example, the encoded TCR Vα domain has the amino acid sequence of SEQ ID NO: 46 and may contain 0 to 10 (or 0 to 5) amino acid substitutions, insertions, or deletions. In this case, the TCR Vα domain includes CDR3 having the amino acid sequence of SEQ ID NO: 42, CDR1 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 40, and CDR2 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 41.

[0336] If the TCR Vα domain has the amino acid sequence of SEQ ID NO: 46, the TCR Vα domain may be encoded by the nucleic acid sequence of SEQ ID NO: 47, or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that codes for the same protein due to genetic code degeneracy).

[0337] In this specification, the term "genetically degenerate sequence" is used synonymously with "derivative."

[0338] To avoid misunderstanding, it should be stated that the nucleic acid sequence encoding the TCR Vα domain may also encode the TCR α chain constant domain. An example of a suitable constant domain (either the TCR α chain or TCR β chain) is encoded in the MP71-TCR-flex retroviral vector. However, the present invention is not limited to this particular constant domain and encompasses all suitable TCR α chain constant domains. The constant domain may be mouse-derived, human-derived, or humanized. Methods for identifying or generating suitable constant domains are well known to those skilled in the art and can be carried out within the bounds of normal capability.

[0339] In some cases, the constant domain may be encoded by or derived from vectors such as retroviruses, lentiviruses, or plasmid vectors, or from adenoviruses, adeno-associated viruses, vaccinia viruses, canary poxviruses, or herpesvirus vectors. These viral vectors have mouse or human constant domains pre-cloned. More recently, TCR gene transfer using minicircles has been reported (using minicircle vectors with non-viral Sleeping Beauty translocations, as published in Monjezi, et al., 2017). Furthermore, naked (synthetic) DNA / RNA can also be used to introduce the TCR. For example, a suitable constant domain can be provided using a pMSGV retroviral vector with pre-cloned TCR-Ca and Cb genes, as reported by LV Coren et al., BioTechniques 2015. In addition, single-stranded or double-stranded DNA / RNA can be inserted into the TCR locus by homologous recombination repair (see Roth et al. 2018 Nature vol 559, p. 405). Furthermore, non-homological end joining is also possible.

[0340] In one example, the nucleic acid composition provided in the present invention includes a nucleic acid sequence encoding a TCR Vα domain, which comprises a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 42.

[0341] In another example, the CDR3 of the Vα domain of the nucleic acid composition provided in the present invention comprises or consists of the amino acid sequence of SEQ ID NO: 42.

[0342] In another example, the Vα domain of the nucleic acid composition provided in the present invention has at least 80% sequence identity with SEQ ID NO: 46, contains SEQ ID NO: 46, or contains an amino acid sequence consisting of SEQ ID NO: 46.

[0343] As stated above, the inventors identified TCR clone TCR-1, and this clone is HLA-A * We confirmed that it interacts with VLAPRVLRA (sequence number 2) in the context of 02:01. The sequences of sequence numbers 40 to 49 provided herein correspond to the TCR clone TCR-1.

[0344] Therefore, an example of a TCR Vβ domain CDR3 amino acid sequence that specifically binds to the RCN1 antigen, particularly to peptides containing the amino acid sequence of SEQ ID NO: 1 (e.g., to SEQ ID NO: 2), is shown in SEQ ID NO: 45. As will be apparent to those skilled in the art, variants of the amino acid sequence shown in SEQ ID NO: 45 are also functional (i.e., retain the ability to specifically bind to peptides containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2) when CDR3 is part of the TCR Vβ domain). Such functional variants are included herein.

[0345] For example, a suitable (functional) Vβ-domain CDR3 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 45. That is, it may have at least 80%, at least 83%, at least 91%, or even 100% sequence identity with SEQ ID NO: 45. In other words, a suitable (functional) Vβ-domain CDR3 amino acid sequence may differ from the sequence shown in SEQ ID NO: 45 by one or more (e.g., two) amino acids.

[0346] In one example, the CDR3 of the Vβ domain contains or consists of the amino acid sequence of SEQ ID NO: 45. If the TCR Vβ domain CDR3 has the amino acid sequence of SEQ ID NO: 45, the CDR3 may be encoded by any suitable nucleic acid sequence.

[0347] The encoded TCR Vβ domain may include, in addition to the specified CDR3, a CDR1 containing the amino acid sequence of SEQ ID NO: 43, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0348] For example, a suitable functional Vβ-domain CDR1 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 43. That is, it may have at least 80% or even 100% sequence identity with SEQ ID NO: 43. In other words, a suitable (functional) Vβ-domain CDR1 amino acid sequence may differ from the sequence shown in SEQ ID NO: 43 by one or more (e.g., two) amino acids.

[0349] In one example, the Vβ domain CDR1 contains or consists of the amino acid sequence of SEQ ID NO: 43. If the TCR Vβ domain CDR1 has the amino acid sequence of SEQ ID NO: 43, CDR1 may be encoded by any suitable nucleic acid sequence.

[0350] The encoded TCR Vβ domain may include, in addition to the specified CDR3 (and the specified CDR1 above), a CDR2 containing the amino acid sequence of Sequence ID No. 44, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to HLA-A*02, most preferably HLA-A*02:01).

[0351] For example, a suitable functional Vβ-domain CDR2 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 44. That is, it may have at least 80%, at least 83%, or even 100% sequence identity with SEQ ID NO: 44. In other words, a suitable (functional) Vβ-domain CDR2 amino acid sequence may differ from the sequence shown in SEQ ID NO: 44 by one or more (e.g., two) amino acids.

[0352] In one example, the CDR2 of the Vβ domain contains or consists of the amino acid sequence of SEQ ID NO: 44. If the TCR Vβ domain CDR2 has the amino acid sequence of SEQ ID NO: 44, the CDR2 may be encoded by any suitable nucleic acid sequence.

[0353] Therefore, the encoded TCR Vβ domain may contain the CDRs described in detail above (i.e., SEQ ID NO: 45, SEQ ID NO: 43, SEQ ID NO: 44, or their functional variants), with appropriate intervening sequences between the CDRs.

[0354] The encoded TCR Vβ domain may have the amino acid sequence of SEQ ID NO: 48, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1, e.g., the peptide of SEQ ID NO: 2, as part of the binding proteins described in this literature).

[0355] In one example, the encoded TCR Vβ domain may have at least 75%, at least 80%, at least 85%, or at least 90% (or 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 to the amino acid sequence of SEQ ID NO: 1 and retain the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide of SEQ ID NO: 2). In other words, this also includes functional TCR Vβ domains in which one or more amino acids are substituted compared to the sequence of SEQ ID NO: 48. Sequence variations compared to SEQ ID NO: 48 may all be present in regions of the TCR Vβ domain that do not form a CDR (i.e., the variant may have the CDRs of SEQ ID NO: 45, SEQ ID NO: 43, and / or SEQ ID NO: 44, and may have 25% (or less) sequence variation compared to SEQ ID NO: 48). In other words, the sequence of the CDR of sequence number 48 is preserved, and the remaining sequence may vary within a range suitable for the "at least 75% identity" parameter mentioned above. Appropriately, the percentage identity may be calculated as the percentage of identity relative to the total length of the reference sequence (e.g., sequence number 48).

[0356] For example, the encoded TCR Vβ domain may have at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) sequence identity with the amino acid sequence of SEQ ID NO: 48, and the TCR Vβ domain may contain a CDR3 having the amino acid sequence of SEQ ID NO: 45. In this example, CDR1 of the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 43, and CDR2 of the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 44.

[0357] In the example where the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 48, the TCR Vβ domain may be encoded by the nucleotide sequence of SEQ ID NO: 49, or its genetically degenerated sequence (i.e., another nucleotide sequence that encodes the same protein through degeneration of the genetic code).

[0358] To avoid any ambiguity, it should be stated that the nucleotide sequence encoding the TCR Vβ domain may also encode the constant domain of the TCR β chain. Examples of suitable constant domains have generally been discussed above.

[0359] In one example, the nucleotide composition provided herein may include a nucleotide sequence encoding a TCR Vβ domain or a functional fragment thereof, comprising a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 45. In another example, the CDR3 of the Vβ domain of the nucleotide composition provided herein may include the amino acid sequence of SEQ ID NO: 45.

[0360] In yet another example, the Vβ domain of the nucleotide composition provided herein may have at least 80% sequence identity with, or contain, or match the amino acid sequence of SEQ ID NO: 48.

[0361] The TCR Vβ domain sequence obtained from the TCR clone TCR-1 discussed above is particularly compatible with the TCR Vα domain sequence obtained from the TCR clone TCR-1 discussed here.

[0362] Accordingly, in one example, a nucleotide composition described herein may encode a peptide-specific binding protein comprising the amino acid sequence of SEQ ID NO: 1, wherein the binding protein may comprise a nucleotide sequence encoding a TCR Vα domain having a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 42, or a functional fragment thereof, and a TCR Vβ domain having a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 45, or a functional fragment thereof.

[0363] In particular, in one example, the nucleotide composition described herein encodes a binding protein specific to a peptide containing the amino acid sequence of SEQ ID NO: 1, wherein the binding protein has a TCR Vα domain having a CDR3 amino acid sequence containing or matching the amino acid sequence of SEQ ID NO: 42, and a TCR Vβ domain having a CDR3 amino acid sequence containing or matching the amino acid sequence of SEQ ID NO: 45. Furthermore, the peptide containing the amino acid sequence of SEQ ID NO: 1 may contain or match the sequence of SEQ ID NO: 2. Moreover, the TCR Vα domain may be part of a TCR α chain having a constant domain, and the TCR Vβ domain may be part of a TCR β chain having a constant domain.

[0364] In this particular example, the Vα domain may have an amino acid sequence that has at least 80% sequence identity to, contains, or matches, the amino acid sequence of SEQ ID NO: 46, and the Vβ domain may have an amino acid sequence that has at least 80% sequence identity to, contains, or matches, the amino acid sequence of SEQ ID NO: 48. In one example, the Vα domain may have the amino acid sequence of SEQ ID NO: 46, and the Vβ domain may have the amino acid sequence of SEQ ID NO: 48. In such a case, the Vα domain is encoded by the nucleotide sequence of SEQ ID NO: 47, and the Vβ domain is encoded by the nucleotide sequence of SEQ ID NO: 49.

[0365] In this particular example, the TCR Vα domain may contain a CDR1 amino acid sequence containing or matching the CDR1 amino acid sequence of SEQ ID NO: 40, and a CDR2 amino acid sequence containing or matching the CDR2 amino acid sequence of SEQ ID NO: 41. Furthermore, the TCR Vβ domain may contain a CDR1 amino acid sequence containing or matching the CDR1 amino acid sequence of SEQ ID NO: 43, and a CDR2 amino acid sequence containing or matching the CDR2 amino acid sequence of SEQ ID NO: 44.

