Binding protein specific to HA-1H and its use

Abstract

HLA-A * Novel nucleic acid compositions, vectors, modified cells, and pharmaceutical compositions useful for treating or preventing the recurrence of hematological malignancies following allogeneic stem cell transplantation (allo-SCT) in O201-positive human subjects are provided. Corresponding methods and uses are also provided.

Description

Technical Field

[0001] HLA-A * Provided are novel nucleic acid compositions, vector systems, modified cells, and pharmaceutical compositions useful for treating or preventing recurrence of haematological malignancies after allogeneic stem cell transplantation (allo-SCT) in HLA-A 0201 positive human subjects. Corresponding methods and uses are also provided.

Background Art

[0002] Haematological malignancies are cancers that affect the blood and lymphatic systems. Cancers can originate within haematopoietic tissues (e.g., bone marrow) or within cells of the immune system. Patients with haematological malignancies may benefit from treatment by allogeneic stem cell transplantation (allo-SCT) matched to the human leukocyte antigen (HLA). To reduce the incidence of graft-versus-host disease (GvHD), donor T cells can be depleted from the stem cell graft and can be reinfused preemptively after allo-SCT. This two-step procedure of T cell-depleted allo-SCT and donor lymphocyte infusion (DLI) reduces the incidence and severity of GvHD compared to non-T cell-depleted allo-SCT, but GvHD remains an important cause of morbidity and mortality, particularly in the context of HLA mismatched transplantation. The risk of inducing GvHD is further increased when DLI is administered early after allo-SCT. Patients with high-risk leukaemia are likely to relapse at the time when early administration of DLI after transplantation results in GvHD. There is a lack of treatment options for this patient population and a new treatment modality is required that allows for early administration of T cells that can exert a graft-versus-leukaemia (GvL) effect without causing GvHD.

[0003] The adoptive transfer of T cells with defined anti-leukemia specificity is a strategy to decouple the graft-versus-host disease (GvHD) response from the graft-versus-leukemia (GvL) response. Donor T cells that recognize minor histocompatibility antigens (MiHA) selectively expressed on hematopoietic cells have been shown to mediate anti-leukemia reactivity without causing severe GvHD after allo-SCT. HA-1 H antigen is a minor histocompatibility antigen that is highly expressed in hematological malignancies and normal hematopoietic cells but not in normal non-hematopoietic cells, making it a promising target for immunotherapy. Also, therefore, HLA-A * 0201 has also been shown to be an appropriate target antigen in a significant proportion of patients with hematological malignancies. A direct association has previously been shown between the appearance of HA-1-specific T cells in donor-recipient pairs that are HA-1 incompatible and the complete elimination of malignant recipient cells (1). Therefore, HA-1 TCR-modified T cells could potentially be used to treat patients with different hematological malignancies, including leukemia and lymphoma, after allo-SCT. However, it has already been shown in a phase I clinical study that HA-1-specific T cells derived from the donor's T cell repertoire cultured in vitro lacked in vivo persistence and in vivo anti-leukemia reactivity (Meij et al., 2012).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

[0005] [Non-Patent Document 1] Singleton and Sainsbury, "Dictionary of Microbiology and Molecular Biology", 2nd Edition, John Wiley and Sons, NY [Non-Patent Document 2] Hale and Marham, "The Harper Collins Dictionary of Biology", Harper Perennial, NY (1991) [Non-Patent Document 3] Scholten et al., Clin. Immunol. 119:135, 2006 [Non-Patent Document 4] Govers et al., Trends Mol. Med., 16(2): 11(2010) [Non-Patent Document 5] Sadelain et al., Cancer Discov., 3(4):388(2013) [Non-Patent Document 6] Harris and Kranz, Trends Pharmacol. Sci., 37(3):220(2016)

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

Non-Patent Document 12

Non-Patent Document 13

Non-Patent Document 14

Non-Patent Document 15

Non-Patent Document 16

Non-Patent Document 17

Non-Patent Document 29

Non-Patent Document 30

Summary of the Invention

Problems to be Solved by the Invention

[0006] There is a need for new immunotherapies for treating hematological malignancies.

Means for Solving the Problems

[0007] HA-1, a minor histocompatibility antigen H is encoded by the polymeric HMHA1 gene (also called low GTPase-activating protein 45). The HMHA1 variant (rs_1801284 A / A or A / G), which is present in 52% of individuals, results in an immunogenic peptide (VLHDDLLEA; SEQ ID NO: 10) containing a histidine residue instead of an arginine, and the presentation of this peptide by HLA is common HLA-A in individuals* (2) Resulting from the 0201 (A2) allele. Thus, HA-1 H T cell therapy targeting is applicable to approximately 25% of the subjects who receive transplantation for hematological malignancies, and requires a T cell donor who is HLA-A2 negative or HA-1 H negative ("HA-1 RH "; VLRDDLLEA; SEQ ID NO: 79).

[0008] The inventors isolated and sequenced a novel TCR specific for the HA-1 H antigen. Such a TCR is useful for treating or preventing recurrence of hematological malignancies after allogeneic stem cell transplantation (allo-SCT) in HLA-A * 0201-positive human subjects.

[0009] The inventors have previously shown that HA-1-specific T cells, derived from the donor's T cell repertoire and cultured in vitro, lack in vivo persistence and in vivo anti-leukemia reactivity (Meij et al., 2012). The inventors have now isolated HA-1 TCRs that are derived from HA-1-specific T cells isolated from patients and have undergone a strong anti-leukemia response mediated by these HA-1-specific T cells (Marijt et al., 2003, van Loenen et al., 2011). Therefore, the HA-1 TCRs described herein are advantageous as they correspond to high-affinity TCRs that approximate the natural T cell response and are associated with a highly effective anti-leukemia reactivity. Surprisingly, the HA-1-specific TCRs described herein exert a more potent anti-tumor reactivity compared to less pure candidate substances generated by in vitro cultures. In addition, these HA-1 TCRs are known to be safe as they are isolated from activated anti-leukemia responses without signs of graft-versus-host disease (GvHD) and do not induce graft-versus-host disease or other toxicities. The strong anti-leukemia reactivity of the HA-1-specific TCRs is supported against primary AML blasts and primary ALL blasts derived from patients at the time of diagnosis, demonstrating the effectiveness of these HA1-TCRs in patients with hematological malignancies, which is advantageous. Even more persuasively, a preclinical in vivo model was used herein to show the strong in vivo anti-tumor reactivity of the HA-1 TCRs, as HA-1 TCR-engineered T cells were extremely effectively eradicating established multiple myeloma after injection.

[0010] The inventors explored which components of the novel TCRs are essential for HA-1 H antigen specificity and TCR functionality. Surprisingly, the inventors found that the CDR1 region of the TCRβ chain variable domain (Vβ) is HA-1 HAlthough it is extremely important for the specificity of [HA-1], it has been found that it is not sufficient for the specificity of HA-1. The inventors have also identified that any CDR3 region of either the TCRβ chain variable domain (Vβ) or the TCRα chain variable domain (Vα) is required, and that the TCRβ chain variable (Vβ) domain must be encoded by the TRBV7-9 gene.

[0011] Therefore, the HA-1 H specific TCR described herein has the following minimum components: (a) HA-1 H A TCR Vα domain containing a specific CDR3 (see, for example, SEQ ID NOs: 1-3); and (b) A TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, the HA-1 H specific CDR3 (see, for example, SEQ ID NOs: 4-6) and the HA-1 H A TCR Vβ domain containing a specific CDR1 (see, for example, SEQ ID NO: 7) is required.

[0012] The CDR3 TCR sequences described herein are different from the CDR3 TCR sequences known in the prior art, for example, those disclosed in WO2018 / 058002. Furthermore, the TRAV region (which contains the CDR1 and CDR2 regions) of the alpha chain sequence of the TCR described herein is completely different from the TRAV region of the alpha chain of the TCR described in WO2018 / 058002. In addition, the TRAJ region is combined with a different TRAV region. Therefore, there are several differences between the TCR claimed by the inventors and the TCR of the cited prior art.

[0013] Exemplified herein are HA-1 H specific binding proteins (e.g., TCRs) that use a combination of a specific CDR3 sequence and a specific CDR1 sequence. For example, (a) A TCR Vα domain containing the CDR3 of SEQ ID NO: 1; and (b) A TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, comprising the CDR3 of SEQ ID NO: 4 and the CDR1 of SEQ ID NO: 7, and herein designated HA-1 H A TCR Vβ domain shown to confer binding specificity, and The combination of. Although these specific CDR sequences are exemplified, the CDRs can also include some variation from the specified sequences (e.g., each specified CDR can have at least 80% sequence identity to the specified SEQ ID NO). The TRBV7-9 gene can be TRBV7-9 * 03 (see, e.g., TCR M7).

[0014] In addition, (a) A TCR Vα domain comprising the CDR3 of SEQ ID NO: 2; and (b) A TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, comprising the CDR3 of SEQ ID NO: 5 and the CDR1 of SEQ ID NO: 7, and herein designated HA-1 H A TCR Vβ domain shown to confer binding specificity, and The combination of. Although these specific CDR sequences are exemplified, the CDRs can also include some variation from the specified sequences (e.g., each specified CDR can have at least 80% sequence identity to the specified SEQ ID NO). The TRBV7-9 gene can be TRBV7-9 * 01 (see, e.g., TCR M2).

[0015] Furthermore, (a) A TCR Vα domain comprising the CDR3 of SEQ ID NO: 3; and (b) A TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, comprising the CDR3 of SEQ ID NO: 6 and the CDR1 of SEQ ID NO: 7, and HA-1 H A TCR Vβ domain shown to confer binding specificity, and is a combination. Although these specific CDR sequences are exemplified, the CDRs can also include some variation from the specified sequences (e.g., each specified CDR can have at least 80% sequence identity to the specified sequence number). The TRBV7-9 gene can be TRBV7-9 * 01 (see, for example, TCR FK47.83).

[0016] The inventors have also shown that a specific binding protein (e.g., a TCR) can be formed by different combinations of the TCR Vα domain and the TCR Vβ domain described herein. For example, a functional TCR was created by combining a TCR Vβ domain equivalent to the M7 clone (SEQ ID NO: 18) with a TCR Vα domain equivalent to the M2 clone (SEQ ID NO: 29). Thus, a functional binding protein (e.g., a TCR) can also be H formed from a combination of (a) a TCR Vα domain containing the CDR3 of SEQ ID NO: 2 and; (b) a TCR Vβ domain having the amino acid sequence encoded by the TRBV7-9 gene, containing the CDR3 of SEQ ID NO: 4 and the CDR1 of SEQ ID NO: 7, and shown to confer HA-1 binding specificity, with the TCR Vβ domain H of the combination from which it can also be made. Although these specific CDR sequences are exemplified, the CDRs can also include some variation from the specified sequences (e.g., each specified CDR can have at least 80% sequence identity to the specified sequence number). The TRBV7-9 gene can be TRBV7-9 01 (see, for example, TCR M7). * 01 (see, for example, TCR M7).

[0017] The data presented herein relates to the specific combinations described above, but other combinations can also be used to form functional binding proteins specific for HA-1 H and are also encompassed herein and described in more detail below.

[0018] The present invention is exemplified by generating a functional TCR specific for HA-1 H However, the present invention also encompasses other binding proteins with the characteristics specified above for conferring HA-1 H antigen specificity. Thus, other binding proteins (e.g., antigen-binding fragments of TCRs (such as single-chain TCRs, etc.), or chimeric antigen receptors (CARs)) are also encompassed. When these binding proteins are expressed by host cells (e.g., immune cells, such as T cells, etc.), they may be used as monotherapy for treating hematological malignancies or preventing their relapse or recurrence, and may also be used as part of a treatment regimen that includes additional treatments or agents (e.g., after allogeneic SCT or in combination with allogeneic SCT).

[0019] In one aspect, the present invention provides an isolated nucleic acid composition encoding an HA-1 H antigen-specific binding protein having a TCR α-chain variable (Vα) domain and a TCR β-chain variable (Vβ) domain, (a) a nucleic acid sequence encoding a TCR Vα domain comprising a CDR3 amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1 to 3; and (b) a nucleic acid sequence encoding a TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, wherein the Vβ domain comprises a CDR3 amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 4 to 6 and a CDR1 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 7, the composition comprising the nucleic acid sequence.

[0020] Suitably, the TRBV7-9 gene may be TRBV7-9 * 01 or TRBV7-9 * 03.

[0021] Suitably, the HA-1 H antigen may comprise the amino acid sequence shown in SEQ ID NO: 10.

[0022] Suitably, the encoded binding protein may be HA-1 H Antigen: HLA-A * and may be capable of specifically binding to the 0201 complex.

[0023] Suitably, the nucleic acid sequence may be codon-optimized for expression in a host cell, and optionally, the host cell is a human T cell.

[0024] Suitably: (i) The CDR3 of the Vα domain may contain or consist of the amino acid sequence of SEQ ID NO: 1, (ii) The CDR3 of the Vβ domain may contain or consist of the amino acid sequence of SEQ ID NO: 4, (iii) The CDR1 of the Vβ domain may contain or consist of the amino acid sequence of SEQ ID NO: 7.

[0025] Suitably: (i) The CDR3 of the Vα domain may be encoded by a nucleic acid sequence containing the sequence of SEQ ID NO: 11 or SEQ ID NO: 12; and / or (ii) The CDR3 of the Vβ domain may be encoded by a nucleic acid sequence containing the sequence of SEQ ID NO: 13 or SEQ ID NO: 14; and / or (iii) The CDR1 of the Vβ domain may be encoded by a nucleic acid sequence containing the sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0026] Suitably: (i) The Vα domain may contain, include, or consist of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 17; and / or (ii) The Vβ domain may contain, include, or consist of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 18.

[0027] Suitably: (i) The Vα domain can be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 19 or SEQ ID NO: 20; and / or (ii) The Vβ domain can be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 21 or SEQ ID NO: 22.

[0028] Preferably: (i) The CDR3 of the Vα domain can comprise or consist of the amino acid sequence of SEQ ID NO: 2, (ii) The CDR3 of the Vβ domain can comprise or consist of the amino acid sequence of SEQ ID NO: 5, (iii) The CDR1 of the Vβ domain can comprise or consist of the amino acid sequence of SEQ ID NO: 7.

[0029] Preferably: (i) The CDR3 of the Vα domain can be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 23 or SEQ ID NO: 24; and / or (ii) The CDR3 of the Vβ domain can be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 25 or SEQ ID NO: 26; and / or (iii) The CDR1 of the Vβ domain can be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0030] Preferably: (i) The Vα domain can comprise an amino acid sequence having at least 90% sequence identity to, including, or consisting of SEQ ID NO: 29; and / or (ii) The Vβ domain can comprise an amino acid sequence having at least 90% sequence identity to, including, or consisting of SEQ ID NO: 30.

[0031] Preferably: (i) The Vα domain can be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 31 or SEQ ID NO: 32; and / or (ii) The Vβ domain can be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

[0032] Suitably: (i) The CDR3 of the Vα domain can include or consist of the amino acid sequence of SEQ ID NO: 3, (ii) The CDR3 of the Vβ domain can include or consist of the amino acid sequence of SEQ ID NO: 6, (iii) The CDR1 of the Vβ domain can include or consist of the amino acid sequence of SEQ ID NO: 7.

[0033] Suitably: (i) The CDR3 of the Vα domain can be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 35 or SEQ ID NO: 36; and / or (ii) The CDR3 of the Vβ domain can be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 37 or SEQ ID NO: 38; and / or (iii) The CDR1 of the Vβ domain can be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0034] Suitably: (i) The Vα domain can include an amino acid sequence having at least 90% sequence identity to, including, or consisting of SEQ ID NO: 41; and / or (ii) The Vβ domain can include an amino acid sequence having at least 90% sequence identity to, including, or consisting of SEQ ID NO: 42.

[0035] Suitably: (i) The Vα domain can be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 43 or SEQ ID NO: 44; and / or (ii) The Vβ domain can be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 45 or SEQ ID NO: 46.

[0036] Optionally, the isolated nucleic acid composition may further comprise a TCRα chain constant domain and / or a TCRβ chain constant domain.

[0037] Optionally, the encoded binding protein may comprise a TCR, an antigen-binding fragment of a TCR, or a chimeric antigen receptor (CAR).

[0038] Optionally, the antigen-binding fragment of the TCR can be a single-chain TCR (scTCR).

[0039] In another aspect, the present invention provides a vector system comprising the nucleic acid composition described herein.

[0040] Optionally, the vector can be a plasmid, a viral vector, or a cosmid, and optionally, the vector is selected from the group consisting of a retrovirus, a lentivirus, an adeno-associated virus, an adenovirus, a vaccinia virus, a canarypox virus, a herpes virus, a minicircle vector, and synthetic DNA or synthetic RNA.

[0041] In another aspect, the present invention provides a modified cell in which the nucleic acid composition described herein, or the vector system described herein, is transfected or transduced, and is HLA-A * 0201 negative and / or HA-1 H negative.

[0042] Optionally, the modified cell can be selected from the group consisting of CD8 T cells, CD4 T cells, NK cells, NK-T cells, gamma-delta T cells, hematopoietic stem cells, progenitor cells, T cell lines, or NK-92 cell lines.

[0043] Optionally, the modified cell can be a human cell.

[0044] In another aspect, the present invention provides a pharmaceutical composition comprising the nucleic acid composition described herein, the vector system described herein, or the modified cell described herein, and a pharmaceutically acceptable excipient, adjuvant, diluent, and / or carrier.

[0045] In another aspect, the present invention relates to HLA-A * A method for treating or preventing recurrence of a hematological malignancy after allogeneic stem cell transplantation (allo-SCT) in an HLA-A 0201-positive human subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein.

[0046] In another aspect, the present invention relates to HLA-A * Provided is the pharmaceutical composition described herein for use in treating or preventing recurrence of a hematological malignancy after allogeneic stem cell transplantation (allo-SCT) in an HLA-A 0201-positive human subject.

[0047] In another aspect, the present invention relates to HLA-A * Provided is the use of the pharmaceutical composition described herein in the manufacture of a medicament for treating or preventing recurrence of a hematological malignancy after allogeneic stem cell transplantation (allo-SCT) in an HLA-A 0201-positive human subject.

[0048] Suitably, the hematological malignancy may include leukemia, lymphoma, myelodysplastic syndrome, or multiple myeloma.

[0049] Suitably: (i) the hematological malignancy can include leukemia, and optionally, the leukemia is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), mixed phenotype acute leukemia (MPAL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, hairy cell leukemia, or chronic lymphocytic leukemia (CLL); or (ii) Hematological malignancies can include lymphomas, and optionally, the lymphomas can be selected from the group consisting of Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), central nervous system lymphoma, small lymphocytic lymphoma (SLL), CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, mucosa-associated (MALT) extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, precursor B-lymphoblastic lymphoma, immunoblastic large cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or Burkitt lymphoma; or (iii) Hematological malignancies can include myelodysplastic syndromes, and optionally, the myelodysplastic syndromes can be selected from refractory cytopenia with single lineage dysplasia (refractory anemia, refractory neutropenia, and refractory thrombocytopenia), refractory anemia with ring sideroblasts (RARS), refractory anemia with ring sideroblasts and thrombocytosis (RARS-t), refractory cytopenia with multilineage dysplasia (RCMD), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS), refractory anemia with excess blasts (RAEB), unclassifiable myelodysplasia, or pediatric refractory cytopenia.

[0050] Appropriately, the subject may have been previously administered lymphodepleting chemotherapy.

[0051] Appropriately, the lymphodepleting chemotherapy can include cyclophosphamide, fludarabine, antithymocyte globulin, or a combination thereof.

[0052] Appropriately, one or more of the modified cells in the composition described herein can be allogeneic to the subject.

[0053] In another aspect, the invention is capable of specifically binding to a peptide containing the HA-1 H antigen, and HA-1 HA method of creating a binding protein that does not bind to a peptide that does not contain an antigen, the method comprising contacting a nucleic acid composition described herein with a cell under conditions such that the nucleic acid composition is incorporated and expressed by the cell.

[0054] Suitably, the method can be an ex vivo method.

[0055] In another aspect, the invention provides an isolated nucleic acid sequence comprising, or consisting of, any one of the nucleotide sequences of SEQ ID NOs: 11-14, 19-26, 31-38, 43-46, 49-51, 54-56, 59-66, 69-71, and 74-76.

[0056] In another aspect, the invention provides an isolated nucleic acid sequence comprising, or consisting of, any one of the nucleotide sequences of SEQ ID NOs: 11-14, 19-26, 31-38, 43-46, 49-51, 54-56, 59-66, 69-71, and 74-76 for use in therapy.

[0057] Throughout the description and claims of this specification, the terms "comprising" and "containing" and variations thereof mean "including but not limited to" and are not intended (and do not) exclude other parts, additives, components, integers, or steps.

[0058] Throughout the description and claims of this specification, unless the context requires otherwise, the singular form encompasses the plural. In particular, when an indefinite article is used, the specification is to be understood as assuming plurality as well as singularity unless the context requires otherwise.

[0059] It is to be understood that features, integers, characteristics, compounds, chemical moieties, or chemical groups described in connection with particular aspects, embodiments, or examples of the invention are applicable to any other aspect, embodiment, or example described herein, unless incompatible therewith.

[0060] The patent documents, scientific documents, and technical documents referred to in this specification establish the knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published patents, and patent applications under prosecution, as well as other publications, cited herein are hereby incorporated by reference in their entirety to the same extent as if each were specifically and individually indicated to be incorporated by reference. In case of any inconsistency, the present disclosure shall prevail.

[0061] Unless otherwise defined herein, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, "Dictionary of Microbiology and Molecular Biology", 2nd edition, John Wiley and Sons, NY; and Hale and Marham, "The Harper Collins Dictionary of Biology", Harper Perennial, NY (1991) provide a general glossary of many of the terms used in the present invention for those skilled in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are fully described by reference to the entire specification. Also, as used herein, the singular terms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Unless otherwise indicated, nucleic acids are written left to right in the 5' to 3' direction; amino acid sequences are written left to right in the amino to carboxy direction. It is understood that the invention is not limited to the specific methods, protocols, and reagents described, as these may vary depending on the context in which they are used by those skilled in the art.

[0062] Various aspects of the present invention are described in further detail below.

[0063] In this specification, embodiments of the present invention will be further described after referring to the accompanying drawings.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0065] The inventors isolated and sequenced a novel TCR specific for the HA-1 H antigen. Such a TCR is useful for treating or preventing the recurrence of hematological malignancies after allogeneic stem cell transplantation (allo-SCT) in HLA-A * 0201-positive human subjects.

[0066] The inventors explored which components of the novel TCR are essential for HA-1 H antigen specificity and TCR functionality. Surprisingly, the inventors found that the CDR1 region of the TCR β-chain variable domain (Vβ) is HA-1 HAlthough it is extremely important for the specificity of [HA-1], it has been found that it is not sufficient for the specificity of HA-1. The inventors have also identified that any CDR3 region of the TCRβ chain variable domain (Vβ) and the TCRα chain variable domain (Vα) is required, and that the TCRβ chain variable (Vβ) domain needs to be encoded by the TRBV7-9 gene.

[0067] Nucleic acid composition encoding a binding protein component The present invention relates to HA-1 H The present invention provides an isolated nucleic acid composition encoding a binding protein comprising a T cell receptor (TCR) component that specifically binds to an antigen. Thus, the encoded binding protein can specifically bind to a peptide containing the HA-1 H antigen and does not bind to a peptide that does not contain the HA-1 H antigen.

[0068] The nucleic acid composition comprises (a) a nucleic acid sequence encoding a TCR Vα domain with the specified characteristics described herein, and (b) a nucleic acid sequence encoding a TCR Vβ domain with the specified characteristics described herein. The components of the encoded TCR form an HA-1 H antigen-specific binding protein.

[0069] The nucleic acid sequences of (a) and (b) above can be significantly different nucleic acid sequences within the nucleic acid composition. Thus, the TCR components of the binding protein can be encoded by two (or more) nucleic acid sequences (with significantly different nucleotide sequences) that together encode all of the TCR components of the binding protein. In other words, a part of the TCR component may be encoded by one nucleic acid sequence within the nucleic acid composition, and other TCR components may be encoded by another (significantly different) nucleic acid sequence within the nucleic acid composition.

[0070] Alternatively, the nucleic acid sequences of (a) and (b) can be part of a single nucleic acid sequence. Thus, all of the TCR components of the binding protein can be encoded by a single nucleic acid sequence (e.g., with a single open reading frame, or with multiple open reading frames (e.g., 2 or more open reading frames, 3 or more open reading frames, etc.)).

[0071] The nucleic acid sequences described herein can form part of a larger nucleic acid sequence that encodes a larger component of a functional binding protein. For example, the nucleic acid sequence encoding a TCR Vα domain with the specified characteristics described herein can be part of a larger nucleic acid sequence that encodes a functional TCRα chain (including the constant domain). As another example, the nucleic acid sequence encoding a TCR Vβ domain with the specified characteristics described herein can be part of a larger nucleic acid sequence that encodes a functional TCRβ chain (including the constant domain). As a further example, either of the above nucleic acid sequences (a) and (b) can be part of a larger nucleic acid sequence that encodes a combination of a functional TCRα chain (including the constant domain) and a functional TCRβ chain (including the constant domain), in which case, optionally, the sequence encoding the functional TCRα chain is separated from the sequence encoding the functional TCRβ chain by a linker sequence within the same nucleic acid sequence that allows for the coordinated expression of the two proteins or polypeptides. Further details on this are presented below.

[0072] Alternatively, the nucleic acid sequences described herein may only encode a small component of the T cell receptor, e.g., only the TCR Vα domain or the TCR Vβ domain. The nucleic acid sequence can be considered as a "building block" that provides the components essential for the binding specificity of the peptide. The nucleic acid sequences described herein, when incorporated into, for example, HA-1 HNew nucleic acid sequences encoding TCRα chains and / or TCRβ chains that specifically bind to an antigen can be incorporated into significantly different nucleic acid sequences (e.g., vectors) encoding other elements of a functional binding protein such as a TCR so as to create such new nucleic acid sequences. Thus, the nucleic acid sequences described herein are HA-1 H Useful as an essential component for conferring binding specificity to an antigen, it can be used to create larger nucleic acid sequences encoding a binding protein with the required antigen-binding activity and binding specificity.

