Specific binding protein against HA-1H and its uses

Abstract

The present invention provides novel nucleic acid compositions, vectors, modified cells and pharmaceutical compositions, which can be used for treating or preventing the recurrence of hematologic malignancies in HLA-A*0201-positive human subjects after allogeneic stem cell transplantation (allo-SCT). Corresponding methods and uses are also provided.

Description

Technical Field

[0001] The present invention provides novel nucleic acid compositions, vector systems, modified cells, and pharmaceutical compositions that can be used to treat or prevent recurrence of hematologic malignancies following allogeneic stem cell transplantation (allo-SCT) in HLA-A*0201 positive human subjects. Corresponding methods and uses are also provided. Background Art

[0002] Hematologic malignancies are cancers that affect the blood and lymphatic systems. The cancers may originate in hematopoietic tissues (such as bone marrow) or in cells of the immune system. Patients with hematologic malignancies can be successfully treated with allogeneic stem cell transplantation (allo-SCT) using human leukocyte antigen (HLA)-matched donors. To reduce the development of graft-versus-host disease (GvHD), donor T cells can be depleted from the stem cell graft and preemptively re-administered after allo-SCT. Although 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, GvHD remains a significant cause of morbidity and mortality, especially in the case of HLA-mismatched transplants. Early application of DLI after allo-SCT carries a higher risk of inducing GvHD. High-risk leukemia patients may relapse early after transplantation, at a time when administration of DLI may cause GvHD. For this patient population, treatment options are limited and new therapeutic approaches are needed to allow early administration of T cells that can exert a graft-versus-leukemia (GvL) effect without causing GvHD.

[0003] Adoptive transfer of T cells with defined anti-leukemia specificity is a strategy for separating the graft-versus-host disease (GvHD) response from the graft-versus-leukemia (GvL) response. It has been demonstrated that donor T cells that recognize minor histocompatibility antigens (MiHA) selectively expressed on hematopoietic cells mediate anti-leukemia responses after allo-SCT without causing severe GvHD. HA-1 HThe antigen is a minor histocompatibility antigen and an important target for immunotherapy because it is highly expressed in hematological malignancies and normal hematopoietic cells but not in normal non-hematopoietic cells. It also exists in the context of HLA-A*0201, a globally common human leukocyte antigen serotype, and is thus a suitable target antigen for a substantial portion of patients with hematological malignancies. Previously, a direct link was found between the emergence of HA-1-specific T cells and the complete disappearance of malignant recipient cells in HA-1-incompatible donor-recipient pairs (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, previous phase I clinical studies have shown that in vitro-cultured HA-1-specific T cells derived from the donor T cell repertoire lack in vivo persistence and anti-leukemia reactivity in vivo (Meij et al., 2012).

[0004] New immunotherapies are needed to treat hematological malignancies. SUMMARY OF THE INVENTION

[0005] Minor histocompatibility antigen HA-1 H is encoded by the HMHA1 gene (also known as Rho GTPase-activating protein 45). The HMHA1 variant (rs_1801284 A / A or A / G) present in 52% of individuals generates an immunogenic peptide (VLHDDLLEA; SEQ ID NO:10) containing a histidine residue instead of arginine (R139H polymorphism), and HLA presentation of this peptide occurs in individuals with the common HLA-A*0201 (A2) allele (2). Therefore, T cell therapy against HA-1 H is applicable to approximately 25% of hematological malignancy transplant recipients and requires HLA-A2-negative or HA-1 H -negative T cell donors ("HA-1 R "; VLRDDLLEA; SEQ ID NO:79).

[0006] The inventors isolated and sequenced novel TCRs specific for the HA-1 H antigen. Such TCRs can be used to treat or prevent recurrence of hematological malignancies after allogeneic stem cell transplantation (allo-SCT) in HLA-A*0201-positive human subjects.

[0007] The inventors have previously shown that in vitro - cultured HA - 1 - specific T cells from a donor T - cell repertoire lack in - vivo persistence and in - vivo anti - leukemia reactivity (Meij et al, 2012). The inventors have now isolated HA - 1 TCRs derived from HA - 1 - specific T cells isolated from patients who experienced an effective anti - leukemia response mediated by these HA - 1 - specific T cells (Marijt et al 2003, van Loenen et al 2011). Advantageously, the HA - 1 TCRs described herein thus resemble natural T - cell responses and correspond to high - affinity TCRs with potent anti - leukemia reactivity. Surprisingly, the HA - 1 - specific TCRs described herein have stronger anti - tumor reactivity compared to less promising candidate cells generated in vitro. In addition, since these HA - 1 TCRs were isolated from activated anti - leukemia responses and show no signs of graft - versus - host disease (GvHD), these TCRs are safe as they do not induce graft - versus - host disease or other toxicities. Advantageously, effective anti - leukemia responses of HA - 1 - specific TCRs against primary AML and ALL leukemias derived from patients were demonstrated at the time of diagnosis, demonstrating the effectiveness of these HA1 - TCRs in patients with hematological malignancies. More convincingly, a pre - clinical in - vivo model was used herein to show that HA - 1 TCR - engineered T cells very effectively eradicated established multiple myeloma after infusion, highlighting the effective in - vivo anti - tumor reactivity of HA - 1 TCRs.

[0008] The inventors have investigated which components of the novel TCRs are required for HA - 1 H antigen specificity and TCR function. Surprisingly, they found that the CDR1 region of the variable domain of the TCRβ - chain is crucial for HA - 1 H specificity but not sufficient for HA - 1 specificity. They also found that the CDR3 regions of the variable domain of the TCRβ - chain (Vβ) and the variable domain of the TCRα - chain (Vα) are required, and the variable domain of the TCRβ - chain (Vβ) needs to be encoded by the TRBV7 - 9 gene.

[0009] Thus, the HA - 1 H specific TCRs described herein require the following minimum components:

[0010] (a) A TCR Vα domain comprising an HA - 1 H specific CDR3 (see, for example, SEQ ID No: 1 to 3); and

[0011] (b) A TCR Vβ domain having an amino acid sequence encoded by the TRBV7 - 9 gene, wherein said Vβ domain comprises HA - 1 HSpecific CDR3 (see, for example, SEQ ID NO: 4-6) and HA-1 H Specific CDR1 (see, for example, SEQ ID No: 7).

[0012] The CDR3 TCR sequences described herein are different from the sequences known in the prior art (e.g., WO2018 / 058002). In addition, the TRAV region (including the CDR1 and CDR2 regions) of the α-chain sequence of the TCR described herein is completely different from the TRAV region of the α-chain of the TCR described in WO2018 / 058002. In addition, the TRAJ region combines with a different TRAV region. Therefore, there are several differences between the TCRs we claim and the TCRs of the cited prior art.

[0013] HA-1 H Specific binding proteins (e.g., TCRs) are exemplified herein using specific combinations of CDR3 and CDR1 sequences. For example, the following combinations:

[0014] (a) The TCR Vα domain comprising the CDR3 of SEQ ID NO: 1; and

[0015] (b) The TCR Vβ domain having the amino acid sequence encoded by the TRBV7-9 gene that confers HA-1 H binding specificity, wherein the Vβ domain comprises the CDR3 of SEQ ID NO: 4 and the CDR1 of SEQ ID NO: 7. Although these specific CDR sequences are exemplified, the CDRs may also contain variability different from the specific sequences (e.g., each specific CDR may have at least 80% sequence identity with a specific SEQ ID NO). The TRBV7-9 gene may be TRBV7-9*03 (see, for example, TCR M7).

[0016] In addition, there are the following combinations:

[0017] (a) The TCR Vα domain comprising the CDR3 of SEQ ID NO: 2; and

[0018] (b) The TCR Vβ domain having the amino acid sequence encoded by the TRBV7-9 gene that confers HA-1 HBinding specific TCRVβ domain having an amino acid sequence encoded by a TRBV7-9 gene, wherein the Vβ domain comprises CDR3 of SEQ ID NO: 5 and CDR1 of SEQ ID NO: 7. Although these specific CDR sequences are exemplified, the CDRs may also comprise variability different from the specific sequences (e.g., each specific CDR may have at least 80% sequence identity with a specific SEQ ID NO). The TRBV7-9 gene may be TRBV7-9*01 (see, e.g., TCR M2).

[0019] In addition, there are the following combinations:

[0020] (a) a TCR Vα domain comprising the CDR3 of SEQ ID NO: 3; and

[0021] (b) shows the conferring of HA-1 H Binding specific TCRVβ domain having an amino acid sequence encoded by a TRBV7-9 gene, wherein the Vβ domain comprises CDR3 of SEQ ID NO: 6 and CDR1 of SEQ ID NO: 7. Although these CDR sequences are exemplified, CDRs may also comprise changes relative to a specific sequence (e.g., each specific CDR may have at least 80% sequence identity with a specific SEQ ID NO). The TRBV7-9 gene may be TRBV7-9*01 (see, e.g., TCR FK47.83).

[0022] The inventors also showed that HA-1 H Specific binding proteins (e.g., TCRs) can be formed by different combinations of TCR Vα and TCR Vβ domains described herein. For example, a TCR Vβ domain equivalent to the M7 clone (SEQ ID NO: 18) is combined with a TCR Vα domain equivalent to the M2 clone (SEQ ID NO: 29) to produce a functional TCR. Therefore, a functional binding protein (e.g., TCR) can also be generated by a combination of the following:

[0023] (a) a TCR Vα domain comprising the CDR3 of SEQ ID NO: 2; and

[0024] (b) shows the conferring of HA-1 HIn combination with a TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, wherein the Vβ domain comprises a CDR3 of SEQ ID NO: 4 and a CDR1 of SEQ ID NO: 7. Although these specific CDR sequences are exemplified, the CDRs may also contain variability different from the specific sequences (e.g., each specific CDR may have at least 80% sequence identity with a specific SEQ ID NO). The TRBV7-9 gene may be TRBV7-9*01 (see, for example, TCR M7).

[0025] Although the data provided herein relates to the above specific combinations, other combinations can also be used to form a functional binding protein specific for HA-1 H This combination is also covered herein and is described in more detail below.

[0026] The present invention has been illustrated by generating a functional TCR receptor specific for HA-1 H However, the present invention also includes other binding proteins having the above characteristics conferring HA-1 H antigen specificity. Thus, other binding proteins are also included (e.g., antigen-binding fragments of TCRs (e.g., single-chain TCRs) or chimeric antigen receptors (CARs)). These binding proteins (e.g., when expressed by a host cell such as an immune cell (e.g., a T cell)) can be used as an independent therapy for treating hematologic malignancies or preventing their recurrence or relapse, or can be used as part of a treatment regimen comprising other therapies or agents (e.g., after or in combination with allogeneic SCT).

[0027] In one aspect, the present invention provides an isolated nucleic acid composition encoding an HA-1 H antigen-specific binding protein having a variable region of the TCR α chain (Vα) and a variable region of the TCR β chain (Vβ), the composition comprising:

[0028] (a) a nucleic acid sequence encoding a TCR Vα domain, the TCR Vα domain comprising a CDR3 amino acid sequence having at least 85% sequence identity with any one of SEQ ID NOs: 1-3; and

[0029] (b) a nucleic acid sequence encoding a TCR Vβ domain, the 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 with any one of SEQ ID NOs: 4-6 and a CDR1 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 7.

[0030] Suitably, the TRBV7-9 gene may be TRBV7-9*01 or TRBV7-9*03.

[0031] Suitably, HA-1 H The antigen may comprise the amino acid sequence shown in SEQ ID NO: 10.

[0032] Suitably, the encoded binding protein may be capable of specifically binding to HA-1 H Antigen: HLA-A*0201 complex.

[0033] Suitably, the nucleic acid sequence may be codon-optimized for expression in a host cell, optionally, wherein the host cell is a human T cell.

[0034] Suitably:

[0035] (i) The CDR3 of the Vα domain may comprise or consist of the amino acid sequence of SEQ ID NO: 1.

[0036] (ii) The CDR3 of the Vβ domain may comprise or consist of the amino acid sequence of SEQ ID NO: 4.

[0037] (iii) The CDR1 of the Vβ domain may comprise or consist of the amino acid sequence of SEQ ID NO: 7.

[0038] Suitably:

[0039] (i) 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 / or

[0040] (ii) 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 / or

[0041] (iii) 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.

[0042] Suitably:

[0043] (i) The Vα domain may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 17, comprising SEQ ID NO: 17, or consisting of SEQ ID NO: 17; and / or

[0044] (ii) The Vβ domain may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 18, comprising SEQ ID NO: 18, or consisting of SEQ ID NO: 18.

[0045] Suitably:

[0046] (i) The Vα domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 19 or SEQ ID NO: 20; and / or

[0047] (ii) The Vβ domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 21 or SEQ ID NO: 22.

[0048] Suitably:

[0049] (i) The CDR3 of the Vα domain may comprise or consist of the amino acid sequence of SEQ ID NO: 2.

[0050] (ii) The CDR3 of the Vβ domain may comprise or consist of the amino acid sequence of SEQ ID NO: 5, and

[0051] (iii) The CDR1 of the Vβ domain may comprise or consist of the amino acid sequence of SEQ ID NO: 7.

[0052] Suitably:

[0053] (i) The CDR3 of the Vα domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 23 or SEQ ID NO: 24; and / or

[0054] (ii) 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; and / or

[0055] (iii) 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.

[0056] Suitably:

[0057] (i) The Vα domain may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 29, comprising SEQ ID NO: 29, or consisting of SEQ ID NO: 29; and / or

[0058] (ii) The Vβ domain may comprise an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 30, comprises SEQ ID NO: 30, or consists of SEQ ID NO: 30.

[0059] Suitably:

[0060] (i) The Vα domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 31 or SEQ ID NO: 32; and / or

[0061] (ii) The Vβ domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 33 or SEQ ID NO: 34.

[0062] Suitably:

[0063] (i) The CDR3 of the Vα domain may comprise the amino acid sequence of SEQ ID NO: 2 or consist of the amino acid sequence of SEQ ID NO: 3,

[0064] (ii) The CDR3 of the Vβ domain may comprise the amino acid sequence of SEQ ID NO: 4 or consist of the amino acid sequence of SEQ ID NO: 6,

[0065] (iii) The CDR1 of the Vβ domain may comprise the amino acid sequence of SEQ ID NO: 7 or consist of the amino acid sequence of SEQ ID NO: 7.

[0066] Suitably:

[0067] (i) 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 / or

[0068] (ii) 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 / or

[0069] (iii) 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.

[0070] Suitably:

[0071] (i) The Vα domain may comprise an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 41, comprises SEQ ID NO: 41, or consists of SEQ ID NO: 41; and / or

[0072] (ii) The Vβ domain may comprise an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 42, comprises SEQ ID NO: 42, or consists of SEQ ID NO: 42.

[0073] Suitably:

[0074] (i) The Vα domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 43 or SEQ ID NO: 44; and / or

[0075] (ii) The Vβ domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 45 or SEQ ID NO: 46.

[0076] Suitably, the isolated nucleic acid composition may further comprise a TCRα chain constant region and / or a TCRβ chain constant region.

[0077] Suitably, the encoded binding protein may comprise a TCR, an antigen-binding fragment of a TCR, or a chimeric antigen receptor (CAR).

[0078] Suitably, the antigen-binding fragment of the TCR may be a single-chain TCR (scTCR).

[0079] On the other hand, the present invention provides a vector system comprising the nucleic acid composition described herein.

[0080] Suitably, the vector may be a plasmid, a viral vector, or a cosmid, optionally, wherein the vector is selected from the group consisting of: retroviruses, lentiviruses, adeno-associated viruses, adenoviruses, vaccinia viruses, canarypox viruses, herpes viruses, minicircles, and synthetic DNA or RNA.

[0081] On the other hand, the present invention provides a modified cell transfected or transformed with the nucleic acid composition described herein, or the vector system described herein, wherein the modified cell is HLA-A*0201 negative and / or HA-1 H negative.

[0082] Suitably, the modified cell may be selected from the group consisting of: CD8 T cells, CD4 T cells, NK cells, NK-T cells, γ-δ T cells, hematopoietic stem cells, progenitor cells, T cell lines, or NK-92 cell lines.

[0083] Suitably, the modified cell may be a human cell.

[0084] On the other hand, the present invention provides a pharmaceutical composition comprising the nucleic acid composition described herein, the vector system described herein, or the modified cells described herein, and a pharmaceutically acceptable excipient, adjuvant, diluent, and / or carrier.

[0085] On the other hand, the present invention provides a method for treating or preventing recurrence of hematologic malignancies after allogeneic stem cell transplantation (allo-SCT) in HLA-A*0201-positive subjects, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition described herein.

[0086] On the other hand, the present invention provides use of the pharmaceutical composition described herein for treating or preventing recurrence of hematologic malignancies after allogeneic stem cell transplantation (allo-SCT) in HLA-A*0201-positive human subjects.

[0087] On the other hand, the present invention provides use of the pharmaceutical composition described herein in the manufacture of a medicament for treating or preventing recurrence of hematologic malignancies after allogeneic stem cell transplantation (allo-SCT) in HLA-A*0201-positive human subjects.

[0088] Suitably, the hematologic malignancies may include leukemia, lymphoma, myelodysplastic syndrome, or myeloma.

[0089] Suitably:

[0090] (i) The hematologic malignancies may include leukemia, optionally wherein 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

[0091] (ii) The hematologic malignancies may include lymphoma, optionally wherein 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 lymphoid tissue (MALT), 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

[0092] (iii) Hematological malignancies may include myelodysplastic disorders, optionally wherein the myelodysplastic disorder is selected from refractory cytopenia with unilineage dysplasia (refractory anemia, refractory neutropenia, and refractory thrombocytopenia), refractory anemia with ring sideroblasts (RARS), refractory anemia with ring sideroblasts - 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), myelodysplasia, unclassifiable, or childhood refractory cytopenia.

[0093] Suitably, the subject may have previously received lymphodepleting chemotherapy.

[0094] Suitably, lymphodepleting chemotherapy may include cyclophosphamide, fludarabine, antithymocyte globulin, or a combination thereof.

[0095] Suitably, one or more of the modified cells in the compositions described herein may be allogeneic to the subject.

[0096] On the other hand, the present invention provides a method for generating a binding protein that is capable of specifically binding to a peptide containing the HA - 1 H antigen and not binding to a peptide lacking the HA - 1 H antigen, the method comprising contacting a nucleic acid composition described herein with a cell under conditions such that the nucleic acid composition is incorporated into and expressed by the cell.

[0097] Suitably, the method may be ex vivo.

[0098] On the other hand, the present invention provides an isolated nucleic acid sequence comprising the nucleic acid sequence of any one of SEQ ID NO: 11 - 14, 19 - 26, 31 - 38, 43 - 46, 49 - 51, 54 - 56, 59 - 66, 69 - 71, and 74 - 76 or consisting of the nucleic acid sequence of any one of SEQ ID NO: 11 - 14, 19 - 26, 31 - 38, 43 - 46, 49 - 51, 54 - 56, 59 - 66, 69 - 71, and 74 - 76.

[0099] On the other hand, the present invention provides a separated nucleic acid sequence for treatment, which comprises a nucleic acid sequence of any one of SEQ ID NO: 11-14, 19-26, 31-38, 43-46, 49-51, 54-56, 59-66, 69-71 and 74-76 or consists of a nucleic acid sequence of any one of SEQ ID NO: 11-14, 19-26, 31-38, 43-46, 49-51, 54-56, 59-66, 69-71 and 74-76.

[0100] Throughout the specification and claims of this specification, the words "comprising" and "including" and their variations mean "including but not limited to", and they are not intended to (nor do they) exclude other parts, additives, components, wholes or steps.

[0101] Throughout the specification and claims of this specification, unless the context requires otherwise, the singular encompasses the plural. In particular, unless the context otherwise requires, when using the indefinite article, the specification should be understood to contemplate both the plural and the singular.

[0102] Features, wholes, characteristics, compounds, chemical moieties or groups described in connection with a particular aspect, embodiment or example of the present invention should be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

[0103] The patent, scientific and technical literature referred to herein establishes the knowledge available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications cited herein are incorporated herein by reference 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.

[0104] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular Biology, 2d Ed., John Wiley and Sons, NY; and Hale and Marham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) provide one of ordinary skill in the art with a general dictionary of many of the terms used in the present invention. Although any methods and materials similar or equivalent to those described herein have been found to be useful in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined below are more fully described by reference to the entire specification. Further, as used herein, the singular terms "a", "an", and "the" include plural references 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 should be understood that the present invention is not limited to the specific methods, protocols, and reagents described, as these may vary according to the context in which one of ordinary skill in the art uses them.