[0366] To avoid any ambiguity, this particular example includes the components of the TCR clone TCR-1 illustrated here. The different components of the TCR clone TCR-1 and their corresponding sequence numbers are summarized in Table 7 below.

[0367] The nucleotide compositions described herein encode TCR Vα and TCR Vβ domains that form binding proteins capable of specifically binding to the RCN1 antigen. When the TCR Vα and TCR Vβ domains are encoded by the same nucleotide sequence, they may be linked by a linker, such as one that allows the expression of two proteins or polypeptides from the same vector. For example, linkers containing porcine Tescovirus type 1 2A (P2A) sequences can be used, such as the 2A sequences of foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), or Tosea signalavirus (T2A), as reported by AL Szymczak et al. (Nature Biotechnology 22, 589-594 (2004)). The 2A and 2A-like sequences are linkers that can be cleaved after the nucleotide molecules have been transcribed and translated. Another example of a linker is the presence of an internal ribosome entry site (IRES), which allows the translation of two proteins or polypeptides from the same transcript. Other suitable linkers may also be used. Furthermore, the nucleotide sequences encoding the TCR Vα domain and the nucleotide sequences encoding the TCR Vβ domain may be cloned into a vector having a dual internal promoter (e.g., S. Jones et al., Human Gene Ther 2009). Identifying suitable linkers and vectors that enable the expression of both the TCR Vα and TCR Vβ domains is within the ordinary capability of those skilled in the art.

[0368] Other suitable polypeptide domains may also be encoded by the nucleatide sequences encoding the TCR Vα domain and / or TCR Vβ domain. For example, the nucleatide sequence may include a membrane targeting sequence that provides transport of the encoded polypeptide to the cell surface membrane of the modified cell. Other suitable additional domains are widely known, as described, for example, in WO2016 / 071758.

[0369] In one example, the nucreatide compositions described herein may encode a soluble TCR. For example, the nucreatide compositions may encode the variable domains of the TCR alpha and beta chains, respectively, and an immunomodulatory molecule such as a CD3 agonist (e.g., anti-CD3 scFv). The CD3 antigen is present in mature human T cells, thymocytes, and some innate immune cells, is associated with the TCR, and is involved in TCR signaling. Antibodies specific to the human CD3 antigen are widely known, one of which is the monoclonal antibody OKT3, the first to be approved by the FDA. Other antibodies specific to CD3 have also been reported (see, e.g., WO2004 / 106380, U.S. Patent Publication No. 2004 / 0202657, U.S. Patent No. 6,750,325). The immunomobilizing mTCR (ImmTAC; Immunocore Limited, Milton Park, Abington, Oxon, United Kingdom) is a dual-function protein that combines affinity monoclonal T cell receptor (mTCR) targeting with a therapeutic mechanism (i.e., anti-CD3 scFv). In another example, the soluble TCR of the present invention can be combined with a radioisotope or a toxic drug. Suitable radioisotopes and / or toxic drugs are widely known to those skilled in the art and can be easily identified by those skilled in the art.

[0370] In one example, the nucleatide composition may encode a chimeric single-chain TCR in which the TCR alpha-chain variable domain is linked to the TCR beta-chain variable domain and a constant domain fused to, for example, a CD3 zeta signaling domain. In this example, the linker is not cleavable. In an alternative embodiment, the nucleatide composition may encode a chimeric double-chain TCR in which the TCR alpha-chain variable domain and the TCR beta-chain variable domain are each linked to a CD3 zeta signaling domain or other transmembrane and intracellular domains. Methods for preparing such single-chain and double-chain TCRs are well known to those skilled in the art; see, for example, RA Willemsen et al, Gene Therapy 2000.

[0371] (ii) TCR components that interact with TCR clone TCR-2:VLAPRVLRA (SEQ ID NO: 2) As provided elsewhere in this specification, the inventors also provide TCR clone TCR-2, which is HLA-A * It has been confirmed to interact with VLAPRVLRA (sequence number 2) in the context of 02:01. The sequence numbers corresponding to the TCR clone TCR-2 provided here are sequence numbers 50 to 59.

[0372] As one embodiment, an isolated nucleatide composition is provided that encodes an RCN1 antigen-specific binding protein having a TCR α-chain variable (Vα) domain and a TCR β-chain variable (Vβ) domain. The composition comprises: a nucleatide sequence encoding a TCR Vα domain or a functional fragment thereof, whose CDR3 amino acid sequence has at least 80% sequence identity to SEQ ID NO: 52; and a nucleatide sequence encoding a TCR Vβ domain or a functional fragment thereof, whose CDR3 amino acid sequence has at least 80% sequence identity to SEQ ID NO: 55; where these CDR3 sequences together specifically bind to a peptide containing SEQ ID NO: 1 (e.g., VLAPRVLRA (SEQ ID NO: 2)) when complexed with HLA.

[0373] Therefore, another example of a suitable TCR Vα domain CDR3 amino acid sequence that specifically binds to the RCN1 antigen, particularly peptides containing SEQ ID NO: 1 (e.g., VLAPRVLRA (SEQ ID NO: 2)), is shown in SEQ ID NO: 52.

[0374] For example, a suitable (functional) Vα-domain CDR3 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 52. That is, it may have at least 80%, at least 83%, at least 91%, or 100% sequence identity. In other words, a suitable (functional) Vα-domain CDR3 amino acid sequence may differ from the sequence shown in SEQ ID NO: 52 by one or more amino acids (e.g., two).

[0375] In one example, the CDR3 of the Vα domain may include or consist of the amino acid sequence shown in SEQ ID NO: 52. In the example where the TCR Vα domain CDR3 has the amino acid sequence shown in SEQ ID NO: 52, the CDR3 may be encoded by any suitable nucleatide sequence.

[0376] The encoded TCR Vα domain may include, in addition to the specified CDR3, a CDR1 having the amino acid sequence of SEQ ID NO: 50, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to the peptide containing SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0377] For example, a suitable functional Vα domain CDR1 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 50, i.e., at least 80%, at least 83%, or 100%. In other words, a suitable (functional) Vα domain CDR1 amino acid sequence may have one or more amino acids mutated from the sequence shown in SEQ ID NO: 50. In one example, the Vα domain CDR1 may contain or consist of the amino acid sequence of SEQ ID NO: 50. In the example where the TCR Vα domain CDR1 has the amino acid sequence of SEQ ID NO: 50, CDR1 may be encoded by any suitable nucleic acid sequence.

[0378] The encoded TCR Vα domain may include, in addition to the specified CDR3 (and the specified CDR1 above), a CDR2 having the amino acid sequence of SEQ ID NO: 51, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to HLA-A*02, most preferably HLA-A*02:01).

[0379] For example, a suitable functional Vα domain CDR2 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 51, i.e., at least 80%, at least 88%, or 100%. In other words, a suitable (functional) Vα domain CDR2 amino acid sequence may have one or more amino acids mutated from the sequence shown in SEQ ID NO: 51. In one example, the Vα domain CDR2 may contain or consist of the amino acid sequence of SEQ ID NO: 51. In the example where the TCR Vα domain CDR2 has the amino acid sequence of SEQ ID NO: 51, the CDR2 may be encoded by any suitable nucleic acid sequence.

[0380] Therefore, the encoded TCR Vα domain includes the CDRs detailed above (i.e., SEQ ID NO: 52, SEQ ID NO: 50, and SEQ ID NO: 51, or their functional variants), and appropriate intermediate sequences may exist between the CDRs.

[0381] The encoded TCR Vα domain may have the amino acid sequence of SEQ ID NO: 56, or it may include a functional variant thereof (i.e., the mutant TCR Vα domain retains the ability to specifically bind to the peptide containing SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0382] In one example, an encoded TCR Vα domain may have an amino acid sequence with at least 75%, at least 80%, at least 85%, or at least 90% (or 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 amino acid sequence of SEQ ID NO: 56, and may retain the ability to specifically bind to the peptide containing SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2). In other words, this also includes functional TCR Vα domains having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 56. All sequence mutations compared to SEQ ID NO: 56 may be in regions that do not form a CDR of the TCR Vα domain (i.e., the variant may have the CDRs of SEQ ID NO: 52, SEQ ID NO: 50, and / or SEQ ID NO: 51, and may have 25% (or less) sequence mutations compared to SEQ ID NO: 56). In other words, the sequence of CDR of sequence number 56 is preserved, and the rest of its sequence may be appropriately mutated within the "at least 75% identity" parameter mentioned above.

[0383] For example, the encoded TCR Vα domain contains an amino acid sequence that has at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) sequence identity with the amino acid sequence of SEQ ID NO: 56, and the TCR Vα domain contains CDR3 having the amino acid sequence of SEQ ID NO: 52. In this example, CDR1 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 50, and CDR2 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 51.

[0384] In another example, the encoded TCR Vα domain contains the amino acid sequence of SEQ ID NO: 56 and has 0 to 10 (or 0 to 5) amino acid substitutions, insertions, or deletions, and the TCR Vα domain contains CDR3 which has the amino acid sequence of SEQ ID NO: 52. In this example, CDR1 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 50, and CDR2 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 51.

[0385] In the example where the TCR Vα domain has the amino acid sequence of SEQ ID NO: 56, the TCR Vα domain may be encoded by the nucleic acid sequence of SEQ ID NO: 57, or a genetically degenerated version thereof (i.e., another nucleic acid sequence that encodes the same protein through degeneration of the genetic code).

[0386] To avoid ambiguity, the nucleic acid sequence encoding the TCR Vα domain may also encode the TCRα chain constant domain. Examples of suitable constant domains are generally discussed above.

[0387] In one example, the nucleic acid composition provided in this document includes a nucleic acid sequence encoding a TCRVα domain or a functional fragment thereof, which comprises a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 52.

[0388] In another example, the CDR3 of the Vα domain of the nucleic acid composition provided in this book contains or matches the amino acid sequence of SEQ ID NO: 52.

[0389] In another example, the Vα domain of the nucleic acid composition provided in this book contains an amino acid sequence that has at least 80% sequence identity with, is contained in, or matches the amino acid sequence of SEQ ID NO: 56.

[0390] As provided herein, the inventors have identified a TCR clone TCR-2 that interacts with VLAPRVLRA (SEQ ID NO: 2) in the context of HLA-A*02:01. The sequences corresponding to the TCR clone TCR-2 provided herein are SEQ ID NOs: 50 to 59.