[0073] The nucleic acid sequences described herein can be codon-optimized for expression in host cells. For example, the nucleic acid sequences described herein can be codon-optimized for expression in human cells such as immune system cells, hematopoietic stem cells, T cells, primary T cells, T cell lines, K cells, or natural killer T cells (Scholten et al., Clin. Immunol. 119:135, 2006). T cells are CD4 + In the case of T cells, it may also be CD8 + In the case of T cells. Codon optimization is a method well known in the art for maximizing the expression of nucleic acid sequences in a particular host cell. As described in the Examples section below, one or more cysteine residues can also be introduced into the encoded TCR alpha chain component and TCR beta chain component (e.g., to reduce the risk of mispairing with endogenous TCR chains).

[0074] In one example, the nucleic acid sequences described herein are codon-optimized for expression in a suitable host cell and / or codons encoding one or more cysteine amino acids (e.g., into the constant domain of the encoded TCR alpha chain and / or the encoded TCR beta chain) are introduced to modify to reduce the risk of mispairing with endogenous TCR chains.

[0075] In certain examples, the constant domain of the TCR is modified to enhance pairing of the desired TCR chains. For example, enhanced pairing of a heterologous TCRα chain and a heterologous TCRβ chain resulting from the modification can result in preferential assembly of a TCR comprising the two heterologous chains over an undesired mispairing of the heterologous TCRα chain with the endogenous TCRα chain (see, e.g., Govers et al., Trends Mol. Med., 16(2):11 (2010)). Exemplary modifications that enhance pairing of heterologous TCR chains include introduction of complementary cysteine residues in each of the heterologous TCRα and TCRβ chains. In some examples, the polynucleotide encoding the heterologous TCRα chain encodes a cysteine at amino acid position 48 (corresponding to the constant region of the full-length, mature human TCRα chain sequence), and the polynucleotide encoding the heterologous TCRβ chain encodes a cysteine at amino acid position 57 (corresponding to the constant region of the full-length, mature human TCRβ chain sequence).

[0076] The binding protein encoded by the nucleic acid composition described herein is specific for HA-1 H antigen and comprises an HA-1 H antigen-specific TCR component. However, the encoded binding protein is not limited to being a TCR. Other suitable binding proteins comprising the designated HA-1 H antigen-specific TCR component are also contemplated. For example, the encoded binding protein can include a TCR, an antigen-binding fragment of a TCR, or a chimeric antigen receptor (CAR). In the art, TCRs, antigen-binding fragments of TCRs, and CARs are well defined. Non-limiting examples of antigen-binding fragments of TCRs are single-chain TCRs (scTCRs), or chimeric dimers composed of an antigen-binding fragment of a TCRα chain and an antigen-binding fragment of a TCRβ chain linked to the transmembrane and intracellular domains of a dimeric complex such that the complex is a chimeric dimeric TCR (cdTCR).

[0077] In certain examples, the antigen-binding fragment of a TCR comprises both a TCR Vα domain and a TCR Vβ domain, and includes a single-chain TCR (scTCR) that includes only the constant domain of a single TCR. In further examples, the antigen-binding fragment of a TCR or chimeric antigen receptor is a chimeric antigen-binding fragment (e.g., including amino acid residues or motifs derived from more than one donor or species), a humanized antigen-binding fragment (e.g., including residues derived from a non-human organism that have been altered or substituted to reduce the risk of immunogenicity in humans), or a human antigen-binding fragment.

[0078] A “chimeric antigen receptor” (CAR) is a fusion protein engineered to contain two or more naturally occurring amino acid sequences that are joined together either not naturally or not naturally occurring in the host cell, and that, when present on the cell surface, can function as a receptor. The CARs described herein include an extracellular portion (see, e.g., Sadelain et al., Cancer Discov., 3(4):388 (2013). See also Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2016), and Stone et al., Cancer Immunol. Immunother., 63(11):1163 (2014)) that is linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing a co-stimulatory domain), and an antigen-binding domain (i.e., an antigen-binding domain obtained from or derived from an immunoglobulin or immunoglobulin-like molecule such as an antibody specific for a cancer antigen or an scFv derived from a TCR, or an antigen-binding domain derived from or obtained from a killer immunoglobulin receptor derived from an NK cell).

[0079] Methods for generating an engineered TCR are described, for example, in Bowerman et al., Mol. Immunol., 5(15):3000 (2009). Methods for making CARs are well known in the art and are described, for example, in U.S. Patent No. 6,410,319; U.S. Patent No. 7,446,191; U.S. Patent Publication No. 2010 / 065818; U.S. Patent No. 8,822,647; PCT Publication No. WO2014 / 031687; U.S. Patent No. 7,514,537; and Brentjens et al., 2007, Clin. Cancer Res., 73:5426.

[0080] The binding proteins described herein can also be expressed as part of a transgene construct that encodes additional accessory proteins such as safety switch proteins, tags, selection markers, the β-chain, α-chain, or both of the CD8 co-receptor, or any combination thereof.

[0081] A T cell receptor (TCR) is a molecule found on the surface of T cells (T lymphocytes) that contributes to the recognition of a peptide (presented by MHC) bound to a major histocompatibility complex (MHC) molecule on the surface of a target cell. The present invention relates to nucleic acid compositions encoding binding proteins that include components of a TCR that interact with a specific peptide in the context of an appropriate serotype of MHC, i.e., HLA-A * in the context of 02:01, HA-1 H interacts with an antigen (in other words, the encoded binding protein is specific for the HA-1 H antigen:HLA-A * 0201 complex), and is directed to nucleic acid compositions encoding binding proteins that include components of a TCR. HLA-A * 02:01 is a human leukocyte antigen serotype within the HLA-A serotype group that is common worldwide. HLA-A * 02:01 is described as "HLA-A * 02:01 restricted" with respect to the peptides presented to the TCR.

[0082] HA-1 to which the binding protein described in this specification specifically binds H The antigen is an antigenic peptide derived from the amino acid sequence shown in SEQ ID NO: 10. The antigen can be an antigenic fragment (i.e., a part) of the sequence shown in SEQ ID NO: 10, may consist of the sequence of SEQ ID NO: 10, or may contain the sequence of SEQ ID NO: 10 (i.e., be included within a longer sequence). HA-1 H The antigen can be presented by HLA-A * 0201. Therefore, the encoded binding protein can specifically bind to the HA-1 H Antigen: HLA-A * 0201 complex, in which case HA-1 H The antigen is an antigenic fragment of the sequence shown in SEQ ID NO: 10, or HA-1 H The antigen contains or consists of the amino acid sequence shown in SEQ ID NO: 10.

[0083] TCR is composed of two different polypeptide chains. In humans, 95% of TCRs consist of an alpha (α) chain and a beta (β) chain (encoded by TRA and TRB, respectively). When TCR engages a peptide in the context of HLA (e.g., in the context of HLA-A * 02:01), the T cell is activated via signal transduction.

[0084] The alpha and beta chains of TCR are highly variable within the sequence. Each chain is composed of a variable domain (V), which are two extracellular domains, and a constant domain (C). While the variable domain binds to the peptide / HLA-A complex, the constant domain is proximal to the transmembrane region and the short cytoplasmic tail that follows the cell membrane of the T cell.

[0085] The variable domain of each lock has three hypervariable regions (also called complementarity-determining regions (CDRs)). Thus, the TCR alpha variable domain (referred to herein as the TCR Vα domain, TCR V alpha domain, Vα domain or V alpha domain, alpha variable domain, etc.) includes a CDR1 region, a CDR2 region, and a CDR3 region. Similarly, the TCR beta variable domain (referred to herein as the TCR Vβ domain, TCR V beta domain, Vβ domain or V beta domain, beta variable domain, etc.) also includes (different) CDR1 region, CDR2 region, and CDR3 region. In each of the alpha variable domain and the beta variable domain, the main factor for recognizing the peptide presented by HLA-A is CDR3.

[0086] As will be apparent to those skilled in the art, the phrase "TCRα chain variable domain" refers to the variable (V) domain (extracellular domain) of the TCR alpha chain, and thus, in addition to the three hypervariable regions (CDR1, CDR2, and the designated CDR3), it includes intervening sequences but does not include the constant (C) domain of the alpha chain that does not form part of the variable domain.

[0087] As will be apparent to those skilled in the art, the phrase "TCRβ chain variable domain" refers to the variable (V) domain (extracellular domain) of the TCR beta chain, and thus, in addition to the three hypervariable regions (CDR1, CDR2, and the designated CDR3), it includes intervening sequences but does not include the constant (C) domain of the beta chain that does not form part of the variable domain.

[0088] As used herein, HA-1 having a TCRα chain variable (Vα) domain and a TCRβ chain variable (Vβ) domain H An isolated nucleic acid composition encoding an antigen-specific binding protein, (a) A nucleic acid sequence encoding a TCR Vα domain comprising a CDR3 amino acid sequence having at least 85% sequence identity to any one of SEQ ID NOs: 1-3; and (b) A nucleic acid sequence encoding a TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, wherein the Vβ domain comprises a CDR3 amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 4-6 and a CDR1 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 7, nucleic acid sequence A composition comprising the same is provided.

[0089] Any of the permutations described for (a) below can be combined with the permutations described for (b) below (e.g., HA-1 having a TCR α-chain variable (Vα) domain and a TCR β-chain variable (Vβ) domain H to form a suitable nucleic acid composition encoding an antigen-specific binding protein). Below, further details regarding suitable combinations are provided.

[0090] Components of the TCR α-chain variable (Vα) domain The isolated nucleic acid composition described herein encodes an HA-1 H antigen-specific binding protein. HA-1 H The antigen-specific binding protein comprises a TCR Vα domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-3.

[0091] (i) A Vα domain comprising the CDR3 amino acid sequence of SEQ ID NO: 1 and functional variants thereof HA-1 H An example of a CDR3 amino acid sequence of a suitable TCR Vα domain that confers specific binding to an antigen is shown in SEQ ID NO: 1. As will be apparent to those skilled in the art, variants of the amino acid sequence shown in SEQ ID NO: 1 may also be functional (i.e., when the CDR3 is part of the TCR Vα domain, HA-1 H retaining their ability to confer specific binding to an antigen (e.g., the peptide shown in SEQ ID NO: 10)). Thus, such functional variants are encompassed herein.

[0092] For example, the CDR3 amino acid sequence of a suitable (functional) Vα domain can have at least 80% sequence identity to SEQ ID NO: 1, i.e., this can have at least 80%, at least 83%, at least 85%, at least 90%, at least 91%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1. Suitably, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 1). In other words, the CDR3 amino acid sequence of a suitable (functional) Vα domain can vary from the sequence shown in SEQ ID NO: 1 by 1 amino acid or several amino acids (e.g., 2 amino acids, etc.).

[0093] As stated above, functional variants of SEQ ID NO: 1 retain their ability to confer specific binding to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10) when the CDR3 is part of the TCR Vα domain.

[0094] Functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 1. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of 1, 2, or more amino acids of SEQ ID NO: 1, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the CDR3.

[0095] Non-functional variants are amino acid sequence variants of SEQ ID NO: 1 that do not specifically bind to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 1, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0096] In one example, the CDR3 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO: 1. In an example where the CDR3 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 1, the CDR3 can be encoded by the nucleic acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12, or their degenerate gene sequences (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 12 is the codon-optimized form of the nucleic acid sequence for the CDR3 of clone M7 (the non-optimized sequence is SEQ ID NO: 11).

[0097] The encoded TCR Vα domain can include, in addition to the specified CDR3, the amino acid sequence of SEQ ID NO: 80, or a functional variant thereof (i.e., in this case, the variant retains the ability to specifically bind to the N-terminus of the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10)) CDR1. Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 80. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 80, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0098] Non-functional variants are amino acid sequence variants of SEQ ID NO: 80 that do not specifically bind to the N-terminus of the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 80, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0099] For example, the CDR1 amino acid sequence of a suitable (functional) Vα domain can have at least 80% sequence identity to SEQ ID NO: 80, i.e., it can have at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 80. Suitably, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 80). In other words, the CDR1 amino acid sequence of a suitable (functional) Vα domain can vary by 1 amino acid or several amino acids from the sequence shown in SEQ ID NO: 80. As already stated, the variant can include amino acid substitutions such as conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 80. As stated above, a functional variant of SEQ ID NO: 80, when the CDR1 is part of a TCR Vα domain, has HA-1 H the ability to specifically bind to the N-terminus of an antigen (e.g., the peptide shown in SEQ ID NO: 10).

[0100] In one example, the CDR1 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO: 80. In an example where the CDR1 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 80, the CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO: 81 or SEQ ID NO: 82, or a degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 82 is the codon-optimized form of the nucleic acid sequence for the CDR1 of clone M7 (the non-optimized sequence is SEQ ID NO: 81).

[0101] For other suitable CDR1 Vα domain amino acid sequences, such as those CDR1 sequences containing the sequence shown in SEQ ID NO: 8, they are described elsewhere in this specification. Thus, it will be apparent to those skilled in the art that when discussing permutations of Vα CDR1 amino acid sequences and nucleotide sequences for combination with the CDR3 sequence of SEQ ID NO: 80 (or the corresponding nucleotide sequences of SEQ ID NO: 11 or SEQ ID NO: 12), the reference to SEQ ID NO: 8 (as well as the corresponding nucleotide sequences of SEQ ID NO: 9 and SEQ ID NO: 27) can be substituted.

[0102] The encoded TCR Vα domain may also include, in addition to the specified CDR3 (and optionally, the CDR1 specified above), the amino acid sequence of SEQ ID NO: 83 or a functional variant thereof (i.e., in this case, the variant retains the ability to specifically bind to HLA-A * 02:01). Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 83. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 83, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0103] Non-functional variants are amino acid sequence variants of SEQ ID NO: 83 that do not specifically bind to HLA-A * 02:01. Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 83, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0104] For example, the CDR2 amino acid sequence of a suitable (functional) Vα domain can have at least 80% sequence identity to SEQ ID NO: 83, i.e., it can have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 83. Suitably, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 83). In other words, the CDR2 amino acid sequence of a suitable (functional) Vα domain can vary by 1 amino acid or several amino acids from the sequence shown in SEQ ID NO: 83. As already stated, the variant can include amino acid substitutions such as conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 83. As stated above, a functional variant of SEQ ID NO: 83 retains the ability to specifically bind to HLA-A * retains the ability to specifically bind to 02:01.

[0105] In one example, the CDR2 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO: 83. In an example where the CDR2 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 83, the CDR2 can be encoded by the nucleic acid sequence of SEQ ID NO: 84 or SEQ ID NO: 85, or a degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 85 is the codon-optimized form of the nucleic acid sequence for the CDR2 of clone M7 (the non-optimized sequence is SEQ ID NO: 84).

[0106] Other suitable CDR2 sequences of the CDR2 Vα domain, such as the CDR2 sequence containing the sequence shown in SEQ ID NO: 28, are described elsewhere in this specification. Thus, it will be apparent to those skilled in the art that the reference to SEQ ID NO: 83 above can be replaced by a reference to SEQ ID NO: 28 (and the corresponding nucleotide sequences of SEQ ID NO: 39 and SEQ ID NO: 40) when discussing permutations of the Vα CDR2 amino acid and nucleotide sequences for combination with the CDR3 sequence of SEQ ID NO: 1 (or the corresponding nucleotide sequences of SEQ ID NO: 11 or SEQ ID NO: 12).

[0107] Thus, the encoded TCR Vα domain can include the CDRs specifically mentioned above (specifically, in SEQ ID NOs., namely SEQ ID NO: 1, SEQ ID NO: 80 (or SEQ ID NO: 8), and SEQ ID NO: 83 (or SEQ ID NO: 28), or functional variants thereof) with appropriate intervening sequences between the CDRs.

[0108] The encoded TCR Vα domain can include the amino acid sequence of SEQ ID NO: 17 or a functional variant thereof (i.e., in this case, if it includes a part of the binding protein described herein, the variant TCR Vα domain is HA-1 H and retains the ability to specifically bind to the antigen (e.g., the peptide shown in SEQ ID NO: 10). Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 17. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 17, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0109] Non-functional variants are HA-1 HIt is an amino acid sequence variant of SEQ ID NO: 17 that does not specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 17, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0110] In one example, the encoded TCR Vα domain is HA-1 H While retaining the ability to specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10), it 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 to the amino acid sequence of SEQ ID NO: 17. In other words, functional TCR Vα domains with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 17 are also encompassed. As already stated, the amino acid substitutions can be conservative amino acid substitutions. Variations within the sequence compared to SEQ ID NO: 17 can all be within regions of the TCR Vα domain that do not form CDRs (i.e., the variant can have the CDRs of SEQ ID NO: 1, SEQ ID NO: 80, and / or SEQ ID NO: 83, and the variation in the sequence compared to SEQ ID NO: 17 is still 25% (or less)). In other words, the sequences of the CDRs of SEQ ID NO: 17 can be retained even if the remaining sequence varies as necessary within the "at least 75% identity" parameter specified above. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 17).

[0111] By way of example, the encoded TCR Vα domain can 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%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 17, in which case the TCR Vα domain includes a CDR3 having the amino acid sequence of SEQ ID NO: 1. In this example, the CDR1 of the TCR Vα domain can have the amino acid sequence of SEQ ID NO: 80, and the CDR2 of the TCR Vα domain can have the amino acid sequence of SEQ ID NO: 83.

[0112] By way of another example, the encoded TCR Vα domain can have the amino acid sequence of SEQ ID NO: 17 and include an amino acid sequence with 0 to 10 (or 0 to 5) amino acid substitutions, insertions, or deletions, in which case the TCR Vα domain includes a CDR3 having the amino acid sequence of SEQ ID NO: 1. In this example, the CDR1 of the TCR Vα domain can have the amino acid sequence of SEQ ID NO: 80, and the CDR2 of the TCR Vα domain can have the amino acid sequence of SEQ ID NO: 83.

[0113] In the example where the TCR Vα domain has the amino acid sequence of SEQ ID NO: 17, the TCR Vα domain can be encoded by the nucleic acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20, or a degenerate sequence of these genes (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 20 is the codon-optimized form of the nucleic acid sequence for the TCR Vα domain of clone M7 (the non-optimized sequence is SEQ ID NO: 19).

[0114] To avoid misunderstanding, it should be stated that the nucleic acid sequence encoding the TCR Vα domain can also encode the TCRα chain constant domain. Examples of suitable constant domains are encoded within the MP71-TCR-flex retroviral vector. However, the present invention is not limited to this specific constant domain and encompasses any suitable TCRα chain constant domain. The constant domain can be of murine origin, of human origin, or humanized. Methods for identifying or generating suitable constant domains are well known to those skilled in the art and are well within the scope of their defined capabilities.

[0115] For illustrative purposes only, the constant domain can be a lentiviral vector, a retroviral vector, or a plasmid vector, etc., in which a murine constant domain or a human constant domain has been pre-cloned. However, it can also be encoded by or derived from a vector that is also an adenoviral vector, an adeno-associated viral vector, a vaccinia viral vector, a canarypox viral vector, or a herpes viral vector. In recent years, minicircles have also been described for TCR gene transfer (R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics, and M Hudecek, Leukemia, published by 2016, non-viral Sleeping Beauty transposition derived from minicircle vectors). Furthermore, naked (synthetic) DNA / RNA can also be used to introduce TCRs. By way of example, the pMSGV retroviral vector, in which the TCR-Ca gene and the TCR-Cb gene have been pre-cloned, as described by LV Coren et al. in BioTechniques in 2015, can be used to provide a suitable constant domain. Alternatively, single-stranded or double-stranded DNA or RNA can be inserted into the TCR locus by homology-directed repair (see Roth et al., 2018, Nature, volume 559, page 405). As a further option, non-homologous end joining is also possible.

[0116] Examples of specific TCRα chain amino acid sequences that include the TCR Vα domains described herein with appropriate constant domains are shown in SEQ ID NO: 47 and SEQ ID NO: 48. It is noted that the constant domain shown in SEQ ID NO: 48 is a mouse constant domain. Also included are suitable functional variants of SEQ ID NO: 47 and SEQ ID NO: 48 (e.g., variants having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 47 or SEQ ID NO: 48, and in this case, if the variant includes a portion of the binding protein described herein, the amino acid sequence of the variant TCRα chain is HA-1 H

[0117] also retain the ability to specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10). In other words, also included are functional TCRα chains with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 47 or SEQ ID NO: 48. As already stated, the amino acid substitutions can be conservative amino acid substitutions. Variations within the sequence compared to SEQ ID NO: 47 or SEQ ID NO: 48 can all be within regions of the TCRα chain that do not form CDRs (i.e., the variant can have the CDRs of SEQ ID NO: 1, SEQ ID NO: 80, and / or SEQ ID NO: 83, and the variation in the sequence compared to SEQ ID NO: 47 or SEQ ID NO: 48 is still 25% (or less)). In other words, the sequences of the CDRs of SEQ ID NO: 47 or SEQ ID NO: 48 can be retained even if the remaining sequence varies as needed within the "at least 75% identity" parameter specified above. Suitably, the percentage identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 47 or SEQ ID NO: 48, as applicable).By way of example, the TCRα chain to be encoded can 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%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 47 or SEQ ID NO: 48. In this case, the TCRα chain includes a CDR3 having the amino acid sequence of SEQ ID NO: 1. In this example, the CDR1 of the TCRα chain can have the amino acid sequence of SEQ ID NO: 80, and the CDR2 of the TCRα chain can have the amino acid sequence of SEQ ID NO: 83.

[0118] In an example where the TCR α chain has the amino acid sequence of SEQ ID NO: 47, the TCRα chain can be encoded by the nucleic acid sequence of SEQ ID NO: 49 or SEQ ID NO: 50, or a degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 50 is the codon-optimized form of the nucleic acid sequence for the TCR Vα domain of clone M7 (the non-optimized sequence is SEQ ID NO: 49).

[0119] In an example where the TCR α chain has the amino acid sequence of SEQ ID NO: 48, the TCRα chain can be encoded by the nucleic acid sequence of SEQ ID NO: 51, or a degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0120] (ii) A Vα domain comprising the CDR3 amino acid sequence of SEQ ID NO: 2 and functional variants thereof HA-1 H An example of the CDR3 amino acid sequence of a suitable TCR Vα domain that confers specific binding to an antigen is shown in SEQ ID NO: 2. As will be apparent to those skilled in the art, variants of the amino acid sequence shown in SEQ ID NO: 2 can also be functional (i.e., when the CDR3 is part of the TCR Vα domain, HA-1 H retains their ability to confer specific binding to an antigen (e.g., the peptide shown in SEQ ID NO: 10)). Thus, such functional variants are encompassed herein.

[0121] For example, the CDR3 amino acid sequence of a suitable (functional) Vα domain can have at least 80% sequence identity to SEQ ID NO: 2, i.e., they can have at least 80%, at least 83%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 2. Suitably, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 2). In other words, the CDR3 amino acid sequence of a suitable (functional) Vα domain can vary from the sequence shown in SEQ ID NO: 2 by 1 amino acid or several amino acids (e.g., 2 amino acids). As stated above, functional variants of SEQ ID NO: 2 retain their ability to confer specific binding to an HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10) when the CDR3 is part of the TCR Vα domain.

[0122] Functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 2. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of 1, 2, or more amino acids of SEQ ID NO: 2, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the CDR3.

[0123] Non-functional variants are amino acid sequence variants of SEQ ID NO: 2 that do not specifically bind to an HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 2, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0124] In one example, the CDR3 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO: 2. In an example where the CDR3 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 2, the CDR3 can be encoded by the nucleic acid sequence of SEQ ID NO: 23 or SEQ ID NO: 24, or a degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 24 is the codon-optimized form of the nucleic acid sequence for the CDR3 of clone M2 (the non-optimized sequence is SEQ ID NO: 23).

[0125] The encoded TCR Vα domain, in addition to the designated CDR3, may include the amino acid sequence of SEQ ID NO: 8, or a functional variant thereof (i.e., in this case, the variant retains the ability to specifically bind to the N-terminus of an HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10)) and may include CDR1. Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 8. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 8, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0126] Non-functional variants are amino acid sequence variants of SEQ ID NO: 8 that do not specifically bind to the N-terminus of an HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 8, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0127] For example, the CDR1 amino acid sequence of a suitable (functional) Vα domain can have at least 80% sequence identity to SEQ ID NO:8, i.e., this can have at least 80%, at least 83%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:8. Suitably, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO:8). In other words, the CDR1 amino acid sequence of a suitable (functional) Vα domain can vary by 1 amino acid or several amino acids from the sequence shown in SEQ ID NO:8. As already stated, the variant can include amino acid substitutions such as conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:8. As stated above, a functional variant of SEQ ID NO:8, when the CDR1 is part of the TCR Vα domain, is HA-1 H retains the ability to specifically bind to the N-terminus of an antigen (e.g., the peptide shown in SEQ ID NO:10).

[0128] In one example, the CDR1 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO:8. In an example where the CDR1 of the TCR Vα domain has the amino acid sequence of SEQ ID NO:8, the CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:27, or a degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO:27 is the codon-optimized form of the nucleic acid sequence for the CDR1 of clone M2 (the non-optimized sequence is SEQ ID NO:9).

[0129] Other suitable CDR1 sequences of the CDR1 Vα domain, such as those containing the sequence shown in SEQ ID NO: 80, are described elsewhere in this specification. Thus, it will be apparent to those skilled in the art that references to SEQ ID NO: 8 above can be replaced by references to SEQ ID NO: 80 (and the corresponding nucleotide sequences of SEQ ID NO: 81 and SEQ ID NO: 82) when discussing permutations of the Vα CDR1 amino acid and nucleotide sequences for combination with the CDR3 sequence of SEQ ID NO: 2 (or the corresponding nucleotide sequence of SEQ ID NO: 23 or SEQ ID NO: 24).