[0105] Aspects of the present invention will be described in further detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0106] Embodiments of the present invention will be further described below with reference to the drawings, wherein:

[0107] Figure 1 Show low cell surface expression of HA-1-TCR due to the inherent properties of the HA-1-TCR β-chain. (A) The pairing properties of the HA-1-TCR α- and TCR β-chains were analyzed by transforming J76 cells with combinations of the HA-1-TCR α or TCR β-chain and 14 other antigen-specific TCR α- and TCR β-chains. The cell surface expression of these different combinations of TCRs was measured by staining with anti-TCRαβ mAb and analyzing eGFP / NGF-R double-positive J76 cells using flow cytometry 5 days after transformation. Described herein are HA-2-, HA-1-, CMV B7 , and CMV A2The mean fluorescence intensity (MFI) of TCRαβ expression in which all TCRα chains of the -specific TCRs are paired with all TCRβ chains of these TCRs. All TCR chains were encoded by the pLZRS retroviral vector, except for the HA-1-TCRβ chain, which was also encoded by the MP71 retroviral vector as shown. Background staining of non-td J76 cells with anti-TCRαβ mAb was minimal (MFI = 16). The parental TCR combinations are indicated by asterisks. (B) Several T cell clones were stained with anti-TCRαβ and anti-CD3 mAbs and analyzed by flow cytometry. The T cell clones shown include 5 different HA-1-specific T cell clones, 6 different HA-2-specific T cell clones, and 2 different CMV-A2-specific T cell clones. The MFI shown is the mean of different T cell clones. (C) Different T cell clones were stained with their respective tetramers, and the MFI was depicted in dot plots. (D) The mRNA levels of TCRα (closed symbols) and TCRβ chains (open symbols) of different T cell clones were analyzed by q-RT-PCR. As a negative control, cDNA from MSCs was included. Staining was performed in duplicate, and the data shown represent two independent experiments.

[0108] Figure 2The CDR1 region was shown not to be the cause of low HA-1-TCR expression, but is indispensable for HA-1-specificity. (A) Different TRBV7 chains showed high or low TCR expression after TCR gene transfer. Different TRBV7 chains were aligned and nucleotide sequence differences were analyzed. Thirty shared nucleotide differences were observed in the 309-aa long variable region between the highly expressed HA-2-TRBV7-8, JBBun-TRBV7-6, 10G5-TRBV7-1 and the low-expressing HA-1-TRBV7-9. The sequences shown are from amino acid 41 to 80 of the TRBV7 chains (total aa 309), including the CDR1- and CDR2-regions of the HA-1 and HA-2 TCRβ chains (SEQ ID NO: 77 and 78). Shared differences between all other TRBV7 chains and the HA-1TRBV7-9 chain are indicated by arrows. (B) To test the role of the HA-1-TCRβ CDR1 region in low HA-1-TCR expression, J76 cells were transfected with combinations of HA-1-TCRα or HA-2-TCRα with several constructs encoding either unmodified HA-1-TCRβ chains or HA-1-TCRβ chains swapped with the HA-2-TCRβ CDR1 region, or constructs encoding unmodified HA-2-TCRβ chains or HA-2-TCRβ chains swapped only with the HA-1-TCRβ CDR1 region or swapped with both the HA-1-TCRβ CDR1 and CDR3 regions. TCR cell surface expression of eGFP / NGF-R double-positive J76 cells was analyzed by flow cytometry. Non-td J76 cells had little background staining with anti-TCRαβ mAb (MFI = 16). Parental TCR combinations are indicated by asterisks. (C) To test the role of the HA-1-TCRβ CDR1 region in HA-1-specificity, virus-specific T cells were transfected with different constructs. HLA-A2 pos and HA-1 neg LCL-IZA, HLA-A2 pos and HA-1 pos LCL-BDV, and LCL-IZA were used to stimulate eGFP and NGF-R double-positive cells, and interferon-γ production was measured by standard ELISA after o / n incubation.

[0109] Figure 3It is shown that the combination of codon optimization and cysteine modification results in optimal HA-1-TCR expression and HA-1 function. The potential of different modification strategies to optimize HA-1-TCR expression and function was tested. (A) Weak (pp50 VTE T cells) and strong competitor phenotype (EBNA3A FLR T cells) virus-specific T cells were transduced with combinations of unmodified (TRAV25*01), cysteine-modified (TRAV25*01SS), codon-optimized (TRAV25*01opt), or codon-optimized and cysteine-modified (TRAV25*01opt SS) HA-1-TCR α-chains with unmodified (TRBV7-9), cysteine-modified (TRBV7-9 SS), or codon-optimized (TRBV7-9 opt) HA-1-TCR β-chains. Dot plots depict eGFP and NGF-R double-positive virus-specific T cells. (B) All these modified HA-1-TCRs were tested in standard IFN-γ ELISAs on weak and strong competitor virus-specific T cells. The numbers in the figure correspond to those shown in the dot plots of A. The targets used were HLA-A2 pulsed with different concentrations of HA-1-peptide pos HA-1 neg LCL-IZA, HLA-A2 pos HA-1 pos LCL-MRJ and LCL-IZA. The data shown represent three independent experiments performed with virus-specific T cells from two different healthy donors

[0110] Figure 4 It is shown that the introduction of codon-optimized and cysteine-modified HA-1-TCR generally results in efficient HA-1-TCR expression and robust HA-1-specific function. (A) Weak competitor phenotype (pp65 RPH) T cells, strong competitor phenotype (pp65 TPR) T cells, and polyclonal peripheral CD8+ T cells were transduced with a single construct encoding unmodified (WT td) or codon-optimized and cysteine-modified HA-1-TCR chains (opt SS td) and analyzed for HA-1 tetramer staining. Dot plots depict HA-1 tetramer staining of NGF-R positive virus-specific T cells and show the percentage of HA-1 tetramer-positive T cells. The dot plots depicted represent two independent experiments performed with T cells from 3 different healthy individuals. (B / C) pp65 RPH and pp65 TPR T cells transduced with a single construct encoding HA-1-TCR WT or HA-1-TCR opt SS or empty vector were tested for HA-1 specificity against different targets (B) cytotoxic responses in a chromium release assay and (C) IFN-γ production. The targets used were HLA-A2 posPrimary LCLs, AML, and ALL cells that are either positive or negative for HA-1. The data presented represent two independent experiments using T cells from 3 different healthy individuals.

[0111] Figure 5 Shown is that compared to HA-1-TCR WT-transduced T cells, MP71HA-1-TCR opt SS-transduced strong competitor phenotype virus-specific T cells showed more potent HA-1-specific IFNγ production against AML and ALL malignant cells.

[0112] To improve the vector for clinical use, a modified HA-1-TCR chain linked to the T2A sequence was expressed in the MP71 vector without a marker gene. pp50 VTE and EBNA3A FLR virus-specific T cells were transduced with the pLZRS vector encoding the unmodified HA-1-TCR chain, which was linked to the T2A sequence and to a marker gene via an IRES sequence (WTTCR), or with the MP71 vector encoding HA-1-TCR opt SS without a marker gene or an empty vector (mock td). One week after transduction, HA-1-specific IFNγ production was measured against different HLA-A2 HA-1-positive or -negative AML and ALL primary cells as shown. The experiments were performed in duplicate and represent two independent experiments. pos AML and ALL primary cells, and HA-1-specific IFNγ production was measured. The experiments were performed in duplicate and represent two independent experiments.

[0113] Figure 6 Shown is that virus-specific T cells can be purified and transduced using a streptavidin-based selection procedure on a Clini-MACS device. To test whether this procedure can be scaled up, virus-specific T cells were purified using Streptamers and a Clini-MACS device and, 2 - 3 days after isolation, transduced with HA-1-TCR (HA-1-TCR td) or not (non td). For this purpose, 1x10 9 PBMCs were thawed from healthy donors JBC (A+E), UPB (B+F), UHO (C+G), and UBQ (D+H). (A - D) Donor PBMCs were stained with tetramers before and after CliniMACS isolation, and the frequency of virus-specific T cells in the starting material and the positively selected fraction was measured using flow cytometry after the D-biotin dissociation step. (E - H) One week after transduction and 12 - 13 days after MACS isolation, the antigen specificity of non td and HA-1-TCR td cell lines was measured using HA-1- and virus-tetramers. Percentages represent the frequency of virus-specific or HA-1-specific T cells in the specific quadrants. *Sensitive multiplexed coding analysis indicated that the leukapheresis material from UBQ contained 0.008% pp65 A2Specific T cells and 0.03% BMLF-1 A2 Specific T cells.

[0114] Figure 7 showed that HA-1-TCR-modified T cells exhibited dose-dependent HA-1-specific reactivity and recognized HA-1-positive primary leukemia cell samples. (A) Two non-transformed (gray symbols; non td) and HA-1-TCR-transformed (black symbols; HA-1-TCR td) virus-specific T cells of three different test procedures (JBC, UPB, and UBQ) were tested for their HA-1-specific reactivity in a standard IFN-γ ELISA using T2 cells pulsed with different concentrations of HA-1 peptide. 5,000 virus-tetramers pos or 5,000 HA-1-tetramers pos were used per 20,000 T2 cells tested. (B) In addition, the ability of the same T cells to recognize HA-1-positive target cells expressing endogenously processed HA-1 was tested. Target cells were unpulsed (white bars) or pulsed with the relevant viral peptide (viral pep; black striped bars) T2 cells, or HA-1 neg (gray bars) or HA-1 pos (black bars) HLA-A2 pos primary ALL cells. As a control, HA-1-specific T cell clones were included. (C) Un-transformed and HA-1-TCR-transformed virus-specific T cells of all four different test procedures (JBC, UPB, UHO, and UBQ) were tested for their HA-1-specific reactivity using different effector-to-target ratios in a chromium release assay. As a representative example, the cytotoxic reactivity of HA-1-TCR-transformed virus-specific T cells from a healthy individual UHO is described. Target cells were unpulsed (white bars) or pulsed with HA-1 peptide (black bars) or the relevant viral peptide (viral pep; gray bars) T2 cells (left panel), or HA-1 neg (white bars) or HA-1 pos (black bars) HLA-A2 pos primary ALL cells, or HA-1 neg (light gray) or HA-1 pos (dark gray) HLA-A2 pos primary ALL cells (right panel).

[0115] Figure 8It was shown that polyclonal CD8+ T cells modified with HA-1 TCR produced effective HA-1-specific responses against various different HA-1-positive AML and MM cell lines. Both untransformed (mock) and clinically grade retroviral vector (HA-1 TCR GMP)-transformed HA-1-TCR-modified CD8+ T cells were tested for their HA-1-specific reactivity against different HA-1-positive and HA-1-negative target cells (shown in the legend) in a standard IFN-γ ELISA. LCL-MRJ, LCL-JY, and LCL-IZA are EBV-transformed B cell lines. AML 2 and AML 3 are acute myeloid leukemia (AML) cell lines. U266 and RPMI are multiple myeloma (MM) cell lines. T2 is a TAP-deficient cell line loaded with HA-1 peptide (T2 + HA-1 peptide) or unloaded with peptide (T2).

[0116] Figure 9 It was shown that polyclonal CD8+ T cells modified with clinically grade HA-1 TCR supernatant exerted effective anti-tumor reactivity in a xenograft model of multiple myeloma. NSG mice were intravenously injected with the luciferase-positive multiple myeloma cell line U266, and 21 days after tumor challenge, the mice were intravenously injected with HA-1 TCR-modified T cells (4×10e6 or 8×10e6 cells) or CMV TCR-modified T cells (4×10e6 or 8×10e6 cells). Both TCR-transformed T cell populations consisted of 30% TCR-transformed T cells.

[0117] Figure 10 Shows the HA-1 H reactive TCRs (TCRs containing the M7 Vα and Vβ domains described herein (see SEQ ID NO: 17 and 18) – referred to herein as “LUMC”; and TCRs containing the TCR2 Vα and Vβ domains described in WO2018 / 058002A1 (see SEQ ID NO: 91 and SEQ ID NO: 92) – referred to herein as “FHCRC”) transduced CD8 + T cells' HA-1 H -MHC multimer binding.

[0118] CD8 T cells were isolated from PBMCs of healthy donors and transformed with the above TCRs. Subsequently, the transformed CD8 T cells were enriched by FACS using the murine constant β region as a marker for transformation. After these cells were expanded, they were stained with HA-1 H -MHC multimers and anti-CD8 antibodies as well as the murine constant β region (mmCb), and analyzed by flow cytometry. The population was gated on viable CD8+ / mCb + cells.

[0119] PBMC from healthy donors were isolated by Ficoll gradient centrifugation. CD8 T cells were enriched by negative magnetic selection (Miltenyi) and stimulated in non-tissue culture 24-well plates pre-coated with anti-CD3 (5 μg / ml) and anti-CD28 (1 μg / ml) mAb (BD Pharmingen, Heidelberg, Germany). Bispecific retroviral particles were generated by transfecting HEK293T cells with a retroviral plasmid encoding the TCR and two expression plasmids, respectively. On the second day after stimulation, CD8 T cells were transduced, and on the twelfth day, the transduced CD8+ cells were enriched by FACS using the murine constant beta region as a transduction marker and then amplified by REP.

[0120] Figure 11 (A) Functional avidity was analyzed by co-culturing HA-1H-TCR transgenic T cells with T2 cells loaded with graded concentrations of HA-1H peptide (10 -12 M - 10 - 5 M). (B) Non-specific recognition of HA-1R peptide by HA-1H TCR-transduced T cells. T2 cells were loaded with graded concentrations of HA-1R peptide (10 -8 M - 10 -5 M) and co-cultured with CD8+ transduced T cells expressing FHCRC or LUMC.

[0121] Figure 12 Different recognition motifs of CD8+ T cells transduced with FHCRC or LUMC are shown. Amino acid substitution scanning was used to define the key amino acids in the epitope sequence, and as long as these residues were exchanged, they would abolish TCR recognition. These "fixed" amino acids can be used to define unique TCR recognition motifs. Serine residues were used to systematically substitute individual amino acids in the HA1 VLH peptide (serine scanning). The first bar in the figure represents the release of IFN-γ in the co-culture of TCR-transduced T cells with T2 cells loaded with 10 -5 M of the original peptide, followed by amino acid substitution via serine at positions 1 - 9. DETAILED DESCRIPTION

[0122] The inventors isolated and sequenced a novel TCR specific for the HA-1 H antigen. Such a TCR 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.

[0123] The inventors have investigated which components of the novel TCR are responsible for the recognition of HA-1 HAntigen specificity and TCR function are essential. Surprisingly, they found that the CDR1 region of the variable domain of the TCRβ chain is crucial for HA-1 H specificity but insufficient for HA-1 specificity. They also found that the CDR3 regions of the variable domain of the TCRβ chain (Vβ) and the variable domain of the TCRα chain (Vα) are essential, and the variable domain of the TCRβ chain (Vβ) needs to be encoded by the TRBV7-9 gene.

[0124] Nucleic acid compositions encoding binding protein components

[0125] The present invention provides an isolated nucleic acid composition encoding a binding protein comprising a T cell receptor (TCR) component that specifically binds to HA-1 H antigen. Thus, the encoded binding protein is capable of specifically binding to a peptide containing the HA-1 H antigen and not binding to a peptide lacking the HA-1 H antigen.

[0126] The nucleic acid composition comprises (a) a nucleic acid sequence encoding a TCR Vα domain having the specific characteristics described herein, and (b) a nucleic acid sequence encoding a TCR Vβ domain having the specific characteristics described herein. The encoded TCR components form an HA-1 H antigen-specific binding protein.

[0127] The nucleic acid sequences of (a) and (b) above can be different nucleic acid sequences in the nucleic acid composition. Thus, the TCR components of the binding protein can be encoded by two (or more) nucleic acid sequences (having different nucleotide sequences) that together encode all the TCR components of the binding protein. In other words, some TCR components can be encoded by one nucleic acid sequence in the nucleic acid composition, while other components can be encoded by another (different) nucleic acid sequence in the nucleic acid composition.

[0128] Alternatively, the nucleic acid sequences of (a) and (b) can be part of a single nucleic acid sequence. Thus, the TCR components of the binding protein can all be encoded by a single nucleic acid sequence (e.g., having a single open reading frame, or having multiple (e.g., 2 or more, 3 or more, etc.) open reading frames).

[0129] The nucleic acid sequences described herein can form part of a larger nucleic acid sequence encoding a larger component part of a functional binding protein. For example, a nucleic acid sequence encoding a TCR Vα domain having the specific characteristics described herein can be part of a larger nucleic acid sequence encoding a functional TCRα chain (including the constant domain). As another example, a nucleic acid sequence encoding a TCR Vβ domain having the specific characteristics described herein can be part of a larger nucleic acid sequence encoding a functional TCRβ chain (including the constant domain). As another example, the above nucleic acid sequences (a) and (b) can be part of a larger nucleic acid sequence encoding a combination of a functional TCRα chain (including the constant region) and a functional TCRβ chain (including the constant region), optionally, wherein the sequence encoding the functional TCRα chain is separated from the sequence encoding the functional TCRβ chain by a linker sequence capable of coordinating the expression of the two proteins or polypeptides in the same nucleic acid sequence. More details are provided below.

[0130] The nucleic acid sequences described herein can also encode a small component of a T cell receptor, such as only the TCR Vα domain, or the TCR Vβ domain. The nucleic acid sequences can be considered "building blocks" that provide the necessary components for peptide binding specificity. The nucleic acid sequences described herein can be integrated into different nucleic acid sequences (such as vectors) that encode other elements of a functional binding protein (such as a TCR), such that when the nucleic acid sequences described herein are incorporated, a new nucleic acid sequence is produced that encodes, for example, a TCRα chain and / or a TCRβ chain that specifically binds to the HA-1 H antigen. Thus, the nucleic acid sequences described herein can be used as an essential component for conferring binding specificity to the HA-1 H antigen and can therefore be used to generate a larger nucleic acid sequence encoding a binding protein having the desired antigen binding activity and specificity.

[0131] The nucleic acid sequences described herein can be codon-optimized for expression in a host cell. For example, they can be codon-optimized for expression in human cells, such as cells of the immune system, 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). The T cells can be CD4+ or CD8+ T cells. Codon optimization is a well-known method in the art for maximizing the expression of a nucleic acid sequence in a particular host cell. As described in the Examples section below, one or more cysteine residues can also be introduced into the encoded TCRα and β chain components (such as to reduce the risk of mismatch with endogenous TCR chains).

[0132] In one embodiment, the nucleic acid sequences described herein are codon-optimized for expression in a suitable host cell and / or are modified to introduce codons encoding one or more cysteine amino acids (e.g., introduced into the constant region of the encoded TCRα chain and / or the encoded TCRβ chain) to reduce the risk of mispairing with endogenous TCR chains.

[0133] In certain embodiments, the TCR constant regions are modified to enhance the pairing of the desired TCR chains. For example, due to the modification, enhanced pairing between a heterologous TCRα chain and a heterologous TCRβ chain may result in preferential assembly of a TCR comprising two heterologous chains rather than an undesired mispairing of a heterologous TCR chain with an endogenous TCR chain (see, e.g., Govers et al, Trends Mol. Med. 16(2):11(2010)). Exemplary modifications that enhance heterologous TCR chain pairing include introducing complementary cysteine residues in each heterologous TCRα and β chain. In some embodiments, 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).

[0134] The binding protein encoded by the nucleic acid compositions described herein H is specific for the HA-1 H antigen and comprises an HA-1 H antigen-specific TCR component. However, the encoded binding protein is not limited to a TCR. Also included are other suitable binding proteins that comprise a specific HA-1 H antigen-specific TCR component. For example, the encoded binding protein can comprise a TCR, an antigen-binding fragment of a TCR, or a chimeric antigen receptor (CAR). TCRs, antigen-binding fragments thereof, and CARs are well defined in the art. A non-limiting example of an antigen-binding fragment of a TCR is a single-chain TCR (scTCR) or a chimeric dimer, which consists of antigen-binding fragments of the TCRα and TCRβ chains linked to the transmembrane and intracellular domains of a dimer complex such that the complex is a chimeric dimer TCR (cdTCR).

[0135] In certain instances, the antigen-binding fragment of a TCR comprises a single-chain TCR (scTCR), which comprises TCR Vα and TCR Vβ domains but only comprises a single TCR constant domain. In other instances, the antigen-binding fragment of a TCR or a chimeric antigen receptor is chimeric (e.g., comprising amino acid residues or motifs from more than one donor or species), humanized (e.g., comprising residues from non-human organisms that have been altered or substituted to reduce the risk of human immunogenicity), or human.

[0136] "Chimeric Antigen Receptor" (CAR) refers to a fusion protein that is engineered to contain two or more naturally occurring amino acid sequences that are joined together in a non-naturally occurring or not naturally present in a host cell manner and can function as a receptor when the fusion protein is present on the cell surface. The CARs described herein include an extracellular portion that contains an antigen-binding domain (i.e., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as an scFv derived from an antibody specific for a cancer antigen or a TCR, or an antigen-binding domain derived from or obtained from a killer immunoglobulin receptor of a NK cell) linked to a transmembrane domain and one or more intracellular signaling domains (optionally including a co-stimulatory domain) (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)).

[0137] Methods for producing engineered TCRs are described, for example, in Bowerman et al, Mol. Immunol, 5(15):3000 (2009). Methods for preparing 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. WO 2014 / 031687; U.S. Patent No. 7,514,537; and Brentjens et al, 2007, Clin. Cancer Res. 73:5426.

[0138] The binding proteins described herein can also be expressed as part of a transgenic construct that encodes other accessory proteins, such as safety switch proteins, tags, selection markers, CD8 co-receptor beta chain, alpha chain or both, or any combination thereof.

[0139] T cell receptor (TCR) is a molecule found on the surface of T cells (T lymphocytes) that is responsible for recognizing peptides that bind to (presented by) major histocompatibility complex (MHC) molecules on target cells. The present invention relates to nucleic acid compositions encoding binding proteins that contain TCR components that interact with specific peptides in the context of a suitable MHC serotype background, i.e., HA-1 in HLA-A*02:01 HAntigen (in other words, the encoded binding protein is capable of specifically binding to HA-1 H Antigen: HLA-A*0201 complex). HLA-A*02:01 is a globally common human leukocyte antigen serotype in the HLA-A serotype group. Peptides presented by HLA-A*02:01 to the TCR are described as "HLA-A*02:01 restricted".