[0391] A suitable example of the TCR Vβ domain CDR3 amino acid sequence that specifically binds to the RCN1 antigen, particularly peptides containing SEQ ID NO: 1 (e.g., SEQ ID NO: 2), is shown in SEQ ID NO: 55. As is obvious to engineers, variants of the amino acid sequence shown in SEQ ID NO: 55 may also be functional (i.e., retain the ability to specifically bind to peptides containing the amino acid sequence of SEQ ID NO: 1 (e.g., SEQ ID NO: 2) if CDR3 is part of the TCR Vβ domain).

[0392] For example, a suitable (functional) Vβ domain CDR3 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 55. That is, it may have at least 80%, at least 84%, at least 92%, or 100% sequence identity with SEQ ID NO: 55. In other words, a suitable (functional) Vβ domain CDR3 amino acid sequence may differ by one or more (e.g., two) amino acids from the sequence shown in SEQ ID NO: 55. In one example, the Vβ domain CDR3 contains or matches the amino acid sequence of SEQ ID NO: 55. If the TCR Vβ domain CDR3 has the amino acid sequence of SEQ ID NO: 55, its CDR3 may be encoded by a suitable nucleic acid sequence.

[0393] The encoded TCR Vβ domain may include, in addition to the specified CDR3, a CDR1 having the amino acid sequence of SEQ ID NO: 53, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0394] For example, a suitable functional Vβ domain CDR1 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 53. That is, it may have at least 80% or 100% sequence identity with SEQ ID NO: 53. In other words, a suitable (functional) Vβ domain CDR1 amino acid sequence may differ from the sequence shown in SEQ ID NO: 53 by one or more amino acids.

[0395] In one example, the CDR1 of the Vβ domain contains or matches the amino acid sequence of SEQ ID NO: 53. If the TCR Vβ domain CDR1 has the amino acid sequence of SEQ ID NO: 53, then its CDR1 may be encoded by an appropriate nucleic acid sequence.

[0396] The encoded TCR Vβ domain may include, in addition to the specified CDR3 (and optionally the specified CDR1 above), a CDR2 having the amino acid sequence of SEQ ID NO: 54 or a functional variant thereof (i.e., the variant retains the ability to specifically bind to HLA-A*02, most preferably HLA-A*02:01).

[0397] For example, a suitable functional Vβ domain CDR2 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 54. That is, it may have at least 80%, at least 83%, or 100% sequence identity with SEQ ID NO: 54. In other words, a suitable (functional) Vβ domain CDR2 amino acid sequence may differ from the sequence shown in SEQ ID NO: 54 by one or more amino acids.

[0398] In one example, the CDR2 of the Vβ domain contains or matches the amino acid sequence of SEQ ID NO: 54. If the TCR Vβ domain CDR2 has the amino acid sequence of SEQ ID NO: 54, its CDR2 may be encoded by an appropriate nucleic acid sequence.

[0399] Therefore, the encoded TCR Vβ domain may include the CDRs described in detail above (i.e., SEQ ID NO: 55, SEQ ID NO: 53, and SEQ ID NO: 54, or their functional variants), and appropriate intervening sequences may be included between the CDRs.

[0400] The encoded TCR Vβ domain may have the amino acid sequence of SEQ ID NO: 58, or a functional variant thereof (i.e., the mutant TCR Vβ domain retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2) as part of the binding protein described herein).

[0401] In one example, the encoded TCR Vβ domain has an amino acid sequence with at least 75%, at least 80%, at least 85%, or at least 90% (or 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 to the amino acid sequence of SEQ ID NO: 58, and retains the ability to specifically bind to peptides containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2). In other words, this also includes functional TCR Vβ domains having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 58. All sequence variations compared to SEQ ID NO: 58 may be in regions that do not form a CDR of the TCR Vβ domain (i.e., the variant may have the CDRs of SEQ ID NO: 55, SEQ ID NO: 53, and / or SEQ ID NO: 54, and have 25% (or less) sequence variation compared to SEQ ID NO: 58). In other words, the sequence of CDR at sequence number 58 is preserved, while the remaining sequences may vary within the "at least 75% identity" parameter mentioned above.

[0402] For example, the encoded TCR Vβ domain may have at least 75% sequence identity (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) to the amino acid sequence of SEQ ID NO: 58, and the TCR Vβ domain may include a CDR3 having the amino acid sequence of SEQ ID NO: 55. In this example, CDR1 of the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 53, and CDR2 of the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 54.

[0403] In the example where the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 58, the TCR Vβ domain may be encoded by the nucleic acid sequence of SEQ ID NO: 59, or a genetically degenerated version thereof (i.e., another nucleic acid sequence that encodes the same protein through genetic coding degeneration).

[0404] To avoid misunderstanding, the nucleic acid sequence encoding the TCR Vβ domain may also encode the TCR β chain constant domain. Examples of suitable constant domains were generally discussed above.

[0405] In one example, the nucleic acid composition described herein includes a nucleic acid sequence encoding a TCR Vβ domain or a functional fragment thereof, which comprises a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 55.

[0406] In another example, the CDR3 of the Vβ domain of the nucleic acid composition described herein includes or consists solely of the amino acid sequence of Sequence ID No. 55.

[0407] In yet another example, the Vβ domain of the nucleic acid composition described herein includes SEQ ID NO: 58 or an amino acid sequence having at least 80% sequence identity to said sequence.

[0408] The TCR Vβ domain sequence derived from the TCR clone TCR-2 described above is particularly compatible with the TCR Vα domain sequence derived from the TCR clone TCR-2 described herein.

[0409] Therefore, in one example, the nucleic acid composition described herein encodes a binding protein specific to a peptide containing the amino acid sequence of SEQ ID NO: 1. Specifically, it includes a nucleic acid sequence or functional fragment thereof that includes a TCR Vα domain containing a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 52, or a TCR Vβ domain containing a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 55.

[0410] In certain examples, the nucleic acid compositions described herein encode a peptide-specific binding protein having a TCR Vα domain having an amino acid sequence of the amino acid sequence of SEQ ID NO: 1 and containing or comprising the amino acid sequence of SEQ ID NO: 52, and a TCR Vβ domain having a CDR3 containing or comprising the amino acid sequence of SEQ ID NO: 55. Furthermore, the peptide containing the amino acid sequence of SEQ ID NO: 1 may contain or comprise the sequence shown in SEQ ID NO: 2. Furthermore, the TCR Vα domain may be part of a TCR α chain having a constant domain, and the TCR Vβ domain may be part of a TCR β chain having a constant domain.

[0411] In this particular example, the Vα domain may contain, include, or consist of an amino acid sequence having at least 80% sequence identity with, or containing, sequence number 56. Similarly, the Vβ domain may contain, include, or consist of an amino acid sequence having at least 80% sequence identity with, or sequence number 58. In one example, the Vα domain contains the amino acid sequence of sequence number 56, and the Vβ domain contains the amino acid sequence of sequence number 58. In such a case, the Vα domain may be encoded by a nucleic acid sequence containing the sequence of sequence number 57, and the Vβ domain may be encoded by a nucleic acid sequence containing the sequence of sequence number 59.

[0412] In this particular example, the TCR Vα domain may include the amino acid sequence of SEQ ID NO: 50, or the CDR1 amino acid sequence consisting of SEQ ID NO: 50, and the amino acid sequence of SEQ ID NO: 51, or the CDR2 amino acid sequence consisting of SEQ ID NO: 51. Furthermore, the TCR Vβ domain may include the amino acid sequence of SEQ ID NO: 53, or the CDR1 amino acid sequence consisting of SEQ ID NO: 53, and the amino acid sequence of SEQ ID NO: 54, or the CDR2 amino acid sequence consisting of SEQ ID NO: 54.

[0413] To avoid any ambiguity, this particular example encompasses the components of the TCR clone TCR-2 illustrated herein. The different components of the TCR clone TCR-2 and their respective sequence numbers are summarized in Table 8 below.

[0414] As described in more detail elsewhere in this specification, the nucleic acid compositions described herein encode both the TCR Vα domain and the TCR Vβ domain, which form binding proteins that can specifically bind to the RCN1 antigen. In examples where the TCR Vα domain and the TCR Vβ domain are encoded by the same nucleic acid sequence, the TCR Vα domain and the TCR Vβ domain may be linked via a linker. Suitable linkers are outlined elsewhere in this specification. Additional suitable polypeptide domains, which may be encoded by the nucleic acid sequences encoding the TCR Vα domain and / or the TCR Vβ domain, are also outlined elsewhere in this specification.

[0415] For example, the nucleic acid compositions described herein may encode a soluble TCR, or a chimeric single-chain TCR in which a TCRα-variable domain is linked to a constant domain fused to a TCRβ-variable domain and, for example, a CD3ζ signaling domain. These are outlined in more detail elsewhere in this specification.

[0416] (iii) TCR components that interact with TCR clone TCR-4:VLAPRVLRA (SEQ ID NO: 2) As described elsewhere in this specification, the inventors of HLA-A * We also identified TCR clone TCR-4, which interacts with VLAPRVLRA (SEQ ID NO: 2) at 02:01. The sequences corresponding to TCR clone TCR-4 provided herein are SEQ ID NOs: 60-69.

[0417] In one embodiment, an isolated nucleic acid composition is provided encoding an RCN1 antigen-specific binding protein having a TCRα chain variable (Vα) domain and a TCRβ chain variable (Vβ) domain, the composition comprising: a nucleic acid sequence encoding a TCR Vα domain or a functional fragment thereof, comprising a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 62; and a nucleic acid sequence encoding a TCR Vβ domain or a functional fragment thereof, comprising a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 65, wherein the CDR3 sequences together specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (for example, when the peptide is complexed with HLA).

[0418] An example of a suitable TCR Vα domain CDR3 amino acid sequence that specifically binds to the RCN1 antigen, particularly peptides containing the amino acid sequence of SEQ ID NO: 1 (e.g., VLAPRVLRA of SEQ ID NO: 2), is shown in SEQ ID NO: 62.

[0419] For example, a suitable (functional) Vα-domain CDR3 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 62. That is, it may have at least 80%, at least 84%, at least 92%, or 100% sequence identity with SEQ ID NO: 62. Appropriately, the identity percentage is calculated as the percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 62). In other words, a suitable (functional) Vα-domain CDR3 amino acid sequence may differ from the sequence shown in SEQ ID NO: 62 by one or more amino acids (e.g., two).

[0420] In one example, the CDR3 of the Vα domain contains or is composed of the amino acid sequence of SEQ ID NO: 62. In the example where the TCR Vα domain CDR3 has the amino acid sequence of SEQ ID NO: 62, CDR3 may be encoded by any suitable nucleic acid sequence.