[0130] The encoded TCR Vα domain may also include, in addition to the designated CDR3 (and optionally the CDR1 designated above), the amino acid sequence of SEQ ID NO: 28 or a functional variant thereof (i.e., in this case, a variant that retains the ability to specifically bind to HLA-A * 02:01). Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 28. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 28, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0131] Non-functional variants are amino acid sequence variants of SEQ ID NO: 28 that do not specifically bind to HLA-A * 02:01. Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 28, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0132] For example, the CDR2 amino acid sequence of a suitable (functional) Vα domain can have at least 80% sequence identity to SEQ ID NO: 28, i.e., this can have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28. Suitably, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 28). In other words, the CDR2 amino acid sequence of a suitable (functional) Vα domain can vary by 1 amino acid or several amino acids from the sequence shown in SEQ ID NO: 28. As already stated, the variant can include amino acid substitutions such as conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 28. As stated above, the functional variant of SEQ ID NO: 28 retains the ability to specifically bind to HLA-A * retains the ability to specifically bind to 02:01.

[0133] In one example, the CDR2 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO: 28. In the example where the CDR2 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 28, the CDR2 can be encoded by the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40, or their degenerate gene sequences (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 40 is the codon-optimized form of the nucleic acid sequence for the CDR2 of clone M2 (the non-optimized sequence is SEQ ID NO: 39).

[0134] For other suitable CDR2 sequences of the CDR2 Vα domain, e.g., the CDR2 sequence containing the sequence shown in SEQ ID NO: 83, they are described elsewhere in this specification. Thus, it will be apparent to those skilled in the art that when discussing permutations of the Vα CDR2 amino acid sequence and nucleotide sequence for combination with the CDR3 sequence of SEQ ID NO: 28 (or the corresponding nucleotide sequences of SEQ ID NO: 2 or SEQ ID NO: 23 or SEQ ID NO: 24), the reference to SEQ ID NO: 28 can be replaced by a reference to SEQ ID NO: 83 (and the corresponding nucleotide sequences of SEQ ID NO: 84 and SEQ ID NO: 85).

[0135] Thus, the encoded TCR Vα domain can include the CDRs (specifically, in terms of SEQ ID NOs., i.e., SEQ ID NO: 2, SEQ ID NO: 8 (or SEQ ID NO: 80), and SEQ ID NO: 28 (or SEQ ID NO: 83), or functional variants thereof) mentioned in detail above, with appropriate intervening sequences between the CDRs.

[0136] The encoded TCR Vα domain can include the amino acid sequence of SEQ ID NO: 29, or a functional variant thereof (i.e., in this case, if it includes a part of the binding protein described herein, the variant TCR Vα domain retains the ability to specifically bind to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10)). Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 29. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 29, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0137] Non-functional variants are HA-1 HIt is an amino acid sequence variant of SEQ ID NO: 29 that does not specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 29, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0138] In one example, the encoded TCR Vα domain is HA-1 H While retaining the ability to specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10), it 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 to the amino acid sequence of SEQ ID NO: 29. In other words, functional TCR Vα domains with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 29 are also encompassed. As already stated, the amino acid substitutions can be conservative amino acid substitutions. Variations within the sequence compared to SEQ ID NO: 29 can all be within regions of the TCR Vα domain that do not form CDRs (i.e., the variant can have the CDRs of SEQ ID NO: 2, SEQ ID NO: 8, and / or SEQ ID NO: 28, and the variation in the sequence compared to SEQ ID NO: 29 is still 25% (or less)). In other words, the sequence of the CDRs of SEQ ID NO: 29 can be retained even if the remaining sequence varies as needed within the "at least 75% identity" parameter specified above. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 29).

[0139] By way of example, the encoded TCR Vα domain can 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%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 29, in which case the TCR Vα domain includes a CDR3 having the amino acid sequence of SEQ ID NO: 2. In this example, the CDR1 of the TCR Vα domain can have the amino acid sequence of SEQ ID NO: 8, and the CDR2 of the TCR Vα domain can have the amino acid sequence of SEQ ID NO: 28.

[0140] By way of another example, the encoded TCR Vα domain can have the amino acid sequence of SEQ ID NO: 29 and include an amino acid sequence with 0 to 10 (or 0 to 5) amino acid substitutions, insertions, or deletions, in which case the TCR Vα domain includes a CDR3 having the amino acid sequence of SEQ ID NO: 2. In this example, the CDR1 of the TCR Vα domain can have the amino acid sequence of SEQ ID NO: 8, and the CDR2 of the TCR Vα domain can have the amino acid sequence of SEQ ID NO: 28.

[0141] In the example where the TCR Vα domain has the amino acid sequence of SEQ ID NO: 29, the TCR Vα domain can be encoded by the nucleic acid sequence of SEQ ID NO: 31 or SEQ ID NO: 32, or a degenerate sequence of these genes (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 32 is the codon-optimized form of the nucleic acid sequence for the TCR Vα domain of clone M2 (the non-optimized sequence is SEQ ID NO: 31).

[0142] To clarify to avoid misunderstanding, the nucleic acid sequence encoding the TCR Vα domain can also encode the TCRα chain constant domain. Examples of suitable constant domains are encoded within the MP71-TCR-flex retroviral vector. However, the invention is not limited to this specific constant domain and encompasses any suitable TCRα chain constant domain. The constant domain can be of murine origin, of human origin, or humanized. Methods for identifying or creating suitable constant domains are well known to those skilled in the art and are well within the scope of their regulatory capabilities.

[0143] For illustrative purposes only, the constant domain can be a lentiviral vector, a retroviral vector, or a plasmid vector, etc., in which a murine constant domain or a human constant domain has been pre-cloned, but can also be encoded by or derived from a vector that is also an adenoviral vector, an adeno-associated viral vector, a vaccinia viral vector, a canarypox viral vector, or a herpes viral vector. In recent years, minicircles have also been described for TCR gene transfer (R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics, and M Hudecek, Leukemia, published by 2016, non-viral Sleeping Beauty transposition derived from minicircle vectors). Furthermore, naked (synthetic) DNA / RNA can also be used to introduce TCRs. For example, the pMSGV retroviral vector, which has the TCR-Ca gene and the TCR-Cb gene pre-cloned as described by LV Coren et al. in BioTechniques in 2015, can be used to provide a suitable constant domain. Alternatively, single-stranded or double-stranded DNA or RNA can be inserted into the TCR locus by homology-directed repair (see Roth et al., 2018, Nature, volume 559, page 405). As a further option, non-homologous end joining is also possible.

[0144] Examples of specific TCRα chain amino acid sequences that include the TCR Vα domains described herein with appropriate constant domains are shown in SEQ ID NO: 57 and SEQ ID NO: 58. It is noted that the constant domain shown in SEQ ID NO: 58 is a mouse constant domain. Functional variants of SEQ ID NO: 57 and SEQ ID NO: 58 (e.g., variants having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 58, and in this case, if it includes a portion of the binding protein described herein, the amino acid sequence of the variant TCRα chain is HA-1 H also encompassed are those that retain the ability to specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10). In other words, functional TCRα chains with one or several amino acid substitutions as compared to the sequence of SEQ ID NO: 57 or SEQ ID NO: 58 are also encompassed. As already stated, the amino acid substitutions can be conservative amino acid substitutions. Variations within the sequence as compared to SEQ ID NO: 57 or SEQ ID NO: 58 can all be within regions of the TCRα chain that do not form CDRs (i.e., the variant can have the CDRs of SEQ ID NO: 2, SEQ ID NO: 8, and / or SEQ ID NO: 28, and the variation in the sequence as compared to SEQ ID NO: 57 or SEQ ID NO: 58 is still 25% (or less)). In other words, the sequences of the CDRs of SEQ ID NO: 57 or SEQ ID NO: 58 can be retained even if the remaining sequence varies as needed within the "at least 75% identity" parameter specified above. Appropriately, the percent identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 57 or SEQ ID NO: 58, as applicable).

[0145] By way of example, the TCRα chain to be encoded can 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%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 57 or SEQ ID NO: 58. In this case, the TCRα chain includes a CDR3 having the amino acid sequence of SEQ ID NO: 2. In this example, the CDR1 of the TCRα chain can have the amino acid sequence of SEQ ID NO: 8, and the CDR2 of the TCRα chain can have the amino acid sequence of SEQ ID NO: 28.

[0146] In an example where the TCR α chain has the amino acid sequence of SEQ ID NO: 57, the TCRα chain can be encoded by the nucleic acid sequence of SEQ ID NO: 59 or SEQ ID NO: 60, or a degenerate sequence of these genes (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 60 is the codon-optimized form of the nucleic acid sequence for the TCR Vα domain of clone M2 (the non-optimized sequence is SEQ ID NO: 59).

[0147] In an example where the TCR α chain has the amino acid sequence of SEQ ID NO: 58, the TCRα chain can be encoded by the nucleic acid sequence of SEQ ID NO: 61, or a degenerate sequence of this gene (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0148] (iii) A Vα domain comprising the CDR3 amino acid sequence of SEQ ID NO: 3 and functional variants thereof HA-1 H An example of the CDR3 amino acid sequence of a suitable TCR Vα domain that confers specific binding to an antigen is shown in SEQ ID NO: 3. As will be apparent to those skilled in the art, variants of the amino acid sequence shown in SEQ ID NO: 3 can also be functional (i.e., when the CDR3 is part of the TCR Vα domain, HA-1 H retains their ability to confer specific binding to an antigen (e.g., the peptide shown in SEQ ID NO: 10)). Accordingly, such functional variants are encompassed herein.

[0149] For example, the CDR3 amino acid sequence of a suitable (functional) Vα domain can have at least 80% sequence identity to SEQ ID NO: 3, i.e., they can have at least 80%, at least 83%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 3. Suitably, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 3). In other words, the CDR3 amino acid sequence of a suitable (functional) Vα domain can vary from the sequence shown in SEQ ID NO: 3 by 1 amino acid or several amino acids (e.g., 2 amino acids). As stated above, functional variants of SEQ ID NO: 3 retain their ability to confer specific binding to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10) when the CDR3 is part of the TCR Vα domain.

[0150] Functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 3. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of 1, 2, or more amino acids of SEQ ID NO: 3, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the CDR3.

[0151] Non-functional variants are amino acid sequence variants of SEQ ID NO: 3 that do not specifically bind to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 3, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0152] In one example, the CDR3 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO: 3. In an example where the CDR3 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 3, the CDR3 can be encoded by the nucleic acid sequence of SEQ ID NO: 35 or SEQ ID NO: 36, or a degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 36 is the codon-optimized form of the nucleic acid sequence for the CDR3 of clone FK47.83 (the non-optimized sequence is SEQ ID NO: 35).

[0153] The encoded TCR Vα domain, in addition to the specified CDR3, can include the amino acid sequence of SEQ ID NO: 8, or a functional variant thereof (i.e., in this case, a variant that retains the ability to specifically bind to the N-terminus of the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10)) CDR1. Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 8. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 8, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0154] A non-functional variant is an amino acid sequence variant of SEQ ID NO: 8 that does not specifically bind to the N-terminus of the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 8, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0155] For example, the CDR1 amino acid sequence of a suitable (functional) Vα domain can have at least 80% sequence identity to SEQ ID NO: 8, i.e., it can have at least 80%, at least 83%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 8. Suitably, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 8). In other words, the CDR1 amino acid sequence of a suitable (functional) Vα domain can vary by 1 amino acid or several amino acids from the sequence shown in SEQ ID NO: 8. As already stated, the variant can include amino acid substitutions such as conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 8. As stated above, a functional variant of SEQ ID NO: 8, when the CDR1 is part of the TCR Vα domain, retains the ability to specifically bind to the N-terminus of an antigen (e.g., the peptide shown in SEQ ID NO: 10). H Retains the ability to specifically bind to the N-terminus of an antigen (e.g., the peptide shown in SEQ ID NO: 10).

[0156] In one example, the CDR1 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO: 8. In an example where the CDR1 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 8, the CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO: 9 or SEQ ID NO: 27, or a degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 27 is the codon-optimized form of the nucleic acid sequence for the CDR1 of clone FK47.83 (the non-optimized sequence is SEQ ID NO: 9).

[0157] For other suitable CDR1 sequences of the CDR1 Vα domain, such as those containing the sequence shown in SEQ ID NO: 80, they are described elsewhere in this specification. Thus, it will be apparent to those skilled in the art that the reference to SEQ ID NO: 8 above can be replaced by a reference to SEQ ID NO: 80 (and the corresponding nucleotide sequences of SEQ ID NO: 81 and SEQ ID NO: 82) when discussing permutations of the Vα CDR1 amino acid and nucleotide sequences for combination with the CDR3 sequence of SEQ ID NO: 3 (or the corresponding nucleotide sequences of SEQ ID NO: 35 or SEQ ID NO: 36).

[0158] The encoded TCR Vα domain may also include, in addition to the specified CDR3 (and optionally the CDR1 specified above), the amino acid sequence of SEQ ID NO: 28 or a functional variant thereof (i.e., in this case, the variant retains the ability to specifically bind to HLA-A * 02:01). Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 28. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 28, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0159] Non-functional variants are amino acid sequence variants of SEQ ID NO: 28 that do not specifically bind to HLA-A * 02:01. Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 28, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0160] For example, the CDR2 amino acid sequence of a suitable (functional) Vα domain can have at least 80% sequence identity to SEQ ID NO: 28, i.e., it can have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 28. Suitably, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 28). In other words, the CDR2 amino acid sequence of a suitable (functional) Vα domain can vary by 1 amino acid or several amino acids from the sequence shown in SEQ ID NO: 28. As already stated, the variant can include amino acid substitutions such as conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 28. As stated above, the functional variant of SEQ ID NO: 28 retains the ability to specifically bind to HLA-A * 02:01.

[0161] In one example, the CDR2 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO: 28. In the example where the CDR2 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 28, the CDR2 can be encoded by the nucleic acid sequence of SEQ ID NO: 39 or SEQ ID NO: 40, or a degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 40 is the codon-optimized form of the nucleic acid sequence for the CDR2 of clone FK47.83 (the non-optimized sequence is SEQ ID NO: 39).

[0162] For other suitable CDR2 sequences of the CDR2 Vα domain, such as the CDR2 sequence containing the sequence shown in SEQ ID NO: 83, they are described elsewhere in this specification. Therefore, it will be apparent to those skilled in the art that the reference to SEQ ID NO: 28 above can be replaced by a reference to SEQ ID NO: 83 (and the corresponding nucleotide sequences of SEQ ID NO: 84 and SEQ ID NO: 85) when discussing permutations of the Vα CDR2 amino acid and nucleotide sequences for combination with the CDR3 sequence of SEQ ID NO: 3 (or the corresponding nucleotide sequences of SEQ ID NO: 35 or SEQ ID NO: 36).

[0163] Therefore, the encoded TCR Vα domain can include the CDRs (specifically, in terms of SEQ ID NOs., i.e., SEQ ID NO: 3, SEQ ID NO: 8 (or SEQ ID NO: 80), and SEQ ID NO: 28 (or SEQ ID NO: 83), or functional variants thereof) mentioned in detail above, with appropriate intervening sequences between the CDRs.

[0164] The encoded TCR Vα domain can include the amino acid sequence of SEQ ID NO: 41, or a functional variant thereof (i.e., in this case, if it includes a part of the binding protein described in this specification, the variant TCR Vα domain H retains the ability to specifically bind to the HA-1 antigen (e.g., the peptide shown in SEQ ID NO: 10)). Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 41. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 41, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0165] Non-functional variants are HA-1 HIt is an amino acid sequence variant of SEQ ID NO: 41 that does not specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 41, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0166] In one example, the encoded TCR Vα domain is HA-1 H While retaining the ability to specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10), it 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 to the amino acid sequence of SEQ ID NO: 41. In other words, functional TCR Vα domains with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 41 are also encompassed. As already stated, the amino acid substitutions can be conservative amino acid substitutions. Variations within the sequence compared to SEQ ID NO: 41 can all be within regions of the TCR Vα domain that do not form CDRs (i.e., the variant can have the CDRs of SEQ ID NO: 3, SEQ ID NO: 8, and / or SEQ ID NO: 28, and the variation in the sequence compared to SEQ ID NO: 41 is still 25% (or less)). In other words, the sequence of the CDRs of SEQ ID NO: 41 can be retained even if the remaining sequence varies as required within the "at least 75% identity" parameter specified above. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 41).

[0167] By way of example, the encoded TCR Vα domain can comprise 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%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 41, in which case the TCR Vα domain comprises a CDR3 having the amino acid sequence of SEQ ID NO: 3. In this example, the CDR1 of the TCR Vα domain can have the amino acid sequence of SEQ ID NO: 8, and the CDR2 of the TCR Vα domain can have the amino acid sequence of SEQ ID NO: 28.

[0168] By way of another example, the encoded TCR Vα domain can have the amino acid sequence of SEQ ID NO: 41 and can comprise an amino acid sequence with 0 to 10 (or 0 to 5) amino acid substitutions, insertions, or deletions, in which case the TCR Vα domain comprises a CDR3 having the amino acid sequence of SEQ ID NO: 3. In this example, the CDR1 of the TCR Vα domain can have the amino acid sequence of SEQ ID NO: 8, and the CDR2 of the TCR Vα domain can have the amino acid sequence of SEQ ID NO: 28.

[0169] In the example where the TCR Vα domain has the amino acid sequence of SEQ ID NO: 41, the TCR Vα domain can be encoded by the nucleic acid sequence of SEQ ID NO: 43 or SEQ ID NO: 44, or a degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 44 is the codon-optimized form of the nucleic acid sequence for the TCR Vα domain of clone FK47.83 (the non-optimized sequence is SEQ ID NO: 43).

[0170] To avoid misunderstanding, it should be noted that the nucleic acid sequence encoding the TCR Vα domain may also encode the TCRα chain constant domain. Examples of suitable constant domains are encoded within the MP71-TCR-flex retroviral vector. However, the present invention is not limited to this specific constant domain and encompasses any suitable TCRα chain constant domain. The constant domain may be of murine origin, of human origin, or humanized. Methods for identifying or creating suitable constant domains are well known to those skilled in the art and are well within the scope of their regulatory capabilities.

[0171] For illustrative purposes only, the constant domain may be a lentiviral vector, a retroviral vector, or a plasmid vector, etc., in which a murine constant domain or a human constant domain has been pre-cloned, but may also be encoded by or derived from a vector that is also an adenoviral vector, an adeno-associated viral vector, a vaccinia viral vector, a canarypox viral vector, or a herpes viral vector. In recent years, minicircles have also been described for TCR gene transfer (R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics, and M Hudecek, Leukemia, published in 2016, non-viral Sleeping Beauty transposition derived from minicircle vectors). Furthermore, naked (synthetic) DNA / RNA may also be used to introduce TCRs. By way of example, the pMSGV retroviral vector, pre-cloned with the TCR-Ca gene and the TCR-Cb gene, described by LV Coren et al. in BioTechniques in 2015, may be used to provide a suitable constant domain. Alternatively, single-stranded or double-stranded DNA or RNA may be inserted into the TCR locus by homology-directed repair (see Roth et al., 2018, Nature, Vol. 559, p. 405). As a further option, non-homologous end joining is also possible.

[0172] Examples of specific TCRα chain amino acid sequences that include the TCR Vα domains described herein with appropriate constant domains are shown in SEQ ID NO: 67 and SEQ ID NO: 68. It is noted that the constant domain shown in SEQ ID NO: 68 is a mouse constant domain. Appropriate functional variants of SEQ ID NO: 67 and SEQ ID NO: 68 (e.g., variants having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 67 or SEQ ID NO: 68, and in this case, if including a part of the binding protein described herein, the amino acid sequence of the variant TCRα chain is HA-1 H also encompassed are those that retain the ability to specifically bind an antigen (e.g., the peptide shown in SEQ ID NO: 10). In other words, functional TCRα chains with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 67 or SEQ ID NO: 68 are also encompassed. As already stated, the amino acid substitutions can be conservative amino acid substitutions. Variations within the sequence compared to SEQ ID NO: 67 or SEQ ID NO: 68 can all be within regions of the TCRα chain that do not form CDRs (i.e., the variant can have the CDRs of SEQ ID NO: 3, SEQ ID NO: 8, and / or SEQ ID NO: 28, and the variation in the sequence compared to SEQ ID NO: 67 or SEQ ID NO: 68 is still 25% (or less)). In other words, the sequences of the CDRs of SEQ ID NO: 67 or SEQ ID NO: 68 can be retained even if the remaining sequence varies as needed within the "at least 75% identity" parameter specified above. Appropriately, the percent identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 67 or SEQ ID NO: 68, as applicable).

[0173] By way of example, the TCRα chain to be encoded can 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%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 67 or SEQ ID NO: 68. In this case, the TCRα chain includes a CDR3 having the amino acid sequence of SEQ ID NO: 3. In this example, the CDR1 of the TCRα chain can have the amino acid sequence of SEQ ID NO: 8, and the CDR2 of the TCRα chain can have the amino acid sequence of SEQ ID NO: 28.

[0174] In an example where the TCR α chain has the amino acid sequence of SEQ ID NO: 67, the TCRα chain can be encoded by the nucleic acid sequence of SEQ ID NO: 69 or SEQ ID NO: 70, or these degenerate gene sequences (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 70 is the codon-optimized form of the nucleic acid sequence for the TCR Vα domain of clone FK47.83 (the non-optimized sequence is SEQ ID NO: 69).

[0175] In an example where the TCR α chain has the amino acid sequence of SEQ ID NO: 68, the TCRα chain can be encoded by the nucleic acid sequence of SEQ ID NO: 71, or this degenerate gene sequence (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0176] Components of the TCRβ chain variable (Vβ) domain The isolated nucleic acid composition described herein encodes a HA-1 H antigen-specific binding protein. The encoded HA-1 H antigen-specific binding protein includes a TCR Vα domain that includes a CDR3 amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1-3 described above. The encoded HA-1 HThe antigen-specific binding protein also includes a TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, wherein the Vβ domain includes a CDR3 amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 4 to 6 and a CDR1 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 7.

[0177] In humans in vivo, the amino acid sequence of the TCR Vβ chain is generated by V(D)J recombination that rearranges available V (variable) gene segments, J (joining) gene segments, and, optionally, D (diversity) gene segments. This generates a new repertoire of nucleic acid sequences encoding unique TCR Vβ chains with significantly different antigen recognition properties. An example of a human V gene segment is the TRBV7-9 gene (T cell receptor beta variable 7-9 gene; UniprotKB unique identifier: P04435). TRBV7-9 has a number of known alleles with highly conserved nucleotide sequences (e.g., TRBV7-9 * 01, TRBV7-9 * 02, TRBV7-9 * 03, TRBV7-9 * 04, TRBV7-9 * 05, TRBV7-9 * 06, and TRBV7-9 * 07 (see Lefranc, M.-P. and Lefranc, G., "The T cell receptor Facts Book", Academic Press, London, UK (2001)).

[0178] In the present specification, the inventors have confirmed that the HA-1 H antigen-specific binding protein possesses a TCRβ chain with a similar Vβ domain. Each HA-1 described in the following examples HWithin the antigen-specific binding protein, the Vβ domain included the amino acid sequence encoded by the TRBV7-9 gene. This sequence has been shown to contribute to the peptide binding specificity of the binding proteins described herein.

[0179] Thus, the encoded HA-1 described herein H The antigen-specific binding protein includes a TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene. The TRBV7-9 gene can be any allele of TRBV7-9, for example, TRBV7-9 * 01, TRBV7-9 * 02, TRBV7-9 * 03, TRBV7-9 * 04, TRBV7-9 * 05, TRBV7-9 * 06, and TRBV7-9 * 07. In one specific example, the TRBV7-9 gene is TRBV7-9 * 01 or TRBV7-9 * 03.

[0180] As described above, the described binding protein includes a TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene. In addition to this, the TCR Vβ domain of the binding protein described herein includes the amino acid sequence of CDR3 (described directly below) having at least 80% sequence identity to any one of SEQ ID NOs: 4-6. To avoid misunderstanding, any of the CDR3 sequences of the TCR Vβ domain described below can then be combined with any of the CDR1 sequences of the TCR Vβ domain described to create a functional TCR Vβ domain.

[0181] (i) A Vβ domain containing the CDR3 amino acid sequence of SEQ ID NO: 4 and functional variants thereof HA-1 HExamples of CDR3 amino acid sequences of suitable TCR Vβ domains that confer specific binding to an antigen are shown in SEQ ID NO: 4. As will be apparent to those skilled in the art, variants of the amino acid sequence shown in SEQ ID NO: 4 may also be functional (i.e., when the CDR3 is part of the TCR Vβ domain, HA-1 H retain their ability to confer specific binding to an antigen (e.g., the peptide shown in SEQ ID NO: 10)). Accordingly, such functional variants are encompassed herein.

[0182] For example, the CDR3 amino acid sequences of suitable (functional) Vβ domains can have at least 80% sequence identity to SEQ ID NO: 4, i.e., they can have at least 80%, at least 84%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 4. Suitably, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 4). In other words, the CDR3 amino acid sequences of suitable (functional) Vβ domains can vary from the sequence shown in SEQ ID NO: 4 by 1 amino acid or several amino acids (e.g., 2 amino acids). As stated above, functional variants of SEQ ID NO: 4 retain their ability to confer specific binding to an HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10) when the CDR3 is part of the TCR Vβ domain.

[0183] Functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 4. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 4, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the CDR3.

[0184] Non-functional variants are HA-1 HAn amino acid sequence variant of SEQ ID NO: 4 that does not specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 4, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions. Those skilled in the art are familiar with methods for identifying functional and non-functional variants.