[0140] HA-1 specifically bound by the binding protein described herein H 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, it can consist of the sequence of SEQ ID NO:10, or it can include (i.e., within a longer sequence) the sequence of SEQ ID NO:10. HA-1 H Antigen can be presented by HLA-A*0201. Therefore, the encoded binding protein is capable of specifically binding to HA-1 H Antigen: HLA-A*0201 complex, wherein HA-1 H Antigen is an antigenic fragment of the sequence shown in SEQ ID NO:10, or wherein HA-1 H Antigen contains or consists of the amino acid sequence shown in SEQ ID NO:10.

[0141] The TCR consists of two different polypeptide chains. In humans, 95% of TCRs consist of an α chain and a β chain (encoded by TRA and TRB respectively). When the TCR binds to a peptide in the context of HLA (e.g., in the case of HLA-A*02:01), the T cell is activated through signal transduction.

[0142] The α and β chains of the TCR are highly variable in sequence. Each chain consists of two extracellular domains, a variable region (V) and a constant region (C). The constant region is close to the cell membrane of the T cell, followed by a transmembrane region and a short cytoplasmic tail, while the variable region binds to the peptide / HLA-A complex.

[0143] The variable region of each chain has three hypervariable regions (also known as complementarity-determining regions (CDRs)). Thus, the TCRα variable region (referred to herein as the TCR Vα region, TCR Vα domain, Vα region or Vα domain, α variable domain, etc.) includes CDR1, CDR2, and CDR3 regions. Similarly, the TCRβ variable region (referred to herein as the TCR Vβ region, TCR Vβ domain, Vβ region or Vβ domain, β variable domain, etc.) also contains (distinct) CDR1, CDR2, and CDR3 regions. In each α and β variable region, CDR3 is primarily responsible for recognizing peptides presented by HLA-A.

[0144] As is known to those skilled in the art, the phrase "TCRα chain variable region" refers to the variable (V) domain (extracellular domain) of the TCRα chain and thus includes three hypervariable regions (CDR1, CDR2, and a specific CDR3), as well as insertion sequences, but does not include the constant (C) region of the α chain, which does not form part of the variable region.

[0145] As is known to those skilled in the art, the phrase "TCRβ chain variable region" refers to the variable (V) domain (extracellular domain) of the TCRβ chain and thus includes three hypervariable regions (CDR1, CDR2, and a specific CDR3), as well as insertion sequences, but does not include the constant (C) region of the β chain, which does not form part of the variable region.

[0146] Provided herein is an isolated nucleic acid composition encoding an HA-1 H antigen-specific binding protein having a TCRα chain variable region (Vα) and a TCRβ chain variable region (Vβ), the composition comprising:

[0147] (a) a nucleic acid sequence encoding a TCR Vα domain, the 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

[0148] (b) a nucleic acid sequence encoding a TCR Vβ domain, the 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.

[0149] The arrangements described for (a) below can be combined with the arrangements for (b) below (e.g., to form a suitable nucleic acid composition encoding an HA-1 H antigen-specific binding protein having a TCRα chain variable (Vα) region and a TCRβ chain variable (Vβ) region). More details regarding suitable combinations are provided below.

[0150] Components of the variable (Vα) region of the TCR α-chain

[0151] The isolated nucleic acid compositions described herein encode HA-1 H antigen-specific binding proteins. The HA-1 H antigen-specific binding protein comprises a TCR Vα domain, the TCR Vα domain comprises a CDR3 amino acid sequence, and the amino acid sequence has at least 80% sequence identity with any one of SEQ ID Nos: 1-3.

[0152] (i) A Vα domain comprising the CDR3 amino acid sequence of SEQ ID NO: 1 and its functional variants.

[0153] Confers specific binding to HA-1 H Examples of suitable TCR Vα domain CDR3 amino acid sequences that confer specific binding to the antigen are shown in SEQ ID NO: 1. It will be apparent to those skilled in the art that variants of the amino acid sequence shown in SEQ ID NO: 1 can also be functional (i.e., when the CDR3 is part of the TCR Vα domain, retain their ability to confer specific binding to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10)). Accordingly, these functional variants are encompassed herein.

[0154] For example, suitable (functional) Vα domain CDR3 amino acid sequences can have at least 80% sequence identity with SEQ ID NO: 1, 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 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 1. Suitably, the percentage identity is calculated as the percentage identity over the full length of the reference sequence (e.g., SEQ ID NO: 1). In other words, suitable (functional) Vα domain CDR3 amino acid sequences can differ from the sequence shown in SEQ ID NO: 1 by one or a few (e.g., two, etc.) amino acids.

[0155] As described above, when the CDR3 is part of the TCR Vα domain, the 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).

[0156] Functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:1. The term "variant" also includes homologs. Functional variants typically contain only one, two, or more conservative substitutions of the amino acids of SEQ ID NO:1, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of CDR3.

[0157] 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 typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO:1, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0158] In one instance, the CDR3 of the Vα domain contains or consists of the amino acid sequence of SEQ ID NO:1. In instances 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 its genetic degeneracy sequence (i.e., other nucleic acid sequences that encode the same protein due to genetic code degeneracy). Note that SEQ ID NO:12 is a codon-optimized version of the nucleic acid sequence of the CDR3 of clone M7 (the unoptimized sequence is SEQ ID NO:11).

[0159] The encoded TCR Vα domain can also contain CDR1 in addition to the specific CDR3, and the CDR1 contains the amino acid sequence of SEQ ID NO:80 or a functional variant thereof (i.e., 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). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:80. The term "variant" also includes homologs. Functional variants typically contain only one or more conservative substitutions of the amino acids of SEQ ID NO:80, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0160] Non-functional variants are those that do not specifically bind to HA-1 HAn amino acid sequence variant of SEQ ID NO:80 at the N-terminus of an antigen (e.g., the peptide shown in SEQ ID NO:10). Non-functional variants generally 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 of key amino acids or key regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0161] For example, a suitable functional Vα domain CDR1 amino acid sequence can have at least 80% sequence identity with 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 with SEQ ID NO:80. Suitably, the percentage identity is calculated as the percentage identity over the full length of the reference sequence (e.g., SEQ ID NO:80). In other words, a suitable functional Vα domain CDR1 amino acid sequence can differ from the sequence shown in SEQ ID NO:80 by one or a few amino acids. As described above, compared to the sequence shown in SEQ ID NO:80, the variant can contain amino acid substitutions, such as conservative amino acid substitutions. As described above, when CDR1 is part of the TCR Vα domain, the functional variant of SEQ ID NO:80 retains the ability to specifically bind HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10) at the N-terminus.

[0162] In one instance, CDR1 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO:80. In instances where the TCR Vα domain CDR1 has the amino acid sequence of SEQ ID NO:80, CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO:81 or SEQ ID NO:82 or its genetic degenerate sequences (i.e., other nucleic acid sequences that encode the same protein due to genetic code degeneracy). Note that SEQ ID NO:82 is a codon-optimized version of the nucleic acid sequence of CDR1 of clone M7 (the unoptimized sequence is SEQ ID NO:81).

[0163] Other suitable CDR1 Vα domain amino acid sequences are described elsewhere herein, such as the CDR1 sequence comprising the sequence shown in SEQ ID NO:8. It will be apparent to those skilled in the art that when discussing the alignment of Vα CDR1 amino acid and nucleotide sequences in 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), SEQ ID NO:80 mentioned above can be replaced with SEQ ID NO:8 (and the corresponding nucleotide sequences of SEQ ID NO:9 and 27).

[0164] The encoded TCR Vα domain can also contain a CDR2 in addition to the specific CDR3 (and optionally the specific CDR1 mentioned above), which CDR2 comprises the amino acid sequence of SEQ ID NO:83 or a functional variant thereof (i.e., wherein said variant retains the ability to specifically bind HLA-A*02:01). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:83. The term "variant" also includes homologs. Functional variants typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:83, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0165] Non-functional variants are amino acid sequence variants of SEQ ID NO:83 that do not specifically bind HLA-A*02:01. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO:83, or premature truncation, or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0166] For example, a suitable functional Vα domain CDR2 amino acid sequence can have at least 80% sequence identity with 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 with SEQ ID NO:83. Suitably, the percentage identity is calculated as the percentage identity over the full length of the reference sequence (e.g., SEQ ID NO:83). In other words, a suitable functional Vα domain CDR2 amino acid sequence can differ from the sequence shown in SEQ ID NO:83 by one or several amino acids. As mentioned above, compared with the sequence shown in SEQ ID NO:83, the variant can contain amino acid substitutions, such as conservative amino acid substitutions. As described above, the functional variant of SEQ ID NO:83 retains the ability to specifically bind HLA-A*02:01.

[0167] In one instance, the CDR2 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO:83. In the instance where the TCR Vα domain CDR2 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 its genetic degenerate sequences (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO:85 is the codon-optimized version of the nucleic acid sequence of the CDR2 of clone M7 (the unoptimized sequence is SEQ ID NO:84).

[0168] Other suitable CDR2 Vα domain amino acid sequences are described elsewhere herein, such as the CDR2 sequence comprising the sequence shown in SEQ ID NO:28. It will be apparent to those skilled in the art that when discussing the arrangement of Vα CDR2 amino acid and nucleotide sequences in 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), SEQ ID NO:83 mentioned above can be replaced by SEQ ID NO:28 (and the corresponding nucleotide sequences of SEQ ID NO:39 and 40).

[0169] The encoded TCR Vα domain can thus contain the CDRs detailed above (specifically by SEQ ID, i.e., 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 insert sequences between the CDRs.

[0170] The encoded TCR Vα domain can contain the amino acid sequence of SEQ ID NO:17 or a functional variant thereof (i.e., when part of a binding protein as described herein, where the variant TCR Vα domain retains the ability to specifically bind HA-1 H antigen (such as the peptide shown in SEQ ID NO:10)). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:17. The term "variant" also includes homologs. Functional variants typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:17, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0171] Non-functional variants are amino acid sequence variants of SEQ ID NO:17 that do not specifically bind HA-1 H antigen (such as the peptide shown in SEQ ID NO:10). Non-functional variants typically contain non-conservative substitutions, deletions, or insertions or premature truncations of the amino acid sequence of SEQ ID NO:17, or substitutions, insertions, or deletions of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0172] In one instance, the encoded TCR Vα domain can 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, while retaining the ability to specifically bind HA-1 HThe ability of an antigen (such as the peptide shown in SEQ ID NO:10). In other words, it also encompasses a functional TCR Vα domain having one or several amino acid substitutions compared to the sequence of SEQ ID NO:17. As mentioned above, the amino acid substitution can be a conservative amino acid substitution. Compared to SEQ ID NO:17, the variability of the sequence may all be in the 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 still have 25% (or less) sequence variability compared to SEQ ID NO:17). In other words, the sequences of the CDRs of SEQ ID NO:17 can be retained while the rest of the sequence varies and is suitable within the above "at least 75% identity" parameter. Suitably, the percentage of identity can be calculated as the percentage of identity relative to the full length of the reference sequence (such as SEQ ID NO:17).

[0173] For example, the encoded TCR Vα domain can comprise an amino acid sequence having at least 75% (such as 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, wherein the TCR Vα domain comprises a CDR3 having the amino acid sequence of SEQ ID NO:1. In this example, the TCR Vα domain CDR1 can have the amino acid sequence of SEQ ID NO:80, while the TCR Vα domain CDR2 can have the amino acid sequence of SEQ ID NO:83.

[0174] As another example, the encoded TCR Vα domain can comprise an amino acid sequence having 0 to 10 (or 0 to 5) amino acid substitutions, insertions, or deletions on the amino acid sequence of SEQ ID NO:17, wherein the TCR Vα domain comprises a CDR3 having the amino acid sequence of SEQ ID NO:1. In this example, the TCR Vα domain CDR1 can have the amino acid sequence of SEQ ID NO:80, while the TCR Vα domain CDR2 can have the amino acid sequence of SEQ ID NO:83.

[0175] In instances 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 genetic degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO:20 is a codon-optimized version of the nucleic acid sequence of the TCR Vα domain of clone M7 (the non-optimized sequence is SEQ ID NO:19).

[0176] For the avoidance of doubt, the nucleic acid sequence encoding the TCR Vα domain may also encode the TCRα chain constant domain. Examples of suitable constant regions are encoded in the MP71-TCR-flex retroviral vector. However, the present invention is not limited to this particular constant region and includes any suitable TCRα chain constant domain. The constant domain can be murine, human, or humanized. Methods for identifying or generating suitable constant domains are well known to those skilled in the art and are entirely within their routine capabilities.

[0177] By way of example only, the constant region can be encoded by a vector or derived from a vector (such as a lentivirus, retrovirus, or plasmid vector, but can also be an adenovirus, adeno-associated virus, vaccinia virus, canarypox virus, or herpesvirus vector, where murine or human constant regions are pre-cloned). More recently, minicircles have also been described for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors published by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics, and M Hudecek in Leukemia 2016). In addition, naked (synthetic) DNA / RNA can also be used to introduce the TCR. For example, the pMSGV retroviral vector with pre-cloned TCR-Ca and Cb genes, as described by LV Coren et al., BioTechniques 2015, can be used to provide an appropriate constant region. 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 vol 559; page 405). As a further option, non-homologous end joining is possible.

[0178] Examples of specific TCRα chain amino acid sequences comprising a TCR Vα domain with a suitable constant domain as described herein are shown in SEQ ID NO: 47 and SEQ ID NO: 48. Notably, the constant region shown in SEQ ID NO: 48 is murine. Also included are suitable functional variants of SEQ ID NO: 47 and SEQ ID NO: 48 (e.g., variants having at least 75% (such as 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, which, when part of a binding protein as described herein, retain their ability to specifically bind the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10)). In other words, functional TCRα chains having one or several amino acid substitutions compared to the sequences of SEQ ID NO: 47 or SEQ ID NO: 48 are also encompassed. As described above, the amino acid substitutions can be conservative amino acid substitutions. Compared to SEQ ID NO: 47 or SEQ ID NO: 48, the variability of the sequence may all be in regions of the TCRα chain that do not form CDRs (i.e., the variant may have the CDRs of SEQ ID NO: 1, SEQ ID NO: 80, and / or SEQ ID NO: 83 and still have 25% (or less) sequence variability compared to SEQ ID NO: 47 or SEQ ID NO: 48). In other words, the CDR sequences of SEQ ID NO: 47 or SEQ ID NO: 48 can be retained while the rest of the sequence varies, being suitable within the above "at least 75% identity" parameter. Suitably, the percentage of identity can be calculated as the percentage of identity relative to the full length of the reference sequence (e.g., the appropriate SEQ ID NO: 47 or SEQ ID NO: 48).

[0179] For example, the encoded TCRα chain can comprise an amino acid sequence having at least 75% (such as 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, wherein the TCRα chain comprises a CDR3 having the amino acid sequence of SEQ ID NO: 1. In this example, the TCRα chain CDR1 can have the amino acid sequence of SEQ ID NO: 80, while the TCRα chain CDR2 can have the amino acid sequence of SEQ ID NO: 83.

[0180] In instances where the TCR α-chain has the amino acid sequence of SEQ ID NO: 47, the TCR α-chain can be encoded by the nucleic acid sequence of SEQ ID NO: 19 or SEQ ID NO: 50 or its genetic degeneracy sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO: 50 is a codon-optimized version of the nucleic acid sequence of the TCR Vα domain of clone M7 (the non-optimized sequence is SEQ ID NO: 49).

[0181] In instances where the TCR α-chain has the amino acid sequence of SEQ ID NO: 48, the TCR α-chain can be encoded by the nucleic acid sequence of SEQ ID NO: 51 or its genetic degeneracy sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0182] (ii) A Vα domain comprising the CDR3 amino acid sequence of SEQ ID NO: 2 and its functional variants.

[0183] Confers specific binding to HA-1 H Examples of suitable TCR Vα domain CDR3 amino acid sequences that confer specific binding to the antigen are shown in SEQ ID NO: 2. It will be apparent to those skilled in the art that 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, retain their ability to confer specific binding to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10)). Accordingly, these functional variants are encompassed herein.

[0184] For example, a suitable (functional) Vα domain CDR1 amino acid sequence can have at least 80% sequence identity with 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 with SEQ ID NO: 2. Suitably, the percentage identity is calculated as the percentage identity over the full length of the reference sequence (e.g., SEQ ID NO: 2). In other words, a suitable (functional) Vα domain CDR3 amino acid sequence can differ from the sequence shown in SEQ ID NO: 2 by one or a few (e.g., 2) amino acids. As described above, the functional variants of SEQ ID NO: 2 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.

[0185] Functional variants can be naturally occurring, synthetic, or synthetically modified functional variants of SEQ ID NO:2. The term "variant" also includes homologs. Functional variants generally contain only one, two, or more conservative substitutions of the amino acids of SEQ ID NO:2, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of CDR3.

[0186] Non-functional variants are amino acid sequence variants of SEQ ID NO:2 that do not specifically bind to HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10). Non-functional variants generally contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO:2, or premature truncations, or substitutions, insertions, or deletions of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0187] In one example, CDR3 of the Vα domain contains 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, CDR3 can be encoded by the nucleic acid sequence of SEQ ID NO:23 or SEQ ID NO:24 or its genetic degeneracy sequence (i.e., other nucleic acid sequences that encode the same protein due to genetic code degeneracy). Note that SEQ ID NO:24 is a codon-optimized version of the nucleic acid sequence of the CDR3 of clone M2 (the unoptimized sequence is SEQ ID NO:23).

[0188] The encoded TCR Vα domain can also contain CDR1 in addition to the specific CDR3, and the CDR1 contains the amino acid sequence of SEQ ID NO:8 or a functional variant thereof (i.e., a variant that retains the ability to specifically bind to the N-terminus of HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:8. The term "variant" also includes homologs. Functional variants generally contain only one or more conservative substitutions of the amino acids of SEQ ID NO:8, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0189] Non-functional variants are those that do not specifically bind to HA-1 HAn amino acid sequence variant of SEQ ID NO:8 at the N-terminus of an antigen (e.g., the peptide shown in SEQ ID NO:10). Non-functional variants generally 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 of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0190] For example, a suitable functional Vα domain CDR1 amino acid sequence can have at least 80% sequence identity with 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 with SEQ ID NO:8. Suitably, the percentage identity is calculated as the percentage identity over the full length of the reference sequence (e.g., SEQ ID NO:8). In other words, a suitable functional Vα domain CDR1 amino acid sequence can differ from the sequence shown in SEQ ID NO:8 by one or a few amino acids. As described above, compared to the sequence shown in SEQ ID NO:8, the variant can contain amino acid substitutions, such as conservative amino acid substitutions. As described above, when CDR1 is part of the TCR Vα domain, a functional variant of SEQ ID NO:8 retains the ability to specifically bind HA-1 H at the N-terminus of an antigen (e.g., the peptide shown in SEQ ID NO:10).

[0191] In one instance, the CDR1 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO:8. In instances where the TCR Vα domain CDR1 has the amino acid sequence of SEQ ID NO:8, CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:27 or its genetic degenerate sequences (i.e., other nucleic acid sequences that encode the same protein due to genetic code degeneracy). Note that SEQ ID NO:27 is a codon-optimized version of the nucleic acid sequence of the CDR1 of clone M2 (the unoptimized sequence is SEQ ID NO:9).

[0192] Other suitable CDR1 Vα domain amino acid sequences are described elsewhere herein, such as the CDR1 sequence comprising the sequence shown in SEQ ID NO:80. It will be apparent to those skilled in the art that when discussing the alignment of Vα CDR1 amino acid and nucleotide sequences in 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), SEQ ID NO:8 mentioned above can be replaced with SEQ ID NO:80 (and the corresponding nucleotide sequences of SEQ ID NO:81 and 82).

[0193] The encoded TCR Vα domain may further comprise a CDR2 in addition to the specific CDR3 (and optionally the specific CDR1 mentioned above), and the CDR2 comprises the amino acid sequence of SEQ ID NO:28 or a functional variant thereof (i.e., a variant that retains the ability to specifically bind HLA-A*02:01). These functional variants may be naturally occurring, synthetic or synthetically improved functional variants of SEQ ID NO:28. The term "variant" also includes homologs. Functional variants typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:28, or substitutions, deletions or insertions of non-critical amino acids in non-critical regions of the protein.

[0194] Non-functional variants are variants of the amino acid sequence of SEQ ID NO:28 that do not specifically bind HLA-A*02:01. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions or premature truncations of the amino acid sequence of SEQ ID NO:28, or substitutions, insertions or deletions of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0195] For example, a suitable functional Vα domain CDR2 amino acid sequence can have at least 80% sequence identity with 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 with SEQ ID NO:28. Suitably, the percentage identity is calculated as the percentage identity over the full length of the reference sequence (e.g., SEQ ID NO:28). In other words, a suitable functional Vα domain CDR2 amino acid sequence can differ from the sequence shown in SEQ ID NO:28 by one or several amino acids. As mentioned above, compared to the sequence shown in SEQ ID NO:28, the variant can contain amino acid substitutions, such as conservative amino acid substitutions. As described above, the functional variant of SEQ ID NO:28 retains the ability to specifically bind HLA-A*02:01.