[0421] The encoded TCR Vα domain may include, in addition to a specific CDR3, a CDR1 containing the amino acid sequence of SEQ ID NO: 60, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0422] For example, a suitable functional Vα domain CDR1 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 60. That is, it may have at least 80%, at least 83%, or 100% sequence identity with SEQ ID NO: 60. Appropriately, the identity percentage is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 60). In other words, a suitable functional Vα domain CDR1 amino acid sequence may differ from the sequence shown in SEQ ID NO: 60, overlapping with the amino acid sequence of 60 by one or more amino acids. In one example, the Vα domain CDR1 contains or consists of the amino acid sequence of SEQ ID NO: 60. In the example where the TCR Vα domain CDR1 has the amino acid sequence of SEQ ID NO: 60, CDR1 may be encoded by any suitable nucleic acid sequence.

[0423] The encoded TCR Vα domain, in addition to the specified CDR3 (and optionally the specified CDR1 above), contains CDR2, or a functional variant thereof, which includes the amino acid sequence of SEQ ID NO: 61 (i.e., the variant is HLA-A * 02, Most preferably HLA-A * It may also include (which retains the ability to specifically bind to 02:01).

[0424] For example, a suitable functional Vα domain CDR2 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 61, i.e., it may have at least 80%, at least 85%, or 100% sequence identity with SEQ ID NO: 61. In other words, a suitable (functional) Vα domain CDR2 amino acid sequence may differ from the sequence shown in SEQ ID NO: 61 by one or more amino acids.

[0425] In one example, the CDR2 of the Vα domain contains or consists of the amino acid sequence of SEQ ID NO: 61. In the example where the TCR Vα domain CDR2 has the amino acid sequence of SEQ ID NO: 61, CDR2 may be encoded by any suitable nucleic acid sequence.

[0426] Therefore, the encoded TCR Vα domain contains the CDRs described in detail above (specifically, SEQ ID NO: 62, SEQ ID NO: 60, and SEQ ID NO: 61, or their functional variants), with appropriate intervening sequences between the CDRs.

[0427] The encoded TCR Vα domain may include the amino acid sequence of SEQ ID NO: 66, or a functional variant thereof (i.e., if the mutant TCR Vα domain is part of the binding protein described herein, it retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0428] In one example, the encoded TCR Vα domain may have an amino acid sequence that has at least 75%, at least 80%, at least 85%, or at least 90% (or 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 amino acid sequence of SEQ ID NO: 66, but retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2). In other words, this also includes functional TCR Vα domains having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 66. All sequence mutations compared to SEQ ID NO: 66 may reside in a region of the TCR Vα domain that does not form a CDR (i.e., the variant has the CDRs of SEQ ID NO: 62, SEQ ID NO: 60, and / or SEQ ID NO: 61, and has 25% (or less) sequence mutations compared to SEQ ID NO: 66). In other words, the sequence of CDR of sequence number 66 is preserved, and the remaining sequence may be modified as appropriate within the range of the "at least 75% identity" parameter defined above.

[0429] In one example, the encoded TCR Vα domain may contain an amino acid sequence having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) sequence identity with the amino acid sequence of SEQ ID NO: 66, and the TCR Vα domain may contain CDR3 having the amino acid sequence of SEQ ID NO: 62. In this example, TCR Vα domain CDR1 may have the amino acid sequence of SEQ ID NO: 60, and TCR Vα domain CDR2 may have the amino acid sequence of SEQ ID NO: 61.

[0430] In another example, the encoded TCR Vα domain contains an amino acid sequence having the amino acid sequence of SEQ ID NO: 66, and has 0 to 10 (or 0 to 5) amino acid substitutions, insertions, or deletions, and the TCR Vα domain contains CDR3 having the amino acid sequence of SEQ ID NO: 62. In this example, TCR Vα domain CDR1 may have the amino acid sequence of SEQ ID NO: 60, and TCR Vα domain CDR2 may have the amino acid sequence of SEQ ID NO: 61.

[0431] In the example where the TCR Vα domain has the amino acid sequence of SEQ ID NO: 66, the TCR Vα domain may be encoded by the nucleic acid sequence of SEQ ID NO: 67, or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of genetic coding degeneracy).

[0432] To avoid ambiguity, the nucleic acid sequence encoding the TCR Vα domain may also encode the TCRα chain constant domain. Examples of suitable constant domains are outlined above.

[0433] In one example, the nucleic acid composition provided herein includes a nucleic acid sequence encoding a TCR Vα domain, or a functional fragment thereof, which comprises a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 62.

[0434] In another example, the CDR3 of the Vα domain of the nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO: 62.

[0435] In another example, the Vα domain of the nucleic acid composition provided herein has at least 80% sequence identity with, contains, or comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 66.

[0436] As described above, the inventors of the present invention have identified HLA-A *At 02:01, we identified TCR clone TCR-4, which interacts with VLAPRVLRA (SEQ ID NO: 2). The sequences corresponding to TCR clone TCR-4 described herein are SEQ ID NOs: 60-69.

[0437] An example of a suitable TCR Vβ domain CDR3 amino acid sequence that specifically binds to the RCN1 antigen, particularly peptides containing the amino acid sequence of SEQ ID NO: 1 (e.g., SEQ ID NO: 2), is shown in SEQ ID NO: 65.

[0438] For example, a suitable (functional) Vβ-domain CDR3 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 65. That is, they may have at least 80%, at least 86%, at least 93%, or 100% sequence identity with SEQ ID NO: 65. Appropriately, the identity percentage is calculated as the percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 65). In other words, a suitable (functional) Vβ-domain CDR3 amino acid sequence may differ from the sequence shown in SEQ ID NO: 65 by only one or more (e.g., two) amino acids.

[0439] In one example, the CDR3 of the Vβ domain contains or is composed of the amino acid sequence of SEQ ID NO: 65. In the example where the TCR Vβ domain CDR3 has the amino acid sequence of SEQ ID NO: 65, CDR3 may be encoded by any suitable nucleic acid sequence.

[0440] The encoded TCR Vβ domain may include, in addition to a specific CDR3, a CDR1 containing the amino acid sequence of SEQ ID NO: 63, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0441] For example, a suitable functional Vβ-domain CDR1 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 63. That is, it may have at least 80% or even 100% sequence identity with SEQ ID NO: 63. Appropriately, the identity percentage is calculated as the percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 63). In other words, a suitable (functional) Vβ-domain CDR1 amino acid sequence may differ from the sequence shown in SEQ ID NO: 63 by one or more amino acids.

[0442] In one example, the CDR1 of the Vβ domain contains or consists of the amino acid sequence of SEQ ID NO: 63. In the example where the CDR1 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 63, CDR1 may be encoded by any suitable nucleic acid sequence.

[0443] The encoded TCR Vβ domain is CDR2 having the amino acid sequence of SEQ ID NO: 64, in addition to the specified CDR3 (and optionally the specified CDR1 above), or a functional variant thereof (i.e., the variant is HLA-A * 02, Most preferably HLA-A * It may also include (which retains the ability to specifically bind to 02:01).

[0444] For example, a suitable functional Vβ domain CDR2 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 64. That is, it may have at least 80%, at least 83%, or 100% sequence identity with SEQ ID NO: 64. In other words, a suitable (functional) Vβ domain CDR2 amino acid sequence may differ from the sequence shown in SEQ ID NO: 64 by one or more amino acids.

[0445] In one example, the CDR2 of the Vβ domain contains or consists of the amino acid sequence of SEQ ID NO: 64. In the example where the CDR2 of the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 64, the CDR2 may be encoded by any suitable nucleic acid sequence.

[0446] Therefore, the encoded TCR Vβ domain contains the CDRs described in detail above (specifically, SEQ ID NO: 65, SEQ ID NO: 63, and SEQ ID NO: 64, or their functional variants), with appropriate intervening sequences between the CDRs.

[0447] The encoded TCR Vβ domain may have the amino acid sequence of SEQ ID NO: 68, or a functional variant thereof (i.e., if the mutant TCR Vβ domain is part of the binding protein described herein, it retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0448] In one example, the encoded TCR Vβ domain may have an amino acid sequence that has at least 75%, at least 80%, at least 85%, or at least 90% (or 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 amino acid sequence of SEQ ID NO: 68, but retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2). In other words, this also includes functional TCR Vβ domains having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 68. Sequence mutations compared to SEQ ID NO: 68 may be present only in regions of the TCR Vβ domain that do not form a CDR (i.e., the variant may have the CDRs of SEQ ID NO: 65, SEQ ID NO: 63, and / or SEQ ID NO: 64, and have 25% (or less) sequence mutations compared to SEQ ID NO: 68). In other words, the sequence of CDR at sequence number 68 may be preserved, but the remaining sequences may be modified as appropriate within the range of the "at least 75% identity" parameter defined above.

[0449] In one example, the encoded TCR Vβ domain may include an amino acid sequence having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) sequence identity with the amino acid sequence of SEQ ID NO: 68, and the TCR Vβ domain may include CDR3 having the amino acid sequence of SEQ ID NO: 65. In this example, TCR Vβ domain CDR1 may have the amino acid sequence of SEQ ID NO: 63, and TCR Vβ domain CDR2 may have the amino acid sequence of SEQ ID NO: 64.

[0450] In the example where the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 68, the TCR Vβ domain has the nucleic acid sequence of SEQ ID NO: 69, or a genetically degenerate sequence thereof (i.e., as a result of genetic coding degeneracy, there are multiple genes encoding the same protein).

[0451] To avoid ambiguity, the nucleic acid sequence encoding the TCR Vβ domain may also encode the TCRβ chain constant domain. Examples of appropriate constant domains are outlined above.

[0452] In one example, the nucleic acid composition provided herein includes a nucleic acid sequence encoding a TCR Vβ domain or a functional fragment thereof, which comprises a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 65.

[0453] In another example, the CDR3 of the Vβ domain of the nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO: 65.

[0454] In further examples, the Vβ domain of the nucleic acid composition provided herein has at least 80% sequence identity with SEQ ID NO: 68, contains SEQ ID NO: 68, or contains an amino acid sequence consisting of SEQ ID NO: 68.

[0455] The TCR Vβ domain sequence derived from the TCR clone TCR-4 described above is particularly compatible with the TCR Vα domain sequence derived from the TCR clone TCR-4 described elsewhere in this specification.