[0185] In one example, the CDR3 of the Vβ domain comprises or consists of the amino acid sequence of SEQ ID NO: 4. In examples where the CDR3 of the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 4, the CDR3 may be encoded by the nucleic acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14, or genetically degenerate sequences thereof (i.e., other nucleic acid sequences that, as a result of the degeneracy of the genetic code, encode the same protein). It is noted that SEQ ID NO: 14 is a codon-optimized version of the nucleic acid sequence for the CDR3 of clone M7 (the non-optimized sequence is SEQ ID NO: 13).

[0186] The encoded TCR Vβ domain comprises, in addition to the designated CDR3, the amino acid sequence of SEQ ID NO:7, or a functional variant thereof (i.e., in this case the variant is H The functional variants may comprise a CDR1 comprising a CDR2 sequence that retains the ability to specifically bind to the C-terminus of an antigen (e.g., a peptide as shown in SEQ ID NO: 10). Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 7. The term "variant" also encompasses homologues. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 7, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0187] The non-functional mutant is HA-1 HIt is an amino acid sequence variant of SEQ ID NO: 7 that does not specifically bind to the C-terminus of an antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 7, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0188] For example, the CDR1 amino acid sequence of a suitable (functional) Vβ domain can have at least 80% sequence identity to SEQ ID NO: 7, i.e., it can have at least 80%, at least 83%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7. Appropriately, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 7). In other words, the CDR1 amino acid sequence of a suitable (functional) Vβ domain can vary by 1 amino acid or several amino acids from the sequence shown in SEQ ID NO: 7. As already stated, variants can include amino acid substitutions such as conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 7. As stated above, a functional variant of SEQ ID NO: 7, when the CDR1 is part of the TCR Vβ domain, is HA-1 H retains the ability to specifically bind to the C-terminus of an antigen (e.g., the peptide shown in SEQ ID NO: 10).

[0189] In one example, the CDR1 of the Vβ domain comprises or consists of the amino acid sequence of SEQ ID NO: 7. In an example where the CDR1 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 7, the CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16, or a degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 16 is the codon-optimized form of the nucleic acid sequence for the CDR1 of clone M7 (the non-optimized sequence is SEQ ID NO: 15).

[0190] The encoded TCR Vβ domain can also include a CDR2 having the amino acid sequence of SEQ ID NO: 86 or a functional variant thereof (i.e., in this case, a variant that retains the ability to specifically bind to HLA-A * 02:01) in addition to the specified CDR3 (and optionally, the CDR1 specified above). Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 86. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 86, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0191] Non-functional variants are amino acid sequence variants of SEQ ID NO: 86 that do not specifically bind to HLA-A * 02:01. Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 86, or premature truncations, or substitutions, insertions, or deletions in essential amino acids or essential regions. Methods for identifying functional and non-functional variants are well known to those of skill in the art.

[0192] For example, the CDR2 amino acid sequence of a suitable (functional) Vβ domain can have at least 80% sequence identity to SEQ ID NO: 86, i.e., it can have at least 80%, at least 83%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 86. Suitably, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 86). In other words, the CDR2 amino acid sequence of a suitable (functional) Vβ domain can vary by 1 amino acid or several amino acids from the sequence shown in SEQ ID NO: 86. As already stated, the variant can include amino acid substitutions such as conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 86. As stated above, the functional variant of SEQ ID NO: 86 retains the ability to specifically bind to HLA-A * 02:01.

[0193] In one example, the CDR2 of the Vβ domain comprises or consists of the amino acid sequence of SEQ ID NO: 86. In the example where the CDR2 of the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 86, the CDR2 can be encoded by the nucleic acid sequence of SEQ ID NO: 87 or SEQ ID NO: 88, or their degenerate gene sequences (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 88 is the codon-optimized form of the nucleic acid sequence for the CDR2 of clone M7 (the non-optimized sequence is SEQ ID NO: 87).

[0194] Accordingly, the encoded TCR Vβ domain can include the CDRs (specifically, in terms of SEQ ID NOs, i.e., SEQ ID NO: 4, SEQ ID NO: 7, and SEQ ID NO: 86, or functional variants thereof) with suitable intervening sequences between the CDRs as detailed above.

[0195] The encoded TCR Vβ domain has the amino acid sequence of SEQ ID NO: 18, or a functional variant thereof (i.e., in this case, if it comprises a part of the binding protein described herein, the variant TCR Vβ domain is HA-1 H and may have the ability to specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10). Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 18. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 18, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0196] Non-functional variants are amino acid sequence variants of SEQ ID NO: 18 that do not specifically bind to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 18, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0197] In one example, the encoded TCR Vβ domain is HA-1 HWhile retaining the ability to specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10), it may have an amino acid sequence having 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: 18. In other words, functional TCR Vβ domains with one or several amino acid substitutions, compared to the sequence of SEQ ID NO: 18, are also encompassed. As already stated, the amino acid substitutions can be conservative amino acid substitutions. Variations within the sequence compared to SEQ ID NO: 18 can all be within regions of the TCR Vβ domain that do not form CDRs (i.e., the variant can have the CDRs of SEQ ID NO: 4, and SEQ ID NO: 7, and optionally SEQ ID NO: 86, and the variation in the sequence compared to SEQ ID NO: 18 is still 25% or less). In other words, the sequences of the CDRs of SEQ ID NO: 18 can be retained even if the remaining sequence varies as needed within the "at least 75% identity" parameter specified above. Appropriately, the percent identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 18).

[0198] By way of example, the encoded TCR Vβ domain can 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%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 18, in which case the TCR Vβ domain includes a CDR3 having the amino acid sequence of SEQ ID NO: 4. In this example, the CDR1 of the TCR Vβ domain can have the amino acid sequence of SEQ ID NO: 7, and the CDR2 of the TCR Vβ domain can have the amino acid sequence of SEQ ID NO: 86.

[0199] In the example where the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 18, the TCR Vβ domain can be encoded by the nucleic acid sequence of SEQ ID NO: 21 or SEQ ID NO: 22, or their degenerate gene sequences (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 22 is the codon-optimized form of the nucleic acid sequence for the TCR Vβ domain of clone M7 (the non-optimized sequence is SEQ ID NO: 21).

[0200] To avoid misunderstanding, the nucleic acid sequence encoding the TCR Vβ domain can also encode the TCRβ chain constant domain. Examples of suitable constant domains are encoded within the MP71-TCR-flex retroviral vector. However, the present invention is not limited to this specific constant domain and encompasses any suitable TCRβ chain constant domain. The constant domain may be of mouse origin, of human origin, or humanized. Methods for identifying or creating suitable constant domains are well known to those skilled in the art and are well within the scope of their defined capabilities.

[0201] For the purpose of illustration only, the constant domain can be a lentiviral vector, a retroviral vector, or a plasmid vector, etc., in which a murine constant domain or a human constant domain has been pre-cloned. However, it can also be encoded by or derived from a vector that is also an adenoviral vector, an adeno-associated viral vector, a vaccinia viral vector, a canarypox viral vector, or a herpes viral vector. In recent years, minicircles have also been described for TCR gene transfer (R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics, and M Hudecek, Leukemia, published in 2016, non-viral Sleeping Beauty transposition derived from minicircle vectors). Furthermore, naked (synthetic) DNA / RNA can also be used to introduce TCRs. For illustration, the pMSGV retroviral vector pre-cloned with the TCR-Ca gene and the TCR-Cb gene, described by LV Coren et al. in BioTechniques in 2015, can be used to provide an appropriate constant domain.

[0202] Examples of specific TCRβ chain amino acid sequences containing the TCR Vβ domain described herein and an appropriate constant domain are shown in SEQ ID NO: 52 and SEQ ID NO: 53. It is noted that the constant domain shown in SEQ ID NO: 53 is a murine constant domain. Appropriate functional variants of SEQ ID NO: 52 and SEQ ID NO: 53 (for example, variants having at least 75% (for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 53), and in this case, if it contains a part of the binding protein described herein, the amino acid sequence of the variant TCRβ chain is HA-1 HAlso included are those that retain the ability to specifically bind to an antigen (e.g., the peptide set forth in SEQ ID NO: 10). In other words, also included are functional TCRβ chains with substitutions of one or several amino acids as compared to the sequences of SEQ ID NO: 52 or SEQ ID NO: 53. As already stated, the amino acid substitutions can be conservative amino acid substitutions. All variations within the sequence as compared to SEQ ID NO: 52 or SEQ ID NO: 53 can be within regions of the TCRβ chain that do not form CDRs (i.e., the variant can have the CDRs of SEQ ID NO: 4, and SEQ ID NO: 7, and optionally SEQ ID NO: 86, and the variation of the sequence as compared to SEQ ID NO: 52 or SEQ ID NO: 53 is still 25% (or less)). In other words, the sequences of the CDRs of SEQ ID NO: 52 or SEQ ID NO: 53 can be retained even if the remaining sequences vary as needed within the "at least 75% identity" parameter specified above. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 52 or SEQ ID NO: 53 when applicable).

[0203] By way of example, the encoded TCRβ chain can 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%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 52 or SEQ ID NO: 53, in which case the TCRβ chain includes a CDR3 having the amino acid sequence of SEQ ID NO: 4. In this example, the CDR1 of the TCRβ chain can have the amino acid sequence of SEQ ID NO: 7, and the CDR2 of the TCRβ chain can have the amino acid sequence of SEQ ID NO: 86.

[0204] In the example where the TCR β chain has the amino acid sequence of SEQ ID NO: 52, the TCRβ chain can be encoded by the nucleic acid sequence of SEQ ID NO: 54 or SEQ ID NO: 55, or a degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 55 is the codon-optimized form of the nucleic acid sequence for the TCR Vβ domain of clone M7 (the non-optimized sequence is SEQ ID NO: 54).

[0205] In the example where the TCR β chain has the amino acid sequence of SEQ ID NO: 53, the TCRβ chain can be encoded by the nucleic acid sequence of SEQ ID NO: 56, or this degenerate gene sequence (i.e., other nucleic acid sequences encoding the same protein as a result of the degeneracy of the genetic code).

[0206] In certain examples, the nucleic acid compositions described herein comprise a TCR Vα domain with a CDR3 amino acid sequence that comprises or consists of the amino acid sequence of SEQ ID NO: 1; and a TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, with a CDR3 that comprises or consists of the amino acid sequence of SEQ ID NO: 4, and a CDR1 that comprises or consists of the amino acid sequence of SEQ ID NO: 7, HA-1 H encodes an antigen-specific binding protein. The TRBV7-9 gene is TRBV7-9 * can be 03. In addition, HA-1 H The antigen can comprise or consist of the sequence shown in SEQ ID NO: 10. Furthermore, the TCR Vα domain can be part of the TCRα chain having a constant domain, and the TCR Vβ domain can be part of the TCRβ chain having a constant domain.

[0207] In this particular example, the CDR3 of the Vα domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 11 or SEQ ID NO: 12; the CDR3 of the Vβ domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 13 or SEQ ID NO: 14; and the CDR1 of the Vβ domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0208] In this particular example, the Vα domain can include an amino acid sequence having at least 90% sequence identity to, including, or consisting of SEQ ID NO: 17; the Vβ domain includes an amino acid sequence having at least 90% sequence identity to, including, or consisting of SEQ ID NO: 18. In one example, the Vα domain includes the amino acid sequence of SEQ ID NO: 17 and the Vβ domain includes the amino acid sequence of SEQ ID NO: 18. In such a case, the Vα domain may be encoded by a nucleic acid sequence including the sequence of SEQ ID NO: 19 or SEQ ID NO: 20; the Vβ domain may be encoded by a nucleic acid sequence including the sequence of SEQ ID NO: 21 or SEQ ID NO: 22.

[0209] In this particular example, the TCR Vα domain can include the CDR1 amino acid sequence including or consisting of the amino acid sequence of SEQ ID NO: 80, and the CDR2 amino acid sequence including or consisting of the amino acid sequence of SEQ ID NO: 83. Further, the TCR Vβ domain can include the CDR2 amino acid sequence including or consisting of the amino acid sequence of SEQ ID NO: 86.

[0210] To avoid misunderstanding, this particular example encompasses the components of TCR clone M7 exemplified herein. The different components of TCR clone M7 and their respective SEQ ID NOs are summarized in Table 1 below.

[0211]

Table 1

[0212] (ii) A Vβ domain comprising the CDR3 amino acid sequence of SEQ ID NO: 5 and functional variants thereof HA-1 H An example of the CDR3 amino acid sequence of a suitable TCR Vβ domain that confers specific binding to an antigen is shown in SEQ ID NO: 5. As will be apparent to those skilled in the art, variants of the amino acid sequence shown in SEQ ID NO: 5 may also be functional (i.e., when the CDR3 is part of the TCR Vβ domain, HA-1 HThose that confer specific binding to an antigen (e.g., the peptide shown in SEQ ID NO: 10) and retain those abilities. Thus, such functional variants are encompassed herein.

[0213] For example, the CDR3 amino acid sequence of a suitable (functional) Vβ domain can have at least 80% sequence identity to SEQ ID NO: 5, i.e., they can have at least 80%, at least 84%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 5. Suitably, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 5). In other words, the CDR3 amino acid sequence of a suitable (functional) Vβ domain can vary from the sequence shown in SEQ ID NO: 5 by 1 amino acid or several amino acids (e.g., 2 amino acids). As stated above, functional variants of SEQ ID NO: 5, when the CDR3 is part of the TCR Vβ domain, are HA-1 H Those that confer specific binding to an antigen (e.g., the peptide shown in SEQ ID NO: 10) and retain those abilities.

[0214] Functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 5. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 5, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the CDR3.

[0215] Non-functional variants are HA-1 HIt is an amino acid sequence variant of SEQ ID NO: 5 that does not specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 5, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0216] In one example, the CDR3 of the Vβ domain comprises or consists of the amino acid sequence of SEQ ID NO: 5. In the example where the CDR3 of the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 5, the CDR3 can be encoded by the nucleic acid sequence of SEQ ID NO: 25 or SEQ ID NO: 26, or their degenerate gene sequences (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 26 is the codon-optimized form of the nucleic acid sequence for the CDR3 of clone M2 (the non-optimized sequence is SEQ ID NO: 25).

[0217] The encoded TCR Vβ domain can include the amino acid sequence of SEQ ID NO: 7, or a functional variant thereof (i.e., in this case, the variant retains the ability to specifically bind to the C-terminus of the antigen (e.g., the peptide shown in SEQ ID NO: 10)), in addition to the designated CDR3. Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 7. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids in SEQ ID NO: 7, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein. H It can include CDR1 that retains the ability to specifically bind to the C-terminus of an antigen (e.g., the peptide shown in SEQ ID NO: 10). Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 7. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids in SEQ ID NO: 7, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0218] Non-functional variants are HA-1 HIt is an amino acid sequence variant of SEQ ID NO: 7 that does not specifically bind to the C-terminus of an antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 7, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0219] For example, the CDR1 amino acid sequence of a suitable (functional) Vβ domain can have at least 80% sequence identity to SEQ ID NO: 7, i.e., it can have at least 80%, at least 83%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7. Suitably, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 7). In other words, the CDR1 amino acid sequence of a suitable (functional) Vβ domain can vary by 1 amino acid or several amino acids from the sequence shown in SEQ ID NO: 7. As already stated, variants can include amino acid substitutions such as conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 7. As stated above, a functional variant of SEQ ID NO: 7, when the CDR1 is part of a TCR Vβ domain, is HA-1 H retains the ability to specifically bind to the C-terminus of an antigen (e.g., the peptide shown in SEQ ID NO: 10).

[0220] In one example, the CDR1 of the Vβ domain comprises or consists of the amino acid sequence of SEQ ID NO: 7. In an example where the CDR1 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 7, the CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16, or their degenerate gene sequences (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 16 is the codon-optimized form of the nucleic acid sequence for the CDR1 of clone M2 (the non-optimized sequence is SEQ ID NO: 15).

[0221] The encoded TCR Vβ domain can also include a CDR2 having the amino acid sequence of SEQ ID NO: 86 or a functional variant thereof (i.e., in this case, the variant retains the ability to specifically bind to HLA-A * 02:01). Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 86. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 86, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0222] Non-functional variants are amino acid sequence variants of SEQ ID NO: 86 that do not specifically bind to HLA-A * 02:01. Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 86, or premature truncations, or substitutions, insertions, or deletions in essential amino acids or essential regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0223] For example, the CDR2 amino acid sequence of a suitable (functional) Vβ domain can have at least 80% sequence identity to SEQ ID NO: 86, i.e., this can have at least 80%, at least 83%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 86. Suitably, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 86). In other words, the CDR2 amino acid sequence of a suitable (functional) Vβ domain can vary by 1 amino acid or several amino acids from the sequence shown in SEQ ID NO: 86. As already stated, the variant can include amino acid substitutions such as conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 86. As stated above, a functional variant of SEQ ID NO: 86 retains the ability to specifically bind to HLA-A * retains the ability to specifically bind to 02:01.

[0224] In one example, the CDR2 of the Vβ domain comprises or consists of the amino acid sequence of SEQ ID NO: 86. In an example where the CDR2 of the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 86, the CDR2 can be encoded by the nucleic acid sequence of SEQ ID NO: 87 or SEQ ID NO: 88, or a degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 88 is the codon-optimized form of the nucleic acid sequence for the CDR2 of clone M2 (the non-optimized sequence is SEQ ID NO: 87).

[0225] Accordingly, the encoded TCR Vβ domain can include the CDRs (specifically, in terms of SEQ ID NOs., i.e., SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 86, or a functional variant thereof) mentioned in detail above, with suitable intervening sequences between the CDRs.

[0226] The encoded TCR Vβ domain has the amino acid sequence of SEQ ID NO: 30, or a functional variant thereof (i.e., in this case, if it includes a part of the binding protein described herein, the variant TCR Vβ domain is HA-1 H and may have the ability to specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10). Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 30. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 30, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0227] Non-functional variants are amino acid sequence variants of SEQ ID NO: 30 that do not specifically bind to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 30, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0228] In one example, the encoded TCR Vβ domain is HA-1 HWhile retaining the ability to specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10), it may have an amino acid sequence having 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: 30. In other words, functional TCR Vβ domains with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 30 are also encompassed. As already stated, the amino acid substitutions can be conservative amino acid substitutions. Variations within the sequence compared to SEQ ID NO: 30 can all be within regions of the TCR Vβ domain that do not form CDRs (i.e., the variant can have the CDRs of SEQ ID NO: 5, and SEQ ID NO: 7, and optionally, SEQ ID NO: 86, and the variation of the sequence compared to SEQ ID NO: 30 is still 25% (or less)). In other words, the sequences of the CDRs of SEQ ID NO: 30 can be retained even if the remaining sequence varies as needed within the "at least 75% identity" parameter specified above. Appropriately, the percent identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 30).

[0229] By way of example, the encoded TCR Vβ domain can 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%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 30, in which case the TCR Vβ domain includes a CDR3 having the amino acid sequence of SEQ ID NO: 5. In this example, CDR1 of the TCR Vβ domain can have the amino acid sequence of SEQ ID NO: 7, and CDR2 of the TCR Vβ domain can have the amino acid sequence of SEQ ID NO: 86.

[0230] In the example where the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 30, the TCR Vβ domain can be encoded by the nucleic acid sequence of SEQ ID NO: 33 or SEQ ID NO: 34, or a degenerate sequence of these genes (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 34 is the codon-optimized form of the nucleic acid sequence for the TCR Vβ domain of clone M2 (the non-optimized sequence is SEQ ID NO: 33).

[0231] To avoid misunderstanding, the nucleic acid sequence encoding the TCR Vβ domain may also encode the TCRβ chain constant domain. Examples of suitable constant domains are encoded within the MP71-TCR-flex retroviral vector. However, the present invention is not limited to this specific constant domain and encompasses any suitable TCRβ chain constant domain. The constant domain may be of mouse origin, human origin, or humanized. Methods for identifying or generating suitable constant domains are well known to those skilled in the art and are well within the scope of their regulatory capabilities.

[0232] For the purpose of illustration only, the constant domain can be a lentiviral vector, a retroviral vector, or a plasmid vector, etc. in which a mouse constant domain or a human constant domain has been pre-cloned. It can also be encoded by or derived from a vector that is also an adenoviral vector, an adeno-associated viral vector, a vaccinia viral vector, a canarypox viral vector, or a herpes viral vector. In recent years, mini-circles have also been described for TCR gene transfer (R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics, and M Hudecek, Leukemia, 2016, non-viral Sleeping Beauty transposition derived from mini-circle vectors). Furthermore, naked (synthetic) DNA / RNA can also be used to introduce TCRs. For example, the pMSGV retroviral vector pre-cloned with the TCR-Ca gene and the TCR-Cb gene, described by LV Coren et al. in BioTechniques, 2015, can be used to provide an appropriate constant domain.

[0233] Examples of specific TCRβ-chain amino acid sequences containing the TCR Vβ domain described herein and an appropriate constant domain are shown in SEQ ID NO: 62 and SEQ ID NO: 63. It is noted that the constant domain shown in SEQ ID NO: 63 is a mouse constant domain. Appropriate functional variants of SEQ ID NO: 62 and SEQ ID NO: 63 (for example, variants having at least 75% (for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63), and in this case, if it contains a part of the binding protein described herein, the amino acid sequence of the variant TCRβ chain is HA-1 HThose that retain the ability to specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10) are also included. In other words, functional TCRβ chains with one or several amino acid substitutions compared to the sequences of SEQ ID NO: 62 or SEQ ID NO: 63 are also included. As already stated, the amino acid substitutions can be conservative amino acid substitutions. Variations within the sequences compared to SEQ ID NO: 62 or SEQ ID NO: 63 can all be within regions of the TCRβ chain that do not form CDRs (i.e., the variants can have the CDRs of SEQ ID NO: 5, and SEQ ID NO: 7, and optionally SEQ ID NO: 86, and the variation of the sequences compared to SEQ ID NO: 62 or SEQ ID NO: 63 is still 25% (or less)). In other words, the sequences of the CDRs of SEQ ID NO: 62 or SEQ ID NO: 63 can be retained even if the remaining sequences vary as needed within the "at least 75% identity" parameter specified above. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 62 or SEQ ID NO: 63 when applicable).

[0234] By way of example, the encoded TCRβ chain can 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%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 62 or SEQ ID NO: 63, in which case the TCRβ chain includes a CDR3 having the amino acid sequence of SEQ ID NO: 5. In this example, the CDR1 of the TCRβ chain can have the amino acid sequence of SEQ ID NO: 7, and the CDR2 of the TCRβ chain can have the amino acid sequence of SEQ ID NO: 86.

[0235] In the example where the TCR β chain has the amino acid sequence of SEQ ID NO: 62, the TCRβ chain can be encoded by the nucleic acid sequence of SEQ ID NO: 64 or SEQ ID NO: 65, or their degenerate gene sequences (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 65 is the codon-optimized form of the nucleic acid sequence for the TCR Vβ domain of clone M7 (the non-optimized sequence is SEQ ID NO: 64).

[0236] In the example where the TCR β chain has the amino acid sequence of SEQ ID NO: 63, the TCRβ chain can be encoded by the nucleic acid sequence of SEQ ID NO: 66, or this degenerate gene sequence (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0237] In certain examples, the nucleic acid composition described herein has a TCR Vα domain with a CDR3 amino acid sequence that includes or consists of the amino acid sequence of SEQ ID NO: 2, and a TCR Vβ domain with an amino acid sequence encoded by the TRBV7-9 gene, having a CDR3 that includes or consists of the amino acid sequence of SEQ ID NO: 5, and a CDR1 that includes or consists of the amino acid sequence of SEQ ID NO: 7, HA-1 H encodes an antigen-specific binding protein. The TRBV7-9 gene is TRBV7-9 * can be 01. In addition, HA-1 H The antigen can include or consist of the sequence shown in SEQ ID NO: 10. Furthermore, the TCR Vα domain can be part of the TCRα chain having a constant domain, and the TCR Vβ domain can be part of the TCRβ chain having a constant domain.

[0238] In this particular example, the CDR3 of the Vα domain may be encoded by a nucleic acid sequence containing the sequence of SEQ ID NO: 23 or SEQ ID NO: 24; the CDR3 of the Vβ domain may be encoded by a nucleic acid sequence containing the sequence of SEQ ID NO: 25 or SEQ ID NO: 26; and the CDR1 of the Vβ domain may be encoded by a nucleic acid sequence containing the sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0239] In this particular example, the Vα domain can include an amino acid sequence having at least 90% sequence identity to, including, or consisting of SEQ ID NO: 29; the Vβ domain includes an amino acid sequence having at least 90% sequence identity to, including, or consisting of SEQ ID NO: 30. In one example, the Vα domain includes the amino acid sequence of SEQ ID NO: 29, and the Vβ domain includes the amino acid sequence of SEQ ID NO: 30. In such a case, the Vα domain may be encoded by a nucleic acid sequence including the sequence of SEQ ID NO: 31 or SEQ ID NO: 32; the Vβ domain may be encoded by a nucleic acid sequence including the sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

[0240] In this particular example, the TCR Vα domain can include a CDR1 amino acid sequence including or consisting of the amino acid sequence of SEQ ID NO: 8, and a CDR2 amino acid sequence including or consisting of the amino acid sequence of SEQ ID NO: 28.

[0241] Furthermore, the TCR Vβ domain can include a CDR2 amino acid sequence including or consisting of the amino acid sequence of SEQ ID NO: 86.

[0242] To avoid misunderstanding, this particular example encompasses the components of TCR clone M2 exemplified herein. The different components of TCR clone M2, and their respective SEQ ID NOs, are summarized in Table 2 below.

[0243]

Table 2

[0244] (iii) A Vβ domain including the CDR3 amino acid sequence of SEQ ID NO: 6 and functional variants thereof HA-1 HExamples of CDR3 amino acid sequences of suitable TCR Vβ domains that confer specific binding to an antigen are shown in SEQ ID NO: 6. As will be apparent to those skilled in the art, variants of the amino acid sequence shown in SEQ ID NO: 6 may also be functional (i.e., when the CDR3 is part of the TCR Vβ domain, HA-1 H retain their ability to confer specific binding to an antigen (e.g., the peptide shown in SEQ ID NO: 10)). Accordingly, such functional variants are encompassed herein.