[0196] In one instance, the CDR2 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO:28. In instances where the TCR Vα domain CDR2 has the amino acid sequence of SEQ ID NO:28, the CDR2 can be encoded by the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:40 or its genetic degenerate sequences (i.e., other nucleic acid sequences that encode the same protein due to genetic code degeneracy). Note that SEQ ID NO:40 is a codon-optimized version of the nucleic acid sequence of the CDR2 of clone M2 (the unoptimized sequence is SEQ ID NO:39).

[0197] Other suitable CDR2 Vα domain amino acid sequences are described elsewhere herein, such as the CDR2 sequence comprising the sequence shown in SEQ ID NO:83. It will be apparent to those skilled in the art that when discussing the alignment of Vα CDR2 amino acid and nucleotide sequences in combination with the CDR3 sequence of SEQ ID NO:2 (or the corresponding nucleotide sequences of SEQ ID NO:23 or SEQ ID NO:24), SEQ ID NO:28 mentioned above can thus be replaced by SEQ ID NO:83 (and the corresponding nucleotide sequences of SEQ ID NO:84 and 85).

[0198] The encoded TCR Vα domain can thus contain the CDRs detailed above (specifically by SEQ ID, namely 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), with appropriate insertional sequences between the CDRs.

[0199] The encoded TCR Vα domain can contain the amino acid sequence of SEQ ID NO:29 or a functional variant thereof (i.e., when part of a binding protein as described herein, where the variant TCR Vα domain retains the ability to specifically bind to HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10)). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:29. The term "variant" also includes homologs. Functional variants typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:29, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0200] Non-functional variants are amino acid sequence variants of SEQ ID NO:29 that do not specifically bind to HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10). Non-functional variants typically contain non-conservative substitutions, deletions, or insertions, or premature truncations of the amino acid sequence of SEQ ID NO:29, or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0201] In one instance, the encoded TCR Vα domain can 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:29, while retaining the ability to specifically bind to HA-1 HThe ability of an antigen (e.g., the peptide shown in SEQ ID NO:10). In other words, it also encompasses a functional TCR Vα domain having one or several amino acid substitutions compared to the sequence of SEQ ID NO:29. As mentioned above, the amino acid substitution can be a conservative amino acid substitution. Compared to SEQ ID NO:29, the variability of the sequence may all be in the 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 still have 25% (or less) sequence variability compared to SEQ ID NO:29). In other words, the sequences of the CDRs of SEQ ID NO:29 can be retained while the rest of the sequence varies and is suitable within the above "at least 75% identity" parameter. Suitably, the percentage of identity can be calculated as the percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO:29).

[0202] For 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:29, wherein the TCR Vα domain comprises a CDR3 having the amino acid sequence of SEQ ID NO:2. In this example, the TCR Vα domain CDR1 can have the amino acid sequence of SEQ ID NO:8, while the TCR Vα domain CDR2 can have the amino acid sequence of SEQ ID NO:28.

[0203] As another example, the encoded TCR Vα domain can comprise an amino acid sequence having 0 to 10 (or 0 to 5) amino acid substitutions, insertions, or deletions on the amino acid sequence of SEQ ID NO:29, wherein the TCR Vα domain comprises a CDR3 having the amino acid sequence of SEQ ID NO:2. In this example, the TCR Vα domain CDR1 can have the amino acid sequence of SEQ ID NO:8, while the TCR Vα domain CDR2 can have the amino acid sequence of SEQ ID NO:28.

[0204] 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 its genetic degenerate sequences (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO:32 is a codon-optimized version of the nucleic acid sequence of the TCR Vα domain of clone M2 (the non-optimized sequence is SEQ ID NO:31).

[0205] For the avoidance of doubt, the nucleic acid sequence encoding the TCR Vα domain may also encode the TCRα chain constant domain. Examples of suitable constant regions are those encoded in the MP71-TCR-flex retroviral vector. However, the present invention is not limited to this particular constant region and includes any suitable TCRα chain constant region. The constant region may be murine, human or humanized. Methods for identifying or generating suitable constant domains are well known to those skilled in the art and are entirely within their routine capabilities.

[0206] By way of example only, the constant region may be encoded by or derived from a vector (such as a lentivirus, retrovirus or plasmid vector, but may also be an adenovirus, adeno-associated virus, vaccinia virus, canarypox virus or herpesvirus vector, where murine or human constant regions are pre-cloned). More recently, minicircles have also been described for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors published by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics and M Hudecek in Leukemia 2016). In addition, naked (synthetic) DNA / RNA may also be used to introduce the TCR. For example, the pMSGV retroviral vector with pre-cloned TCR-Ca and Cb genes, as described by LV Coren et al., BioTechniques 2015, may be used to provide a suitable constant region. Alternatively, single-stranded or double-stranded DNA or RNA may be inserted into the TCR locus by homologous directed repair (see Roth et al 2018 Nature vol 559; page 405). As a further option, non-homologous end joining is possible.

[0207] Examples of specific TCRα chain amino acid sequences comprising the TCR Vα domain with a suitable constant domain as described herein are shown in SEQ ID NO: 57 and SEQ ID NO: 58. Notably, the constant region shown in SEQ ID NO: 58 is murine. Also included are suitable functional variants of SEQ ID NO: 57 and SEQ ID NO: 58 (such as 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), which, when part of a binding protein as described herein, retain their specific binding to HA-1 HThe ability of an antigen (such as the peptide shown in SEQ ID NO:10). In other words, it also encompasses a functional TCRα chain having one or several amino acid substitutions compared to the sequences of SEQ ID NO:57 or SEQ ID NO:58. As mentioned above, the amino acid substitutions can be conservative amino acid substitutions. Compared to SEQ ID NO:57 or SEQ ID NO:58, the variability of the sequence may all be in the regions of the TCRα chain that do not form CDRs (i.e., this variant may have the CDRs of SEQ ID NO:2, SEQ ID NO:8, and / or SEQ ID NO:28 and still have 25% (or less) sequence variability compared to SEQ ID NO:57 or SEQ ID NO:58). In other words, the CDR sequences of SEQ ID NO:57 or SEQ ID NO:58 can be retained while the rest of the sequence varies, being suitable within the above "at least 75% identity" parameter. Suitably, the percentage of identity can be calculated as the percentage of identity relative to the full length of a reference sequence (e.g., the appropriate SEQ ID NO:57 or SEQ ID NO:58).

[0208] For example, the encoded TCRα chain 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:57 or SEQ ID NO:58, wherein the TCRα chain comprises a CDR3 having the amino acid sequence of SEQ ID NO:2. In this instance, the TCRα chain CDR1 can have the amino acid sequence of SEQ ID NO:8, while the TCRα chain CDR2 can have the amino acid sequence of SEQ ID NO:28.

[0209] In the instance where the TCRα 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 its genetic degeneracy sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO:60 is a codon-optimized version of the nucleic acid sequence of the TCR Vα domain of clone M2 (the non-optimized sequence is SEQ ID NO:59).

[0210] In the instance where the TCRα 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 its genetic degeneracy sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0211] (iii) A Vα domain comprising the CDR3 amino acid sequence of SEQ ID NO:3 and functional variants thereof.

[0212] endowed with HA-1 H Examples of suitable TCRVα domain CDR3 amino acid sequences that confer specific binding to the HA-1 antigen are shown in SEQ ID NO: 3. It will be apparent to those skilled in the art that 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 TCRVα domain, retain their ability to confer specific binding to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10)). Accordingly, these functional variants are encompassed herein.

[0213] For example, suitable functional Vα domain CDR1 amino acid sequences can have at least 80% sequence identity with 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 with SEQ ID NO: 3. Suitably, the percentage identity is calculated as the percentage identity over the full length of the reference sequence (e.g., SEQ ID NO: 3). In other words, suitable (functional) Vα domain CDR3 amino acid sequences can differ from the sequence shown in SEQ ID NO: 3 by one or a few (e.g., 2) amino acids. As described above, when the CDR3 is part of the TCR Vα domain, functional variants of SEQ ID NO: 2 retain their ability to confer specific binding to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10).

[0214] Functional variants can be naturally occurring, synthetic, or synthetically modified functional variants of SEQ ID NO: 3. The term "variant" also includes homologs. Functional variants generally contain only one, two, or more conservative substitutions of the amino acids of SEQ ID NO: 3, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the CDR3.

[0215] 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 generally contain non-conservative substitutions, deletions, or insertions or premature truncations of the amino acid sequence of SEQ ID NO: 3, or substitutions, insertions, or deletions of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0216] 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 TCR Vα domain CDR3 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 its genetic degenerate sequences (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO: 36 is the codon-optimized version of the nucleic acid sequence of the CDR3 of clone FK47.83 (the unoptimized sequence is SEQ ID NO: 35).

[0217] The encoded TCR Vα domain can further comprise CDR1 in addition to the specific CDR3, and the CDR1 comprises the amino acid sequence of SEQ ID NO: 8 or a functional variant thereof (i.e., 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). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 8. The term "variant" also includes homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 8, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0218] A non-functional variant is a variant of the amino acid sequence 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 generally 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 of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0219] For example, a suitable functional Vα domain CDR1 amino acid sequence can have at least 80% sequence identity with 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 with SEQ ID NO:8. Suitably, the percent identity is calculated as the percent identity over the full length of the reference sequence (e.g., SEQ ID NO:8). In other words, a suitable functional Vα domain CDR1 amino acid sequence can differ from the sequence shown in SEQ ID NO:8 by one or several amino acids. As mentioned above, compared to the sequence shown in SEQ ID NO:8, the variant can contain amino acid substitutions, such as conservative amino acid substitutions. As described above, when CDR1 is part of the TCR Vα domain, a functional variant of SEQ ID NO:8 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).

[0220] In one instance, the CDR1 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO:8. In instances where the TCR Vα domain CDR1 has the amino acid sequence of SEQ ID NO:8, CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:27 or its genetic degeneracy sequence (i.e., other nucleic acid sequences that encode the same protein due to genetic code degeneracy). Note that SEQ ID NO:27 is a codon-optimized version of the nucleic acid sequence cloning the CDR1 of FK47.83 (the unoptimized sequence is SEQ ID NO:9).

[0221] Other suitable CDR1 Vα domain amino acid sequences are described elsewhere herein, such as the CDR1 sequence comprising the sequence shown in SEQ ID NO:80. It will be apparent to those skilled in the art that when discussing the arrangement of Vα CDR1 amino acid and nucleotide sequences in 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), SEQ ID NO:8 mentioned above can be replaced by SEQ ID NO:80 (and the corresponding nucleotide sequences of SEQ ID NO:81 and 82).

[0222] The encoded TCR Vα domain can contain CDR2 in addition to the specific CDR3 (and optionally the above-mentioned specific CDR1), and the CDR2 contains the amino acid sequence of SEQ ID NO:28 or a functional variant thereof (i.e., where the variant retains the ability to specifically bind HLA-A*02:01). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:28. The term "variant" also includes homologs. Functional variants typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:28, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0223] A non-functional variant is a variant of the amino acid sequence of SEQ ID NO:28 that does not specifically bind HLA-A*02:01. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO:28, or premature truncation, or substitutions, insertions, or deletions of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0224] For example, a suitable functional Vα domain CDR2 amino acid sequence can have at least 80% sequence identity with 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 with SEQ ID NO:28. Suitably, the percentage identity is calculated as the percentage identity over the full length of the reference sequence (e.g., SEQ ID NO:28). In other words, a suitable functional Vα domain CDR2 amino acid sequence can differ from the sequence shown in SEQ ID NO:28 by one or a few amino acids. As described above, compared to the sequence shown in SEQ ID NO:28, the variant can contain amino acid substitutions, such as conservative amino acid substitutions. As described above, the functional variant of SEQ ID NO:28 retains the ability to specifically bind HLA-A*02:01.

[0225] In one example, the CDR2 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO: 28. In an 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: 9 or SEQ ID NO: 40 or its genetic degenerate sequences (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO: 40 is the codon-optimized version of the nucleic acid sequence of the CDR2 of clone FK47.83 (the unoptimized sequence is SEQ ID NO: 39).

[0226] Other suitable CDR2 Vα domain amino acid sequences are described elsewhere herein, such as the CDR2 sequence containing the sequence shown in SEQ ID NO: 83. It will be apparent to those skilled in the art that when discussing the alignment of the Vα CDR2 amino acid and nucleotide sequences in 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), SEQ ID NO: 28 mentioned above can thus be replaced with SEQ ID NO: 83 (and the corresponding nucleotide sequences of SEQ ID NOs: 84 and 85).

[0227] The encoded TCR Vα domain can thus contain the CDRs mentioned in detail above (specifically by SEQ ID, 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), with appropriate insertion sequences between the CDRs.

[0228] The encoded TCR Vα domain can contain the amino acid sequence of SEQ ID NO: 41 or a functional variant thereof (i.e., when part of a binding protein as described herein, where 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)). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 41. The term "variant" also includes homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO: 41, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0229] Non-functional variants do not specifically bind to HA-1 HAn amino acid sequence variant of SEQ ID NO: 41 of an antigen (such as the peptide shown in SEQ ID NO: 10). Non-functional variants generally contain non-conservative substitutions, deletions, or insertions or premature truncations of the amino acid sequence of SEQ ID NO: 41, or substitutions, insertions, or deletions of key amino acids or key regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0230] In one example, the encoded TCR Vα domain can 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: 41, while maintaining the ability to specifically bind HA-1 H antigen (such as the peptide shown in SEQ ID NO: 10). In other words, also covered are functional TCR Vα domains having one or several amino acid substitutions compared to the sequence of SEQ ID NO: 41. As described above, the amino acid substitutions can be conservative amino acid substitutions. Compared to SEQ ID NO: 41, the variability of the sequence may all be in 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 still have 25% (or less) sequence variability compared to SEQ ID NO: 41). In other words, the sequences of the CDRs of SEQ ID NO: 41 can be retained while the rest of the sequence varies, within the "at least 75% identity" parameter described above. Suitably, the percentage of identity can be calculated as the percentage of identity relative to the full length of the reference sequence (such as SEQ ID NO: 41).

[0231] For example, the encoded TCR Vα domain can contain an amino acid sequence having at least 75% (such as 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, wherein the TCR Vα domain contains a CDR3 having the amino acid sequence of SEQ ID NO: 3. In this example, the TCR Vα domain CDR1 can have the amino acid sequence of SEQ ID NO: 8, while the TCR Vα domain CDR2 can have the amino acid sequence of SEQ ID NO: 28.

[0232] As another example, the encoded TCR Vα domain can comprise an amino acid sequence having 0 to 10 (or 0 to 5) amino acid substitutions, insertions, or deletions on the amino acid sequence of SEQ ID NO: 41, wherein the TCR Vα domain comprises a CDR3 having the amino acid sequence of SEQ ID NO: 3. In this example, the TCR Vα domain CDR1 can have the amino acid sequence of SEQ ID NO: 8, while the TCR Vα domain CDR2 can have the amino acid sequence of SEQ ID NO: 28.

[0233] In an 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 genetic degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO: 44 is a codon-optimized version of the nucleic acid sequence of the TCR Vα domain cloned from FK47.83 (the non-optimized sequence is SEQ ID NO: 43).

[0234] For the avoidance of doubt, the nucleic acid sequence encoding the TCR Vα domain can also encode the TCRα chain constant domain. Examples of suitable constant regions are encoded in the MP71-TCR-flex retroviral vector. However, the present invention is not limited to this particular constant region and includes any suitable TCRα chain constant domain. The constant domain can be murine, human, or humanized. Methods for identifying or generating suitable constant domains are well known to those skilled in the art and are entirely within their routine capabilities.

[0235] For example only, the constant region can be encoded by or derived from a vector (such as a lentivirus, retrovirus or plasmid vector, but can also be an adenovirus, adeno-associated virus, vaccinia virus, canarypox virus or herpesvirus vector, where murine or human constant regions are pre-cloned). Recently, minicircles have also been described for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors published by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics and M Hudecek in Leukemia 2016). Additionally, naked (synthetic) DNA / RNA can also be used to introduce TCRs. For example, the pMSGV retroviral vector with pre-cloned TCR-Ca and Cb genes, as described by LV Coren et al., BioTechniques 2015, can be used to provide an appropriate constant region. 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 vol 559; page 405). As a further option, non-homologous end joining is possible.

[0236] Examples of specific TCRα chain amino acid sequences including the TCR Vα domain with an appropriate constant domain as described herein are shown in SEQ ID NO: 67 and SEQ ID NO: 68. Notably, the constant region shown in SEQ ID NO: 68 is murine. Also included are suitable functional variants of SEQ ID NO: 67 and SEQ ID NO: 68 (such as 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), which, when part of a binding protein as described herein, maintain their specific binding to HA-1 HThe ability of an antigen (e.g., the peptide shown in SEQ ID NO:10). In other words, it also encompasses a functional TCRα chain having one or several amino acid substitutions compared to the sequences of SEQ ID NO:67 or SEQ ID NO:68. As mentioned above, the amino acid substitutions can be conservative amino acid substitutions. Compared to SEQ ID NO:67 or SEQ ID NO:68, the variability of the sequence may all be in the regions of the TCRα chain that do not form CDRs (i.e., this variant may have the CDRs of SEQ ID NO:3, SEQ ID NO:8, and / or SEQ ID NO:28 and still have 25% (or less) sequence variability compared to SEQ ID NO:67 or SEQ ID NO:68). In other words, the CDR sequences of SEQ ID NO:67 or SEQ ID NO:68 can be retained while the rest of the sequence varies and is suitable within the above "at least 75% identity" parameter. Suitably, the percentage of identity can be calculated as the percentage of identity relative to the full length of the reference sequence (e.g., the appropriate SEQ ID NO:67 or SEQ ID NO:68).

[0237] For example, the encoded TCRα chain 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:67 or SEQ ID NO:68, wherein the TCRα chain comprises a CDR3 having the amino acid sequence of SEQ ID NO:3. In this example, the TCRα chain CDR1 can have the amino acid sequence of SEQ ID NO:8, while the TCRα chain CDR2 can have the amino acid sequence of SEQ ID NO:28.

[0238] In the example where the TCRα 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 its genetic degeneracy sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO:70 is a codon-optimized version of the nucleic acid sequence of the TCR Vα domain cloned from FK47.83 (the non-optimized sequence is SEQ ID NO:69).

[0239] In the example where the TCRα 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 its genetic degeneracy sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0240] Composition of the variable (Vβ) region of the TCR β-chain

[0241] The isolated nucleic acid compositions described herein encode HA-1 H antigen-specific binding protein. The encoded HA-1 H antigen-specific binding protein comprises a TCR Vα domain that comprises a CDR3 amino acid sequence that has at least 80% sequence identity with any one of SEQ ID NOs: 1 to 3 above. The encoded HA-1 H antigen-specific binding protein further comprises 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 80% sequence identity with any one of SEQ ID NOs: 4 to 6 and a CDR1 amino acid sequence having at least 80% sequence identity with SEQ ID NO: 7.

[0242] In humans, TCR Vβ chain amino acid sequences are generated in vivo by V(D)J recombination, which rearranges available variable region (V), joining region (J), and in some cases diversity region (D) gene segments. This generates a new repertoire of nucleic acid sequences encoding unique TCR Vβ chains with 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 (e.g., TRBV7-9*01, TRBV7-9*02, TRBV7-9*03, TRBV7-9*04, TRBV7-9*05, TRBV7-9*06, and TRBV7-9*07, with highly conserved nucleotide sequences (see Lefranc, M.-P. and Lefranc, G. The T cell receptor Facts Book Academic Press, London, UK (2001)).

[0243] The inventors have herein demonstrated that HA-1 H antigen-specific binding protein contains a TCRβ chain with a similar Vβ domain. In each of the HA-1 H antigen-specific binding proteins described in the examples below, the Vβ domain comprises an 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.

[0244] Thus, the encoded HA-1 described herein HThe antigen - specific binding protein comprises a TCR Vβ domain having an amino acid sequence encoded by the TRBV7 - 9 gene. The TRBV7 - 9 gene can be any TRBV7 - 9 allele, 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 a particular instance, the TRBV7 - 9 gene is TRBV7 - 9*01 or TRBV7 - 9*03.

[0245] As described above, the binding protein comprises a TCR Vβ domain having an amino acid sequence encoded by the TRBV7 - 9 gene. In addition, the TCR Vβ domain of the binding protein described herein comprises a CDR3 amino acid sequence that has at least 80% sequence identity with any of SEQ ID NOs: 4 to 6 (described directly below). For the avoidance of doubt, any TCR Vβ domain CDR3 sequence described below can be combined with any TCR Vβ domain CDR1 sequence described subsequently to produce a functional TCR Vβ domain.

[0246] (i) A Vβ domain comprising the CDR3 amino acid sequence of SEQ ID NO:4 and functional variants thereof.

[0247] Confers specific binding to HA - 1 H Examples of suitable TCR Vβ domain CDR3 amino acid sequences that confer specific binding to the HA - 1 H antigen (e.g., the peptide shown in SEQ ID NO: 10) are shown in SEQ ID NO: 4. It will be apparent to those skilled in the art that variants of the amino acid sequence shown in SEQ ID NO: 4 can also be functional (i.e., when CDR3 is part of the TCR Vβ domain, retain their ability to confer specific binding to the HA - 1 H antigen (such as the peptide shown in SEQ ID NO: 10)). Accordingly, these functional variants are encompassed herein.