[0456] Therefore, in one example, the nucleic acid composition described herein encodes a peptide comprising the amino acid sequence of SEQ ID NO: 1 having a TCR Vα domain having at least 80% sequence identity with SEQ ID NO: 62, or a functional fragment thereof, and a nucleic acid sequence-specific binding protein encoding a TCR Vβ domain having at least 80% sequence identity with SEQ ID NO: 65, or a functional fragment thereof.

[0457] In certain examples, the nucleic acid compositions described herein encode a peptide-specific binding protein having a TCR Vα domain having a CDR3 amino acid sequence containing or comprising the amino acid sequence of SEQ ID NO: 62, and a TCR Vβ domain having a CDR3 containing or comprising the amino acid sequence of SEQ ID NO: 65. Furthermore, the peptide having the amino acid sequence of SEQ ID NO: 1 contains or comprises the sequence shown in SEQ ID NO: 2. In addition, the TCR Vα domain may be part of a TCRα chain having a constant domain, and the TCR Vβ domain may be part of a TCRβ chain having a constant domain.

[0458] In this particular example, the Vα domain may have at least 80% sequence identity with SEQ ID NO: 66, contain SEQ ID NO: 66, or contain an amino acid sequence consisting of SEQ ID NO: 66, and the Vβ domain may have at least 80% sequence identity with SEQ ID NO: 68, contain SEQ ID NO: 68, or contain an amino acid sequence consisting of SEQ ID NO: 68. In one example, the Vα domain contains the amino acid sequence of SEQ ID NO: 66, and the Vβ domain contains the amino acid sequence of SEQ ID NO: 68. In such a case, the Vα domain may be encoded by a nucleic acid sequence containing the sequence of SEQ ID NO: 67. The Vβ domain may also be encoded by a nucleic acid sequence containing the sequence of SEQ ID NO: 69.

[0459] In this particular example, the TCR Vα domain may include the amino acid sequence of SEQ ID NO: 60, or the CDR1 amino acid sequence consisting of SEQ ID NO: 60, and the amino acid sequence of SEQ ID NO: 61, or the CDR2 amino acid sequence consisting of SEQ ID NO: 61. Furthermore, the TCR Vβ domain may include the amino acid sequence of SEQ ID NO: 63, or the CDR1 amino acid sequence consisting of SEQ ID NO: 63, and the amino acid sequence of SEQ ID NO: 64, or the CDR2 amino acid sequence consisting of SEQ ID NO: 64.

[0460] To avoid any ambiguity, this particular example encompasses the components of the TCR clone TCR-4 illustrated herein. The different components of the TCR clone TCR-4 and their respective sequence numbers are summarized in Table 9 below.

[0461] As described in more detail elsewhere in this specification, the nucleic acid compositions described herein encode both the TCR Vα domain and the TCR Vβ domain, which form binding proteins that can specifically bind to the RCN1 antigen. In examples where the TCR Vα domain and the TCR Vβ domain are encoded by the same nucleic acid sequence, the TCR Vα domain and the TCR Vβ domain may be linked via a linker. Suitable linkers are outlined elsewhere in this specification. Additional suitable polypeptide domains, which may be encoded by the nucleic acid sequences encoding the TCR Vα domain and / or the TCR Vβ domain, are also outlined elsewhere in this specification.

[0462] In one example, the nucleic acid compositions described herein may encode a soluble TCR or a chimeric single-chain TCR in which the TCRα-chain variable domain is linked to a constant domain fused to the TCRβ-chain variable domain and, for example, a CD3ζ signaling domain. These are outlined elsewhere in this specification.

[0463] (iv) TCR clone TCR-5:VLAPRVLRA (SEQ ID NO: 2) TCR components that interact with TCR clone TCR-5:VLAPRVLRA As described elsewhere in this specification, the inventors of HLA-A * In 02:01, we also identified TCR clone TCR-5, which interacts with VLAPRVLRA (sequence number 2). In this specification, sequences corresponding to TCR clone TCR-5 are sequence numbers 70-79.

[0464] (iv) TCR clone TCR-5:VLAPRVLRA (SEQ ID NO: 2) TCR components that interact with TCR clone TCR-5:VLAPRVLRA As described elsewhere in this specification, the inventors have also identified TCR clone TCR-5, which interacts with VLAPRVLRA (SEQ ID NO: 2) at HLA-A*02:01. The sequences corresponding to TCR clone TCR-5 provided herein are SEQ ID NOs: 70-79.

[0465] In one embodiment, an isolated nucleic acid composition is provided that encodes an RCN1 antigen-specific binding protein having a TCRα chain variable (Vα) domain and a TCRβ chain variable (Vβ) domain, the composition comprising: A nucleic acid sequence encoding a TCR Vα domain, or a functional fragment thereof, containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 72; and a nucleic acid sequence encoding a TCR Vβ domain, or a functional fragment thereof, containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 75, where the CDR3 sequence together specifically binds to a peptide containing the amino acid sequence of SEQ ID NO: 1 (for example, when the peptide is complexed with HLA).

[0466] An example of a suitable TCR Vα domain CDR3 amino acid sequence that confers specific binding to the RCN1 antigen, particularly to peptides containing the amino acid sequence of SEQ ID NO: 1 (e.g., VLAPRVLRA (SEQ ID NO: 2)), is shown in SEQ ID NO: 72.

[0467] For example, a suitable (functional) Vα domain CDR3 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 72. That is, it may have at least 80%, at least 84%, at least 92%, or 100% sequence identity with SEQ ID NO: 72. Appropriately, the identity percentage is calculated as the percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 72). In other words, a suitable (functional) Vα domain CDR3 amino acid sequence may differ from the sequence shown in SEQ ID NO: 72 by one or more amino acids (e.g., two).

[0468] In one example, the CDR3 of the Vα domain contains or is composed of the amino acid sequence of SEQ ID NO: 72. In the example where the TCR Vα domain CDR3 has the amino acid sequence of SEQ ID NO: 72, CDR3 may be encoded by any suitable nucleic acid sequence.

[0469] The encoded TCR Vα domain may include, in addition to a specific CDR3, a CDR1 containing the amino acid sequence of SEQ ID NO: 70, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0470] For example, a suitable functional Vα domain CDR1 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 70. That is, it may have at least 80%, at least 83%, or 100% sequence identity with SEQ ID NO: 70. Appropriately, the identity percentage is calculated as the percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 70). In other words, a suitable functional Vα domain CDR1 amino acid sequence may differ from the sequence shown in SEQ ID NO: 70 by one or more amino acids.

[0471] In one example, the CDR1 of the Vα domain contains or consists of the amino acid sequence of SEQ ID NO: 70. In the example where the TCR Vα domain CDR1 has the amino acid sequence of SEQ ID NO: 70, CDR1 may be encoded by any suitable nucleic acid sequence.

[0472] The encoded TCR Vα domain may also include, in addition to the specified CDR3 (and optionally the specified CDR1 above), a CDR2 containing the amino acid sequence of SEQ ID NO: 71, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to HLA-A*02, most preferably HLA-A*02:01).

[0473] For example, a suitable functional Vα domain CDR2 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 71. That is, it may have at least 80%, at least 83%, or 100% sequence identity with SEQ ID NO: 71. In other words, a suitable (functional) Vα domain CDR2 amino acid sequence may differ from the sequence shown in SEQ ID NO: 71 by only one or more amino acids.

[0474] In one example, the CDR2 of the Vα domain contains or is composed of the amino acid sequence of SEQ ID NO: 71. In the example where the TCR Vα domain CDR2 has the amino acid sequence of SEQ ID NO: 71, CDR2 may be encoded by any suitable nucleic acid sequence.

[0475] Therefore, the encoded TCR Vα domain may contain the CDRs described in detail above (specifically, SEQ ID NO: 72, SEQ ID NO: 70, and SEQ ID NO: 71, or their functional variants), with appropriate intervening sequences between the CDRs.

[0476] The encoded TCR Vα domain may include the amino acid sequence of SEQ ID NO: 76, or a functional variant thereof (i.e., if the mutant TCR Vα domain is part of the binding protein described herein, it retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0477] In one example, the encoded TCR Vα domain may have an amino acid sequence that has at least 75%, at least 80%, at least 85%, or at least 90% (or 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 amino acid sequence of SEQ ID NO: 76, but retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2). In other words, this also includes functional TCR Vα domains that have one or more amino acid substitutions compared to the sequence of SEQ ID NO: 76. Sequence mutations compared to SEQ ID NO: 76 may only be present in regions of the TCR Vα domain that do not form a CDR (i.e., the variant may have the CDRs of SEQ ID NO: 72, SEQ ID NO: 70, and / or SEQ ID NO: 71, and may have 25% (or less) sequence mutations compared to SEQ ID NO: 76). In other words, the sequence of CDR at sequence number 76 may be preserved, but the remaining sequences may be modified as appropriate within the range of the "at least 75% identity" parameter defined above.

[0478] In one example, the encoded TCR Vα domain may contain an amino acid sequence having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) sequence identity with the amino acid sequence of SEQ ID NO: 76, where the TCR Vα domain may contain CDR3 having the amino acid sequence of SEQ ID NO: 72. In this example, TCR Vα domain CDR1 may have the amino acid sequence of SEQ ID NO: 70, and TCR Vα domain CDR2 may have the amino acid sequence of SEQ ID NO: 71.

[0479] In another example, the encoded TCR Vα domain may include an amino acid sequence having the amino acid sequence of SEQ ID NO: 76, having 0 to 10 (or 0 to 5) amino acid substitutions, insertions, or deletions, where the TCR Vα domain includes CDR3 having the amino acid sequence of SEQ ID NO: 72. In this example, TCR Vα domain CDR1 has the amino acid sequence of SEQ ID NO: 70, and TCR Vα domain CDR2 has the amino acid sequence of SEQ ID NO: 71.

[0480] In the example where the TCR Vα domain has the amino acid sequence of SEQ ID NO: 76, the TCR Vα domain may be encoded by the nucleic acid sequence of SEQ ID NO: 77, or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that codes for the same protein as a result of genetic code degeneracy).

[0481] To avoid ambiguity, the nucleic acid sequence encoding the TCR Vα domain may also encode the TCRα chain constant domain. Examples of appropriate constant domains are outlined above.

[0482] In one example, the nucleic acid composition provided herein includes a nucleic acid sequence encoding a TCR Vα domain or a functional fragment thereof, which comprises a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 72.

[0483] In another example, the CDR3 of the Vα domain of the nucleic acid composition provided herein contains or consists of the amino acid sequence of SEQ ID NO: 72.

[0484] In another example, the Vα domain of the nucleic acid composition provided herein has at least 80% sequence identity with SEQ ID NO: 76, or contains SEQ ID NO: 76, or contains an amino acid sequence consisting of SEQ ID NO: 76.