[0245] For example, the CDR3 amino acid sequences of suitable (functional) Vβ domains can have at least 80% sequence identity to SEQ ID NO: 6, i.e., they can have at least 80%, at least 84%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 6. Suitably, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 6). In other words, the CDR3 amino acid sequences of suitable (functional) Vβ domains can vary from the sequence shown in SEQ ID NO: 6 by 1 amino acid or several amino acids (e.g., 2 amino acids). As stated above, functional variants of SEQ ID NO: 6 retain their ability to confer specific binding to an HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10) when the CDR3 is part of the TCR Vβ domain.

[0246] Functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 6. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 6, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the CDR3.

[0247] Non-functional variants are HA-1 HIt is an amino acid sequence variant of SEQ ID NO: 6 that does not specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 6, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0248] In one example, the CDR3 of the Vβ domain comprises or consists of the amino acid sequence of SEQ ID NO: 6. In the example where the CDR3 of the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 6, the CDR3 can be encoded by the nucleic acid sequence of SEQ ID NO: 37 or SEQ ID NO: 38, or their degenerate sequences (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 38 is the codon-optimized form of the nucleic acid sequence for the CDR3 of clone FK47.83 (the non-optimized sequence is SEQ ID NO: 37).

[0249] The encoded TCR Vβ domain can include, in addition to the designated CDR3, the amino acid sequence of SEQ ID NO: 7, or a functional variant thereof (i.e., in this case, the variant retains the ability to specifically bind to the C-terminus of an antigen (e.g., the peptide shown in SEQ ID NO: 10)) and can include CDR1. Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 7. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids in SEQ ID NO: 7, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein. H It can include CDR1 that contains a functional variant (i.e., in this case, the variant retains the ability to specifically bind to the C-terminus of an antigen (e.g., the peptide shown in SEQ ID NO: 10)) of the amino acid sequence of SEQ ID NO: 7. Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 7. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids in SEQ ID NO: 7, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0250] Non-functional variants are HA-1 HIt is an amino acid sequence variant of SEQ ID NO: 7 that does not specifically bind to the C-terminus of an antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 7, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0251] For example, the CDR1 amino acid sequence of a suitable (functional) Vβ domain can have at least 80% sequence identity to SEQ ID NO: 7, i.e., it can have at least 80%, at least 83%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7. Appropriately, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 7). In other words, the CDR1 amino acid sequence of a suitable (functional) Vβ domain can vary by 1 amino acid or several amino acids from the sequence shown in SEQ ID NO: 7. As already stated, variants can include amino acid substitutions such as conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 7. As stated above, a functional variant of SEQ ID NO: 7, when the CDR1 is part of the TCR Vβ domain, is HA-1 H retains the ability to specifically bind to the C-terminus of an antigen (e.g., the peptide shown in SEQ ID NO: 10).

[0252] In one example, the CDR1 of the Vβ domain comprises or consists of the amino acid sequence of SEQ ID NO: 7. In an example where the CDR1 of the TCR Vα domain has the amino acid sequence of SEQ ID NO: 7, the CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16, or a degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 16 is the codon-optimized form of the nucleic acid sequence for the CDR1 of clone FK47.83 (the non-optimized sequence is SEQ ID NO: 15).

[0253] The encoded TCR Vβ domain can also include a CDR2 having the amino acid sequence of SEQ ID NO: 86 or a functional variant thereof (i.e., in this case, the variant retains the ability to specifically bind to HLA-A * 02:01) in addition to the specified CDR3 (and optionally, the CDR1 specified above). Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 86. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 86, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0254] Non-functional variants are amino acid sequence variants of SEQ ID NO: 86 that do not specifically bind to HLA-A * 02:01. Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 86, or premature truncations, or substitutions, insertions, or deletions in essential amino acids or essential regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0255] For example, the CDR2 amino acid sequence of a suitable (functional) Vβ domain can have at least 80% sequence identity to SEQ ID NO: 86, i.e., it can have at least 80%, at least 83%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 86. Suitably, the percent identity is calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 86). In other words, the CDR2 amino acid sequence of a suitable (functional) Vβ domain can vary by 1 amino acid or several amino acids from the sequence shown in SEQ ID NO: 86. As already stated, the variant can include amino acid substitutions such as conservative amino acid substitutions compared to the sequence shown in SEQ ID NO: 86. As stated above, the functional variant of SEQ ID NO: 86 retains the ability to specifically bind to HLA-A * 02:01.

[0256] In one example, the CDR2 of the Vβ domain comprises or consists of the amino acid sequence of SEQ ID NO: 86. In the example where the CDR2 of the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 86, the CDR2 can be encoded by the nucleic acid sequence of SEQ ID NO: 87 or SEQ ID NO: 88, or a degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 88 is the codon-optimized form of the nucleic acid sequence for the CDR2 of clone FK47.83 (the non-optimized sequence is SEQ ID NO: 87).

[0257] Thus, the encoded TCR Vβ domain can include the CDRs (specifically, in terms of SEQ ID NOs, i.e., SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 86, or functional variants thereof) mentioned in detail above, with suitable intervening sequences between the CDRs.

[0258] The encoded TCR Vβ domain has the amino acid sequence of SEQ ID NO: 42, or a functional variant thereof (i.e., in this case, if it includes a part of the binding protein described herein, the variant TCR Vβ domain is HA-1 H and may have the ability to specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10). Such functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 42. The term "variant" also encompasses homologs. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 42, or substitutions, deletions, or insertions of non-essential amino acids within non-essential regions of the protein.

[0259] Non-functional variants are amino acid sequence variants of SEQ ID NO: 42 that do not specifically bind to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants will typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO: 42, or premature truncations, or substitutions, insertions, or deletions in the most important amino acids or most important regions. Methods for identifying functional and non-functional variants are well known to those skilled in the art.

[0260] In one example, the encoded TCR Vβ domain is HA-1 HWhile retaining the ability to specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10), it may have an amino acid sequence having 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: 42. In other words, functional TCR Vβ domains with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 42 are also encompassed. As already stated, the amino acid substitutions can be conservative amino acid substitutions. Variations within the sequence compared to SEQ ID NO: 42 can all be within regions of the TCR Vβ domain that do not form CDRs (i.e., the variant can have the CDRs of SEQ ID NO: 6, and SEQ ID NO: 7, and optionally SEQ ID NO: 86, and the variation in the sequence compared to SEQ ID NO: 42 is still 25% (or less)). In other words, the sequences of the CDRs of SEQ ID NO: 42 can be retained even if the remaining sequence varies as required within the "at least 75% identity" parameter specified above. Appropriately, the percent identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 42).

[0261] By way of example, the encoded TCR Vβ domain can 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%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 42, in which case the TCR Vβ domain includes a CDR3 having the amino acid sequence of SEQ ID NO: 6. In this example, the CDR1 of the TCR Vβ domain can have the amino acid sequence of SEQ ID NO: 7, and the CDR2 of the TCR Vβ domain can have the amino acid sequence of SEQ ID NO: 86.

[0262] In the example where the TCR Vβ domain has the amino acid sequence of SEQ ID NO: 42, the TCR Vβ domain can be encoded by the nucleic acid sequence of SEQ ID NO: 45 or SEQ ID NO: 46, or their degenerate gene sequences (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 46 is the codon-optimized form of the nucleic acid sequence for the TCR Vβ domain of clone FK47.83 (the non-optimized sequence is SEQ ID NO: 45).

[0263] To avoid misunderstanding, the nucleic acid sequence encoding the TCR Vβ domain can also encode the TCRβ chain constant domain. Examples of suitable constant domains are encoded within the MP71-TCR-flex retroviral vector. However, the present invention is not limited to this specific constant domain and encompasses any suitable TCRβ chain constant domain. The constant domain can be of murine origin, human origin, or humanized. Methods for identifying or creating suitable constant domains are well known to those skilled in the art and are well within the scope of their defined capabilities.

[0264] For the purposes of illustration only, a constant domain may be a lentiviral vector, a retroviral vector, or a plasmid vector, etc., in which a murine constant domain or a human constant domain has been pre-cloned, but may also be encoded by or derived from a vector that is also an adenoviral vector, an adeno-associated viral vector, a vaccinia viral vector, a canarypox viral vector, or a herpes viral vector. In recent years, minicircles have also been described for TCR gene transfer (R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics, and M Hudecek, Leukemia, 2016, non-viral Sleeping Beauty transposition derived from minicircle vectors). Furthermore, naked (synthetic) DNA / RNA may also be used to introduce TCRs. For example, the pMSGV retroviral vector, in which the TCR-Ca gene and the TCR-Cb gene have been pre-cloned as described by LV Coren et al. in BioTechniques, 2015, may be used to provide an appropriate constant domain.

[0265] Examples of specific TCRβ chain amino acid sequences that include the TCR Vβ domain described herein and an appropriate constant domain are shown in SEQ ID NO: 72 and SEQ ID NO: 73. It is noted that the constant domain shown in SEQ ID NO: 73 is a murine constant domain. Appropriate functional variants of SEQ ID NO: 72 and SEQ ID NO: 73 (e.g., variants having at least 75% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 72 or SEQ ID NO: 73), and in this case, if the variant contains a portion of the binding protein described herein, the amino acid sequence of the variant TCRβ chain is HA-1 HAlso included are those that retain the ability to specifically bind to an antigen (e.g., the peptide shown in SEQ ID NO: 10). In other words, also included are functional TCRβ chains with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 72 or SEQ ID NO: 73. As already stated, the amino acid substitutions can be conservative amino acid substitutions. Variations within the sequence compared to SEQ ID NO: 72 or SEQ ID NO: 73 can all be within regions of the TCRβ chain that do not form CDRs (i.e., the variant can have the CDRs of SEQ ID NO: 6, and SEQ ID NO: 7, and optionally SEQ ID NO: 86, and the variation in the sequence compared to SEQ ID NO: 72 or SEQ ID NO: 73 is still 25% (or less)). In other words, the sequences of the CDRs of SEQ ID NO: 72 or SEQ ID NO: 73 can be retained even if the remaining sequence varies as required within the "at least 75% identity" parameter specified above. Appropriately, the percentage of identity can be calculated as the percentage of identity to the full length of the reference sequence (e.g., SEQ ID NO: 72 or SEQ ID NO: 73, where applicable).

[0266] By way of example, the encoded TCRβ chain can 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%, etc.) sequence identity to the amino acid sequence of SEQ ID NO: 72 or SEQ ID NO: 73, in which case the TCRβ chain includes a CDR3 having the amino acid sequence of SEQ ID NO: 6. In this example, the CDR1 of the TCRβ chain can have the amino acid sequence of SEQ ID NO: 7, and the CDR2 of the TCRβ chain can have the amino acid sequence of SEQ ID NO: 86.

[0267] In the example where the TCR β chain has the amino acid sequence of SEQ ID NO: 72, the TCRβ chain can be encoded by the nucleic acid sequence of SEQ ID NO: 74 or SEQ ID NO: 75, or their gene degenerate sequences (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 75 is the codon-optimized form of the nucleic acid sequence for the TCR Vβ domain of clone M7 (the non-optimized sequence is SEQ ID NO: 74).

[0268] In the example where the TCR β chain has the amino acid sequence of SEQ ID NO: 73, the TCRβ chain can be encoded by the nucleic acid sequence of SEQ ID NO: 76, or this degenerate gene sequence (i.e., other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).

[0269] In certain examples, the nucleic acid compositions described herein comprise a TCR Vα domain with a CDR3 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and a TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, with a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 6, and a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 7, HA-1 H encoding an antigen-specific binding protein. The TRBV7-9 gene is TRBV7-9 * can be 01. In addition, HA-1 H The antigen can comprise or consist of the sequence shown in SEQ ID NO: 10. Furthermore, the TCR Vα domain can be part of the TCRα chain having a constant domain, and the TCR Vβ domain can be part of the TCRβ chain having a constant domain.

[0270] In this particular example, the CDR3 of the Vα domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 35 or SEQ ID NO: 36; the CDR3 of the Vβ domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 37 or SEQ ID NO: 38; and the CDR1 of the Vβ domain can be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0271] In this particular example, the Vα domain can include an amino acid sequence having at least 90% sequence identity to, including, or consisting of SEQ ID NO: 41; the Vβ domain includes an amino acid sequence having at least 90% sequence identity to, including, or consisting of SEQ ID NO: 42. In one example, the Vα domain includes the amino acid sequence of SEQ ID NO: 41 and the Vβ domain includes the amino acid sequence of SEQ ID NO: 42. In such a case, the Vα domain may be encoded by a nucleic acid sequence including the sequence of SEQ ID NO: 43 or SEQ ID NO: 44; the Vβ domain may be encoded by a nucleic acid sequence including the sequence of SEQ ID NO: 45 or SEQ ID NO: 46.

[0272] In this particular example, the TCR Vα domain can include the CDR1 amino acid sequence including or consisting of the amino acid sequence of SEQ ID NO: 8, and the CDR2 amino acid sequence including or consisting of the amino acid sequence of SEQ ID NO: 28. Further, the TCR Vβ domain can include the CDR2 amino acid sequence including or consisting of the amino acid sequence of SEQ ID NO: 86.

[0273] To avoid misunderstanding, this particular example encompasses the components of the TCR clone FK47.83 exemplified herein. The different components of the TCR clone FK47.83 and their respective SEQ ID NOs are summarized in Table 3 below.

[0274]

Table 3

[0275] Any of the TCR Vα domains (or TCR α chains) described herein can be combined with any of the TCR Vα domains (or TCR α chains).

[0276] a) The components of the Vα domain of the TCR clone M2, accompanied by the components of the Vβ domain of the TCR clone M7 (or FK47.83) For example, the nucleic acid composition described in this specification comprises a TCR Vα domain with a CDR3 amino acid sequence that includes or consists of the amino acid sequence of SEQ ID NO: 2, and a TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, with a CDR3 that includes or consists of the amino acid sequence of SEQ ID NO: 4 (or SEQ ID NO: 6) and a CDR1 that includes or consists of the amino acid sequence of SEQ ID NO: 7, HA-1 H encodes an antigen-specific binding protein. The TRBV7-9 gene is TRBV7-9 * 03 (or TRBV7-9 * 01). Additionally, the HA-1 H antigen may include or consist of the sequence shown in SEQ ID NO: 10. Furthermore, the TCR Vα domain can be part of the TCRα chain having a constant domain, and the TCR Vβ domain can be part of the TCRβ chain having a constant domain.

[0277] In this particular example, the CDR3 of the Vα domain may be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO: 23 or SEQ ID NO: 24, and the CDR3 of the Vβ domain may be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO: 13 or SEQ ID NO: 14 (or SEQ ID NO: 37 or SEQ ID NO: 38); the CDR1 of the Vβ domain may be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0278] In this particular example, the Vα domain can include an amino acid sequence having at least 90% sequence identity to, including, or consisting of SEQ ID NO: 29; the Vβ domain includes an amino acid sequence having at least 90% sequence identity to, including, or consisting of SEQ ID NO: 18 (or SEQ ID NO: 42). In one example, the Vα domain includes the amino acid sequence of SEQ ID NO: 29, and the Vβ domain includes the amino acid sequence of SEQ ID NO: 18 (or SEQ ID NO: 42). In such a case, the Vα domain may be encoded by a nucleic acid sequence including the sequence of SEQ ID NO: 31 or SEQ ID NO: 32; the Vβ domain may be encoded by a nucleic acid sequence including the sequence of SEQ ID NO: 21 or SEQ ID NO: 22 (or SEQ ID NO: 45 or SEQ ID NO: 46).

[0279] In this particular example, the TCR Vα domain can include the CDR1 amino acid sequence including or consisting of the amino acid sequence of SEQ ID NO: 8, and the CDR2 amino acid sequence including or consisting of the amino acid sequence of SEQ ID NO: 28. Further, the TCR Vβ domain can include the CDR2 amino acid sequence including or consisting of the amino acid sequence of SEQ ID NO: 86.

[0280] b) Components of the Vα domain of TCR clone M7, accompanied by components of the Vβ domain of TCR clone M2 (or TCR clone FK47.83) For example, the nucleic acid composition described herein includes a TCR Vα domain with a CDR3 amino acid sequence including or consisting of the amino acid sequence of SEQ ID NO: 1, and a TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, including or consisting of the CDR3 of the amino acid sequence of SEQ ID NO: 5 (or SEQ ID NO: 6) and the CDR1 of the amino acid sequence including or consisting of the amino acid sequence of SEQ ID NO: 7, HA-1 H encodes an antigen-specific binding protein. The TRBV7-9 gene can be TRBV7-9 * 01. In addition, HA-1 HThe antigen may comprise or consist of the sequence shown in SEQ ID NO: 10. Further, the TCR Vα domain can be part of the TCRα chain having a constant domain, and the TCR Vβ domain can be part of the TCRβ chain having a constant domain.

[0281] In this particular example, the CDR3 of the Vα domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 11 or SEQ ID NO: 12, and the CDR3 of the Vβ domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 25 or SEQ ID NO: 26 (or SEQ ID NO: 37 or SEQ ID NO: 38); the CDR1 of the Vβ domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0282] In this particular example, the Vα domain can comprise an amino acid sequence having at least 90% sequence identity to, comprising, or consisting of SEQ ID NO: 17; the Vβ domain comprises an amino acid sequence having at least 90% sequence identity to, comprising, or consisting of SEQ ID NO: 30 (or SEQ ID NO: 42). In one example, the Vα domain comprises the amino acid sequence of SEQ ID NO: 17, and the Vβ domain comprises the amino acid sequence of SEQ ID NO: 30 (or SEQ ID NO: 42). In such a case, the Vα domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 19 or SEQ ID NO: 20; the Vβ domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 33 or SEQ ID NO: 34 (or SEQ ID NO: 45 or SEQ ID NO: 46).

[0283] In this particular example, the TCR Vα domain can comprise a CDR1 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 80, and a CDR2 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 83. Further, the TCR Vβ domain can comprise a CDR2 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 86.

[0284] c) Components of the Vα domain of TCR clone FK47.8, along with components of the Vβ domain of TCR clone M2 (or M7) For example, the nucleic acid composition described herein comprises a TCR Vα domain with a CDR3 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 3, and a TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, with a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 5 (or SEQ ID NO: 4) and a CDR1 comprising or consisting of the amino acid sequence of SEQ ID NO: 7, having HA-1 H Encoding an antigen-specific binding protein. The TRBV7-9 gene is TRBV7-9 * 01 (or TRBV7-9 * 03). Additionally, HA-1 H The antigen may comprise or consist of the sequence shown in SEQ ID NO: 10. Furthermore, the TCR Vα domain can be part of the TCRα chain having a constant domain, and the TCR Vβ domain can be part of the TCRβ chain having a constant domain.

[0285] In this particular example, the CDR3 of the Vα domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 35 or SEQ ID NO: 36, and the CDR3 of the Vβ domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 25 or SEQ ID NO: 26 (or SEQ ID NO: 13 or SEQ ID NO: 14); the CDR1 of the Vβ domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 15 or SEQ ID NO: 16.

[0286] In this particular example, the Vα domain can include an amino acid sequence having at least 90% sequence identity to, including, or consisting of SEQ ID NO: 41; the Vβ domain includes an amino acid sequence having at least 90% sequence identity to, including, or consisting of SEQ ID NO: 30 (or SEQ ID NO: 18). In one example, the Vα domain includes the amino acid sequence of SEQ ID NO: 41, and the Vβ domain includes the amino acid sequence of SEQ ID NO: 30 (or SEQ ID NO: 18). In such a case, the Vα domain may be encoded by a nucleic acid sequence including the sequence of SEQ ID NO: 43 or SEQ ID NO: 44; the Vβ domain may be encoded by a nucleic acid sequence including the sequence of SEQ ID NO: 33 or SEQ ID NO: 34 (or SEQ ID NO: 21 or SEQ ID NO: 22).

[0287] In this particular example, the TCR Vα domain can include a CDR1 amino acid sequence including, or consisting of, the amino acid sequence of SEQ ID NO: 8, and a CDR2 amino acid sequence including, or consisting of, the amino acid sequence of SEQ ID NO: 28. Further, the TCR Vβ domain can include a CDR2 amino acid sequence including, or consisting of, the amino acid sequence of SEQ ID NO: 86.

[0288] As described in more detail elsewhere herein, the nucleic acid compositions described herein are HA-1 HIt encodes both the TCR Vα domain and the TCR Vβ domain, which form a binding protein capable of specifically binding to an antigen. In an example 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 can be integrally connected via a linker, for example, a linker that enables the expression of two proteins or polypeptides from the same vector. For illustrative purposes, a porcine teschovirus 12A (P2A) sequence, such as the 2A sequence derived from the following: foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), or Thosea asigna virus (T2A) (as published by A.L. Szymczak et al., Nature Biotechnology, 22, 589-594 (2004)), or a linker containing a 2A-like sequence can be used. The 2A sequence and the 2A-like sequence are linkers that can be cleaved when the nucleic acid molecule is transcribed and translated. Another example of a linker is an internal ribosome entry site (IRES) that enables the translation of two proteins or polypeptides from the same transcript. Any other suitable linker can also be used. As a further example, the nucleic acid sequence encoding the TCR Vα domain and the nucleic acid sequence encoding the TCR Vβ domain can be cloned into a vector with a dual internal promoter (see, for example, S Jones et al., Human Gene Ther, 2009). Identifying suitable linkers and vectors that enable the expression of both the TCR Vα domain and the TCR Vβ domain is within the purview of those of ordinary skill in the art.

[0289] Additional suitable polypeptide domains can also be encoded by the nucleic acid sequence encoding the TCR Vα domain and / or the TCR Vβ domain. For illustrative purposes only, the nucleic acid sequence can include a membrane targeting sequence that results in the transport of the encoded polypeptide to the cell surface membrane of the modified cell. Other suitable additional domains are well known and are described, for example, in WO2016 / 071758.

[0290] In one example, the nucleic acid compositions described herein may encode a soluble TCR. For example, the nucleic acid composition may encode the variable domains of each of the TCR alpha and beta chains in combination with an immunomodulatory molecule such as a CD3 agonist (e.g., anti-CD3 scFv). The CD3 antigen is present in subsets of mature human T cells, thymocytes, and natural killer cells. The CD3 antigen is associated with the TCR and is involved in TCR signaling. Antibodies specific for the human CD3 antigen are well known. One such antibody is the murine monoclonal antibody OKT3, the first monoclonal antibody approved by the FDA. Other antibodies specific for CD3 have also been reported (see, e.g., WO2004 / 106380; US Patent Application Publication No. 2004 / 0202657; US Patent No. 6,750,325). ImmTAC (Immune mobilising mTCR Against Cancer; Immunocore Limited, Milton Partk, Abington, Oxon, United Kingdom) is a bifunctional protein that combines an affinity monoclonal T cell receptor (mTCR) target with a therapeutic mechanism of action (i.e., anti-CD3 scFv). In another example, the soluble TCR of the invention may be combined with a radioisotope or a toxic drug. Suitable radioisotopes and / or toxic drugs are well known in the art and can be readily identified by one of ordinary skill in the art.

[0291] In one example, the nucleic acid composition is capable of encoding a chimeric single-chain TCR, in which case the TCR alpha-chain variable domain is linked to a constant domain fused to the variable domain of the TCR beta-chain and, for example, the CD3 zeta signaling domain. In this example, the linker is not cleavable. In an alternative embodiment, the nucleic acid composition can encode a chimeric double-stranded TCR in which each of the TCR alpha-chain variable domain and the TCR beta-chain variable domain is linked to a CD3 zeta signaling domain, or other transmembrane and intracellular domains. Methods for preparing such single-chain and double-stranded TCRs are well known in the art; see, for example, R A Willemsen et al., Gene Therapy, 2000.

[0292] Vector system Also provided is a vector system comprising the nucleic acid composition described herein. The vector system can have one or more vectors. As already discussed, the binding protein component encoded by the nucleic acid composition can be encoded by one or more nucleic acid sequences within the nucleic acid composition. In an example where all of the binding protein components are encoded by a single nucleic acid sequence, the nucleic acid sequence can be present within a single vector (thus, the vector system described herein can comprise only one vector). In an example where the binding protein component is encoded by two or more nucleic acid sequences (wherein the plurality of nucleic acid sequences together encode all of the components of the binding protein), these two or more nucleic acid sequences can be present within one vector (e.g., different open reading frames of the vector), or can be distributed across two or more vectors. In this example, the vector system will comprise a plurality of significantly different vectors (i.e., vectors with different nucleotide sequences).

[0293] Any suitable vector can be used. For illustrative purposes only, the vector can be a plasmid, cosmid, or viral vector such as a retroviral vector or a lentiviral vector. Adenovirus, adeno-associated virus, vaccinia virus, canarypox virus, herpes virus, minicircle vector, and naked (synthetic) DNA / RNA can also be used (for details on minicircle vectors, see, for example, R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics, and M Hudecek, Leukemia, 2016, which published non-viral Sleeping Beauty transposition). Alternatively, single-stranded or double-stranded DNA or RNA can be used to transfect lymphocytes with the TCR of interest (see Roth et al., 2018, Nature 559, p. 405).

[0294] As used herein, the term "vector" refers to a nucleic acid sequence capable of transporting another nucleic acid sequence to which it is operably linked. The vector may be capable of autonomous replication or may be integrated into the host DNA. The vector may contain restriction enzyme sites for inserting recombinant DNA and may contain one or more selectable markers or suicide genes. The vector can be a nucleic acid sequence in the form of a plasmid, bacteriophage, or cosmid. Preferably, the vector is suitable for expression in cells (i.e., the vector is an "expression vector"). Preferably, the vector is suitable for expression in human T cells such as in CD8 + T cells or CD4 + T cells. In certain embodiments, the vector is a viral vector such as a retroviral vector, a lentiviral vector, or an adeno-associated vector. Optionally, the vector is selected from the group consisting of adenovirus, vaccinia virus, canarypox virus, herpes virus, minicircle vector, and synthetic DNA or synthetic RNA.