[0248] For example, a suitable functional Vα domain CDR1 amino acid sequence can have at least 80% sequence identity with 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 with SEQ ID NO:4. Suitably, the percentage identity is calculated as the percentage identity over the full length of the reference sequence (e.g., SEQ ID NO:4). In other words, a suitable (functional) Vβ domain CDR3 amino acid sequence can differ from the sequence shown in SEQ ID NO:4 by one or a few (e.g., 2) amino acids. As described above, when CDR3 is part of the TCR Vβ domain, functional variants of SEQ ID NO:4 retain their ability to confer specific binding to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10).

[0249] Functional variants can be naturally occurring, synthetic, or synthetically modified functional variants of SEQ ID NO:4. The term "variant" also includes homologs. Functional variants generally contain only one or more conservative substitutions of the amino acids of SEQ ID NO:4, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of CDR3.

[0250] Non-functional variants are amino acid sequence variants of SEQ ID NO:4 that do not specifically bind to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10). Non-functional variants generally contain non-conservative substitutions, deletions, or insertions or premature truncations of the amino acid sequence of SEQ ID NO:4, or substitutions, insertions, or deletions of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0251] In one example, the CDR3 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO:4. In instances where the TCR Vβ domain CDR3 has the amino acid sequence of SEQ ID NO:4, CDR3 can be encoded by the nucleic acid sequence of SEQ ID NO:13 or SEQ ID NO:14 or a genetic degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein due to genetic code degeneracy). Note that SEQ ID NO:14 is a codon-optimized version of the nucleic acid sequence of CDR3 of clone M7 (the unoptimized sequence is SEQ ID NO:13).

[0252] The encoded TCR Vβ domain may contain CDR1 in addition to a specific CDR3, and the CDR1 contains the amino acid sequence of SEQ ID NO:7 or a functional variant thereof (i.e., a variant that retains the ability to specifically bind to the C-terminus of the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10)). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:7. The term "variant" also includes homologs. Functional variants typically contain only one or more conservative substitutions of the amino acids of SEQ ID NO:7, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0253] A non-functional variant is a variant of the amino acid sequence of SEQ ID NO:7 that does not specifically bind to the C-terminus of the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10). Non-functional variants typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO:7, or premature truncations, or substitutions, insertions, or deletions of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0254] For example, a suitable functional Vβ domain CDR1 amino acid sequence can have at least 80% sequence identity with 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 with SEQ ID NO:7. Suitably, the percentage identity is calculated as the percentage identity over the full length of the reference sequence (e.g., SEQ ID NO:7). In other words, a suitable functional Vβ domain CDR1 amino acid sequence can differ from the sequence shown in SEQ ID NO:7 by one or a few amino acids. As mentioned above, compared with the sequence shown in SEQ ID NO:7, the variant can contain amino acid substitutions, such as conservative amino acid substitutions. As described above, when CDR1 is part of the TCR Vβ domain, the functional variant of SEQ ID NO:7 retains the ability to specifically bind to the C-terminus of the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10).

[0255] In one instance, CDR1 of the Vα domain comprises the amino acid sequence of SEQ ID NO:7 or consists of the amino acid sequence of SEQ ID NO:7. In instances where the CDR1 of the TCR Vα domain has the amino acid sequence of SEQ ID NO:7, CDR1 can be encoded by the nucleic acid sequence of SEQ ID NO:15 or SEQ ID NO:16 or its genetic degenerate sequences (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO:16 is a codon-optimized version of the nucleic acid sequence of CDR1 of clone M7 (the unoptimized sequence is SEQ ID NO:15).

[0256] The encoded TCR Vβ domain can comprise a CDR2 in addition to a specific CDR3 (and optionally the above-mentioned specific CDR1), which CDR2 has the amino acid sequence of SEQ ID NO:86 or a functional variant thereof (i.e., a variant that retains the ability to specifically bind HLA-A*02:01). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:86. The term "variant" also includes homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO:86, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0257] Non-functional variants are variants of the amino acid sequence of SEQ ID NO:86 that do not specifically bind HLA-A*02:01. Non-functional variants generally 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 of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0258] For example, a suitable functional Vβ domain CDR2 amino acid sequence can have at least 80% sequence identity with 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 with SEQ ID NO:86. Suitably, the percent identity is calculated as the percent identity over the full length of the reference sequence (e.g., SEQ ID NO:86). In other words, a suitable functional Vβ domain CDR2 amino acid sequence can differ from the sequence shown in SEQ ID NO:86 by one or a few amino acids. As mentioned above, compared to the sequence shown in SEQ ID NO:86, the variant can contain amino acid substitutions, such as conservative amino acid substitutions. As described above, the functional variant of SEQ ID NO:86 retains the ability to specifically bind HLA-A*02:01.

[0259] In one instance, the CDR2 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO:86. In instances where the TCR Vβ domain CDR2 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 its genetic degeneracy sequence (i.e., other nucleic acid sequences that encode the same protein due to genetic code degeneracy). Note that SEQ ID NO:88 is a codon-optimized version of the nucleic acid sequence of the CDR2 of clone M7 (the unoptimized sequence is SEQ ID NO:87).

[0260] The encoded TCR Vβ domain can thus contain the CDRs detailed above (specific to SEQ ID, i.e., SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO:86, or their functional variants), with appropriate insertion sequences between the CDRs.

[0261] The encoded TCR Vβ domain can have the amino acid sequence of SEQ ID NO:18 or its functional variant (i.e., when part of a binding protein as described herein, wherein the variant TCR Vβ domain retains the ability to specifically bind HA-1 Hthe ability of an antigen (e.g., the peptide shown in SEQ ID NO: 10). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 18. The term "variant" also includes homologs. Functional variants typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 18, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0262] Non-functional variants are those that do not specifically bind to HA-1 H amino acid sequence variants of SEQ ID NO: 18 of an antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 18, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0263] In one instance, the encoded TCR Vβ domain can 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, while retaining the ability to specifically bind to HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10). In other words, functional TCR Vβ domains having one or several amino acid substitutions compared to the sequence of SEQ ID NO: 18 are also encompassed. As mentioned above, the amino acid substitutions can be conservative amino acid substitutions. The variability of the sequence compared to SEQ ID NO: 18 may all be in 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 still have 25% (or less) sequence variability compared to SEQ ID NO: 18). In other words, the sequences of the CDRs of SEQ ID NO: 18 can be retained while the rest of the sequence varies, being suitable within the "at least 75% identity" parameter described above. Suitably, the percentage of identity can be calculated as the percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 18).

[0264] For example, the encoded TCR Vβ 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: 18, wherein the TCR Vβ domain comprises a CDR3 having the amino acid sequence of SEQ ID NO: 4. In this example, the TCR Vβ domain CDR1 can have the amino acid sequence of SEQ ID NO: 7, while the TCR Vβ domain CDR2 can have the amino acid sequence of SEQ ID NO: 86.

[0265] In an 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 a genetic degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO: 22 is a codon-optimized version of the nucleic acid sequence of the TCR Vβ domain of clone M7 (the non-optimized sequence is SEQ ID NO: 21).

[0266] For the avoidance of doubt, the nucleic acid sequence encoding the TCR Vβ domain can also encode a TCRβ chain constant domain. An example of a suitable constant region is encoded in the MP71-TCR-flex retroviral vector. However, the present invention is not limited to this particular constant region and includes any suitable TCRβ chain constant domain. The constant region can be murine, human, or humanized. Methods for identifying or generating a suitable constant domain are well known to those skilled in the art and are entirely within their routine capabilities.

[0267] Merely by way of example, the constant region can be encoded by or derived from a vector (such as a lentivirus, retrovirus or plasmid vector, but can also be an adenovirus, adeno-associated virus, vaccinia virus, canarypox virus or herpesvirus vector, where murine or human constant regions are pre-cloned). More recently, minicircles have also been described for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors published by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics and M Hudecek in Leukemia 2016). Additionally, naked (synthetic) DNA / RNA can also be used to introduce TCRs. For example, the pMSGV retroviral vector with pre-cloned TCR-Ca and Cb genes, as described by LV Coren et al., BioTechniques 2015, can be used to provide an appropriate constant region.

[0268] Examples of specific TCRβ chain amino acid sequences comprising the TCR Vβ domain and an appropriate constant domain as described herein are shown in SEQ ID NO: 52 and SEQ ID NO: 53. Notably, the constant region shown in SEQ ID NO: 53 is murine. Also included are suitable functional variants of SEQ ID NO: 52 and SEQ ID NO: 53 (such as 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: 52 or SEQ ID NO: 53), which, when part of a binding protein as described herein, wherein the variant TCRβ chain amino acid sequence retains its specific binding to HA-1 HThe ability of an antigen (e.g., the peptide shown in SEQ ID NO: 10). In other words, it also encompasses a functional TCRβ chain having one or several amino acid substitutions as compared to the sequences of SEQ ID NO: 52 or SEQ ID NO: 53. As described above, the amino acid substitutions can be conservative amino acid substitutions. As compared to SEQ ID NO: 52 or SEQ ID NO: 53, the variability of the sequence may all be in the regions of the TCRβ chain that do not form CDRs (i.e., the variant may have the CDRs of SEQ ID NO: 4 and SEQ ID NO: 7 and optionally SEQ ID NO: 86, and still have 25% (or less) sequence variability as compared to SEQ ID NO: 52 or SEQ ID NO: 53). In other words, the CDR sequences of SEQ ID NO: 52 or SEQ ID NO: 53 can be retained while the rest of the sequence varies, being suitable within the above "at least 75% identity" parameter. Suitably, the percentage of identity can be calculated as the percentage of identity relative to the full length of a reference sequence (e.g., the appropriate SEQ ID NO: 52 or SEQ ID NO: 53).

[0269] For example, the encoded TCRβ chain 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: 52 or SEQ ID NO: 53, wherein the TCRβ chain comprises a CDR3 having the amino acid sequence of SEQ ID NO: 4. In this example, the TCRβ chain CDR1 can have the amino acid sequence of SEQ ID NO: 7, while the TCRβ chain CDR2 can have the amino acid sequence of SEQ ID NO: 86.

[0270] In the example where the TCRβ 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 its genetic degeneracy sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO: 55 is the codon-optimized version of the nucleic acid sequence of the TCR Vβ domain of clone M7 (the non-optimized sequence is SEQ ID NO: 54).

[0271] In the example where the TCRβ 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 its genetic degeneracy sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0272] In a particular instance, the nucleic acid compositions described herein encode an HA-1H antigen-specific binding protein having a TCR Vα domain and a TCR Vβ domain, wherein the TCR Vα domain has a CDR3 amino acid sequence that comprises or consists of the amino acid sequence of SEQ ID NO:1; the TCR Vβ domain has an amino acid sequence encoded by the TRBV7-9 gene, the TCR Vβ domain has a CDR3 and a CDR1, the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO:4, and the CDR1 comprises or consists of the amino acid sequence of SEQ ID NO:7. The TRBV7-9 gene may be TRBV7-9*03. Further, the HA-1 H antigen may comprise or consist of the sequence set forth in SEQ ID NO:10. Further, the TCR Vα domain may be part of a TCRα chain having a constant domain, and the TCR Vβ domain may be part of a TCRβ chain having a constant domain.

[0273] In this particular instance, 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.

[0274] In this particular instance, the Vα domain may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO:17, the Vα domain may comprise or consist of SEQ ID NO:17; and the Vβ domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:18, the Vβ domain comprises or consists of SEQ ID NO:18. In one instance, 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:18. 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; and the Vβ domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO:21 or SEQ ID NO:22.

[0275] In this particular instance, the TCR Vα domain can include a CDR1 amino acid sequence and a CDR2 amino acid sequence, where the CDR1 amino acid sequence comprises the amino acid sequence of SEQ ID NO:80 or consists of the amino acid sequence of SEQ ID NO:80, and the CDR2 amino acid sequence comprises the amino acid sequence of SEQ ID NO:83 or consists of the amino acid sequence of SEQ ID NO:83. Additionally, the TCR Vβ domain can include a CDR2 amino acid sequence, where the CDR2 amino acid sequence comprises the amino acid sequence of SEQ ID NO:86 or consists of the amino acid sequence of SEQ ID NO:86.

[0276] For the avoidance of doubt, this particular instance includes the components of TCR clone M7 illustrated herein. The different components of TCR clone M7 and their respective SEQ ID Nos are summarized in Table 1 below.

[0277]

[0278]

[0279]

[0280] Table 1 - Components of clone M7 and their respective SEQ ID Nos.

[0281] (ii) A Vβ domain comprising the CDR3 amino acid sequence of SEQ ID NO:5 and functional variants thereof.

[0282] Conferring specific binding to the HA-1 H Examples of suitable TCR Vβ domain CDR3 amino acid sequences that confer specific binding to the antigen are shown in SEQ ID NO:5. It will be apparent to those skilled in the art that variants of the amino acid sequence shown in SEQ ID NO:5 can also be functional (i.e., when the CDR3 is part of the TCR Vβ domain, retain their ability to confer specific binding to the HA-1 H antigen (such as the peptide shown in SEQ ID NO:10)). Accordingly, these functional variants are encompassed herein.

[0283] For example, a suitable functional Vα domain CDR1 amino acid sequence can have at least 80% sequence identity with 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 with SEQ ID NO:5. Suitably, the percentage identity is calculated as the percentage identity over the full length of the reference sequence (e.g., SEQ ID NO:5). In other words, a suitable (functional) Vβ domain CDR3 amino acid sequence can differ from the sequence shown in SEQ ID NO:5 by one or a few (e.g., 2) amino acids. As described above, when CDR3 is part of the TCR Vβ domain, functional variants of SEQ ID NO:5 retain their ability to confer specific binding to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10).

[0284] Functional variants can be naturally occurring, synthetic, or synthetically modified functional variants of SEQ ID NO:5. The term "variant" also includes homologs. Functional variants generally contain only one or more conservative substitutions of the amino acids of SEQ ID NO:5, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of CDR3.

[0285] Non-functional variants are amino acid sequence variants of SEQ ID NO:5 that do not specifically bind to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10). Non-functional variants generally contain non-conservative substitutions, deletions, or insertions or premature truncations of the amino acid sequence of SEQ ID NO:5, or substitutions, insertions, or deletions of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0286] In one instance, the CDR3 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO:5. In instances where the TCR Vβ domain CDR3 has the amino acid sequence of SEQ ID NO:5, CDR3 can be encoded by the nucleic acid sequence of SEQ ID NO:25 or SEQ ID NO:26 or its genetic degeneracy sequence (i.e., other nucleic acid sequences that encode the same protein due to genetic code degeneracy). Note that SEQ ID NO:26 is a codon-optimized version of the nucleic acid sequence of the CDR3 of clone M2 (the unoptimized sequence is SEQ ID NO:25).

[0287] The encoded TCR Vβ domain, in addition to containing a specific CDR3, may also contain CDR1, which contains the amino acid sequence of SEQ ID NO:7 or a functional variant thereof (i.e., a variant that retains the ability to specifically bind to the C-terminus of the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10)). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:7. The term "variant" also includes homologs. Functional variants generally contain only one or more conservative substitutions of the amino acids of SEQ ID NO:7, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0288] Non-functional variants are variants of the amino acid sequence of SEQ ID NO:7 that do not specifically bind to the C-terminus of the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10). Non-functional variants generally contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO:7, or premature truncations, or substitutions, insertions, or deletions of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0289] For example, a suitable functional Vβ domain CDR1 amino acid sequence can have at least 80% sequence identity with 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 with SEQ ID NO:7. Suitably, the percentage identity is calculated as the percentage identity over the full length of the reference sequence (e.g., SEQ ID NO:7). In other words, a suitable functional Vβ domain CDR1 amino acid sequence can differ from the sequence shown in SEQ ID NO:7 by one or a few amino acids. As described above, compared to the sequence shown in SEQ ID NO:7, the variant can contain amino acid substitutions, such as conservative amino acid substitutions. As described above, when CDR1 is part of the TCR Vβ domain, the functional variant of SEQ ID NO:7 retains the ability to specifically bind to the C-terminus of the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10).

[0290] In one instance, the CDR1 of the Vα domain comprises the amino acid sequence of SEQ ID NO:7 or consists of the amino acid sequence of SEQ ID NO:7. In instances 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 its genetic degeneracy sequences (i.e., other nucleic acid sequences that encode the same protein due to genetic code degeneracy). Note that SEQ ID NO:16 is the codon-optimized version of the nucleic acid sequence of the CDR1 of clone M2 (the unoptimized sequence is SEQ ID NO:15).

[0291] The encoded TCR Vβ domain can also comprise a CDR2, in addition to a specific CDR3 (and optionally the above-mentioned specific CDR1), which has the amino acid sequence of SEQ ID NO:86 or a functional variant thereof (i.e., a variant that retains the ability to specifically bind HLA-A*02:01). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:86. The term "variant" also includes homologs. Functional variants generally contain only conservative substitutions of one or more amino acids of SEQ ID NO:86, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0292] A non-functional variant is a variant of the amino acid sequence of SEQ ID NO:86 that does not specifically bind HLA-A*02:01. Non-functional variants generally 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 of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0293] For example, a suitable functional Vβ domain CDR2 amino acid sequence can have at least 80% sequence identity with 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 with SEQ ID NO:86. Suitably, the percent identity is calculated as the percent identity over the full length of the reference sequence (e.g., SEQ ID NO:86). In other words, a suitable functional Vβ domain CDR2 amino acid sequence can differ from the sequence shown in SEQ ID NO:86 by one or several amino acids. As mentioned above, compared to the sequence shown in SEQ ID NO:86, the variant can contain amino acid substitutions, such as conservative amino acid substitutions. As described above, the functional variant of SEQ ID NO:86 retains the ability to specifically bind HLA-A*02:01.

[0294] In one instance, the CDR2 of the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO:86. In instances where the TCR Vβ domain CDR2 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 its genetic degenerate sequences (i.e., other nucleic acid sequences that encode the same protein due to genetic code degeneracy). Note that SEQ ID NO:88 is a codon-optimized version of the nucleic acid sequence of the CDR2 of clone M2 (the unoptimized sequence is SEQ ID NO:87).

[0295] The encoded TCR Vβ domain can thus contain the CDRs detailed above (specifically by SEQ ID, i.e., SEQ ID NO:5, SEQ ID NO:7 and SEQ ID NO:86, or their functional variants), with appropriate insertion sequences between the CDRs.

[0296] The encoded TCR Vβ domain can have the amino acid sequence of SEQ ID NO:30 or its functional variant (i.e., when part of a binding protein as described herein, wherein the variant TCR Vβ domain retains the ability to specifically bind HA-1 HThe ability of an antigen (e.g., the peptide shown in SEQ ID NO:10). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:30. The term "variant" also includes homologs. Functional variants typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:30, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0297] A non-functional variant is one that does not specifically bind to HA-1 H An amino acid sequence variant of SEQ ID NO:30 of an antigen (e.g., the peptide shown in SEQ ID NO:10). Non-functional variants typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO:30, or premature truncation, or substitutions, insertions, or deletions of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0298] In one instance, the encoded TCR Vβ domain can 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, while retaining the ability to specifically bind to HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10). In other words, functional TCR Vβ domains having one or several amino acid substitutions compared to the sequence of SEQ ID NO:30 are also encompassed. As mentioned above, the amino acid substitutions can be conservative amino acid substitutions. The variability of the sequence compared to SEQ ID NO:30 may all be in 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 still have 25% (or less) sequence variability compared to SEQ ID NO:30). In other words, the sequences of the CDRs of SEQ ID NO:30 can be retained while the rest of the sequence varies, within the "at least 75% identity" parameter described above. Suitably, the percentage of identity can be calculated as the percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO:30).

[0299] For example, the encoded TCR Vβ 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: 30, wherein the TCR Vβ domain comprises a CDR3 having the amino acid sequence of SEQ ID NO: 5. In this instance, the TCR Vβ domain CDR1 can have the amino acid sequence of SEQ ID NO: 7, while the TCR Vβ domain CDR2 can have the amino acid sequence of SEQ ID NO: 86.

[0300] In instances 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 genetic degenerate sequence thereof (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO: 34 is a codon-optimized version of the nucleic acid sequence of the TCR Vβ domain of clone M2 (the non-optimized sequence is SEQ ID NO: 33).

[0301] For the avoidance of doubt, the nucleic acid sequence encoding the TCR Vβ domain can also encode a TCRβ chain constant domain. An example of a suitable constant region is encoded in the MP71-TCR-flex retroviral vector. However, the invention is not limited to this particular constant region and includes any suitable TCRβ chain constant domain. The constant domain can be murine, human, or humanized. Methods for identifying or generating suitable constant domains are well known to those skilled in the art and are entirely within their routine capabilities.

[0302] Merely by way of example, the constant region can be encoded by or derived from a vector (such as a lentivirus, retrovirus or plasmid vector, but can also be an adenovirus, adeno-associated virus, vaccinia virus, canarypox virus or herpesvirus vector, where murine or human constant regions are pre-cloned). More recently, minicircles have also been described for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors published by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics and M Hudecek in Leukemia 2016). Additionally, naked (synthetic) DNA / RNA can also be used to introduce TCRs. For example, the pMSGV retroviral vector with pre-cloned TCR-Ca and Cb genes, as described by LV Coren et al., BioTechniques 2015, can be used to provide an appropriate constant region.