[0485] As described above, the inventors identified TCR clone TCR-5, which interacts with VLAPRVLRA (SEQ ID NO: 2) at HLA-A*02:01. In this specification, the sequences corresponding to TCR clone TCR-5 are SEQ ID NOs: 70-79.

[0486] An example of a suitable TCR Vβ domain CDR3 amino acid sequence that confers specific binding to a peptide containing the amino acid sequence of SEQ ID NO: 1 (for example, the peptide shown in SEQ ID NO: 2) is shown in SEQ ID NO: 75.

[0487] For example, a suitable (functional) Vβ-domain CDR3 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 75. That is, they may have at least 80%, at least 85%, at least 92%, or 100% sequence identity with SEQ ID NO: 75. Appropriately, the identity percentage is calculated as the percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 75). In other words, a suitable (functional) Vβ-domain CDR3 amino acid sequence may differ from the sequence shown in SEQ ID NO: 75 by only one or more (e.g., two) amino acids.

[0488] In one example, the CDR3 of the Vβ domain contains or is composed of the amino acid sequence of SEQ ID NO: 75. In the example where the TCR Vβ domain CDR3 has the amino acid sequence of SEQ ID NO: 75, CDR3 may be encoded by any suitable nucleic acid sequence.

[0489] The encoded TCR Vβ domain may include, in addition to a specific CDR3, a CDR1 containing the amino acid sequence of SEQ ID NO: 73, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0490] For example, a suitable functional Vβ domain CDR1 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 73. That is, it may have at least 80%, at least 83%, or 100% sequence identity with SEQ ID NO: 73. In other words, a suitable (functional) Vβ domain CDR1 amino acid sequence may differ from the sequence shown in SEQ ID NO: 73 by only one or more amino acids.

[0491] In one example, the CDR1 of the Vβ domain has the same amino acid sequence as SEQ ID NO: 73. In the example where the TCR Vα domain CDR1 has the amino acid sequence of SEQ ID NO: 73, CDR1 may be encoded by any suitable nucleic acid sequence.

[0492] The encoded TCR Vβ domain, in addition to the specified CDR3 (and optionally the specified CDR1 above), is CDR2 having the amino acid sequence of SEQ ID NO: 74, or a functional variant thereof (i.e., the variant is HLA-A * 02, Most preferably HLA-A * It may also include (which retains the ability to specifically bind to 02:01).

[0493] For example, a suitable functional Vβ-domain CDR2 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 74. That is, it may have at least 80%, at least 83%, or 100% sequence identity with SEQ ID NO: 74. Appropriately, the identity percentage is the percentage of identity with respect to the full length of the reference sequence (e.g., SEQ ID NO: 74). In other words, a suitable (functional) Vβ-domain CDR2 amino acid sequence may differ by one or more amino acids from the sequence shown in SEQ ID NO: 74.

[0494] In one example, the CDR2 of the Vβ domain contains or consists of the amino acid sequence of SEQ ID NO: 74. In the example where the CDR2 of the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 74, the CDR2 may be encoded by any suitable nucleic acid sequence.

[0495] Therefore, the encoded TCR Vβ domain contains the CDRs described in detail above (specifically, SEQ ID NO: 75, SEQ ID NO: 73, and SEQ ID NO: 74, or their functional variants), with appropriate intervening sequences between the CDRs.

[0496] The encoded TCR Vβ domain may have the amino acid sequence of SEQ ID NO: 78, or a functional variant thereof (i.e., if the mutant TCR Vβ domain is part of the binding protein described herein, it retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0497] In one example, the encoded TCR Vβ domain may have an amino acid sequence that has at least 75%, at least 80%, at least 85%, or at least 90% (or 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 amino acid sequence of SEQ ID NO: 78, but retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2). In other words, this also includes functional TCR Vβ domains that have one or more amino acid substitutions compared to the sequence of SEQ ID NO: 78. Sequence mutations compared to SEQ ID NO: 78 may only be present in regions of the TCR Vβ domain that do not form a CDR (i.e., the variant may have the CDRs of SEQ ID NO: 75, SEQ ID NO: 73, and / or SEQ ID NO: 74, and may have 25% (or less) sequence mutations compared to SEQ ID NO: 78). In other words, the sequence of CDR at sequence number 78 may be preserved, but the remaining sequences may be modified as appropriate within the range of the "at least 75% identity" parameter defined above.

[0498] For example, the encoded TCR Vβ domain may contain an amino acid sequence having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) sequence identity with the amino acid sequence of SEQ ID NO: 78, and the TCR Vβ domain may contain CDR3 having the amino acid sequence of SEQ ID NO: 75. In this example, TCR Vβ domain CDR1 may have the amino acid sequence of SEQ ID NO: 73, and TCR Vβ domain CDR2 may have the amino acid sequence of SEQ ID NO: 74.

[0499] In the example where the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 78, the TCR Vβ domain may be encoded by the nucleic acid sequence of SEQ ID NO: 79, or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence). As a result of genetic coding degeneracy, there are multiple genes that encode the same protein.

[0500] To avoid ambiguity, the nucleic acid sequence encoding the TCR Vβ domain may also encode the TCR β chain constant domain. Examples of appropriate constant domains are outlined above.

[0501] In one example, the nucleic acid composition provided herein includes a nucleic acid sequence encoding a TCR Vβ domain, or a functional fragment thereof, which includes a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 75.

[0502] In another example, the CDR3 of the Vβ domain of the nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO: 75.

[0503] In further examples, the Vβ domain of the nucleic acid composition described herein has at least 80% sequence identity with SEQ ID NO: 78, contains SEQ ID NO: 78, or contains an amino acid sequence consisting of SEQ ID NO: 78.

[0504] The TCR Vβ domain sequence derived from the aforementioned TCR clone TCR-5 is particularly compatible with the TCR Vα domain sequence derived from the TCR clone TCR-5 described elsewhere in this specification.

[0505] Therefore, in one example, the nucleic acid composition described herein encodes a peptide comprising the amino acid sequence of SEQ ID NO: 1 having a TCR Vα domain comprising a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 72, or a functional fragment thereof, and a nucleic acid sequence encoding a TCR Vβ domain comprising a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 75, or a binding protein specific to that functional fragment thereof.

[0506] In certain examples, the nucleic acid compositions described herein encode a peptide-specific binding protein having a TCR Vα domain having a CDR3 amino acid sequence containing or comprising the amino acid sequence of SEQ ID NO: 72, and a TCR Vβ domain having a CDR3 containing or comprising the amino acid sequence of SEQ ID NO: 75. Furthermore, the peptide containing the amino acid sequence of SEQ ID NO: 1 contains or comprises the sequence shown in SEQ ID NO: 2. In addition, the TCR Vα domain may be part of a TCRα chain having a constant domain, and the TCR Vβ domain may be part of a TCRβ chain having a constant domain.

[0507] In this particular example, the Vα domain may have at least 80% sequence identity with SEQ ID NO: 76, contain SEQ ID NO: 76, or contain an amino acid sequence consisting of SEQ ID NO: 76, and the Vβ domain may have at least 80% sequence identity with SEQ ID NO: 78, contain SEQ ID NO: 78, or contain an amino acid sequence consisting of SEQ ID NO: 78. In one example, the Vα domain contains the amino acid sequence of SEQ ID NO: 76, and the Vβ domain contains the amino acid sequence of SEQ ID NO: 78. In such a case, the Vα domain may be encoded by a nucleic acid sequence containing the sequence of SEQ ID NO: 77. The Vβ domain may also be encoded by a nucleic acid sequence containing the sequence of SEQ ID NO: 79.

[0508] In this particular example, the TCR Vα domain may include the amino acid sequence of SEQ ID NO: 70, or the CDR1 amino acid sequence consisting of SEQ ID NO: 70, and the amino acid sequence of SEQ ID NO: 71, or the CDR2 amino acid sequence consisting of SEQ ID NO: 71. Furthermore, the TCR Vβ domain may include the amino acid sequence of SEQ ID NO: 73, or the CDR1 amino acid sequence consisting of SEQ ID NO: 73, and the amino acid sequence of SEQ ID NO: 74, or the CDR2 amino acid sequence consisting of SEQ ID NO: 74.

[0509] To avoid any ambiguity, this particular example encompasses the components of the TCR clone TCR-5 illustrated herein. The different components of the TCR clone TCR-5 and their respective sequence numbers are summarized in Table 10 below.

[0510] As described in more detail elsewhere in this specification, the nucleic acid compositions described herein encode both the TCR Vα domain and the TCR Vβ domain, which form binding proteins that can specifically bind to the RCN1 antigen. In examples where the TCR Vα domain and the TCR Vβ domain are encoded by the same nucleic acid sequence, the TCR Vα domain and the TCR Vβ domain may be linked via a linker. Suitable linkers are outlined elsewhere in this specification. Additional suitable polypeptide domains, which may be encoded by nucleic acid sequences encoding the TCR Vα domain and / or the TCR Vβ domain, are also outlined elsewhere in this specification.

[0511] In one example, the nucleic acid compositions described herein may encode a soluble TCR or a chimeric single-chain TCR in which the TCRα-chain variable domain is linked to a constant domain fused to the TCRβ-chain variable domain and, for example, a CD3ζ signaling domain. These are outlined elsewhere in this specification.

[0512] (v) TCR clone TCR-9:VLAPRVLRA (SEQ ID NO: 2) TCR components that interact with TCR clone TCR-9. As described elsewhere in this specification, the inventors of HLA-A * In 02:01, we also identified TCR clone TCR-9, which interacts with VLAPRVLRA (sequence number 2). In this specification, sequences corresponding to TCR clone TCR-9 are sequence numbers 80-89.

[0513] In one embodiment, an isolated nucleic acid composition is provided that encodes an RCN1 antigen-specific binding protein having a TCRα chain variable (Vα) domain and a TCRβ chain variable (Vβ) domain. This composition comprises: A nucleic acid sequence or functional fragment thereof encoding a TCR Vα domain containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 82; and a nucleic acid sequence or functional fragment thereof encoding a TCR Vβ domain containing a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 85; where the CDR3 sequences together specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (for example, when the peptide forms a complex with HLA).

[0514] An example of a suitable TCR Vα domain CDR3 amino acid sequence that confers specific binding to the RCN1 antigen, particularly to peptides containing the amino acid sequence of SEQ ID NO: 1 (e.g., VLAPRVLRA (SEQ ID NO: 2)), is shown in SEQ ID NO: 82.