[0295] Preferably, the (expression) vector is capable of propagating in the host cell and is stably propagated to the progeny.

[0296] The vector may contain regulatory sequences. As used herein, the term "regulatory sequence" refers to an element of DNA or RNA capable of controlling gene expression. Examples of expression control sequences include promoters, enhancers, silencers, TATA boxes, internal ribosome entry sites (IRESs), junction sites for transcription factors, transcription terminators, polyadenylation sites, and the like. Optionally, the vector contains one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include tissue-specific, regulatory sequences and / or inducible sequences in addition to regulatory sequences that direct constitutive expression.

[0297] Optionally, the vector contains a nucleic acid sequence of interest operably linked to a promoter. As used herein, the term "promoter" refers to a nucleotide sequence in DNA to which RNA polymerase binds to initiate transcription. The promoter may be expressed inductively or constitutively. Alternatively, the promoter is under the control of a repressor protein or a stimulatory protein. The promoter may be a promoter that is not naturally found in the host cell (e.g., the promoter may be an exogenous promoter). Those skilled in the art are well aware of suitable promoters for use in the expression of a target protein, and in this case, the promoter selected will depend on the host cell.

[0298] "Operably linked" refers to a single control element described below, or a combination thereof, that is in a functional relationship with each other and with the coding sequence so as to direct the expression of the coding sequence, for example, in a linked relationship and integrated with the coding sequence.

[0299] The vector may contain a transcription terminator. As used herein, "transcription terminator" refers to a DNA element that terminates the function of RNA polymerase, which is a factor in transcribing DNA into RNA. Preferred transcription terminators are characterized by a run of T residues following a GC-rich, dyad-symmetric region.

[0300] The vector may contain a translation control element. As used herein, "translation control element" refers to a DNA or RNA element that controls the translation of mRNA. Preferred translation control elements are ribosome binding sites. Preferably, the translation control element is derived from a promoter, e.g., a promoter, and a system homologous to the ribozyme binding site associated therewith. Preferred ribosome binding sites are known and depend on the selected host cell.

[0301] The vector may contain a restriction enzyme recognition site. As used herein, "restriction enzyme recognition site" refers to a motif on DNA that is recognized by a restriction enzyme.

[0302] The vector may contain a selectable marker. As used herein, "selectable marker" refers to a protein that, when expressed in a host cell, confers a phenotype that allows for the selection of cells that express the selectable marker gene. Generally, this may be a protein that confers a new beneficial property (e.g., antibiotic resistance) to the host cell, or a protein that is expressed on the cell surface and is thus accessible for binding to an antibody. Suitable selectable markers are well known in the art.

[0303] Optionally, the vector may also contain a suicide gene. As used herein, "suicide gene" refers to a protein that induces the death of modified cells when treated with a specific drug. By way of example, the suicide of cells modified with the herpes simplex virus thymidine kinase gene when treated with a specific nucleoside analogue containing ganciclovir, the suicide of cells modified with human CD20 when treated with an anti-CD20 monoclonal antibody, and the suicide of cells modified with inducible caspase 9 (iCasp9) when treated with AP1903 can be induced (reviewed by BS Jones, LS Lamb, F Goldman, A Di Stasi, "Improving the safety of cell therapy products by suicide gene transfer", Front Pharmacol. (2014), 5:254). Suitable suicide genes are well known in the art.

[0304] Preferably, the vector contains the genetic elements necessary for the expression of the binding protein described herein by the host cell. Elements required for transcription and translation in the host cell include a promoter, the coding region of the protein of interest, and a transcription terminator.

[0305] Those skilled in the art will be well aware of the molecular methods available for preparing (expression) vectors and how (expression) vectors can be transduced or transfected into appropriate host cells (thereby creating the modified cells further described below). The (expression) vector systems described herein can be introduced into cells by conventional techniques such as transformation, transfection, or transduction. "Transformation", "transfection", and "transduction" generally refer to techniques for introducing foreign (exogenous) nucleic acid sequences into host cells, and thus include methods such as electroporation, microinjection, delivery by gene gun, transduction by retroviral vectors, lentiviral vectors, or adeno-associated viral vectors, lipofection, superfection, etc. The specific method used typically depends on both the type of vector and the type of cell. In the art, suitable methods for introducing nucleic acid sequences and vectors into host cells such as human cells are well known; see, for example, Sambrook et al. (1989), "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; 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. Further conventional methods suitable for preparing expression vectors and introducing them into appropriate host cells are described in detail, for example, in WO2016 / 071758.

[0306] In some examples, it is understood that the host cell contacts the vector system (e.g., viral vector) in vitro or ex vivo, and in some examples, the host cell contacts the vector system (e.g., viral vector) in vivo.

[0307] The term "host cell" includes any cell into which a nucleic acid composition or vector system described herein can be introduced (e.g., transduced). As used herein, when a nucleic acid molecule or vector system is introduced into a cell, this cell may be referred to as a "modified cell". When a nucleic acid molecule or vector is introduced into a host cell, the resulting modified cell is capable of expressing the encoded binding protein (and, for example, appropriately localizing the encoded binding protein for its intended function, e.g., transporting the encoded binding protein to the cell surface).

[0308] The term "modified cell" refers to a genetically altered (e.g., transformed or transfected) cell. The term refers to a particular target cell, but also to the progeny, or potential progeny, of such a cell. In subsequent generations, such progeny may not actually be identical to the parental cell due to mutations or environmental influences that can give rise to a particular modification, but are still included within the scope of the term as used herein.

[0309] Host cells (and thus modified cells) are typically eukaryotic cells, and in particular, human cells (e.g., CD8 + T cells or CD4 + T cells, or T cells such as mixtures thereof). Host cells (and thus modified cells) can be allogeneic cells (e.g., CD8 + T cells or CD4 + T cells, or allogeneic T cells such as mixtures thereof) that refer to cells derived from an individual different from the individual into which they are later administered. In other words, host cells (and thus modified cells) can be T cells isolated from an individual significantly different from the subject being treated.

[0310] A host cell (and thus, a modified cell) can be any cell capable of conferring anti-tumor immunity after TCR gene transfer. 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, hematopoietic stem cells or other progenitor cells, and any other autologous or allogeneic cells or cell lines (e.g., the NK-92 cell line, or a T cell line) that are capable of conferring anti-tumor immunity after TCR gene transfer.

[0311] In the context of the treatment methods described herein, a host cell (and thus, a modified cell) is typically a host cell for administration to an HLA-A * 0201-positive human subject. In this regard, a host cell (and thus, a modified cell) is typically HLA-A * 0201-negative and / or HA-1 H negative. (i.e., does not express either HLA-A * 0201 or HA-1 H ).

[0312] The modified cell is advantageous because it can express a binding protein (i.e., a component of the TCR) encoded by the nucleic acid composition or vector system described herein, resulting in an immunotherapy that specifically targets hematological malignancies and can be used to treat or prevent the recurrence of hematological malignancies after allogeneic stem cell transplantation (allo-SCT) in HLA-A * 0201-positive human subjects. Further details regarding this use are provided below.

[0313] Pharmaceutical composition The nucleic acid composition, vector system, or modified cell described herein can be provided as part of a pharmaceutical composition. Such a composition is advantageous because it can be administered to a human subject having a hematological malignancy after allogeneic stem cell transplantation (allo-SCT) so as to treat or prevent recurrence (e.g., by inducing or enhancing an HA-1 H antigen-targeted immune response).

[0314] The pharmaceutical composition may comprise a nucleic acid composition, a vector system, or a modified cell as described herein, together with a pharmaceutically acceptable excipient, adjuvant, diluent, and / or carrier.

[0315] The composition may, by regulation, contain salts, buffers, preservatives, compatible carriers, adjuvants, and auxiliary immunopotentiators such as cytokines at pharmaceutically acceptable concentrations, and optionally, other therapeutic agents or compounds.

[0316] As used herein, "pharmaceutically acceptable" refers to a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual without causing an undesirable biological effect or interacting in a harmful manner with any of the other components of the pharmaceutical composition in which it is contained, together with the selected nucleic acid composition, vector system, or modified cell.

[0317] An excipient is a natural or synthetic substance formulated with an active ingredient (e.g., a nucleic acid sequence, vector, modified cell, or isolated peptide presented herein) and incorporated for the purpose of bulking up the target formulation or imparting a therapeutic enhancement to the active ingredient in the final dosage form, such as promoting the absorption or solubility of the drug. Excipients can also assist in the manipulation of the active substance of interest during the manufacturing process, for example, by promoting the flowability or non-sticking properties of powders, and in addition, can be useful in supporting in vitro stability, such as preventing degradation over the shelf life. Pharmaceutically acceptable excipients are well known in the art. Therefore, suitable excipients can be readily identified by those skilled in the art. By way of example, suitable pharmaceutically acceptable excipients include water, physiological saline, aqueous dextrose solution, glycerol, ethanol, and the like.

[0318] An adjuvant is a pharmacological agent and / or an immunological agent in a formulation that modifies the effect of other agents. Pharmaceutically acceptable adjuvants are well known in the art. Thus, suitable adjuvants can be readily identified by those skilled in the art.

[0319] A diluent is a diluting agent. Pharmaceutically acceptable diluents are well known in the art. Thus, suitable diluents can be readily identified by those skilled in the art.

[0320] A carrier is non-toxic to the recipient at the dosages and concentrations employed and is compatible with the other ingredients of the formulation. The term "carrier" denotes a natural or synthetic, organic or inorganic ingredient with which the active ingredient is combined to facilitate its application. Pharmaceutically acceptable carriers are well known in the art. Thus, suitable carriers can be readily identified by those skilled in the art.

[0321] Treatment of a subject The pharmaceutical composition HLA-A described herein * It may be advantageous that it can be used to treat or prevent recurrence of hematological malignancies after allogeneic stem cell transplantation (allo-SCT) in HLA-A0201 positive human subjects.

[0322] In one example, a method of treating or preventing recurrence of a hematological malignancy described herein results in the induction or enhancement of an immune response (e.g., a cell-mediated response) in a subject (e.g., an immune response targeted to malignant cells presenting the HA-1 H -HLA-A * 0201 binding peptide).

[0323] The phrase "induction or enhancement of an immune response" refers to an increase in the immune response of a subject (e.g., a cell-mediated immune response such as a T cell-mediated immune response) during or after treatment, as compared to those immune responses before treatment. Thus, "induction or enhancement of an immune response" encompasses any measurable increase in the immune response that is directly or indirectly targeted to the hematological malignancy to be treated (or prevented).

[0324] One of ordinary skill in the art will be well aware of hematological malignancies that can be treated according to the present invention. By way of example, suitable hematological malignancies include leukemia, lymphoma, myelodysplastic syndrome, or myeloma.

[0325] For example, when the hematological malignancy includes leukemia, the leukemia can be acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), mixed phenotype acute leukemia (MPAL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, hairy cell leukemia, or chronic lymphocytic leukemia (CLL).

[0326] As another example, when the hematological malignancy includes lymphoma, the lymphoma can be Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), central nervous system lymphoma, small lymphocytic lymphoma (SLL), CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, mucosa-associated (MALT) extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, precursor B-lymphoblastic lymphoma, immunoblastic large cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or Burkitt lymphoma.

[0327] As a further example, when the hematological malignancy includes myelodysplastic syndrome, the myelodysplastic syndrome can be refractory cytopenia with single lineage dysplasia (refractory anemia, refractory neutropenia, and refractory thrombocytopenia), refractory anemia with ring sideroblasts (RARS), refractory anemia with ring sideroblasts and thrombocytosis (RARS-t), refractory cytopenia with multilineage dysplasia (RCMD), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS), refractory anemia with excess blasts (RAEB), unclassifiable myelodysplasia, or pediatric refractory cytopenia.

[0328] In certain cases, the subject has been previously administered lymphodepleting chemotherapy, such as lymphodepleting chemotherapy comprising cyclophosphamide, fludarabine, antithymocyte globulin, or combinations thereof.

[0329] Typically, the modified cells administered to the subject are allogeneic modified cells.

[0330] As used herein, the terms "treating," "treatment of," and "treatment" are understood to include interventions that are intended to prevent the onset of a condition, disorder, or symptom (i.e., in this case, a hematological malignancy) or alter the disease state. Thus, "treatment" refers to both therapeutic treatment and prophylactic or preventive measures that are aimed at preventing or alleviating (inhibiting) a targeted condition, disorder, or symptom. Thus, "treatment" encompasses a reduction, alleviation, or inhibition of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the amount or concentration of malignant cells, as measured, for example, in a sample obtained from the subject, compared to the amount or concentration of malignant cells prior to treatment. Methods for measuring the amount or concentration of malignant cells include, for example, qRT-PCR and quantification of hematological malignancy-specific biomarkers in a sample obtained from the subject.

[0331] As used herein, the term "subject" refers to an individual, e.g., a human, who has or is at risk of having a specified condition, disorder, or symptom. A subject can be a patient, i.e., a subject in need of treatment according to the present invention. A subject may have been treated for a condition, disorder, or symptom. Alternatively, a subject may not have been treated prior to treatment according to the present invention.

[0332] The compositions described herein can be administered to a subject by any conventional route, including injection or slow infusion over time. Administration can be, for example, by injection, intramuscular, intravascular, intracavitary, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, or transdermal administration.

[0333] The compositions described herein can be in any form suitable for the above-described modes of administration. For example, a composition containing modified cells can be in any form suitable for injection. As a further example, forms suitable for parenteral injection (including subcutaneous, intramuscular, intravascular injection, or infusion) include sterile solutions, suspensions, or emulsions. Alternatively, the route of administration can be by direct injection into the target area, by regional delivery, or by local delivery. Identification of an appropriate dosage of the compositions of the present invention is well within the purview of one of ordinary skill in the art.

[0334] The compositions described herein are a rapid, reliable method of generating a large number of T cells with specificity for an HA-1 H antigenic peptide (e.g., the peptide shown in SEQ ID NO: 10), and are advantageously formulated for use in T cell receptor (TCR) gene transfer. Using TCR gene transfer, modified allogeneic cells suitable for injection can be generated within a few days.

[0335] The compositions described herein are compositions for administration in an effective amount. An "effective amount" is an amount that, alone or in combination with further dosages, provides a desired response (a therapeutic response or a non-therapeutic response). The effective amount used depends, for example, on the therapeutic (or non-therapeutic) purpose, the route of administration, and the patient / subject condition. For example, the dosage of the composition of the present invention suitable for a given patient / subject will be determined by the attending physician (or the person responsible for administering the composition), taking into account various factors known to modify the action of the composition of the present invention, such as the severity and type of the hematological malignancy, body weight, gender, diet, number of administrations, and route of administration, other medications, and other relevant clinical factors. The dosage and schedule may vary according to the patient's / subject's particular condition, disorder, or symptom, and general condition. The effective dosage may be determined by in vitro methods or in vivo methods in some cases.

[0336] The pharmaceutical compositions described herein are advantageously presented in unit dosage form.

[0337] Method for generating TCR Also, HA-1 H A method for generating a binding protein that can specifically bind to a peptide containing an HA-1 antigen and does not bind to a peptide not containing the HA-1 antigen, the method comprising contacting a nucleic acid composition (or vector system) described herein with a cell under conditions such that the nucleic acid composition is incorporated and expressed by the cell is also provided. H The method can be carried out ex vivo or in vitro on host cells. Alternatively, the method may be carried out in vivo, in which case the nucleic acid composition (or vector system) is administered to the subject and the nucleic acid sequence is contacted with the host cells in vivo under conditions such that it is incorporated and expressed by the host cells to generate the binding protein. In one example, the method is not a method of treating the human or animal body.

[0338]

[0339] ​As described elsewhere in this specification, suitable in vivo, in vitro, and ex vivo methods for contacting a nucleic acid sequence (or vector system) with a host cell under conditions such that the nucleic acid sequence (or vector) is incorporated and expressed by the cell are well known.

[0340] General Definitions As used herein, the terms "nucleic acid sequence", "polynucleotide", "nucleic acid", and "nucleic acid molecule" are used interchangeably to refer to an oligonucleotide sequence or polynucleotide sequence. The nucleotide sequence may be of genomic origin, synthetic origin, recombinant origin, double-stranded origin, or single-stranded origin (representing the sense strand or the antisense strand). The term "nucleotide sequence" includes genomic DNA, cDNA, synthetic DNA, and RNA (e.g., mRNA), and analogs of DNA or RNA made, for example, by the use of nucleotide analogs.

[0341] As used herein, an "isolated nucleic acid sequence" or "isolated nucleic acid composition" refers to a nucleic acid sequence that is not in its natural environment when linked to the sequences to which it is naturally associated in its natural environment. In other words, an isolated nucleic acid sequence / composition is not a natural nucleotide sequence / composition, where "natural nucleotide sequence / composition" refers to the entire nucleotide sequence that is in its natural environment and operably linked to all promoters that are naturally associated and also in their natural environment. Such nucleic acids may be part of a vector and / or such nucleic acids or polypeptides may be part of a composition (e.g., a cell lysate), but are still isolated in the sense that such a vector or composition is not part of the natural environment for the nucleic acid or polypeptide. The term "gene" refers to a segment of DNA involved in the production of a polypeptide chain and includes intervening sequences (introns) between individual coding segments (exons), as well as regions preceding and following the coding region ("leader and trailer").

[0342] As used herein, "specifically binds to" or "specific for" refers to the binding of a binding protein (e.g., a TCR receptor) or a binding domain (or a fusion protein thereof) to a target molecule with an affinity of 10 5 M -1 or greater or a K a (i.e., the equilibrium association constant for a particular binding interaction, with units of 1 / M), which is equal to the ratio of the on-rate [k on to the off-rate [k Off for this association reaction), and which means that it does not associate or integrate appreciably with any other molecule or component in the sample. The binding protein or binding domain (or a fusion protein thereof) may be classified as a "high-affinity" binding protein or binding domain (or a fusion protein thereof) or as a "low-affinity" binding protein or binding domain (or a fusion protein thereof). A "high-affinity" binding protein or binding domain refers to a binding protein or binding domain having a K 7 M -1 of at least 10 8 M -1 of at least 10 9 M -1 of at least 10 10 M -1 of at least 10 11 M -1 of at least 10 12 M -1 or of at least 10 13 M -1 . A "low-affinity" binding protein or binding domain refers to a binding protein or binding domain having a K a of at most 10 7 M -1 of at most 10 6 M -1 or of at most 10 5 M -1 . Alternatively, the affinity may be expressed in units of M (e.g., 10 a 10 -5 M to 10-13 M), for a particular binding interaction, the equilibrium dissociation constant (K d ) can also be defined as.

[0343] In certain embodiments, the receptor or binding domain is "enhanced in affinity", which refers to a selected or engineered receptor or binding domain in which binding to the target antigen is more potent than the wild-type (or parental) binding domain. For example, enhanced affinity may be due to a K a (equilibrium association constant) for the target antigen that is higher than that of the wild-type binding domain equilibrium association, a K d (dissociation constant) for the target antigen that is lower than that of the wild-type binding domain dissociation, a k Off off-rate (k Off ) for the target antigen that is lower than that of the wild-type binding domain, or combinations of these. In certain embodiments, to enhance affinity, the TCR can be codon-optimized to enhance expression in a particular host cell, such as an immune system cell, hematopoietic stem cell, T cell, primary T cell, T cell line, K cell, or natural killer T cell (Scholten et al., Clin. Immunol. 119:135, 2006). The T cell can be a CD4+ T cell or a CD8+ T cell.

[0344] As used herein, "HA-1 H antigen" or "HA-1 H peptide antigen" or "HA-1 H containing peptide antigen" (or "minor HA-1 <h>"antigen" or "minor HA-1" H "peptide antigen" or "minor HA-1" H "peptide antigen-containing" or "minor histocompatibility HA-1" H The term "(antigen peptide)" refers to a peptide moiety of the HMHA1 protein that is either naturally produced or synthetically made and is in the range of about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, up to about 20 amino acids in length, contains the R139H substitution polymorphism, and is capable of forming a complex with an MHC (e.g., HLA) molecule, HA-1 H The binding proteins of the present disclosure that are specific for a peptide:MHC (e.g., HLA) complex can specifically bind to such a complex. Exemplary HA-1 H peptide antigens include a peptide having the amino acids VLHDDLLEA (SEQ ID NO: 10), where the histidine in bold in the sequence represents the R139H polymorphism.

[0345] As used herein, "HA-1" H The term "specific binding protein" refers to HA-1 H a peptide antigen (or, e.g., on the cell surface, HA-1 H peptide antigen:HLA complex) that specifically binds and does not bind to an HMHA peptide that does not contain the HA-1 polymorphism (e.g., a peptide containing the amino acid sequence shown in SEQ ID NO: 79) or to an HLA complex containing such an HMHA peptide, and refers to a protein or polypeptide such as a TCR or CAR. H In certain embodiments, the HA-1

[0346] specific binding protein binds to an HA-1-containing peptide (or an HA-1 H peptide:HLA complex) with a K H of less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M, or less than about 10 -13 M of K d or, for example, HA-1 as presented herein, measured by the same assay H exhibited by an exemplary HA-1 specific binding protein as presented herein, such as any of the specific TCRs, binds specifically with an affinity that is equal to, substantially the same as, at least substantially the same as, or greater than this affinity. In certain embodiments, the HA-1 specific binding protein comprises a binding protein of the HA-1 specific immunoglobulin superfamily, or binding portions thereof.

[0347] HA-1 H In the context of antigen presentation, selective binding can be binding by HLA-A * 02:01. In other words, in certain embodiments, a binding protein that "binds specifically to an antigen" is such only if it binds (i.e., is presented by) HLA-A H 02:01 or is in a structural form equivalent to that presented by HLA-A * 02:01. *

[0348] A "non-essential" (or "least important") amino acid residue is a residue that can be changed from the wild-type sequence (e.g., the sequence identified by the SEQ ID NOs herein) without abrogating biological activity, or more preferably, without substantially altering it, whereas an "essential" (or "most important") amino acid residue results in such a change. For example, amino acid residues that are conserved are generally not expected to be amenable to change, except where amino acid residues within the hydrophobic core of a domain can be replaced by other residues having substantially equivalent hydrophobicity without substantially altering activity.

[0349] ​"Conservative amino acid substitution" refers to an amino acid substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. In the art, families of amino acid residues having similar side chains are defined. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, non-essential (or least important) amino acid residues within a protein are preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations are introduced randomly and the resulting mutants are screened for activity to identify mutants that retain activity.

[0350] Calculation of sequence homology or sequence identity (the terms are used interchangeably herein) between sequences is performed as follows.

[0351] To determine the percent identity of two amino acid sequences, or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., for optimal alignment, gaps may be introduced into one or both of the first amino acid or nucleic acid sequence, and the second amino acid or nucleic acid sequence, and non-homologous sequences may be rejected for the purpose of comparison). In preferred embodiments, the length of the reference sequence aligned for comparison is at least 30%, preferably at least 40%, more preferably at least 50%, still more preferably 60%, still more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. Next, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, "identity" of an amino acid or nucleic acid is synonymous with "homology" of an amino acid or nucleic acid). The percent identity between two sequences is a function of the number of identical positions shared by the sequences when considering the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap.

[0352] The comparison of sequences between two arrays and the determination of the percent identity can be achieved using mathematical algorithms. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the BLOSUM 62 matrix or the PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, using the algorithm by Needleman et al. ((1970), J. Mol. Biol., 48:444-453) incorporated into the GAP program within the GCG software package (available at http: / / www.gcg.com). In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the NWSgapdna.CMP matrix, and gap weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6, using the GAP program within the GCG software package (available at http: / / www.gcg.com). A particularly preferred set of parameters (and the set of parameters to be used when the practitioner is unsure which parameters should be applied to determine whether a molecule is within the limits of sequence identity or sequence homology of the present invention) is the BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0353] Alternatively, the percent identity between two amino acid sequences or nucleotide sequences can be determined using the algorithm by Meyers et al. (1989), CABIOS, 4:11-17 incorporated into the ALIGN program (version 2.0) using the PAM120 weighted residue table with a gap length penalty of 12 and a gap penalty of 4.

[0354] The nucleic acid and protein sequences described in this specification can also be used as "query sequences" for performing searches against publicly available databases to identify, for example, other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) by Altschul et al. (1990), J. Mol. Biol., 215:403-410. To obtain nucleotide sequences homologous to the nucleic acid sequences of the present invention, BLAST nucleotide searches can be performed using the NBLAST program with score = 100 and wordlength = 12. To obtain amino acid sequences homologous to the protein molecules of the present invention, BLAST protein searches can be performed using the XBLAST program with score = 50 and wordlength = 3. For purposes of comparison, gapped BLAST, as described in Altschul et al. (1997, Nucl. Acids Res., 25:3389-3402), can be used to obtain gapped alignments. When utilizing the BLAST and gapped BLAST programs, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. See <http: / / www.ncbi.nlm.nih.gov>.

[0355] The polypeptide molecules and nucleic acid molecules described herein may have an amino acid sequence or a nucleic acid sequence that is fully or substantially identical to the sequence identified by the sequence number. As used herein, the terms "fully identical" or "substantially identical" mean that the first amino acid sequence or the first nucleotide sequence and the second amino acid sequence or the second nucleotide sequence have a sufficient number or a minimal number of identical or equivalent (e.g., with similar side chains) amino acid residues or nucleotides such that the second amino acid sequence or the second nucleotide sequence has a general structural domain or a general functional activity. In other words, an amino acid sequence or a nucleic acid sequence having one or several (e.g., two, three, four, etc.) amino acid substitutions or nucleic acid substitutions as compared to the corresponding sequence identified by the sequence number may be fully or substantially identical to the sequence identified by the sequence number, provided that they retain the essential functionality. In such examples, one or several (e.g., two, three, four, etc.) amino acid substitutions or nucleic acid substitutions may be conservative substitutions. For example, as used herein, an amino acid sequence or a nucleotide sequence containing a general structural domain that has at least about 60%, or 65% identity, is likely to have 75% identity, and is more likely to have 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity is defined as being fully or substantially identical.