[0303] Examples of specific TCRβ chain amino acid sequences comprising the TCR Vβ domain and an appropriate constant domain described herein are shown in SEQ ID NO: 62 and SEQ ID NO: 63. Notably, the constant region shown in SEQ ID NO: 63 is murine. Also included are suitable functional variants of SEQ ID NO: 62 and SEQ ID NO: 63 (such as 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: 62 or SEQ ID NO: 63), which, when part of a binding protein described herein, wherein the variant TCRβ chain amino acid sequence retains its specific binding to HA-1 HThe ability of an antigen (e.g., the peptide shown in SEQ ID NO: 10). In other words, it also encompasses a functional TCRβ chain having one or several amino acid substitutions compared to the sequences of SEQ ID NO: 62 or SEQ ID NO: 63. As mentioned above, the amino acid substitutions can be conservative amino acid substitutions. Compared to SEQ ID NO: 62 or SEQ ID NO: 63, the variability of the sequence may all be in the regions of the TCRβ chain that do not form CDRs (i.e., this variant may have the CDRs of SEQ ID NO: 5 and SEQ ID NO: 7 and optionally SEQ ID NO: 86, and still have 25% (or less) sequence variability compared to SEQ ID NO: 62 or SEQ ID NO: 63). In other words, the CDR sequences of SEQ ID NO: 62 or SEQ ID NO: 63 can be retained while the rest of the sequence varies, being suitable within the above "at least 75% identity" parameter. Suitably, the percentage of identity can be calculated as the percentage of identity relative to the full length of a reference sequence (e.g., the appropriate SEQ ID NO: 62 or SEQ ID NO: 63).

[0304] For example, the encoded TCRβ chain 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: 62 or SEQ ID NO: 63, wherein the TCRβ chain comprises a CDR3 having the amino acid sequence of SEQ ID NO: 5. In this instance, the TCRβ chain CDR1 can have the amino acid sequence of SEQ ID NO: 7, while the TCRβ chain CDR2 can have the amino acid sequence of SEQ ID NO: 86.

[0305] In the instance where the TCRβ 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 its genetic degeneracy sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO: 65 is the codon-optimized version of the nucleic acid sequence of the TCR Vβ domain of clone M7 (the non-optimized sequence is SEQ ID NO: 64).

[0306] In the instance where the TCRβ 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 its genetic degeneracy sequence (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0307] In a particular instance, the nucleic acid compositions described herein encode an HA-1H antigen-specific binding protein having a TCR Vα domain and a TCR Vβ domain, wherein the TCR Vα domain has a CDR3 amino acid sequence that comprises or consists of the amino acid sequence of SEQ ID NO:2; the TCR Vβ domain has an amino acid sequence encoded by the TRBV7-9 gene, and the TCR Vβ domain has a CDR3 and a CDR1, wherein the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO:5, and the CDR1 comprises or consists of the amino acid sequence of SEQ ID NO:7. The TRBV7-9 gene may be TRBV7-9*01. Additionally, the HA-1 H antigen may comprise or consist of the sequence shown in SEQ ID NO:10. Additionally, the TCR Vα domain may be part of a TCRα chain having a constant domain, and the TCR Vβ domain may be part of a TCRβ chain having a constant domain.

[0308] In this particular instance, the CDR3 of the Vα domain may be encoded by a nucleic acid sequence comprising 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 comprising 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 comprising the sequence of SEQ ID NO:15 or SEQ ID NO:16.

[0309] In this particular instance, the Vα domain may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO:29, the Vα domain may comprise or consist of SEQ ID NO:29; and the Vβ domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:30, the Vβ domain comprises or consists of SEQ ID NO:30. In one instance, the Vα domain comprises the amino acid sequence of SEQ ID NO:29, and the Vβ domain comprises the amino acid sequence of SEQ ID NO:30. In such a case, the Vα domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO:31 or SEQ ID NO:32; and the Vβ domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO:33 or SEQ ID NO:34.

[0310] In this particular instance, the TCR Vα domain can include a CDR1 amino acid sequence and a CDR2 amino acid sequence, where the CDR1 amino acid sequence comprises the amino acid sequence of SEQ ID NO:8 or consists of the amino acid sequence of SEQ ID NO:8, and the CDR2 amino acid sequence comprises the amino acid sequence of SEQ ID NO:28 or consists of the amino acid sequence of SEQ ID NO:28. Additionally, the TCR Vβ domain can include a CDR2 amino acid sequence, where the CDR2 amino acid sequence comprises the amino acid sequence of SEQ ID NO:86 or consists of the amino acid sequence of SEQ ID NO:86.

[0311] For the avoidance of doubt, this particular instance includes the components of TCR clone M2 illustrated herein. The different components of TCR clone M2 and their respective SEQ ID Nos are summarized in Table 2 below.

[0312]

[0313]

[0314] Table 2 - Components of clone M2 and their respective SEQ ID Nos.

[0315] (iii) A Vβ domain comprising the CDR3 amino acid sequence of SEQ ID NO:6 and functional variants thereof.

[0316] Confers specific binding to HA-1 H An example of a suitable TCR Vβ domain CDR3 amino acid sequence that confers specific binding to the antigen is shown in SEQ ID NO:6. It will be apparent to those skilled in the art that variants of the amino acid sequence shown in SEQ ID NO:6 can also be functional (i.e., when the CDR3 is part of the TCR Vβ domain, retain their ability to confer specific binding to the HA-1 H antigen (such as the peptide shown in SEQ ID NO:10)). Accordingly, these functional variants are encompassed herein.

[0317] For example, a suitable (functional) Vβ domain CDR1 amino acid sequence can have at least 80% sequence identity with 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 with SEQ ID NO:6. Suitably, the percentage identity is calculated as the percentage identity over the full length of the reference sequence (e.g., SEQ ID NO:6). In other words, a suitable (functional) Vβ domain CDR3 amino acid sequence can differ from the sequence shown in SEQ ID NO:6 by one or a few (e.g., 2) amino acids. As described above, the functional variants of SEQ ID NO:6 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 Vβ domain.

[0318] Functional variants can be naturally occurring, synthetic, or synthetically modified functional variants of SEQ ID NO:6. The term "variant" also includes homologs. Functional variants generally contain only one or more conservative substitutions of the amino acids of SEQ ID NO:6, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of CDR3.

[0319] Non-functional variants are amino acid sequence variants of SEQ ID NO:6 that do not specifically bind to the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10). Non-functional variants generally contain non-conservative substitutions, deletions, or insertions or premature truncations of the amino acid sequence of SEQ ID NO:6, or substitutions, insertions, or deletions of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0320] In one instance, the CDR3 of the Vβ domain comprises or consists of the amino acid sequence of SEQ ID NO:6. In instances where the TCR Vβ domain CDR3 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 its genetic degeneracy sequence (i.e., other nucleic acid sequences that encode the same protein due to genetic code degeneracy). Note that SEQ ID NO:38 is a codon-optimized version of the nucleic acid sequence of the CDR3 of clone FK47.83 (the non-optimized sequence is SEQ ID NO:37).

[0321] The encoded TCR Vβ domain can contain CDR1 in addition to a specific CDR3, and the CDR1 contains the amino acid sequence of SEQ ID NO:7 or a functional variant thereof (i.e., a variant that retains the ability to specifically bind to the C-terminus of the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10)). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:7. The term "variant" also includes homologs. Functional variants 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.

[0322] A non-functional variant is a variant of the amino acid sequence of SEQ ID NO:7 that does not specifically bind to the C-terminus of the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10). Non-functional variants typically contain non-conservative substitutions, deletions, or insertions of the amino acid sequence of SEQ ID NO:7, or premature truncation, or substitutions, insertions, or deletions of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0323] For example, a suitable functional Vβ domain CDR1 amino acid sequence can have at least 80% sequence identity with 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 with SEQ ID NO:7. Suitably, the percentage identity is calculated as the percentage identity over the full length of the reference sequence (e.g., SEQ ID NO:7). In other words, a suitable functional Vβ domain CDR1 amino acid sequence can differ from the sequence shown in SEQ ID NO:7 by one or a few amino acids. As described above, compared with the sequence shown in SEQ ID NO:7, the variant can contain amino acid substitutions, such as conservative amino acid substitutions. As described above, when CDR1 is part of the TCR Vβ domain, the functional variant of SEQ ID NO:7 retains the ability to specifically bind to the C-terminus of the HA-1 H antigen (e.g., the peptide shown in SEQ ID NO:10).

[0324] In one instance, the CDR1 of the Vβ domain comprises or consists of the amino acid sequence of SEQ ID NO:7. In instances 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 its genetic degenerate sequences (i.e., other nucleic acid sequences that encode the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO:16 is the codon-optimized version of the nucleic acid sequence of the CDR1 of clone FK47.83 (the unoptimized sequence is SEQ ID NO:15).

[0325] The encoded TCR Vβ domain can further comprise a CDR2, in addition to a specific CDR3 (and optionally the above-mentioned specific CDR1), which has the amino acid sequence of SEQ ID NO:86 or a functional variant thereof (i.e., a variant that retains the ability to specifically bind HLA-A*02:01). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:86. The term "variant" also includes homologs. Functional variants typically contain only one or more conservative substitutions of the amino acids of SEQ ID NO:86, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0326] Non-functional variants are variants of the amino acid sequence of SEQ ID NO:86 that do not specifically bind HLA-A*02:01. Non-functional variants typically contain non-conservative substitutions, deletions, or insertions, or premature truncations of the amino acid sequence of SEQ ID NO:86, or substitutions, insertions, or deletions of critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0327] For example, a suitable functional Vβ domain CDR2 amino acid sequence can have at least 80% sequence identity with 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 with SEQ ID NO:86. Suitably, the percentage identity is calculated as the percentage identity over the full length of the reference sequence (e.g., SEQ ID NO:86). In other words, a suitable functional Vβ domain CDR2 amino acid sequence can differ from the sequence shown in SEQ ID NO:86 by one or several amino acids. As mentioned above, compared with the sequence shown in SEQ ID NO:86, the variant can contain amino acid substitutions, such as conservative amino acid substitutions. As described above, the functional variant of SEQ ID NO:86 retains the ability to specifically bind HLA-A*02:01.

[0328] In one instance, the CDR2 of the Vβ domain comprises or consists of the amino acid sequence of SEQ ID NO:86. In the instance where the TCR Vβ domain CDR2 has the amino acid sequence of SEQ ID NO:86, CDR2 can be encoded by the nucleic acid sequence of SEQ ID NO:87 or SEQ ID NO:88 or its genetic degeneracy sequence (i.e., other nucleic acid sequences that encode the same protein due to genetic code degeneracy). Note that SEQ ID NO:88 is a codon-optimized version of the nucleic acid sequence of the CDR2 of clone FK47.83 (the unoptimized sequence is SEQ ID NO:87).

[0329] The encoded TCR Vβ domain can thus contain the CDRs mentioned in detail above (specifically by SEQ ID, i.e., SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:86, or their functional variants), with appropriate insertion sequences between the CDRs.

[0330] The encoded TCR Vβ domain can have the amino acid sequence of SEQ ID NO:42 or its functional variant (i.e., when part of a binding protein as described herein, where the variant TCR Vβ domain retains the ability to specifically bind HA-1 HThe ability of an antigen (e.g., the peptide shown in SEQ ID NO: 10). These functional variants can be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 42. The term "variant" also includes homologs. Functional variants typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 42, or substitutions, deletions, or insertions of non-critical amino acids in non-critical regions of the protein.

[0331] A non-functional variant does not specifically bind to HA-1 H An amino acid sequence variant of SEQ ID NO: 42 that is an antigen (e.g., the peptide shown in SEQ ID NO: 10). Non-functional variants typically contain non-conservative substitutions, deletions, or insertions in the amino acid sequence of SEQ ID NO: 42, or premature truncations, or substitutions, insertions, or deletions in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to those of ordinary skill in the art.

[0332] In one instance, the encoded TCR Vβ domain can 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, while maintaining the ability to specifically bind to HA-1 H antigen (e.g., the peptide shown in SEQ ID NO: 10). In other words, functional TCR Vβ domains having one or several amino acid substitutions compared to the sequence of SEQ ID NO: 42 are also encompassed. As mentioned above, the amino acid substitutions can be conservative amino acid substitutions. The variability of the sequence compared to SEQ ID NO: 42 can all be in 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 still have 25% (or less) sequence variability compared to SEQ ID NO: 42). In other words, the sequences of the CDRs of SEQ ID NO: 42 can be retained while the rest of the sequence varies, being suitable within the "at least 75% identity" parameter described above. Suitably, the percentage of identity can be calculated as the percentage of identity relative to the full length of the reference sequence (e.g., SEQ ID NO: 42).

[0333] For example, the encoded TCR Vβ 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: 42, wherein the TCR Vβ domain comprises a CDR3 having the amino acid sequence of SEQ ID NO: 6. In this example, the TCR Vβ domain CDR1 can have the amino acid sequence of SEQ ID NO: 7, while the TCR Vβ domain CDR2 can have the amino acid sequence of SEQ ID NO: 86.

[0334] In an 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 a genetic degenerate sequence thereof (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO: 46 is a codon-optimized version of the nucleic acid sequence of the TCR Vβ domain cloned from FK47.83 (the non-optimized sequence is SEQ ID NO: 45).

[0335] For the avoidance of doubt, the nucleic acid sequence encoding the TCR Vβ domain can also encode a TCRβ chain constant domain. An example of a suitable constant region is encoded in the MP71-TCR-flex retroviral vector. However, the present invention is not limited to this particular constant region and includes any suitable TCRβ chain constant domain. The constant domain can be murine, human, or humanized. Methods for identifying or generating suitable constant domains are well known to those skilled in the art and are entirely within their routine capabilities.

[0336] By way of example only, the constant region can be encoded by or derived from a vector (such as a lentivirus, retrovirus or plasmid vector, but can also be an adenovirus, adeno-associated virus, vaccinia virus, canarypox virus or herpesvirus vector, where murine or human constant regions are pre-cloned). More recently, minicircles have also been described for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors published by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics and M Hudecek in Leukemia 2016). In addition, naked (synthetic) DNA / RNA can also be used to introduce TCRs. For example, the pMSGV retroviral vector with pre-cloned TCR-Ca and Cb genes as described by LV Coren et al., BioTechniques 2015, can be used to provide an appropriate constant region.

[0337] Examples of specific TCRβ chain amino acid sequences comprising the TCR Vβ domain and an appropriate constant domain as described herein are shown in SEQ ID NO: 72 and SEQ ID NO: 73. Notably, the constant region shown in SEQ ID NO: 73 is murine. Also included are suitable functional variants of SEQ ID NO: 72 and SEQ ID NO: 73 (such as variants having at least 75% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc.) sequence identity to the amino acid sequences of SEQ ID NO: 62 or SEQ ID NO: 73), when part of a binding protein as described herein, wherein the variant TCRβ chain amino acid sequence retains its specific binding to HA-1 HThe ability of an antigen (e.g., the peptide shown in SEQ ID NO: 10). In other words, it also encompasses a functional TCRβ chain having one or several amino acid substitutions compared to the sequences of SEQ ID NO: 72 or SEQ ID NO: 73. As mentioned above, the amino acid substitutions can be conservative amino acid substitutions. Compared to SEQ ID NO: 72 or SEQ ID NO: 73, the variability of the sequence may all be in the regions of the TCRβ chain that do not form CDRs (i.e., this variant may have the CDRs of SEQ ID NO: 6 and SEQ ID NO: 7 and optionally SEQ ID NO: 86, and still have 25% (or less) sequence variability compared to SEQ ID NO: 72 or SEQ ID NO: 73). In other words, the CDR sequences of SEQ ID NO: 72 or SEQ ID NO: 73 can be retained while the rest of the sequence varies, being suitable within the above "at least 75% identity" parameter. Suitably, the percentage of identity can be calculated as the percentage of identity relative to the full length of a reference sequence (e.g., the appropriate SEQ ID NO: 72 or SEQ ID NO: 73).

[0338] For example, the encoded TCRβ chain 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: 72 or SEQ ID NO: 73, wherein the TCRβ chain comprises a CDR3 having the amino acid sequence of SEQ ID NO: 6. In this example, the TCRβ chain CDR1 can have the amino acid sequence of SEQ ID NO: 7, while the TCRβ chain CDR2 can have the amino acid sequence of SEQ ID NO: 86.

[0339] In the example where the TCRβ 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 its genetic degenerate sequences (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code). Note that SEQ ID NO: 75 is a codon-optimized version of the nucleic acid sequence of the TCR Vβ domain of clone M7 (the non-optimized sequence is SEQ ID NO: 74).

[0340] In the example where the TCRβ 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 its genetic degenerate sequences (i.e., other nucleic acid sequences encoding the same protein due to the degeneracy of the genetic code).

[0341] In one particular instance, the nucleic acid compositions described herein encode an HA-1H antigen-specific binding protein having a TCR Vα domain and a TCR Vβ domain, wherein the TCR Vα domain has a CDR3 amino acid sequence that comprises or consists of the amino acid sequence of SEQ ID NO:3; the TCR Vβ domain has an amino acid sequence encoded by the TRBV7-9 gene, the TCR Vβ domain has a CDR3 and a CDR1, the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO:6, and the CDR1 comprises or consists of the amino acid sequence of SEQ ID NO:7. The TRBV7-9 gene may be TRBV7-9*01. Additionally, the HA-1 H antigen may comprise or consist of the sequence shown in SEQ ID NO:10. Additionally, the TCR Vα domain may be part of a TCRα chain having a constant domain, and the TCR Vβ domain may be part of a TCRβ chain having a constant domain.

[0342] In this particular instance, 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 may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO:15 or SEQ ID NO:16.

[0343] In this particular instance, the Vα domain may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO:41, the Vα domain may comprise or consist of SEQ ID NO:41; and the Vβ domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:42, the Vβ domain comprises or consists of SEQ ID NO:42. In one instance, the Vα domain comprises the amino acid sequence of SEQ ID NO:41, and the Vβ domain comprises the amino acid sequence of 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:43 or SEQ ID NO:44; and the Vβ domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO:45 or SEQ ID NO:46.

[0344] In this particular instance, the TCR Vα domain can include a CDR1 amino acid sequence and a CDR2 amino acid sequence, where the CDR1 amino acid sequence comprises the amino acid sequence of SEQ ID NO:8 or consists of the amino acid sequence of SEQ ID NO:8, and the CDR2 amino acid sequence comprises the amino acid sequence of SEQ ID NO:28 or consists of the amino acid sequence of SEQ ID NO:28. In addition, the TCR Vβ domain can include a CDR2 amino acid sequence, where the CDR2 amino acid sequence comprises the amino acid sequence of SEQ ID NO:86 or consists of the amino acid sequence of SEQ ID NO:86.

[0345] For the avoidance of doubt, this particular instance includes the components of the TCR clone FK47.83 illustrated herein. The different components of the TCR clone FK47.83 and their respective SEQ ID Nos are summarized in Table 3 below.

[0346]

[0347]

[0348] Table 3 - Components of clone FK47.83 and their respective SEQ ID Nos.

[0349] Any TCR Vα domain (or TCR α chain) described herein can bind to any TCR Vα domain (or TCR α chain).

[0350] a) Components of the TCR clone M2 Vα domain and components of the TCR clone M7 (or FK47.83) Vβ domain.

[0351] For example, the nucleic acid composition described herein encodes an HA-1H antigen-specific binding protein having a TCR Vα domain and a TCR Vβ domain, where the TCR Vα domain has a CDR3 amino acid sequence that comprises the amino acid sequence of SEQ ID NO:3 or consists of the amino acid sequence of SEQ ID NO:2; the TCR Vβ domain has an amino acid sequence encoded by the TRBV7-9 gene, the TCR Vβ domain has a CDR3 and a CDR1, the CDR3 comprises the amino acid sequence of SEQ ID NO:4 (or SEQ ID NO:6) or consists of the amino acid sequence of SEQ ID NO:4 (or SEQ ID NO:6), and the CDR1 comprises the amino acid sequence of SEQ ID NO:7 or consists of the amino acid sequence of SEQ ID NO:7. The TRBV7-9 gene may be TRBV7-9*03 (or TRBV7-9*01). In addition, HA-1H The antigen may comprise the sequence shown in SEQ ID NO:10 or consist of the sequence shown in SEQ ID NO:10. In addition, the TCR Vα domain may be part of a TCRα chain having a constant domain, and the TCR Vβ domain may be part of a TCRβ chain having a constant domain.

[0352] In this particular instance, the CDR3 of the Vα domain may be encoded by a nucleic acid sequence comprising 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 comprising the sequence of SEQ ID NO:13 or SEQ ID NO:14 (or SEQ ID NO:37 or SEQ ID NO:38); 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.

[0353] In this particular instance, the Vα domain may comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO:29, the Vα domain may comprise SEQ ID NO:29 or consist of SEQ ID NO:29; and the Vβ domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:18 (or SEQ ID NO:42), the Vβ domain comprises SEQ ID NO:18 (or SEQ ID NO:42) or consists of SEQ ID NO:18 (or SEQ ID NO:42). In one instance, the Vα domain comprises the amino acid sequence of SEQ ID NO:29, and the Vβ domain comprises 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 comprising the sequence of SEQ ID NO:31 or SEQ ID NO:32; and the Vβ domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO:21 or SEQ ID NO:22 (or SEQ ID NO:45 or SEQ ID NO:46).