[0515] For example, a suitable (functional) Vα domain CDR3 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 82. That is, it may have at least 80%, at least 82%, at least 88%, at least 94%, or 100% sequence identity with SEQ ID NO: 82.

[0516] In one example, the CDR3 of the Vα domain contains or consists of the amino acid sequence of SEQ ID NO: 82. In an example where the TCR Vα domain CDR3 has an amino acid sequence, the CDR3 of SEQ ID NO: 82 may be encoded by any suitable nucleic acid sequence.

[0517] The encoded TCR Vα domain may include, in addition to a specific CDR3, a CDR1 containing the amino acid sequence of SEQ ID NO: 80, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0518] For example, a suitable functional Vα domain CDR1 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 80. That is, it may have at least 80%, at least 85%, or 100% sequence identity with SEQ ID NO: 80. In other words, a suitable functional Vα domain CDR1 amino acid sequence may differ from the sequence shown in SEQ ID NO: 80 by one or more amino acids.

[0519] In one example, the CDR1 of the Vα domain contains or consists of the amino acid sequence of SEQ ID NO: 80. In the example where the TCR Vα domain CDR1 has the amino acid sequence of SEQ ID NO: 80, CDR1 may be encoded by any suitable nucleic acid sequence.

[0520] The encoded TCR Vα domain, in addition to the specified CDR3 (and optionally the specified CDR1 above), includes CDR2 containing the amino acid sequence of SEQ ID NO: 81, or its functional variant (i.e., the variant is HLA-A * 02, Most preferably HLA-A * It may also include those that possess the ability to specifically bind to 02:01.

[0521] For example, a suitable functional Vα domain CDR2 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 81. That is, it may have at least 80%, at least 87%, or 100% sequence identity with SEQ ID NO: 81. In other words, a suitable (functional) Vα domain CDR2 amino acid sequence may differ from the sequence shown in SEQ ID NO: 81 by only one or more amino acids. In one example, the Vα domain CDR2 contains or is composed of the amino acid sequence of SEQ ID NO: 81. In the example where the TCR Vα domain CDR2 has the amino acid sequence of SEQ ID NO: 81, CDR2 may be encoded by any suitable nucleic acid sequence.

[0522] Therefore, the encoded TCR Vα domain contains the CDRs described in detail above (specifically, SEQ ID NO: 82, SEQ ID NO: 80, and SEQ ID NO: 81, or their functional variants), and appropriate intervening sequences may be present between the CDRs.

[0523] The encoded TCR Vα domain may contain the amino acid sequence of SEQ ID NO: 86, or a functional variant thereof (i.e., if the mutant TCR Vα domain is part of the binding protein described herein, it retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0524] In one example, the encoded TCR Vα domain may have an amino acid sequence that has at least 75%, at least 80%, at least 85%, or at least 90% (or 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 amino acid sequence of SEQ ID NO: 86, but retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2). In other words, this also includes functional TCR Vα domains having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 86. All sequence mutations compared to SEQ ID NO: 86 may reside in a region of the TCR Vα domain that does not form a CDR (i.e., the variant has the CDRs of SEQ ID NO: 82, SEQ ID NO: 80, and / or SEQ ID NO: 81, and has 25% (or less) sequence mutations compared to SEQ ID NO: 86). In other words, the sequence of CDR at sequence number 86 is preserved, and the remaining sequence may be modified as appropriate within the range of the "at least 75% identity" parameter defined above.

[0525] For example, an encoded TCR Vα domain may contain an amino acid sequence having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) sequence identity with the amino acid sequence of SEQ ID NO: 86, where the TCR Vα domain may contain CDR3 having the amino acid sequence of SEQ ID NO: 82. In this example, TCR Vα domain CDR1 may have the amino acid sequence of SEQ ID NO: 80, and TCR Vα domain CDR2 may have the amino acid sequence of SEQ ID NO: 81.

[0526] As another example, an encoded TCR Vα domain may contain an amino acid sequence having the amino acid sequence of SEQ ID NO: 86, having 0 to 10 (or 0 to 5) amino acid substitutions, insertions, or deletions, where the TCR Vα domain contains CDR3 having the amino acid sequence of SEQ ID NO: 82. In this example, TCR Vα domain CDR1 has the amino acid sequence of SEQ ID NO: 80, and TCR Vα domain CDR2 has the amino acid sequence of SEQ ID NO: 81.

[0527] In the example where the TCR Vα domain has the amino acid sequence of SEQ ID NO: 86, the TCR Vα domain may be encoded by the nucleic acid sequence of SEQ ID NO: 87, or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that codes for the same protein as a result of genetic code degeneracy).

[0528] To avoid ambiguity, the nucleic acid sequence encoding the TCR Vα domain may also encode the TCRα chain constant domain. Examples of appropriate constant domains are outlined above.

[0529] In one example, the nucleic acid composition provided herein includes a nucleic acid sequence encoding a TCRVα domain, or a functional fragment thereof, which includes a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 82.

[0530] In another example, the CDR3 of the Vα domain of the nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO: 82.

[0531] In another example, the Vα domain of the nucleic acid composition provided herein has at least 80% sequence identity with, contains, or comprises an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 86.

[0532] As described above, the inventors identified TCR clone TCR-9 that interacts with VLAPRVLRA (SEQ ID NO: 2) at HLA-A*02:01. The sequences corresponding to TCR clone TCR-9 provided herein are SEQ ID NOs: 80-89.

[0533] An example of a suitable TCR Vβ domain CDR3 amino acid sequence that confers specific binding to the RCN1 antigen, particularly to peptides containing the amino acid sequence of SEQ ID NO: 1 (e.g., VLAPRVLRA (SEQ ID NO: 2)), is shown in SEQ ID NO: 85. As will be apparent to those skilled in the art, variants of the amino acid sequence shown in SEQ ID NO: 85 may also be functional (i.e., retain the ability to confer specific binding to peptides containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2) when CDR3 is part of the TCR Vβ domain).

[0534] For example, a suitable (functional) Vβ domain CDR3 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 85. That is, they may have at least 80%, at least 83%, at least 91%, or 100% sequence identity with SEQ ID NO: 85. In other words, a suitable (functional) Vβ domain CDR3 amino acid sequence may differ from the sequence shown in SEQ ID NO: 85 by only one or more (e.g., two) amino acids.

[0535] In one example, the CDR3 of the Vβ domain contains or is composed of the amino acid sequence of SEQ ID NO: 85. In the example where the TCR Vβ domain CDR3 has the amino acid sequence of SEQ ID NO: 85, CDR3 may be encoded by any suitable nucleic acid sequence.

[0536] In one example, the CDR3 of the Vβ domain contains or is composed of the amino acid sequence of SEQ ID NO: 85. In the example where the TCR Vβ domain CDR3 has the amino acid sequence of SEQ ID NO: 85, CDR3 may be encoded by any suitable nucleic acid sequence.

[0537] For example, a suitable functional Vβ domain CDR1 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 83. That is, it may have at least 80% or even 100% sequence identity with SEQ ID NO: 83. In other words, a suitable (functional) Vβ domain CDR1 amino acid sequence may differ from the sequence shown in SEQ ID NO: 83 by one or more amino acids.

[0538] In one example, the CDR1 of the Vβ domain contains or consists of the amino acid sequence of SEQ ID NO: 83. In the example where the CDR1 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 83, CDR1 may be encoded by any suitable nucleic acid sequence. The encoded TCR Vβ domain, in addition to the specified CDR3 (and optionally the specified CDR1 above), is CDR2 having the amino acid sequence of SEQ ID NO: 84, or a functional variant thereof (i.e., the variant is HLA-A * 02, Most preferably HLA-A * It may also include those that possess the ability to specifically bind to 02:01.

[0539] For example, a suitable functional Vβ domain CDR2 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 84. That is, it may have at least 80%, at least 83%, or 100% sequence identity with SEQ ID NO: 84. In other words, a suitable (functional) Vβ domain CDR2 amino acid sequence may differ from the sequence shown in SEQ ID NO: 84 by one or more amino acids.

[0540] In one example, the CDR2 of the Vβ domain contains or is composed of the amino acid sequence of SEQ ID NO: 84. In the example where the TCR Vβ domain CDR2 has the amino acid sequence of SEQ ID NO: 84, CDR2 may be encoded by any suitable nucleic acid sequence.

[0541] Therefore, the encoded TCR Vβ domain may contain the CDRs described in detail above (specifically, SEQ ID NO: 85, SEQ ID NO: 83, and SEQ ID NO: 84, or their functional variants), with appropriate intervening sequences between the CDRs.

[0542] The encoded TCR Vβ domain may have the amino acid sequence of SEQ ID NO: 88, or a functional variant thereof (i.e., if the mutant TCR Vβ domain is part of the binding protein described herein, it retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0543] In one example, the encoded TCR Vβ domain may have an amino acid sequence that has at least 75%, at least 80%, at least 85%, or at least 90% (or 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 amino acid sequence of SEQ ID NO: 88, but retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2). In other words, this also includes functional TCR Vβ domains that have one or more amino acid substitutions compared to the sequence of SEQ ID NO: 88. Sequence mutations compared to SEQ ID NO: 88 may only be present in regions of the TCR Vβ domain that do not form a CDR (i.e., the variant may have the CDRs of SEQ ID NO: 85, SEQ ID NO: 83, and / or SEQ ID NO: 84, and may have 25% (or less) sequence mutations compared to SEQ ID NO: 88). In other words, the sequence of CDR at sequence number 88 may be preserved, but the remaining sequences may change as appropriate within the range of the "at least 75% identity" parameter defined above.

[0544] For example, the encoded TCR Vβ domain may contain an amino acid sequence having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) sequence identity with the amino acid sequence of SEQ ID NO: 88, where the TCR Vβ domain may contain CDR3 having the amino acid sequence of SEQ ID NO: 85. In this example, TCR Vβ domain CDR1 may have the amino acid sequence of SEQ ID NO: 83, and TCR Vβ domain CDR2 may have the amino acid sequence of SEQ ID NO: 84.

[0545] In the example where the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 88, the TCR Vβ domain may be encoded by the nucleic acid sequence of SEQ ID NO: 89, or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that encodes the same protein as a result of the degeneration of the genetic code).

[0546] To avoid ambiguity, the nucleic acid sequence encoding the TCR Vβ domain may also encode the TCR β chain constant domain. Examples of suitable constant domains are outlined above.

[0547] In one example, the nucleic acid composition provided herein includes a nucleic acid sequence encoding a TCR Vβ domain, or a functional fragment thereof, which comprises a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 85.

[0548] In another example, the CDR3 of the Vβ domain of the nucleic acid composition described herein comprises or consists of the amino acid sequence of SEQ ID NO: 85.