[0356] TCR sequences are defined according to IMGT. For further details, see the references by LeFranc in this specification, namely, [1] Lefranc M.-P., "Unique database numbering system for immunogenetic analysis", Immunology Today, 18:509 (1997); [2] Lefranc M.-P., "The IMGT unique numbering for immunoglobulins, T cell Receptors and Ig-like domains", The immunologist, 7, 132-136 (1999); [3] Lefranc M.-P. et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", Dev. Comp. Immunol., 27, 55-77 (2003); [4] Lefranc M.-P. et al., "IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains", Dev. Comp. Immunol., 2005, 29, 185-203, PMID: 15572068.

[0357] Aspects of the present invention are supported by the following non-limiting examples.

Example

[0358] The introduction of TCR genes is an attractive strategy to modify T cells to have well-defined specificity in a short time. In recent years, the effectiveness of TCR introduction has been demonstrated in patients with melanoma or synovial sarcoma treated with TCR-modified autologous T cells. To engineer T cells that exert selective GvL without GvHD, the inventors prioritize the introduction of HA-1-TCR. To expand the applicability of adoptive T cell therapy in hematological malignancies, the inventors initiated a clinical study using T cells specific for the virus into which HA-1-TCR was introduced. The inventors sequenced the TCR chains of three HA-1-specific T cell clones, M2, M7, and FK47.83 (Table 4).

[0359]

Table 4

[0360] Previously, as already described, the inventors again observed that all three HA-1-specific T cell clones expressed a beta chain (TRBV7-9) with a similar V region (4). LZRS retroviral constructs encoding the TCR alpha and TCR beta chains of M2 and M7 were generated. The HA-1-TCRβ chain of M7 was also cloned into the retroviral vector, MP71. The TCR alpha chain was linked via an IRES to the marker eGFP, and the TCR beta chain was linked via an IRES to the marker, truncated nerve growth factor receptor (NGF-R). Both HA-1-TCR chains were also linked to the T2A sequence and expressed in the pLZRS vector in combination with NGF-R or in the MP71 vector without a marker gene. An MP71 construct encoding a codon-optimized and cysteine-modified HA-1-TCR was also generated.

[0361] Based on the low cell surface expression of HA-1-TCR after gene transfer, as described by the inventors in three ways, the inventors explored whether this low expression was due to the inability of TCR chains to efficiently pair with each other or due to the intrinsic properties of the TCR chains. Individual HA-1-TCRα chains and HA-1-TCRβ chains, combined with 17 different TCRα chains and TCRβ chains, were transduced (td) into TCRαβ-deficient J76 cells, and anti-TCRαβ mAb was used to measure surface expression in TCR cells. In Figure 1A, TCR cell surface expression is shown for HA-1-TCRαβ, CMV B7 -TCRαβ, HA-2-TCRαβ, CMV A2 -TCRαβ, and combinations of mixed TCRα chains / TCRβ chains. HA-2-TCRαβ td J76 cells (MFI 330) and CMV A2 -TCRαβ td J76 cells (MFI 274) demonstrated high TCR expression. TCR expression by HA-1-TCRαβ td J76 cells (MFI 129) was low compared to HA-2-TCRαβ td J76 cells. Furthermore, no recovery of TCR cell surface expression was observed when combinations of HA-1-TCRβ with HA-2-TCRα or CMV A2 -TCRα were transduced into J76 cells (Figure 1A). Additionally, no recovery of TCR expression was observed in any of the transductions of HA-1-TCRβ chains with one of the other 14 TCRα chains (data not shown). In contrast, HA-1-TCRα chains combined with HA-2-TCRβ chains or CMV A2 -TCRβ chains resulted in TCR cell surface expression equivalent to that of the parental HA-2-TCR complex and CMV A2 -TCR complex, indicating that the reduction in HA-1-TCR cell surface expression was due to HA-1-TCRβ chains rather than HA-1-TCRα chains.

[0362] HA-1-TCRβ also maintained low cell surface expression of the TCR with all 14 other test TCR α-chains. Therefore, the inventors concluded that the low cell surface expression of HA-1-TCR was not due, in particular, to inefficient pairing of the HA-1-TCR α-chain with the HA-1-TCRβ chain. To rule out the case where the LZRS vector used to introduce the TCR chains selectively caused low HA-1-TCR expression, the HA-1-TCRβ gene was inserted into the MP71 vector, which is described as mediating high transgene expression. As can be seen in Figure 1A, the use of the MP71 vector encoding the HA-1-TCRβ chain did not improve the cell surface expression of HA-1-TCR, indicating that the low cell surface expression of HA-1-TCR by td J76 cells was not due to vector-specific properties. To explore whether the introduction of the HA-1-TCRβ chain resulted in low cell surface expression due to the sequence-specific properties of the always identical variable region of the HA-1-TRBV7-9 chain, CMV B7 -TCRβ with the same variable TRBV7-9 as the HA-1-TCRβ chain but with a completely different CDR3 region was analyzed for cell surface expression. As shown in Figure 1A, the parental CMV B7 -TCR complex confirmed low cell surface expression equivalent to that of the parental HA-1-TCR complex. This low TCR expression was also not restored when the CMV B7 -TCRβ chain was combined with the HA-2-TCRα chain or the CMV A2 -TCRα chain, whereas the CMV A2 -TCRα chain combined with the HA-2-TCRβ chain or the CMV B7 -TCRβ chain resulted in high TCR cell surface expression equivalent to that of the parental HA-2-TCR or CMV A2 -TCR. These results imply that the low expression of the HA-1-TCRβ chain and the CMV B7 -TCRβ chain was due to the sequence-specific properties of the variable region. These data together indicate that the low cell surface expression of HA-1-TCR is due to the intrinsic properties of the HA-1-TCRβ chain.

[0363] To confirm that the suboptimal cell surface expression of HA-1-TCR after gene transfer was due to the endogenous properties of the TCRβ chain, the cell surface expression of HA-1-TCR by different parental HA-1-specific T cell clones, as well as the mRNA levels of HA-1-TCRα chain and HA-1-TCRβ chain, were determined. As supported by Figure 1B, FACS analysis with an antibody directed against the TCRαβ / CD3 complex showed that HA-1-specific T cell clones as well as CMV B7 specific T cell clones expressed lower levels of the TCR-CD3 complex on the cell surface compared to HA-2-specific T cell clones and CMV A2 specific T cell clones. However, HA-1-specific T cell clones were stained with equal intensity for their respective tetramers compared to other T cell clones (Figure 1C) and were fully functional T cells based on cytokine production and cytotoxicity (data not shown). To rule out the possibility that the low TCRαβ expression was due to low transcriptional activity, the TCRα mRNA level and TCRβ mRNA level of HA-1-specific T cell clones were determined and compared with the TCRα mRNA level and TCRβ mRNA level of other T cell clones. As supported by Figure 1D, no significant difference in the expression level of HA-1-TCRα mRNA or HA-1-TCRβ mRNA was detected compared to other T cell clones. In conclusion, parental HA-1-specific T cell clones exhibit low cell surface expression of TCR despite normal TCRαβ mRNA levels. These results indicate that the low HA-1-TCR expression observed in HA-1-TCR-transduced T cells, since the TCR expression of parental HA-1-specific T cell clones is already low, is an endogenous feature of HA-1-TCR.

[0364] To enable improvement of HA-1-TCR expression after gene transfer, the present inventors determined a specific region of HA-1-TCRβ that contributes to the low TCR cell surface expression, and explored whether modification of this region could improve the expression of HA-1-TCR (6). For this purpose, the sequences of several TCRβ chains belonging to the TRBV7 variable domain family, which are known to exhibit high cell surface expression after gene transfer, namely, HA-2-TRBV7-8, JBBun-TRBV7-6, and 10G5-TRBV7-1, were used for HA-1 and CMV B7 -Aligned against the sequence of -TRBV7-9. As depicted in Figure 2A, in total, more than 30 common differences are scattered throughout the variable region that is 309 amino acids (aa) in length, among which 9 nucleotide differences formed clusters within the 18-nucleotide-long CDR1 region. Based on these results, the inventors hypothesized that, among HA-1-TCR TRBV7-9, mainly the CDR1 region might affect the cell surface expression of the HA-1-TCR β-chain. To study this, different constructs were made in which the CDR1 region of HA-1-TCRβ was exchanged with the CDR1 region of HA-2-TCRβ, and different constructs with the reverse exchange were also made. Transduced J76 cells with modified HA-1-TCR and modified HA-2-TCR were analyzed for TCR cell surface expression using anti-TCRαβ specific mAb. As supported by Figure 2B, the exchange of the HA-1-TCRβ CDR1 region with the CDR1 region of HA-2-TCRβ did not result in a significant improvement in the cell surface expression of TCR on J76 cells. Similarly, the exchange of the HA-2-TCRβ CDR1 region with the HA-1-TCRβ CDR1 region did not result in a significant reduction in the cell surface expression of TCR on J76 cells. These results indicate that the CDR1 region is not solely responsible for the low TCR cell surface expression. In addition, the inventors confirmed that the exchange of the CDR1 region of HA-1-TCRβ with the CDR1 region of HA-2-TCRβ by transduction of different modified TCR chains into virus-specific T cells resulted in a complete inactivation of HA-1-specific IFN-γ production (Figure 2C), thus also exemplifying that the HA-1-TCRβ CDR1 region is extremely important for the specificity of HA-1. However, the exchange of the HA-2-TCRβ CDR1 region with the HA-1-TCRβ CDR1 region confirmed that the exchange of this region alone is not sufficient for the introduction of the specificity of HA-1. The exchange of both the CDR1 region and the CDR3 region of HA-2-TCRβ with these regions of HA-1-TCRβ resulted in the specificity of HA-1 (Figure 2C).However, since only very low recognition of endogenously processed HA-1 (LCL-BDV) was observed in these td T cells, they were still inefficient compared to the parental HA-1-TCR td T cells (Figure 2C). Stated as a conclusion, the HA-1-TCRβ CDR1 region is extremely important for the specificity of HA-1, but is not sufficient for the specificity of HA-1. In addition, the CDR3 region, and also the CDR3 region of the HA-1-TCRβ chain, are also extremely important for the specificity of HA-1.

[0365] Based on the similar TRBV7-9 chain usage (4) by all of the HA-1-specific T cell clones, the inventors also explored whether a combination of chimeric TCRs of M2 TCR and M7 TCR is also HA-1-specific. A combination of chimeric TCRs of two HA-1 TCRs and one CMV-B7 TCR (all with the TRB7-9 chain) was transduced into peripheral T cells, and the functionality of the introduced chimeric TCRs was compared with that of the original HA-1 TCR combination. As shown in Table 5, the results indicate that the M7 beta chain can form a functional HA-1 TCR complex with either * M7 TRAV25 * 01 or M2 TRAV13-1 * 02, but not with CMV TRAV17 * 01. In addition, M2 TRAV13-1 * 02 and M7 TRAV25

[0366]

Table 5

[0367] Since the expression of HA-1-TCR could not be improved by modification of the specific sequence of the HA-1-TCR β chain, other strategies described for improving the cell surface expression of TCR by gene-introduced TCR were explored. The inventors studied whether codon optimization or incorporation of cysteine residues (7) within the constant domains of both the HA-1-TCR α chain and the HA-1-TCR β chain would result in potent HA-1-specific T cells after gene introduction. The inventors analyzed the cell surface expression of HA-1-TCR after introduction of different constructs into virus-specific T cells known to possess weakly competing endogenous TCRs (weak competitors; pp50 VTE-specific T cells, Figure 3) with respect to cell surface expression, and virus-specific T cells known to possess strongly competing endogenous TCRs (strong competitors; EBNA3A FLR-specific T cells, Figure 3). As evidenced in Figure 3A, after introduction of the unmodified HA-1-TCR complex and using tetramers, introduction of the unmodified HA-1-TCR complex into weak competitor T cells resulted in 40% HA-1 tetramer-positive T cells, whereas no appreciable HA-1-TCR expression could be measured after introduction of the unmodified HA-1-TCR complex into strong competitor T cells. Incorporation of cysteine residues into both HA-1-TCR chains improved the expression of HA-1-TCR, especially in virus-specific T cells, which are strong competitors. As expected, incorporation of cysteine residues in only one of the two HA-1-TCR chains markedly abrogated the expression of HA-1-TCR. In addition, codon optimization also improved HA-1-TCR expression in both virus-specific T cells, which are weak competitors, and virus-specific T cells, which are strong competitors.However, since T cells transfected with codon-optimized HA-1-TCR α-chain in combination with wild-type HA-1-TCR β-chain showed a similar improvement in the percentage of HA-1-tetramer-positive T cells compared to T cells transfected with both codon-optimized HA-1-TCR α-chain and codon-optimized HA-1-TCR β-chain, it was considered that the increase in HA-1-TCR expression was due to the improvement in the expression of HA-1-TCR α-chain rather than the improvement in the expression of HA-1-TCR β-chain. In both virus-specific T cells, which are weak competitors, and virus-specific T cells, which are strong competitors, the combination of codon-optimized and cysteine-modified HA-1-TCR α-chain with cysteine-modified HA-1-TCR β-chain most strikingly improved the expression of HA-1-TCR (Figure 3A).

[0368] To examine whether the improvement in HA-1-TCR expression resulted in an improvement in HA-1-specific functionality, HA-1-TCR td of virus-specific T cells, which are weak competitors, and virus-specific T cells, which are strong competitors, were examined against target cells loaded with HA-1 peptide and target cells that endogenously express the HA-1 antigen (Figure 3B). In virus-specific T cells, which are weak competitors, the combination of codon-optimized and cysteine-modified HA-1-TCR α-chain with cysteine-modified HA-1-TCR β-chain (combination #8) supported the highest IFN-γ production against target cells loaded with peptide and target cells presenting endogenously processed HA-1 antigen. Most notably, in strong competitor T cells, this TCR combination was the only T cell capable of eliciting a significant HA-1-specific reactivity. In conclusion, the combination of codon optimization of the HA-1-TCR α-chain with cysteine modification of the HA-1-TCR chains resulted in efficient HA-1-TCR expression after gene transfer and robust HA-1-specific functionality even in strong competitor T cells.

[0369] To confirm the generality of these data, a single retroviral vector encoding both unmodified or codon-optimized and cysteine-modified HA-1-TCRα chain and HA-1-TCRβ chain, linked to a self-cleaving 2A sequence derived from picornavirus, was transduced into polyclonal, peripheral CD8 + T cells, as well as other, weak competitor T cells and strong competitor T cells, and examined for cell surface expression of HA-1-TCR (Figure 4A). Also, the HA-1-TCRβ chain was codon-optimized, but we did not observe an improvement in cell surface expression of the codon-optimized HA-1-TCRβ chain, justifying that the mRNA stability of the TCRβ chain does not negatively affect the expression of the TCRα chain. Thus, transduction with the modified HA-1-TCR resulted in the most efficient cell surface expression in both weak competitor T cells and strong competitor T cells. Polyclonal CD8 + T cells, similar to strong competitor T cells, also demonstrated significant HA-1-TCR cell surface expression after introduction of the modified HA-1-TCR (Figure 4A).

[0370] To investigate whether this improvement in HA-1-TCR cell surface expression correlates with clinically relevant HA-1-specific functionality, T cells of the weak competitor phenotype and T cells of the strong competitor phenotype transduced with unmodified HA-1-TCR or codon-optimized and cysteine-modified HA-1-TCR were analyzed for HA-1-specific cytotoxic activity (Figure 4B) and IFN-γ production (Figure 4C). Weak competitor T cells transduced with unmodified HA-1-TCR exerted HA-1-specific cytotoxic reactivity and production of IFN-γ against AML and ALL, whereas introduction of the modified TCR enhanced HA-1-specific reactivity (Figure 4B and Figure 4C, respectively). In addition, strong competitor T cells transduced with the modified HA-1-TCR were HA-1 + It was possible to confirm a marked cytotoxic activity and IFN-γ production directed against malignant cells (Figure 4B and Figure 4C, respectively). Stated as a conclusion, these results confirm the generality of the improvement of HA-1-TCR expression by the modified HA-1-TCR introduced into both T cells with a weak competitor phenotype and T cells with a strong competitor phenotype, as well as polyclonal CD8+ T cells, and thus support that the inventors can generate redirected, potent HA-1-specific T cells.

[0371] For use in clinical treatment, the introduced TCR must be encoded by a retroviral construct without potentially immunogenic marker genes. Thus, the inventors constructed an MP71 vector encoding the modified HA-1-TCRα chain and the modified HA-1-TCRβ chain but without marker genes, and analyzed whether this clinically useful vector transduced weak competitor T cells (Figure 5) and strong competitor T cells (Figure 5) would support a similar improvement in anti-leukemia reactivity (8). One week after transduction, IFN-γ ELISA was used to analyze weak competitor T cells and strong competitor T cells for HA-1-specific reactivity against malignant target cells (Figure 5). The transduction efficiencies of the pLZRS vector and the MP71 vector were confirmed to be 15 and 2%, respectively, based on NGF-R or HA-1 tetramer staining. While sufficient recognition of malignant cells by weak competitor T cells transduced with unmodified HA-1-TCR or weak competitor T cells transduced with modified HA-1-TCR was equivalent (Figure 5), strong competitor T cells transduced with modified HA-1-TCR supported a marked improvement in IFNγ production compared to T cells transduced with unmodified HA-1-TCR against AML target cells and ALL target cells. Stated as a conclusion, TCR introduction by codon-optimized and cysteine-modified HA-1-TCR resulted in efficient expression of the introduced HA-1-TCR and robust HA-1-specific functionality against clinically relevant target cells in both weak competitor T cells and strong competitor T cells.

[0372] Based on previous results, the inventors investigated whether this procedure could result in a rapid procedure for manipulating a therapeutically relevant number of pure, virus-specific T cells transduced with HA-1-TCR as a result of scaling up for clinical purposes. After the entire procedure, to obtain the number of therapeutic cells, donor leukocytes were incubated in the donor with one or two Streptamers consisting of the relevant CMV peptide-HLA complex and EBV peptide-HLA complex for which there is a large T cell population against them. For this purpose, the inventors used PBMC 1×10 derived from leukapheresis products of 4 healthy individuals, donors JBC, UPB, UHO, and UBQ 9 Four test procedures were performed using [number of items] (Figure 6). White blood cells were incubated with the relevant Streptamer and purified using CliniMACS (Figures 6A - 6D). Immediately after isolation, the T cells were incubated with D - biotin and analyzed for purity using flow cytometry. As depicted in Figures 6A - 6D, all positive fractions contained ≥60% virus - specific T cells even when the starting material had low - frequency virus - specific T cells (Figure 6D). For all four test procedures, the recovery rate of virus - specific T cells present in the starting material by the positive fraction was approximately 60%. After isolation by CliniMACS, the positive fractions were cultured in T - cell medium containing irradiated autologous feeders (1:5 ratio) and cytokines. While a part of the Streptamer - enriched cell line was not transduced, 2 - 3 days after isolation, the cell line was transduced with a GMP - grade retroviral supernatant encoding HA - 1 - TCR, produced by Eufets (Germany), into the largest fraction of the cell line. After an additional culture period of 8 - 12 days, the transduced T cells were analyzed for transduction efficiency and purity using HA - 1 and virus tetramers. All four Streptamer - enriched cell lines that were not transduced were ≥97% pure as measured by the virus tetramer (Figures 6E - 6H). The transduction efficiency of the four Streptamer - enriched cell lines transduced with HA - 1 - TCR ranged between 22.5% - 54.2% (Figures 6E - 6H). Among the virus - specific T cells transduced with HA - 1 - TCR, the T cells positively stained by the HA - 1 tetramer mainly expressed HA - 1 - TCR and, due to competition for cell - surface expression, expressed a reduced level of virus - TCR. At the end of the culture period (14 days after isolation), all T - cell products were harvested and viable cells were counted. The test procedures JBC, UPB, and UHO resulted in ≥15×10 6 cells of highly pure antigen - specific T cells. The test procedure UBQ, which had low - frequency virus - specific T cells in the starting material, resulted in 2×10 6 These results demonstrated that using the GMP-grade isolation method, virus-specific T cells can be efficiently concentrated and transduced from thawed PBMC materials with high recovery rates.

[0373] HA-1-specific functionality was examined in three of the virus-specific T cells (JBC, UHO, UBQ) transduced with HA-1-TCR in a peptide titration assay by measuring IFN-γ production. All three transduced virus-specific T cell lines demonstrated HA-1-specific, dose-dependent IFN-γ production equivalent to that of the HA-1-specific control T cell clone. In addition, HA-1-specific IFN-γ production by non-td T cells was not observed. The virus-specific T cells transduced with HA-1-TCR were able to recognize primary malignant leukemia cells presenting endogenously processed HA1 H antigen. To investigate whether virus-specific T cells transduced with HA-1-TCR could recognize primary ALL cells presenting HA1 H pos or HA1 H neg presented by HLA-A2 pos which is HA1 pos primary ALL cells were examined. As observed in Figure 7B, untransduced virus-specific T cells did not, but all virus-specific T cell lines transduced with HA-1-TCR were able to produce IFN-γ after stimulation with primary ALL cells, whereas IFN-γ was not produced after stimulation with HA-1 neg primary ALL cells. Both the virus-specific T cell lines transduced with HA-1-TCR and untransduced virus-specific T cells produced IFN-γ after stimulation with T2 cells pulsed with viral peptides. In addition, all four virus-specific T cells transduced with HA-1-TCR demonstrated HA-1-specific cytotoxic reactivity against T2 cells pulsed with viral peptides or HA-1 peptides, or HA1 H pos or HA1 H neg is HLA-A2 pos The HA-1 specific cytotoxic reactivity against primary ALL cells and primary AML cells was examined (Figure 7C). The results showed that virus-specific T cells transduced with HA-1-TCR recognized HLA-A2 pos HA1 H 1 pos confirmed efficient lysis of primary ALL samples and primary AML samples. In addition, virus-specific T cells transduced with HA-1-TCR showed cytotoxic reactivity equivalent to that of untransduced virus-specific T cells against T2 cells pulsed with viral peptides. These results confirm the feasibility of generating HA-1-TCR modified T cells with potent anti-leukemia reactivity in a reproducible manner using GMP grade manufacturing processes.

[0374] Recently, it has been confirmed that patients treated with CD19 CAR modified T cells derived from patients after allogeneic SCT do not induce GvHD. Therefore, the inventors examined whether polyclonal CD8+ T cells could be transduced to efficiently express HA1-TCR on the cell surface. For this purpose, the inventors isolated CD8+ T cells from healthy individuals by MACS and transduced them with a GMP grade retroviral supernatant encoding HA1-TCR (codon-optimized and cysteine-modified) 2 days after specific stimulation with PHA in IL-2 supplemented medium. Transduction of polyclonal CD8+ T cells resulted in 50% HA1-TCR positive T cells, and the modified T cells recognized HA1 H expressing, diverse, different HLA-A * HA1H positive target cells (EBV-MRJ, U266, AML3) very efficiently, as evidenced by high IFN-γ production after stimulation with 0201+ target cells, whereas HLA-A2 H 0201+ target cells (EBV-IZA, EBV-JY, AML2) that do not express immunogenic HA1 * were not recognized (Figure 8).

[0375] In addition, since these HA-1 TCR-engineered CD8+ T cells effectively mediated an anti-leukemia response in a multiple myeloma xenograft model (Figure 9), these HA-1 TCR-engineered T cells are highly antitumor-reactive in vitro and in vivo, which is evidence. Therefore, these data indicate that patients with relapsed or refractory hematological malignancies can be effectively treated with these potent HA1-TCR-modified T cells.

[0376] Assay setup for Figures 10 and 11: Two different HA1-H-specific TCRs (TCR LUMC and TCR FHCRC from WO2018058002A1) were transduced into CD8+ T cells of two healthy HLA-A * 02:01+ donors that are homozygous for the HA-1R variant. The TRBV-CDR3-TRBJ and TRAV-CDR3-TRBJ of FHCRC and LUMC with the mouse alpha C region and beta C region were cloned into the pES.12-6 vector (see SEQ ID NO: 89 and SEQ ID NO: 90 below).

[0377] Thereafter, the transduced cells were sorted and expanded for mouse Cβ+CD8+ cells (rapid expansion protocol).

[0378] To analyze functional avidity, T2 cells were loaded with increasing concentrations of HA-1H peptide (VLHDDLLEA (SEQ ID NO: 10), 10 -12 M to 10 -5 M), incubated at 37°C for 1-2 hours, washed with PBS, and resuspended in culture medium. The transduced T cells were co-cultured with the peptide-loaded T2 cells at an effector-to-target ratio of 2:1 (20,000 effector cells per 96-well). IFN-γ in the supernatant was measured by ELISA 20 hours after co-culture.

[0379] To analyze the non-specific recognition of the HA-1R peptide, the same effector cell preparation as described above was co-cultured with increasing concentrations of the HA-1R peptide (VLRDDLLEA (SEQ ID NO: 79), 10 -8 M to 10 -5 M). Peptide loading, co-culture, and readout were performed as described above.

[0380] Nucleic acid and amino acid sequences of interest: SEQ ID NO: 1 (amino acid sequence of CDR3 of the Vα domain of HA-1 H TCR M7):

Chem.

[0381] SEQ ID NO: 2 (amino acid sequence of CDR3 of the Vα domain of HA-1 H TCR M2):

Chem.

[0382] SEQ ID NO: 3 (amino acid sequence of CDR3 of the Vα domain of HA-1 H TCR FK47.83):

Chem.

[0383] SEQ ID NO: 4 (amino acid sequence of CDR3 of the Vβ domain of HA-1 H TCR M7):

Chem.

[0384] SEQ ID NO: 5 (amino acid sequence of CDR3 of the Vβ domain of HA-1 H TCR M2):

Chem.