[0354] In this particular instance, the TCR Vα domain can include a CDR1 amino acid sequence and a CDR2 amino acid sequence, where the CDR1 amino acid sequence comprises the amino acid sequence of SEQ ID NO:8 or consists of the amino acid sequence of SEQ ID NO:8, and the CDR2 amino acid sequence comprises the amino acid sequence of SEQ ID NO:28 or consists of the amino acid sequence of SEQ ID NO:28. In addition, the TCR Vβ domain can include a CDR2 amino acid sequence, where the CDR2 amino acid sequence comprises the amino acid sequence of SEQ ID NO:86 or consists of the amino acid sequence of SEQ ID NO:86.

[0355] b) Components of the Vα domain of TCR clone M7 and components of the Vβ domain of TCR clone M2 (or FK47.83).

[0356] For example, the nucleic acid compositions described herein encode an HA-1H antigen-specific binding protein having a TCR Vα domain and a TCR Vβ domain, where the TCR Vα domain has a CDR3 amino acid sequence that comprises the amino acid sequence of SEQ ID NO:1 or consists of the amino acid sequence of SEQ ID NO:1; the TCR Vβ domain has an amino acid sequence encoded by the TRBV7-9 gene, the TCR Vβ domain has a CDR3 and a CDR1, the CDR3 comprises the amino acid sequence of SEQ ID NO:5 (or SEQ ID NO:6) or consists of the amino acid sequence of SEQ ID NO:5 (or SEQ ID NO:6), and the CDR1 comprises the amino acid sequence of SEQ ID NO:7 or consists of the amino acid sequence of SEQ ID NO:7. The TRBV7-9 gene may be TRBV7-9*01. In addition, the HA-1 H antigen can comprise the sequence shown in SEQ ID NO:10 or consist of the sequence shown in SEQ ID NO:10. In addition, the TCR Vα domain can be part of a TCRα chain having a constant domain, and the TCR Vβ domain can be part of a TCRβ chain having a constant domain.

[0357] In this particular instance, the CDR3 of the Vα domain can 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 can 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); 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.

[0358] In this particular instance, the Vα domain can comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO:17, the Vα domain can comprise SEQ ID NO:17 or consist of SEQ ID NO:17; and the Vβ domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:30 (or SEQ ID NO:42), the Vβ domain comprises SEQ ID NO:30 (or SEQ ID NO:42) or consists of SEQ ID NO:30 (or SEQ ID NO:42). In one instance, the Vα domain comprises the amino acid sequence of SEQ ID NO:17, while 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 can be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO:19 or SEQ ID NO:20; and the Vβ domain can 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).

[0359] In this particular instance, the TCR Vα domain can include a CDR1 amino acid sequence and a CDR2 amino acid sequence, the CDR1 amino acid sequence comprising the amino acid sequence of SEQ ID NO:80 or consisting of the amino acid sequence of SEQ ID NO:80, and the CDR2 amino acid sequence comprising the amino acid sequence of SEQ ID NO:83 or consisting of the amino acid sequence of SEQ ID NO:83. In addition, the TCR Vβ domain can include a CDR2 amino acid sequence, the CDR2 amino acid sequence comprising the amino acid sequence of SEQ ID NO:86 or consisting of the amino acid sequence of SEQ ID NO:86.

[0360] c) Components of the Vα domain of TCR clone FK47.8 and components of the Vβ domain of TCR clone M2 (or M7).

[0361] For example, the nucleic acid compositions described herein encode an HA-1H antigen-specific binding protein having a TCR Vα domain and a TCR Vβ domain, wherein the TCR Vα domain has a CDR3 amino acid sequence that comprises or consists of the amino acid sequence of SEQ ID NO:3; the TCR Vβ domain has an amino acid sequence encoded by the TRBV7-9 gene, the TCR Vβ domain has a CDR3 and a CDR1, the CDR3 comprises or consists of the amino acid sequence of SEQ ID NO:5 (or SEQ ID NO:4), and the CDR1 comprises or consists of the amino acid sequence of SEQ ID NO:7. The TRBV7-9 gene may be TRBV7-9*01 (or TRBV7-9*03). Additionally, the HA-1 H antigen may comprise or consist of the sequence shown in SEQ ID NO:10. Additionally, the TCR Vα domain may be part of a TCRα chain having a constant domain, and the TCR Vβ domain may be part of a TCRβ chain having a constant domain.

[0362] In this particular instance, 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:25 or SEQ ID NO:26 (or 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.

[0363] In this particular instance, the Vα domain can comprise an amino acid sequence having at least 90% sequence identity with SEQ ID NO:41, the Vα domain can comprise SEQ ID NO:41 or consist of SEQ ID NO:41; and the Vβ domain comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:30 (or SEQ ID NO:18), the Vβ domain comprises SEQ ID NO:30 (or SEQ ID NO:18) or consists of SEQ ID NO:30 (or SEQ ID NO:18). In one instance, the Vα domain comprises the amino acid sequence of SEQ ID NO:41, while the Vβ domain comprises the amino acid sequence of SEQ ID NO:30 (or SEQ ID NO:18). In such a case, 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 the Vβ domain can be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO:33 or SEQ ID NO:34 (or SEQ ID NO:21 or SEQ ID NO:22).

[0364] In this particular instance, the TCR Vα domain can include a CDR1 amino acid sequence and a CDR2 amino acid sequence, the CDR1 amino acid sequence comprising the amino acid sequence of SEQ ID NO:8 or consisting of the amino acid sequence of SEQ ID NO:8, and the CDR2 amino acid sequence comprising the amino acid sequence of SEQ ID NO:28 or consisting of the amino acid sequence of SEQ ID NO:28. In addition, the TCR Vβ domain can include a CDR2 amino acid sequence, the CDR2 amino acid sequence comprising the amino acid sequence of SEQ ID NO:86 or consisting of the amino acid sequence of SEQ ID NO:86.

[0365] As described in more detail elsewhere herein, the nucleic acid compositions described herein encode a TCR Vα domain and a TCR Vβ domain that form a TCR capable of specifically binding to HA-1 HBinding proteins of antigens. In instances 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 joined together by a linker, e.g., a linker capable of expressing two proteins or polypeptides from the same vector. For example, a linker containing the porcine teschovirus-1 2A (P2A) sequence can be used, e.g., the 2A sequence from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), or Theophila obfuscata virus (T2A) published by A.L. Szymczak et al., Nature Biotechnology 22, 589-594 (2004), or a 2A-like sequence. 2A and 2A-like sequences are linkers that can be cleaved once the nucleic acid molecule is transcribed and translated. Another example of a linker is an internal ribosome entry site (IRES), which is capable of translating two proteins or polypeptides from the same transcript. Any other suitable linker can also be used. As another 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, e.g., S Jones et al., Human Gene Ther 2009). Identification of suitable linkers and vectors capable of expressing the TCR Vα domain and the TCR Vβ domain is well within the routine capabilities of those skilled in the art.

[0366] Other suitable polypeptide domains can also be encoded by the nucleic acid sequence encoding the TCR Vα domain and / or the TCR Vβ domain. By way of example only, the nucleic acid sequence can include a membrane targeting sequence that provides for 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, e.g., in WO2016 / 071758.

[0367] In one instance, the nucleic acid compositions described herein can encode a soluble TCR. For example, the nucleic acid composition can encode the variable domains of the TCR α and β chains, respectively, and an immunomodulatory molecule, such as a CD3 agonist (e.g., an anti-CD3 scFv). The CD3 antigen is present on mature human T cells, thymocytes, and a subset of natural killer cells. It is associated with the TCR and is involved in TCR signal transduction. Human CD3 antigen-specific antibodies are well known. One such antibody is the murine monoclonal antibody OKT3, which was the first monoclonal antibody approved by the FDA. Other antibodies specific for CD3 have also been reported (see, e.g., WO2004 / 106380; U.S. Patent Application Publication No. 2004 / 0202657; U.S. Pat. No. 6,750,325). The Immune Mobilizing mTCR against Cancer (ImmTAC; Immunocore Limited, Milton Partk, Abingdon, Oxon, UK) is a bifunctional protein that combines an affinity monoclonal T cell receptor (mTCR) targeting mechanism of action (i.e., an anti-CD3 scFv). In another instance, the soluble TCR of the invention can be conjugated to 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.

[0368] In one embodiment, the nucleic acid composition can encode a chimeric single-chain TCR, wherein the TCR α-chain variable domain is linked to the TCR β-chain variable domain, and the constant domain is fused, for example, to a CD3ζ signaling domain. In this instance, the linker is non-cleavable. In another embodiment, the nucleic acid composition can encode a chimeric double-chain TCR, wherein the TCR α-chain variable domain and the TCR β-chain variable domain are each linked to a CD3ζ signaling domain or other transmembrane and intracellular domains. Methods for preparing such single-chain and double-chain TCRs are well known in the art; see, e.g., RAWillemsen et al, Gene Therapy 2000.

[0369] Vector system

[0370] Also provided is a vector system comprising the nucleic acid compositions described herein. The vector system can have one or more vectors. As described above, the binding protein components encoded by the nucleic acid compositions can be encoded by one or more nucleic acid sequences in the nucleic acid compositions. In instances where all of the binding protein components are encoded by a single nucleic acid sequence, the nucleic acid sequence can be present in a single vector (and thus the vector system described herein can comprise only one vector). In instances where the binding protein components are encoded by two or more nucleic acid sequences (where the multiple nucleic acid sequences together encode all of the components of the binding protein), the two or more nucleic acid sequences can be present in one vector (e.g., in different open reading frames of the vector), or can be distributed on two or more vectors. In this instance, the vector system will comprise multiple different vectors (i.e., vectors having different nucleotide sequences).

[0371] Any suitable vector can be used. By way of example 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, non-viral Sleeping Beauty transposition from minicircle vectors published by R Monjezi, C Miskey, T Gogishvili, M Schleef, M Schmeer, H Einsele, Z Ivics and M Hudecek in Leukemia 2016). Alternatively, single-stranded or double-stranded DNA or RNA can be used to transfect lymphocytes with a TCR of interest (see Roth et al 2018 Nature vol 559; page 405).

[0372] 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 is capable of autonomous replication or integration into the host DNA. The vector can include restriction enzyme cleavage sites for insertion of recombinant DNA and can include one or more selectable markers or suicide genes. The vector can be a nucleic acid sequence in the form of a plasmid, phage or cosmid. Preferably, the vector is suitable for expression in a cell (i.e., the vector is an "expression vector"). Preferably, the vector is suitable for expression in human T cells, such as CD8 + T cells or CD4 + T cells). In certain aspects, 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.

[0373] Preferably, the (expression) vector is capable of proliferating in a host cell and being stably transmitted to progeny.

[0374] The vector may comprise regulatory sequences. As used herein, "regulatory sequence" refers to a DNA or RNA element capable of controlling gene expression. Examples of expression control sequences include promoters, enhancers, silencers, TATA boxes, internal ribosome entry sites (IRES), transcription factor attachment sites, transcription terminators, polyadenylation sites, etc. Optionally, the vector comprises one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed. Regulatory sequences include those that direct constitutive expression, as well as tissue-specific regulation and / or inducible sequences.

[0375] Optionally, the vector contains a nucleic acid sequence of interest operably linked to a promoter. As used herein, "promoter" refers to a nucleotide sequence in DNA to which RNA polymerase binds to initiate transcription. The promoter can be inducible or constitutively expressed. Alternatively, the promoter is under the control of a repressor or stimulatory protein. The promoter can be a promoter not naturally occurring in the host cell (e.g., it can be a foreign promoter). Suitable promoters for the expression of target proteins are well known to those skilled in the art, and the promoter selected depends on the host cell.

[0376] "Operably linked" means that a single or combination of the following control elements are together with the coding sequence and have a functional relationship (e.g., a linkage relationship) with each other so as to direct the expression of the coding sequence.

[0377] The vector may contain a transcription terminator. As used herein, "transcription terminator" refers to a DNA element that terminates the function of RNA polymerase responsible for transcribing DNA into RNA. Preferred transcription terminators are characterized by a series of T residues followed by a GC-rich dyad symmetry region.

[0378] The vector may include translation control elements. As used herein, "translation control element" refers to a DNA or RNA element that controls mRNA translation. A preferred translation control element is a ribosome binding site. Preferably, the translation control element is from a homologous system as the promoter, such as the promoter and its associated ribozyme binding site. Preferred ribosome binding sites are known and depend on the host cell selected.

[0379] The vector may contain restriction enzyme recognition sites. As used herein, "restriction endonuclease recognition site" refers to a motif on DNA recognized by a restriction endonuclease.

[0380] The vector may contain a selectable marker. As used herein, "selectable marker" refers to a protein that confers a phenotype to a cell when expressed in a host cell, and this phenotype allows selection of cells expressing the selectable marker gene. Typically, this can be a protein that confers a new beneficial property to the host cell (e.g., antibiotic resistance) or a protein that is expressed on the cell surface and can thus be used for antibody binding. Suitable selectable markers are well known in the art.

[0381] Optionally, the vector may also contain a suicide gene. As used herein, "suicide gene" refers to a protein that induces modified cell death upon treatment with a specific drug. For example, cells modified with the herpes simplex virus thymidine kinase gene can be induced to commit suicide after treatment with specific nucleoside analogs including ganciclovir; cells modified with human CD20 can be induced to commit suicide after treatment with an anti-CD20 monoclonal antibody; cells modified with inducible Caspase9 (iCasp9) can be induced to commit suicide after treatment with AP1903 (revised 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.

[0382] Preferably, the vector contains those genetic elements necessary for the host cell to express the binding protein described herein. The 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.

[0383] Those skilled in the art will be well aware of the molecular techniques available for preparing (expressing) vectors, and how to transduce or transfect the (expressing) vector into a suitable host cell (thereby generating the modified cells further described below). The (expressing) 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, gene gun delivery, transduction with retroviruses, lentiviruses or adeno-associated vectors, liposome transfection, superfection, etc. The specific method used usually depends on the type of vector and cell. Suitable methods for introducing nucleic acid sequences and vectors into host cells such as human cells are well known in the art; for example, see 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. Other conventional methods suitable for preparing expression vectors and introducing them into suitable host cells are described in detail in, for example, WO2016 / 071758.

[0384] It should be understood that in some embodiments, the host cell is contacted with the vector system (e.g., a viral vector) in vitro, and in some embodiments, the host cell is contacted with the vector system (e.g., a viral vector) in vivo.

[0385] The term "host cell" includes any cell into which the nucleic acid composition or vector system described herein can be introduced (e.g., transduced). Once the nucleic acid molecule or vector system has been introduced into a cell, it can be referred to herein as a "modified cell". Once the nucleic acid molecule or vector has been introduced into a host cell, the resulting modified cell should be able to express the encoded binding protein (and, for example, correctly localize the encoded binding protein to achieve its intended function, such as transporting the encoded binding protein to the cell surface).

[0386] The term "modified cell" refers to a genetically altered (e.g., transformed or transfected) cell. This term refers to a particular cell being tested, as well as the progeny or potential progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not actually be identical to the parental cell, but are still included within the scope of the term as used herein.

[0387] The host cell (and thus the modified cell) is typically a eukaryotic cell, particularly a human cell (e.g., a T cell, such as a CD8 + T cell or a CD4 + T cell, or a mixture thereof). The host cell (and thus the modified cell) can be an allogeneic cell (e.g., an allogeneic T cell, such as a CD8 + T cell or a CD4 + T cell, or a mixture thereof), which refers to a cell derived from a different individual, which individual is different from the individual to whom it is subsequently administered. In other words, compared to the subject to be treated, the host cell (and thus the modified cell) can be isolated T cells from a different individual.

[0388] The host cell (and thus the modified cell) can be any cell that is 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, γ-δ T cells, hematopoietic stem cells or other progenitor cells, and any other autologous or allogeneic cells or cell lines (e.g., NK-92 or a T cell line) that are capable of conferring anti-tumor immunity after TCR gene transfer.

[0389] In the context of the treatment methods described herein, the host cell (and thus the modified cell) is typically used for administration to a human subject who is HLA-A*0201 positive. With this in mind, the host cell (and thus the modified cell) is typically HLA-A*0201 negative and / or HA-1 H negative (i.e., it does not express HLA-A*0201 and HA-1 H ).

[0390] Advantageously, the modified cell is capable of expressing the binding protein (i.e., the TCR component part) encoded by the nucleic acid composition or vector system described herein, and thus the modified cell provides an immunotherapy that specifically targets hematological malignancies and can therefore be used to treat or prevent the recurrence of hematological malignancies after allogeneic stem cell transplantation (allo-SCT) in HLA-A*0201 positive subjects. More details regarding such use are given below.

[0391] Pharmaceutical composition

[0392] The nucleic acid compositions, vector systems or modified cells described herein can be provided as part of a pharmaceutical composition. Advantageously, such a composition can be administered to a human subject with a hematological malignancy after allogeneic stem cell transplantation (allo-SCT) to treat or prevent relapse (e.g., by inducing or enhancing HA-1 H antigen-targeted specific immune responses).

[0393] The pharmaceutical composition can comprise the nucleic acid composition, vector system or modified cell described herein, and a pharmaceutically acceptable excipient, adjuvant, diluent, and / or carrier.

[0394] The composition can generally comprise pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, co-immunostimulatory agents (such as adjuvants and cytokines), and optionally other therapeutic agents or compounds.

[0395] As used herein, "pharmaceutically acceptable" means a material that is not biologically or otherwise undesirable, i.e., the substance can be administered to an individual together with the selected nucleic acid composition, vector system or modified cell without causing any undesirable biological effects or interacting in a harmful manner with any other component of the pharmaceutical composition in which it is contained.

[0396] An excipient is a natural or synthetic substance formulated with an active ingredient (such as the nucleic acid sequences, vectors, modified cells or isolated peptides provided herein), with the aim of bulking up the formulation or conferring a therapeutic enhancing effect on the active ingredient in the final dosage form, e.g., facilitating drug absorption or solubility. Excipients are also useful in the manufacturing process, helping to handle the relevant active substances, e.g., by promoting powder flowability or non-stick properties, and also contributing to in vitro stability, e.g., preventing denaturation during the intended shelf life. Pharmaceutically acceptable excipients are well known in the art. Thus, a person of ordinary skill in the art can readily identify suitable excipients. By way of example, suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose solutions, glycerol, ethanol, and the like.

[0397] An adjuvant is a pharmacological and / or immunological reagent that can modify the action of other reagents in a formulation. Pharmaceutically acceptable adjuvants are well known in the art. Thus, a person of ordinary skill in the art can readily identify suitable adjuvants.

[0398] A diluent is a reagent used for dilution. Pharmaceutically acceptable diluents are well known in the art. Thus, a person of ordinary skill in the art can readily identify suitable diluents.

[0399] At the dosages and concentrations used, the carrier is non-toxic to the recipient and is compatible with the other components of the formulation. The term "carrier" denotes an organic or inorganic, natural or synthetic component that is combined with the active ingredient to facilitate its application. Pharmaceutically acceptable carriers are well known in the art. Accordingly, a suitable carrier can be readily identified by one of ordinary skill in the art.

[0400] Treatment of a subject

[0401] The pharmaceutical compositions described herein can be advantageously used for treating or preventing recurrence of hematological malignancies in HLA-A*0201-positive human subjects after allogeneic stem cell transplantation (allo-SCT).

[0402] In one example, the methods for treating or preventing recurrence of hematological malignancies described herein result in an induced or enhanced immune response (e.g., a cell-mediated response) (e.g., a targeted immune response against malignant cells presenting HA-1 H -HLA-A*0201-restricted peptides) in a subject.

[0403] The phrase "induced or enhanced immune response" refers to an increase in the immune response (e.g., a cell-mediated immune response, e.g., a T cell-mediated immune response) of a subject during or after treatment as compared to the immune response of the subject prior to treatment. Thus, an "induced or enhanced" immune response includes any measurable increase in the immune response that is directly or indirectly directed against the hematological malignancy being treated (or prevented).

[0404] Those skilled in the art will be fully aware of the hematological malignancies that can be treated in accordance with the present invention. By way of example, suitable hematological malignancies include leukemia, lymphoma, myelodysplastic syndromes or myeloma.

[0405] 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).

[0406] As another example, when the hematologic 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, extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue (MALT), 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.

[0407] As another example, when the hematologic malignancy includes myelodysplastic syndrome, the myelodysplastic syndrome can be refractory cytopenia with unilineage dysplasia (refractory anemia, refractory neutropenia, and refractory thrombocytopenia), refractory anemia with ring sideroblasts (RARS), refractory anemia with ring sideroblasts - 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), myelodysplasia, unclassifiable, or refractory cytopenia of childhood.

[0408] In some cases, the subject has previously received lymphodepleting chemotherapy, such as lymphodepleting chemotherapy comprising cyclophosphamide, fludarabine, antithymocyte globulin, or a combination thereof.

[0409] Typically, the modified cells administered to the subject are allogeneic.