[0549] In further examples, the Vβ domain of the nucleic acid composition described herein has at least 80% sequence identity with SEQ ID NO: 88, contains SEQ ID NO: 88, or contains an amino acid sequence consisting of SEQ ID NO: 88.

[0550] The TCR Vβ domain sequence derived from the TCR clone TCR-9 described above is particularly compatible with the TCR Vα domain sequence derived from the TCR clone TCR-9 described elsewhere in this specification.

[0551] Accordingly, in one example, the nucleic acid composition described herein may include a nucleic acid sequence encoding a peptide-specific binding protein comprising the amino acid sequence of SEQ ID NO: 1, and having a TCR Vα domain or a functional fragment thereof comprising a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 82, and further comprising a nucleic acid sequence encoding a TCR Vβ domain or a functional fragment thereof comprising a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 85.

[0552] In certain examples, the nucleic acid compositions described herein encode a peptide-specific binding protein having a TCR Vα domain having a CDR3 amino acid sequence comprising or consisting thereof the amino acid sequence of SEQ ID NO: 82, and a TCR Vβ domain having a CDR3 comprising or consisting thereof the amino acid sequence of SEQ ID NO: 85. Furthermore, the peptide having the amino acid sequence of SEQ ID NO: 1 comprises or consists of the sequence shown in SEQ ID NO: 2. In addition, the TCR Vα domain may be part of a TCR α chain having a constant domain, and the TCR Vβ domain may be part of a TCR β chain having a constant domain.

[0553] In this particular example, the Vα domain may have at least 80% sequence identity with SEQ ID NO: 86, contain SEQ ID NO: 86, or contain an amino acid sequence consisting of SEQ ID NO: 86, and the Vβ domain may have at least 80% sequence identity with SEQ ID NO: 88, contain SEQ ID NO: 88, or contain an amino acid sequence consisting of SEQ ID NO: 88. In one example, the Vα domain contains the amino acid sequence of SEQ ID NO: 86, and the Vβ domain contains the amino acid sequence of SEQ ID NO: 88. In such a case, the Vα domain may be encoded by a nucleic acid sequence containing the sequence of SEQ ID NO: 87. The Vβ domain may also be encoded by a nucleic acid sequence containing the sequence of SEQ ID NO: 89.

[0554] In this particular example, the TCR Vα domain may include the amino acid sequence of SEQ ID NO: 80, or the CDR1 amino acid sequence consisting of SEQ ID NO: 80, and the amino acid sequence of SEQ ID NO: 81, or the CDR2 amino acid sequence consisting of SEQ ID NO: 81. Furthermore, the TCR Vβ domain may include the amino acid sequence of SEQ ID NO: 83, or the CDR1 amino acid sequence consisting of SEQ ID NO: 83, and the amino acid sequence of SEQ ID NO: 84, or the CDR2 amino acid sequence consisting of SEQ ID NO: 84.

[0555] To avoid any ambiguity, this particular example encompasses the components of the TCR clone TCR-9 illustrated herein. The different components of the TCR clone TCR-9 and their respective sequence numbers are summarized in Table 11 below.

[0556] As described in more detail elsewhere in this specification, the nucleic acid compositions described herein encode both the TCR Vα domain and the TCR Vβ domain, which form binding proteins that can specifically bind to the RCN1 antigen. In examples where the TCR Vα domain and the TCR Vβ domain are encoded by the same nucleic acid sequence, the TCR Vα domain and the TCR Vβ domain may be linked via a linker. Suitable linkers are outlined elsewhere in this specification. Additional suitable polypeptide domains, which may be encoded by nucleic acid sequences encoding the TCR Vα domain and / or the TCR Vβ domain, are also outlined elsewhere in this specification.

[0557] In one example, the nucleic acid compositions described herein may encode a soluble TCR or a chimeric single-chain TCR in which the TCRα-chain variable domain is linked to a constant domain fused to the TCRβ-chain variable domain and, for example, a CD3ζ signaling domain. These are outlined elsewhere in this specification.

[0558] (vi) TCR clone TCR-12:TCR components that interact with VLAPRVLRA (SEQ ID NO: 2) As described elsewhere in this specification, the inventors of HLA-A * We also identified TCR clone TCR-12, which interacts with VLAPRVLRA (sequence number 2) at 02:01. In this specification, sequences corresponding to TCR clone TCR-12 are sequence numbers 90-99.

[0559] In one embodiment, an isolated nucleic acid composition is provided that encodes an RCN1 antigen-specific binding protein having a TCRα chain variable (Vα) domain and a TCRβ chain variable (Vβ) domain. The composition comprises: A nucleic acid sequence encoding a TCR Vα domain or a functional fragment thereof, comprising a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 92; and a nucleic acid sequence encoding a TCR Vβ domain or a functional fragment thereof, comprising a CDR3 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 95, wherein the CDR3 sequence together specifically binds to a peptide comprising the amino acid sequence of SEQ ID NO: 1 (for example, when the peptide is complexed with HLA).

[0560] An example of a suitable TCR Vα domain CDR3 amino acid sequence that confers specific binding of the RCN1 antigen, particularly peptides containing the amino acid sequence of SEQ ID NO: 1 (e.g., VLAPRVLRA (SEQ ID NO: 2)), is shown in SEQ ID NO: 92.

[0561] For example, a suitable (functional) Vα domain CDR3 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 92. That is, it may have at least 80%, at least 84%, at least 92%, or 100% sequence identity with SEQ ID NO: 92. In other words, a suitable (functional) Vα domain CDR3 amino acid sequence may differ from the sequence shown in SEQ ID NO: 92 by one or more amino acids (e.g., two).

[0562] In one example, the CDR3 of the Vα domain contains or consists of the amino acid sequence of SEQ ID NO: 92. In the example where the TCR Vα domain CDR3 has the amino acid sequence of SEQ ID NO: 92, CDR3 may be encoded by any suitable nucleic acid sequence.

[0563] The encoded TCR Vα domain may include, in addition to a specific CDR3, a CDR1 containing the amino acid sequence of SEQ ID NO: 90, or a functional variant thereof (i.e., the variant retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0564] For example, a suitable functional Vα domain CDR1 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 90. That is, it may have at least 80%, at least 83%, or 100% sequence identity with SEQ ID NO: 90. Appropriately, the identity percentage is calculated as the percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 90). In other words, a suitable functional Vα domain CDR1 amino acid sequence may differ from the sequence shown in SEQ ID NO: 90 by only one or more amino acids.

[0565] In one example, the CDR1 of the Vα domain contains or is composed of the amino acid sequence of SEQ ID NO: 90. In the example where the TCR Vα domain CDR1 has the amino acid sequence of SEQ ID NO: 90, CDR1 may be encoded by any suitable nucleic acid sequence.

[0566] The encoded TCR Vα domain, in addition to specific CDR3 (and optionally specific CDR1 mentioned above), contains CDR2, which includes the amino acid sequence of SEQ ID NO: 91, or its functional variant (i.e., the variant is HLA-A * 02, Most preferably HLA-A * It may also include those that possess the ability to specifically bind to 02:01.

[0567] For example, a suitable functional Vα domain CDR2 amino acid sequence may have at least 80% sequence identity with SEQ ID NO: 91. That is, it may have at least 80%, at least 83%, or 100% sequence identity with SEQ ID NO: 91. Appropriately, the identity percentage is calculated as the percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 91). In other words, a suitable (functional) Vα domain CDR2 amino acid sequence may differ by one or more amino acids from the sequence shown in SEQ ID NO: 91.

[0568] In one example, the CDR2 of the Vα domain contains or is composed of the amino acid sequence of SEQ ID NO: 91. In the example where the TCR Vα domain CDR2 has the amino acid sequence of SEQ ID NO: 91, CDR2 may be encoded by any suitable nucleic acid sequence.

[0569] Therefore, the encoded TCR Vα domain may contain the CDRs described in detail above (specifically, SEQ ID NO: 92, SEQ ID NO: 90, and SEQ ID NO: 91, or their functional variants) along with appropriate intervening sequences between the CDRs.

[0570] The encoded TCR Vα domain may include the amino acid sequence of SEQ ID NO: 96, or a functional variant thereof (i.e., the mutant TCR Vα domain, if it is part of a binding protein described herein, retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2)).

[0571] In one example, the encoded TCR Vα domain may have an amino acid sequence that has at least 75%, at least 80%, at least 85%, or at least 90% (or 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 amino acid sequence of SEQ ID NO: 96, but retains the ability to specifically bind to a peptide containing the amino acid sequence of SEQ ID NO: 1 (e.g., the peptide shown in SEQ ID NO: 2). In other words, this also includes functional TCR Vα domains having one or more amino acid substitutions compared to the sequence of SEQ ID NO: 96. All sequence mutations compared to SEQ ID NO: 96 may reside in a region of the TCR Vα domain that does not form a CDR (i.e., the variant has the CDRs of SEQ ID NO: 92, SEQ ID NO: 90, and / or SEQ ID NO: 91 and has 25% (or less) sequence mutations compared to SEQ ID NO: 96). In other words, the sequence of CDR at sequence number 96 is preserved, and the remaining sequences may be modified as appropriate within the range of the "at least 75% identity" parameter defined above.

[0572] For example, the encoded TCR Vα domain may include an amino acid sequence having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%) sequence identity with the amino acid sequence of SEQ ID NO: 96, where the TCR Vα domain may include CDR3 having the amino acid sequence of SEQ ID NO: 92. In this example, TCR Vα domain CDR1 may have the amino acid sequence of SEQ ID NO: 90, and TCR Vα domain CDR2 may have the amino acid sequence of SEQ ID NO: 91.

[0573] As another example, the encoded TCR Vα domain may include an amino acid sequence having the amino acid sequence of SEQ ID NO: 96, which has 0 to 10 (or 0 to 5) amino acid substitutions, insertions, or deletions, where the TCR Vα domain includes CDR3 having the amino acid sequence of SEQ ID NO: 92. In this example, TCR Vα domain CDR1 has the amino acid sequence of SEQ ID NO: 90, and TCR Vα domain CDR2 has the amino acid sequence of SEQ ID NO: 91.

[0574] In the example where the TCR Vα domain has the amino acid sequence of SEQ ID NO: 96, the TCR Vα domain may be encoded by the nucleic acid sequence of SEQ ID NO: 97, or a genetically degenerate sequence thereof (i.e., another nucleic acid sequence that codes for the same protein as a result of genetic code degeneracy).

[0575] To avoid ambiguity, the nucleic acid sequence encoding the TCR Vα domain may also e...

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