[0385] Sequence number 6 (HA-1 H Amino acid sequence of CDR3 of the Vβ domain of TCR FK47.83):

Chem.

[0386] Sequence number 7 (HA-1 H TCR M7, HA-1 H TCR M2, or HA-1 H Amino acid sequence of CDR1 of the Vβ domain of TCR FK47.83):

Chem.

[0387] Sequence number 8 (HA-1 H TCR M2 or HA-1 H Amino acid sequence of CDR1 of the Vα domain of TCR FK47.83):

Chem.

[0388] Sequence number 9 (HA-1 H TCR M2 or HA-1 H Nucleic acid sequence of CDR1 of the Vα domain of TCR FK47.83):

Chem.

[0389] Sequence number 10 (HA-1 H Amino acid sequence for the antigen):

Chem.

[0390] Sequence number 11 (HA-1 H Nucleic acid sequence of the CDR3 of the Vα domain of TCR M7:

Chem.

[0391] SEQ ID NO: 12 (HA-1 H Codon-optimized nucleic acid sequence of the CDR3 of the Vα domain of TCR M7:

Chem.

[0392] SEQ ID NO: 13 (HA-1 H Nucleic acid sequence of the CDR3 of the Vβ domain of TCR M7:

Chem.

[0393] SEQ ID NO: 14 (HA-1 H Codon-optimized nucleic acid sequence of the CDR3 of the Vβ domain of TCR M7:

Chem.

[0394] SEQ ID NO: 15 (HA-1 H TCR M7, HA-1 H TCR M2, or HA-1 H Nucleic acid sequence of the CDR1 of the Vβ domain of TCR FK47.83:

Chem.

[0395] SEQ ID NO: 16 (HA-1 H TCR M7; HA-1 H TCR M2, or HA-1 H Codon-optimized nucleic acid sequence of the CDR1 of the Vβ domain of TCR FK47.83:

Chem.

[0396] Sequence number 17 (HA-1 H Amino acid sequence of the Vα (VJ) domain of TCR M7): [Chemical formula]

[0397] Sequence number 18 (HA-1 H Amino acid sequence of the Vβ (VDJ) domain of TCR M7): [Chemical formula]

[0398] Sequence number 19 (HA-1 H Nucleic acid sequence of the Vα (VJ) domain of TCR M7): [Chemical formula]

[0399] Sequence number 20 (HA-1 H Codon-optimized nucleic acid sequence of the Vα (VJ) domain of TCR M7): [Chemical formula]

[0400] Sequence number 21 (HA-1 H Nucleic acid sequence of the Vβ (VDJ) domain of TCR M7): [Chemical formula]

[0401] Sequence number 22 (HA-1 H Codon-optimized nucleic acid sequence of the Vβ (VDJ) domain of TCR M7): [Chemical formula]

[0402] Accession number 23 (HA-1 H Nucleic acid sequence of CDR3 of the Vα domain of TCR M2):

Chemical formula

[0403] Accession number 24 (HA-1 H Codon-optimized nucleic acid sequence of CDR3 of the Vα domain of TCR M2):

Chemical formula

[0404] Accession number 25 (HA-1 H Nucleic acid sequence of CDR3 of the Vβ domain of TCR M2):

Chemical formula

[0405] Accession number 26 (HA-1 H Codon-optimized nucleic acid sequence of CDR3 of the Vβ domain of TCR M2):

Chemical formula

[0406] Accession number 27 (HA-1 H TCR M2 or HA-1 H Codon-optimized nucleic acid sequence of CDR1 of the Vα domain of TCR FK47.83):

Chemical formula

[0407] Accession number 28 (HA-1 H TCR M2 or HA-1 H Amino acid sequence of the CDR2 of the Vα domain of TCR FK47.83):

Chem.

[0408] SEQ ID NO: 29 (HA-1 H Amino acid sequence of the Vα (VJ) domain of TCR M2):

Chem.

[0409] SEQ ID NO: 30 (HA-1 H Amino acid sequence of the Vβ (VDJ) domain of TCR M2):

Chem.

[0410] SEQ ID NO: 31 (HA-1 H Nucleic acid sequence of the Vα (VJ) domain of TCR M2):

Chem.

[0411] SEQ ID NO: 32 (HA-1 H Codon-optimized nucleic acid sequence of the Vα (VJ) domain of TCR M2):

Chem.

[0412] SEQ ID NO: 33 (HA-1 H Nucleic acid sequence of the Vβ (VDJ) domain of TCR M2):

Chem.

[0413] SEQ ID NO: 34 (HA-1 H Codon-optimized nucleic acid sequence of TCR M2 for the Vβ (VDJ) domain: [Chem.]

[0414] SEQ ID NO: 35 (HA-1 H Nucleic acid sequence of TCR FK47.83 for CDR3 of the Vα domain: [Chem.]

[0415] SEQ ID NO: 36 (HA-1 H Codon-optimized nucleic acid sequence of TCR FK47.83 for CDR3 of the Vα domain: [Chem.]

[0416] SEQ ID NO: 37 (HA-1 H Nucleic acid sequence of TCR FK47.83 for CDR3 of the Vβ domain: [Chem.]

[0417] SEQ ID NO: 38 (HA-1 H Codon-optimized nucleic acid sequence of TCR FK47.83 for CDR3 of the Vβ domain: [Chem.]

[0418] SEQ ID NO: 39 (HA-1 H TCR M2 or HA-1 H Nucleic acid sequence of TCR FK47.83 for CDR2 of the Vα domain: [Chem.]

[0419] SEQ ID NO: 40 (HA-1 H TCR M2 or HA-1 H Codon-optimized nucleic acid sequence for the CDR2 of the Vα domain of TCR FK47.83):

Chemical formula

[0420] SEQ ID NO: 41 (HA-1 H Amino acid sequence for the Vα (VJ) domain of TCR FK47.83):

Chemical formula

[0421] SEQ ID NO: 42 (HA-1 H Amino acid sequence for the Vβ (VDJ) domain of TCR FK47.83):

Chemical formula

[0422] SEQ ID NO: 43 (HA-1 H Nucleic acid sequence for the Vα (VJ) domain of TCR FK47.83):

Chemical formula

[0423] SEQ ID NO: 44 (HA-1 H Codon-optimized nucleic acid sequence for the Vα (VJ) domain of TCR FK47.83):

Chemical formula

[0424] SEQ ID NO: 45 (HA-1 H Nucleic acid sequence for the Vβ (VDJ) domain of TCR FK47.83):

Chemical formula

[0425] Sequence number 46 (HA-1 H Codon-optimized nucleic acid sequence for the Vβ (VDJ) domain of TCR FK47.83: [Chemical formula]

[0426] Sequence number 47 (HA-1 H Amino acid sequence for the Vα (VJ) domain and the constant domain of TCR M7: [Chemical formula]

[0427] Sequence number 48 (HA-1 H Amino acid sequence for the Vα (VJ) domain and the constant domain (mouse) of TCR M7: [Chemical formula]

[0428] Sequence number 49 (HA-1 H Nucleic acid sequence for the Vα (VJ) domain and the constant domain of TCR M7: [Chemical formula]

[0429] Sequence number 50 (HA-1 H Codon-optimized nucleic acid sequence for the Vα (VJ) domain and the constant domain of TCR M7: [Chemical formula]

[0430] Sequence number 51 (HA-1 H Codon-optimized nucleic acid sequence for the Vα (VJ) domain and the constant domain (mouse) of TCR M7: [Chemical formula]

[0431] Accession number 52 (HA-1 H Amino acid sequences of the Vβ (VDJ) domain and the constant domain of TCR M7: [Chemical formula]

[0432] Accession number 53 (HA-1 H Amino acid sequences of the Vβ (VDJ) domain and the constant domain (mouse) of TCR M7: [Chemical formula]

[0433] Accession number 54 (HA-1 H Nucleic acid sequences of the Vβ (VDJ) domain and the constant domain of TCR M7: [Chemical formula]

[0434] Accession number 55 (HA-1 H Codon-optimized nucleic acid sequences of the Vβ (VDJ) domain and the constant domain of TCR M7: [Chemical formula]

[0435] Accession number 56 (HA-1 H Codon-optimized nucleic acid sequences of the Vβ (VDJ) domain and the constant domain (mouse) of TCR M7: [Chemical formula]

[0436] Accession number 57 (HA-1 H Amino acid sequences of the Vα (VJ) domain and the constant domain of TCR M2:

Chem.

[0437] SEQ ID NO: 58 (HA-1 H Amino acid sequences of the Vα (VJ) domain and the constant domain (mouse) of TCR M2:

Chem.

[0438] SEQ ID NO: 59 (HA-1 H Nucleic acid sequences of the Vα (VJ) domain and the constant domain of TCR M2:

Chem.

[0439] SEQ ID NO: 60 (HA-1 H Codon-optimized nucleic acid sequences of the Vα (VJ) domain and the constant domain of TCR M2:

Chem.

[0440] SEQ ID NO: 61 (HA-1 H Codon-optimized nucleic acid sequences of the Vα (VJ) domain and the constant domain (mouse) of TCR M2:

Chem.

[0441] SEQ ID NO: 62 (HA-1 H Amino acid sequences of the Vβ (VDJ) domain and the constant domain of TCR M2:

Chem.

[0442] Accession number 63 (HA-1 H Amino acid sequences of the Vβ (VDJ) domain and the constant domain (mouse) of TCR M2:

Chemical formula

[0443] Accession number 64 (HA-1 H Nucleic acid sequences of the Vβ (VDJ) domain and the constant domain of TCR M2:

Chemical formula

[0444] Accession number 65 (HA-1 H Codon-optimized nucleic acid sequences of the Vβ (VDJ) domain and the constant domain of TCR M2:

Chemical formula

[0445] Accession number 66 (HA-1 H Codon-optimized nucleic acid sequences of the Vβ (VDJ) domain and the constant domain (mouse) of TCR M2:

Chemical formula

[0446] Accession number 67 (HA-1 H Amino acid sequences of the Vα (VJ) domain and the constant domain of TCR FK47.83:

Chemical formula

[0447] Accession number 68 (HA-1 H Amino acid sequences of the Vα (VJ) domain and the constant domain (mouse) of TCR FK47.83: [Chemical formula]

[0448] Accession number 69 (HA-1 H Nucleic acid sequences for the Vα (VJ) domain and the constant domain of TCR FK47.83: [Chemical formula]

[0449] Accession number 70 (HA-1 H Codon-optimized nucleic acid sequences for the Vα (VJ) domain and the constant domain of TCR FK47.83: [Chemical formula]

[0450] Accession number 71 (HA-1 H Codon-optimized nucleic acid sequences for the Vα (VJ) domain and the constant domain (mouse) of TCR FK47.83: [Chemical formula]

[0451] Accession number 72 (HA-1 H Amino acid sequences for the Vβ (VDJ) domain and the constant domain of TCR FK47.83: [Chemical formula]

[0452] Accession number 73 (HA-1 H Amino acid sequences for the Vβ (VDJ) domain and the constant domain (mouse) of TCR FK47.83: [Chemical formula]

[0453] Accession number 74 (HA-1 H Nucleic acid sequences for the Vβ (VDJ) domain and the constant domain of TCR FK47.83):

Chemical formula

[0454] Accession number 75 (HA-1 H Codon-optimized nucleic acid sequences for the Vβ (VDJ) domain and the constant domain of TCR FK47.83):

Chemical formula

[0455] Accession number 76 (HA-1 H Codon-optimized nucleic acid sequences for the Vβ (VDJ) domain and the constant domain (mouse) of TCR FK47.83):

Chemical formula

[0456] Accession number 77 (Nucleotide sequence encoding amino acids 1-80 (Figure 2A) of HA-1 TCR BV7-9):

Chemical formula

[0457] Accession number 78 (Nucleotide sequence encoding amino acids 1-80 (Figure 2A) of HA-2 TCR BV7-8):

Chemical formula

[0458] Accession number 79 (Amino acid sequence of HA-1R):

Chemical formula

[0459] Accession number 80 (HA-1 H Amino acid sequence of CDR1 of the Vα domain of TCR M7):

Chem.

[0460] SEQ ID NO: 81 (HA-1 H Nucleic acid sequence of CDR1 of the Vα domain of TCR M7):

Chem.

[0461] SEQ ID NO: 82 (HA-1 H Codon-optimized nucleic acid sequence of CDR1 of the Vα domain of TCR M7):

Chem.

[0462] SEQ ID NO: 83 (HA-1 H Amino acid sequence of CDR2 of the Vα domain of TCR M7):

Chem.

[0463] SEQ ID NO: 84 (HA-1 H Nucleic acid sequence of CDR2 of the Vα domain of TCR M7):

Chem.

[0464] SEQ ID NO: 85 (HA-1 H Codon-optimized nucleic acid sequence of CDR2 of the Vα domain of TCR M7):

Chem.

[0465] SEQ ID NO: 86 (HA-1 H TCR M7, HA-1 H TCR M2, or HA-1 H Amino acid sequence of the CDR2 of the Vβ domain of TCR FK47.83):

Chem.

[0466] SEQ ID NO: 87 (HA-1 H TCR M7, HA-1 H TCR M2, or HA-1 H Nucleic acid sequence of the CDR2 of the Vβ domain of TCR FK47.83):

Chem.

[0467] SEQ ID NO: 88 (HA-1 H TCR M7, HA-1 H TCR M2, or HA-1 H Codon-optimized nucleic acid sequence of the CDR2 of the Vβ domain of TCR FK47.83):

Chem.

[0468] SEQ ID NO: 89 (Mouse C beta region):

Chem.

[0469] SEQ ID NO: 90 (Mouse C alpha region):

Chem.

[0470] SEQ ID NO: 91 (Vβ domain of Seattle-type TCR2 according to WO2018 / 058002):

Chem.

[0471] Accession number 92 (Vα domain of Seattle-type TCR2 according to WO2018 / 058002):

Chemical formula

[0472] (References) TIFF0007704675000098.tif125167TIFF0007704675000099.tif147168< / h>

Claims

Claim 1 HA-1 having a TCRα-chain variable (Vα) domain and a TCRβ-chain variable (Vβ) domain H An isolated nucleic acid composition encoding an antigen-specific binding protein, wherein said composition is (i) (a) A nucleic acid sequence encoding a TCR Vα domain comprising a CDR3 amino acid sequence, a CDR1 amino acid sequence, and a CDR2 amino acid sequence, wherein the CDR3 amino acid sequence consists of the sequence of SEQ ID NO: 1, the CDR1 amino acid sequence consists of the sequence of SEQ ID NO: 80, and the CDR2 amino acid sequence consists of the sequence of SEQ ID NO: 83; and (b) A nucleic acid sequence encoding a TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, wherein the Vβ domain comprises a CDR3 amino acid sequence, a CDR1 amino acid sequence, and a CDR2 amino acid sequence, the CDR3 amino acid sequence consists of the sequence of SEQ ID NO: 4, the CDR1 amino acid sequence consists of the sequence of SEQ ID NO: 7, and the CDR2 amino acid sequence consists of the sequence of SEQ ID NO: 86; (ii) (a) A nucleic acid sequence encoding a TCR Vα domain comprising a CDR3 amino acid sequence, a CDR1 amino acid sequence, and a CDR2 amino acid sequence, wherein the CDR3 amino acid sequence consists of the sequence of SEQ ID NO: 2, the CDR1 amino acid sequence consists of the sequence of SEQ ID NO: 8, and the CDR2 amino acid sequence consists of the sequence of SEQ ID NO: 28; and (b) A nucleic acid sequence encoding a TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, wherein the Vβ domain comprises a CDR3 amino acid sequence, a CDR1 amino acid sequence, and a CDR2 amino acid sequence, the CDR3 amino acid sequence consists of the sequence of SEQ ID NO: 5, the CDR1 amino acid sequence consists of the sequence of SEQ ID NO: 7, and the CDR2 amino acid sequence consists of the sequence of SEQ ID NO: 86; or (iii) (a) A nucleic acid sequence encoding a TCR Vα domain comprising a CDR3 amino acid sequence, a CDR1 amino acid sequence, and a CDR2 amino acid sequence, wherein the CDR3 amino acid sequence consists of the sequence of SEQ ID NO: 3, the CDR1 amino acid sequence consists of the sequence of SEQ ID NO: 8, and the CDR2 amino acid sequence consists of the sequence of SEQ ID NO: 28; and (b) A nucleic acid sequence encoding a TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, wherein the Vβ domain comprises a CDR3 amino acid sequence, a CDR1 amino acid sequence, and a CDR2 amino acid sequence, the CDR3 amino acid sequence consists of the sequence of SEQ ID NO: 6, the CDR1 amino acid sequence consists of the sequence of SEQ ID NO: 7, and the CDR2 amino acid sequence consists of the sequence of SEQ ID NO: 86; or (iv) (a) A nucleic acid sequence encoding a TCR Vα domain comprising a CDR3 amino acid sequence, a CDR1 amino acid sequence, and a CDR2 amino acid sequence, wherein the CDR3 amino acid sequence consists of the sequence of SEQ ID NO: 2, the CDR1 amino acid sequence consists of the sequence of SEQ ID NO: 8, and the CDR2 amino acid sequence consists of the sequence of SEQ ID NO: 28; and (b) A nucleic acid sequence encoding a TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, wherein the Vβ domain comprises a CDR3 amino acid sequence, a CDR1 amino acid sequence, and a CDR2 amino acid sequence, the CDR3 amino acid sequence consists of the sequence of SEQ ID NO: 4, the CDR1 amino acid sequence consists of the sequence of SEQ ID NO: 7, and the CDR2 amino acid sequence consists of the sequence of SEQ ID NO: 86 A composition comprising the same. Claim 2 The TRBV7-9 gene is TRBV7-9 * 01 or TRBV7-9 * The isolated nucleic acid composition according to claim 1, wherein it is 03. Claim 3 the aforementioned HA-1 H The isolated nucleic acid composition according to claim 1 or 2, wherein the antigen comprises the amino acid sequence shown in SEQ ID NO:

10. Claim 4 The encoded binding protein is HA-1 H Antigen: HLA-A * The isolated nucleic acid composition according to any one of claims 1 to 3, wherein the encoded binding protein is capable of specifically binding to the 0201 complex Claim 5 The isolated nucleic acid composition according to any one of claims 1 to 4, wherein the nucleic acid sequence is codon-optimized for expression in a host cell. Claim 6 The isolated nucleic acid composition according to claim 5, wherein the host cell is a human T cell. Claim 7 (i) The Vα domain comprises an amino acid sequence having at least 90% sequence identity to, including, or consisting of SEQ ID NO: 17; and / or (ii) The Vβ domain comprises an amino acid sequence having at least 90% sequence identity to, including, or consisting of SEQ ID NO: 18 The isolated nucleic acid composition according to claim 1. Claim 8 (i) The Vα domain is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 19 or SEQ ID NO: 20; and / or (ii) The Vβ domain is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 21 or SEQ ID NO: 22 The isolated nucleic acid composition according to claim 7. Claim 9 (i) the Vα domain comprises an amino acid sequence having at least 90% sequence identity to, comprising, or consisting of SEQ ID NO: 29; and / or (ii) the Vβ domain comprises an amino acid sequence having at least 90% sequence identity to, comprising, or consisting of SEQ ID NO: 30, The isolated nucleic acid composition according to claim 1.

10. (i) the Vα domain is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 31 or SEQ ID NO: 32; and / or (ii) the Vβ domain is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 33 or SEQ ID NO: 34, The isolated nucleic acid composition according to claim 9.

11. (i) the Vα domain comprises an amino acid sequence having at least 90% sequence identity to, comprising, or consisting of SEQ ID NO: 41; and / or (ii) the Vβ domain comprises an amino acid sequence having at least 90% sequence identity to, comprising, or consisting of SEQ ID NO: 42, The isolated nucleic acid composition according to claim 1.

12. (i) the Vα domain is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 43 or SEQ ID NO: 44; and / or (ii) the Vβ domain is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 45 or SEQ ID NO: 46, The isolated nucleic acid composition according to claim 11.

13. The isolated nucleic acid composition according to any one of claims 1 to 12, further comprising a TCRα chain constant domain and / or a TCRβ chain constant domain.

14. The isolated nucleic acid composition according to any one of claims 1 to 13, wherein the encoded binding protein comprises a TCR, an antigen-binding fragment of a TCR, or a chimeric antigen receptor (CAR).

15. The isolated nucleic acid composition according to claim 14, wherein the antigen-binding fragment of the TCR is a single-chain TCR (scTCR).

16. One or more vectors comprising the nucleic acid composition according to any one of claims 1 to 15.

17. The one or more vectors according to claim 16, wherein the vector is a plasmid, a viral vector, or a cosmid.

18. The one or more vectors according to claim 17, wherein the vector is selected from the group consisting of a retrovirus, a lentivirus, an adeno-associated virus, an adenovirus, a vaccinia virus, a canarypox virus, a herpes virus, and a minicircle vector.

19. A modified cell comprising the nucleic acid composition according to any one of claims 1 to 15, or one or more vectors according to claim 16, 17 or 18, wherein the modified cell is HLA-A * 0201 negative and / or HA-1 H negative, modified cell.

20. The modified cell according to claim 19, wherein the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, a NK cell, a NK-T cell, a gamma-delta T cell, a hematopoietic stem cell, a progenitor cell, a T cell line, or a NK-92 cell line.

21. The modified cell according to claim 19 or 20, wherein the modified cell is a human cell.

22. A pharmaceutical composition comprising the nucleic acid composition according to any one of claims 1 to 15, the one or more vectors according to claim 16, 17 or 18, or the modified cell according to any one of claims 19 to 21, and a pharmaceutically acceptable excipient, adjuvant, diluent, and / or carrier.

23. HLA-A * The pharmaceutical composition according to claim 22 for use in the treatment of hematological malignancies or the prevention of their recurrence after allogeneic stem cell transplantation (allo-SCT) in HLA-A 0201 positive human subjects.

24. HLA-A * Use of the pharmaceutical composition according to claim 22 in the manufacture of a medicament for treating or preventing recurrence of a hematological malignancy after allogeneic stem cell transplantation (allo-SCT) in HLA-A 0201 positive human subjects.

25. The pharmaceutical composition for use according to claim 23, wherein the hematological malignancy comprises leukemia, lymphoma, myelodysplastic syndrome, or multiple myeloma.

26. (i) the hematological malignancy comprises leukemia, and the leukemia is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), mixed phenotype acute leukemia (MPAL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, hairy cell leukemia, or chronic lymphocytic leukemia (CLL); or (ii) the hematological malignancy comprises lymphoma, and the lymphoma is selected from the group consisting of Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), central nervous system lymphoma, small lymphocytic lymphoma (SLL), CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, extranodal marginal zone B-cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, precursor B-lymphoblastic lymphoma, immunoblastic large cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or Burkitt lymphoma; or (iii) The hematological malignancy includes myelodysplastic syndrome, and the myelodysplastic syndrome is refractory cytopenia with single lineage dysplasia (refractory anemia, refractory neutropenia, and refractory thrombocytopenia), refractory anemia with ring sideroblasts (RARS), refractory anemia with ring sideroblasts and thrombocytosis (RARS-t), refractory cytopenia with multilineage dysplasia (RCMD), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS), refractory anemia with excess blasts (RAEB), unclassifiable myelodysplasia, or pediatric refractory cytopenia, selected from The pharmaceutical composition for use according to claim 25.

27. The pharmaceutical composition for use according to claim 23, 25 or 26, wherein the subject has been previously administered lymphodepleting chemotherapy.

28. The pharmaceutical composition for use according to claim 27, wherein the lymphodepleting chemotherapy comprises cyclophosphamide, fludarabine, antithymocyte globulin, or a combination thereof.

29. The pharmaceutical composition for use according to any one of claims 23 and 25 to 28, wherein one or more of the modified cells in the composition according to claim 22 are allogeneic to the subject.

30. HA-1 H It is capable of specifically binding to a peptide containing an antigen, and HA-1 H An in vitro or ex vivo method for producing a binding protein that does not bind to a peptide not containing an antigen, the method comprising contacting the nucleic acid composition according to any one of claims 1 to 15 with a cell under conditions such that the nucleic acid composition is incorporated and expressed by the cell.

31. The use according to claim 24, wherein the hematological malignancy includes leukemia, lymphoma, myelodysplastic syndrome, or multiple myeloma.

32. (i) The hematological malignancy includes leukemia, and the leukemia is selected from the group consisting of acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), mixed phenotype acute leukemia (MPAL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, hairy cell leukemia, or chronic lymphocytic leukemia (CLL); or (ii) the hematological malignancy includes lymphoma, and the lymphoma is selected from the group consisting of Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), central nervous system lymphoma, small lymphocytic lymphoma (SLL), CD37+ dendritic cell lymphoma, lymphoplasmacytic lymphoma, splenic marginal zone lymphoma, extranodal marginal zone B-cell lymphoma of mucosa-associated (MALT) lymphoid tissue, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, precursor B-lymphoblastic lymphoma, immunoblastic large cell lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, or Burkitt lymphoma; or (iii) the hematological malignancy includes myelodysplastic syndrome, and the myelodysplastic syndrome is selected from refractory cytopenia with single lineage dysplasia (refractory anemia, refractory neutropenia, and refractory thrombocytopenia), refractory anemia with ring sideroblasts (RARS), refractory anemia with ring sideroblasts and thrombocytosis (RARS-t), refractory cytopenia with multilineage dysplasia (RCMD), refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS), refractory anemia with excess blasts (RAEB), unclassifiable myelodysplasia, or pediatric refractory cytopenia The use according to claim 31.

33. The use according to any one of claims 24, 31, and 32, wherein the subject has been previously administered lymphodepleting chemotherapy.

34. The use according to claim 33, wherein the lymphodepleting chemotherapy comprises cyclophosphamide, fludarabine, antithymocyte globulin, or a combination thereof.

35. The use according to any one of claims 24 and 31 to 34, wherein one or more of the modified cells in the composition according to claim 22 are allogeneic to the subject.

Images