[0410] As used herein, the term "treatment" is considered to include an intervention performed to prevent the development of a condition, disorder, or symptom or to alter its pathological state (i.e., a hematologic malignancy in this case). Thus, "treatment" refers to therapeutic treatment and prophylactic or preventive measures, where the goal is to prevent or slow down (mitigate) the target condition, disorder, or symptom. Thus, "treatment" includes a reduction, slowing, or inhibition of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in the amount or concentration of malignant cells compared to the amount or concentration of malignant cells prior to treatment, e.g., as measured in a sample obtained from the subject. Methods of measuring the number or concentration of malignant cells include, for example, qRT-PCR, and quantification of hematologic malignancy-specific biomarkers in a sample obtained from the subject.

[0411] As used herein, the term "subject" refers to an individual. A person suffering from or at risk of suffering from a particular condition, disorder, or symptom. A subject can be a patient, i.e., a subject in need of treatment according to the present invention. The subject may have received treatment for the condition, disorder, or symptom. Alternatively, the subject has not received treatment prior to treatment according to the present invention.

[0412] The compositions described herein can be administered to a subject by any conventional route, including injection or gradual infusion over time. Administration can be, for example, by infusion or by intramuscular, intravascular, intracavitary, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, transdermal administration.

[0413] The compositions described herein can be in any form suitable for the above-described modes of administration. For example, a composition comprising modified cells can be in any form suitable for infusion. As a further example, suitable forms for parenteral injection (including subcutaneous, intramuscular, intravascular, or infusion) include sterile solutions, suspensions, or emulsions. Alternatively, the route of administration can be by direct injection into the target area, or by regional or by topical administration. Determination of a suitable dose of the compositions of the present invention is entirely within the routine capabilities of those skilled in the art.

[0414] Advantageously, the compositions described herein can be formulated for T cell receptor (TCR) gene transfer, a method that is rapid, reliable, and capable of generating large numbers of T cells specific for an HA-1 H antigenic peptide (such as the peptide shown in SEQ ID NO:10), independent of the patient's pre-existing immune system. Using TCR gene transfer, modified allogeneic cells suitable for infusion can be generated within a few days.

[0415] The compositions described herein are administered in an effective amount. "Effective amount" means an amount that produces the desired (therapeutic or non-therapeutic) response, either alone or in combination with other doses. The effective amount used will depend, for example, on the (therapeutic or non-therapeutic) objective, the route of administration, and the condition of the patient / subject. For example, the appropriate dose of the compositions of the present invention for a given patient / subject will be determined by the attending physician (or the person administering the composition), taking into account various factors known to modify the action of the compositions of the present invention, such as the severity and type of hematological malignancy, body weight, sex, diet, time and route of administration, other medications, and other relevant clinical factors. The dose and schedule can vary according to the overall condition of the specific condition, disorder, or symptom of the patient / subject. The effective dose can be determined by in vitro or in vivo methods.

[0416] The pharmaceutical compositions described herein are advantageously in unit dosage form.

[0417] Method for generating TCR

[0418] Also provided is a method for producing a binding protein that is capable of specifically binding to a peptide containing the HA-1 H antigen, but not binding to a peptide without the HA-1 H antigen, the method comprising contacting a nucleic acid composition (or vector system) as described herein with a cell under conditions in which the nucleic acid composition is incorporated into and expressed by the cell.

[0419] The method can be carried out ex vivo or in vitro on host cells. Alternatively, the method can be carried out in vivo, wherein the nucleic acid composition (or vector system) is administered to a subject under conditions in which the nucleic acid sequence is introduced into and expressed by the host cell to produce the binding protein, and contacts the host cell in vivo. In one example, the method is not a method of treating the human body or an animal body.

[0420] Suitable in vivo, in vitro and ex vivo methods for contacting a nucleic acid sequence (or vector) with a host cell under conditions in which the nucleic acid sequence (or vector) is incorporated into and expressed by the cell are well known and are described elsewhere herein.

[0421] General definitions

[0422] As used herein, the terms "nucleic acid sequence", "polynucleotide", "nucleic acid" and "nucleic acid molecule" are used interchangeably to refer to oligonucleotide sequences or polynucleotide sequences. The nucleotide sequence can be genomic, synthetic or recombinant and can be double-stranded or single-stranded (representing the sense or antisense strand). The term "nucleotide sequence" includes genomic DNA, cDNA, synthetic DNA and RNA (e.g., mRNA) and analogues of DNA or RNA produced, for example, by using nucleotide analogues.

[0423] 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 the nucleic acid sequence is linked to the naturally associated sequences in its natural environment. In other words, an isolated nucleic acid sequence / composition is not a natural nucleotide sequence / composition, where a "natural nucleotide sequence / composition" refers to the complete nucleotide sequence in its natural environment, when operably linked to the complete promoter that is naturally associated with it and that is also in its natural environment. Such a nucleic acid can be part of a vector and / or such a nucleic acid or polypeptide can be part of a composition (e.g., a cell lysate), and still be isolated because such a vector or composition is not part of the natural environment of the nucleic acid or polypeptide. The term "gene" refers to a DNA segment involved in producing a polypeptide chain; it includes the regions before and after the coding region ("leader and tail") and the intervening sequences (introns) between the individual coding segments (exons).

[0424] As used herein, "specifically binds" or "specifically targets" refers to the association or 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 or K a (i.e., the equilibrium association constant of a specific binding interaction, in units of 1 / M) equal to or greater than 10 5 M -1 (which is equal to the association rate [k on of the association reaction divided by the dissociation rate [k off ) and that does not significantly associate or bind with any other molecule or component in the sample. A binding protein or binding domain (or a fusion protein thereof) can be classified as a "high-affinity" binding protein or binding domain (or a fusion protein thereof) or a "low-affinity" binding protein or binding domain (or a fusion protein thereof). A "high-affinity" binding protein or binding domain refers to a K a of at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1 of those binding proteins or binding domains. A "low-affinity" binding protein or binding domain refers to a K a of up to 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 of those binding proteins or binding domains. Alternatively, affinity can be defined as the equilibrium dissociation constant (K d ) of a specific binding interaction in units of M (e.g., 10 -5 M to 10 -13 M).

[0425] In certain embodiments, a receptor or binding domain can have "enhanced affinity", which refers to a selected or engineered receptor or binding domain that binds more strongly to a target antigen compared to a wild-type (or parental) binding domain. For example, enhanced affinity may be due to a higher K a (equilibrium association constant) of the target antigen compared to the wild-type binding domain, due to a lower K d (dissociation constant) of the target antigen compared to the wild-type binding domain, due to a lower dissociation rate (k offlower than the wild-type binding domain, or combinations thereof. In certain embodiments, the affinity-enhanced TCR can be codon-optimized to enhance expression in a particular host cell, such as a cell of the immune system, 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+ or CD8+ T cell.

[0426] As used herein, the term "HA-1" H antigen" or "HA-1 H peptide antigen" or "peptide antigen containing HA-1 H " (or "minor HA-1" <h>"antigen" or "Minor HA-1" H "peptide antigen" or "Minor HA-1-containing" H "peptide antigen" or "Minor histocompatibility HA-1" H "antigen peptide") refers to a naturally or synthetically produced peptide moiety of the HMHA1 protein having a length range of from about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, up to about 20 amino acids and containing the R139H substitution polymorphism, which can form a complex with an MHC (e.g., HLA) molecule and a binding protein of the present disclosure specific for the HA-1H peptide: the MHC (e.g., HLA) complex can specifically bind to such a complex. Exemplary HA-1 H HA-1 peptide antigens include peptides having the amino acids VLHDDLLEA (SEQ ID NO:10), where the bold histidine in the sequence represents the R139H polymorphism.

[0427] As used herein, the term "HA-1" H "specific binding protein" refers to a protein or polypeptide (e.g., TCR or CAR) that specifically binds to an HA-1 H peptide antigen (or HA-1 H peptide antigen:HLA complex, e.g., on the cell surface), and does not bind to an HMHA peptide lacking the HA-1 H polymorphism (e.g., a peptide comprising the amino acid sequence shown in SEQ ID NO:79), and does not bind to an HLA complex comprising such an HMHA peptide.

[0428] In certain embodiments, the HA-1 H specific binding protein specifically binds to an HA-1-containing peptide (or HA-1 H peptide:HLA complex) having a K -8 d 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, or having an affinity (e.g., measured by the same assay) approximately equal to, at least approximately equal to, or greater than or approximately equal to that exhibited by an exemplary HA-1 specific binding protein provided herein (e.g., any HA-1 d specific TCR provided herein). In certain embodiments, the HA-1 specific binding protein comprises an HA-1 specific immunoglobulin superfamily binding protein or a binding portion thereof. H

[0429] ​Selective binding can occur in the context of HA-1H antigen presentation by HLA-A*02:01. In other words, in certain embodiments, a binding protein that "specifically binds HA-1" can selectively bind only when it is presented by HLA-A*02:01 (i.e., when it is bound by HLA-A*02:01), or when it is in the same structural form as when it is presented by HLA-A*02:01. H "Non-essential" (or non-critical) amino acid residues are residues that can be altered from the wild-type sequence (e.g., the sequence represented by SEQ ID NO) without eliminating, or more preferably without substantially altering, biological activity, while "essential" (or "critical") amino acid residues result in such an alteration. For example, conservative amino acid residues are predicted to be particularly unlikely to be altered, except that amino acid residues within the hydrophobic core of a domain can generally be replaced by other residues having approximately equal hydrophobicity without significantly altering activity.

[0430] A "conservative amino acid substitution" refers to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, non-essential (or non-critical) amino acid residues in a protein are preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly, and the activity of the resulting mutants can be screened to identify mutants that retain activity.

[0431] The calculation of sequence homology or identity between sequences (the terms are used interchangeably herein) is performed as follows.

[0432] The calculation of sequence homology or identity between sequences (the terms are used interchangeably herein) is performed as follows.

[0433] To determine the percent identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and for comparison purposes, non-homologous sequences can be disregarded). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even 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. Then the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. When 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 at that position are identical (as used herein, "identity" of amino acids or nucleic acids is equivalent to "homology" of amino acids or nucleic acids). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which are introduced for optimal alignment of the two sequences.

[0434] Comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the algorithm described in Needleman et al. (1970) J. Mol. Biol. 48:444-453, which has been incorporated into the GAP program in the GCG software package (available at http: / / www.gcg.com), using the BLOSUM 62 matrix or the PAM250 matrix, with gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. In another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com), using the NWSgapdna.CMP matrix, with gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (which should be used if the practitioner is unsure which parameters to use to determine whether a molecule is within the sequence identity or homology limitations 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.

[0435] Alternatively, the percent identity between two amino acid or nucleotide sequences may be determined using the algorithm of Meyers et al. (1989) CABIOS 4:11-17, which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0436] The nucleic acid and protein sequences described herein can be used as a "query sequence" to search against public databases, for example, to identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-410. A BLAST nucleotide search can be performed with the NBLAST program, score = 100, wordlength = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. A BLAST protein search can be performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al. (1997, Nucl. Acids Res. 25:3389-3402). When using the BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See <http: / / www.ncbi.nlm.nih.gov>.

[0437] The polypeptide and nucleic acid molecules described herein can have an amino acid sequence or nucleic acid sequence that is sufficiently identical or substantially identical to the sequence represented by SEQ ID NO. The terms "sufficiently identical" or "substantially identical" are used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimal number of identical or equivalent (e.g., having similar side chains) amino acid residues or nucleotides to the second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common domain or common functional activity. In other words, an amino acid sequence or nucleic acid sequence having one or a few (e.g., two, three, four, etc.) amino acid or nucleic acid substitutions compared to the corresponding sequence represented by SEQ ID NO can be sufficiently or substantially identical to the sequence represented by SEQ ID NO (as long as they retain the necessary function). In such instances, one or a few (e.g., two, three, four, etc.) amino acid or nucleic acid substitutions can be conservative substitutions. For example, an amino acid or nucleotide sequence containing a common domain having at least about 60%, or 65% identity, possibly 75% identity, more likely 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity is defined herein as sufficiently or substantially identical.

[0438] The TCR sequences are defined according to IMGT. For more details, see the LeFranc references in this article, 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).

[0439] [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).

[0440] [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.

[0441] Aspects of the present invention are illustrated by the following non - limiting examples.

[0442] Examples

[0443] TCR gene transfer is an attractive strategy to modify T cells with well - defined specificity in a short time. Recently, the efficacy of TCR transfer has been demonstrated in patients with melanoma or synovial cell sarcoma treated with autologous T cells modified with TCR. To design T cells that can exert selective GvL without GvHD, we more preferably transfer HA - 1 - TCR. To expand the applicability of adoptive T cell therapy in hematological malignancies, we initiated a clinical study using virus - specific T cells with HA - 1 - TCR transfer. We sequenced the TCR chains of three HA - 1 - specific T cell clones M2, M7, and FK47.83 (Table 4).

[0444]

[0445]

[0446] Table 4 HA - 1 - TCR sequences of clones M2, M7, and FK47.83.

[0447] As previously described, we again observed that all three HA - 1 - specific T cell clones expressed a β - chain (TRBV7 - 9) with a similar V - region (4). LZRS retroviral constructs were prepared that encoded the TCRα and β chains of M2 and M7. The HA - 1 - TCRβ chain of M7 was also cloned into the retroviral vector MP71. The TCRα chain was linked to the marker eGFP via an IRES, and the TCRβ chain was linked to the marker truncated nerve growth factor receptor (NGF - R) via an IRES. The two HA - 1 - TCR chains were also linked to the T2A sequence and expressed in the pLZRS vector bound to NGF - R or the MP71 vector without a marker gene. MP71 constructs encoding codon - optimized and cysteine - modified HA - 1 - TCR were also prepared.

[0448] Based on the low cell surface expression of HA-1-TCR after gene transfer as described by us (3), we investigated whether this low expression was due to the inability of the TCR chains to pair effectively with each other or due to the intrinsic properties of the TCR chains. J76 cells lacking TCRαβ were co-transduced (td) with individual HA-1-TCRα and HA-1-TCRβ chains and 17 different TCRα and TCRβ chains, and TCR cell surface expression was measured using an anti-TCRαβ monoclonal antibody. In Figure 1 A, the TCR cell surface expression of HA-1-TCRαβ, CMV B7 -TCRαβ, HA-2-TCRαβ, CMV A2 -TCRαβ, and a mixed TCRα and β chain combination is shown. HA-2-TCRαβ td J76 cells (MFI 330) and CMV A2 -TCRαβ td J76 cells (MFI 274) showed high TCR expression. The TCR expression of HA-1-TCRαβ td J76 cells (MFI 129) was lower than that of HA-2-TCRαβ td J76 cells. In addition, when J76 cells were co-transduced with HA-1-TCRβ and a combination of HA-2- or CMV A2 -TCRα, no restored TCR cell surface expression was observed ( Figure 1 A). In addition, no restored TCR expression was observed in any transduction of the HA-1-TCRβ chain with any of the other 14 TCRα chains (data not shown). In contrast, binding of the HA-1-TCRα chain to HA-2- or CMV A2 -TCRβ chains resulted in TCR cell surface expression comparable to that of the parental HA-2- and CMV A2 -TCR complexes, indicating that the reduced HA-1-TCR cell surface expression was not due to the HA-1-TCRα chain, but due to the HA-1-TCRβ chain.

[0449] Since the TCR cell surface expression of HA-1-TCRβ remained low with all 14 other TCRα chains tested, we concluded that the low HA-1-TCR cell surface expression was not due to low pairing efficiency specific to the HA-1-TCRα and HA-1-TCRβ chains. To rule out the possibility that the LZRS vector used to introduce the TCR chains caused selective low expression of HA-1-TCR, the HA-1-TCRβ gene was inserted into the MP71 vector, which is described as mediating high transgene expression. From Figure 1 It can be seen that the MP71 vector encoding the HA-1-TCR β-chain did not increase the cell surface expression of HA-1-TCR, indicating that the low cell surface expression of HA-1-TCR was not due to the specificity of the vector. To investigate whether the transfer of the HA-1-TCR β-chain resulted in low cell surface expression due to the sequence specificity of the constantly identical variable region of the HA-1-TRBV7-9 chain, the cell surface expression of CMV B7 -TCRβ with the same variable region TRBV7-9 as the HA-1-TCR β-chain but with a completely different CDR3 region was analyzed. As Figure 1 shown in B7 A, the parental CMV B7 -TCR complex exhibited comparable low cell surface expression to the parental HA-1-TCR complex. When the CMV A2 -TCR β-chain was combined with the HA2- or CMV B7 -TCR α-chain, this low TCR expression was not restored, while the combination of the CMV A2 -TCR α-chain with the HA2- or CMV A2 -TCR α-chain led to high cell surface expression of the TCR, comparable to the expression of the parental HA-2- or CMV B7 -TCR. These results indicate that the low expression of the HA-1- and CMV

[0450] -TCR β-chains was due to the sequence specificity of the variable region. Collectively, these data indicate that the low cell surface expression of HA-1-TCR is due to the intrinsic properties of the HA-1-TCR β-chain. Figure 1 To confirm that the suboptimal cell surface expression of HA-1-TCR after gene transfer was due to the intrinsic properties of the HA-1-TCR β-chain, the cell surface expression of HA-1-TCR and the mRNA levels of the HA-1-TCR α- and β-chains were determined in different parental HA-1-specific T cell clones. As Figure 1 shown in Figure 1 As shown in Panel D, there was no significant difference in the mRNA expression levels of HA-1-TCRα or β compared to other T cell clones. In summary, although the mRNA levels of TCRαβ were normal, the parental HA-1-specific T cell clone showed lower TCR cell surface expression. These results suggest that the low expression of HA-1-TCR observed in HA-1-TCR-transferred T cells is an inherent feature of HA-1-TCR, as the TCR expression of the parental HA-1-specific T cell clone was already low.

[0451] To be able to increase the expression of HA-1-TCR after gene transfer, we investigated whether it was possible to identify a specific region of HA-1-TCRβ responsible for the low cell surface expression of this TCR and to increase HA-1-TCR expression by modifying this region (6). For this purpose, the sequences of several TCRβ chains belonging to the TRBV7 variable domain family and known to exhibit high cell surface expression after gene transfer (i.e., HA-2-TRBV7-8, JBBun-TRBV7-6, and 10G5-TRBV7-1) were aligned with the sequences of HA-1 and CMV B7 -TRBV7-9. A total of 30 consensus differences were scattered over the 309 amino acid (aa)-long variable region, with 9 nucleotide differences clustering in the 18-nucleotide-long CDR1 region, as Figure 2 shown in Panel A. Based on these results, we hypothesized that the major CDR1 region of HA-1-TCR TRBV7-9 might influence the cell surface expression of the HA-1-TCRβ chain. To investigate this, we constructed different constructs in which the HA-1-TCRβ CDR1 region was exchanged with the HA-2-TCRβ CDR1 region and vice versa. The TCR cell surface expression of J76 cells transduced with the modified HA-1- and HA-2-TCRs was analyzed using an anti-TCRαβ-specific monoclonal antibody. As Figure 2 shown in Panel B, the exchange of the HA-1-TCRβ CDR1 region with the CDR1 region of HA-2-TCRβ did not result in a significant increase in TCR cell surface expression 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 decrease in TCR cell surface expression on J76 cells. These results suggest that the CDR1 region is not the sole cause of the low TCR cell surface expression. In addition, we demonstrated by transduction of virus-specific T cells with different modified TCR chains that the exchange of the CDR1 region of HA-1-TCRβ with the CDR1 region of HA-2-TCRβ led to a complete elimination of HA-1-specific IFN-γ production ( Figure 2 C), indicating that the HA-1-TCRβ CDR1 region is crucial for HA-1 specificity. However, the exchange of the HA-2-TCRβ CDR1 region with the HA-1-TCRβ CDR1 region showed that exchanging only this region was not sufficient to transfer HA-1 specificity. The exchange of the HA-2-TCRβ CDR1 and CDR3 regions with the regions of HA-1-TCRβ led to HA-1-specificity ( Figure 2 C). However, compared to the parental HA-1-TCR td T cells, the efficiency of these td T cells was still lower because only a very low recognition rate of endogenously processed HA-1 (LCL-BDV) was observed ( Figure 2 C). In summary, the HA-1-TCRβ CDR1 region is crucial for HA-1 specificity but not sufficient to achieve HA-1 specificity. In addition, the CDR3 region and the HA-1-TCRβ chain are also crucial for the specificity of HA-1.

[0452] Based on the similar usage of the TRBV7-9 chain in all HA-1-specific T cell clones (4), we investigated whether chimeric TCR combinations of M2 and M7 TCRs could also be HA-1-specific. Peripheral T cells were transduced with chimeric TCR combinations of 2 HA-1 TCRs and 1 CMV-B7 TCR (both with the TRB7-9 chain), and the functionality of the transferred chimeric TCRs was compared with the original HA-1 TCR combination. As shown in Table 5, the results indicated that the M7β chain could form a functional HA-1 TCR complex with both M7 TRAV25*01 and M2 TRAV13-1*02, but not with CMV TRAV17*01. In addition, M2TRAV13-1*02 and M7 TRAV25*01 did not form a functional HA-1 TCR complex with the TRBV7-9 of the CMV-B7 TCR. These results suggest (as described above) that the combination of TCRα and TCRβ determines HA-1 specificity.

[0453] TCR M2 M7 CMV TRAV13-1*02 TRAV25*01 TRAV17*01 M2 TRBV7-9 24,2 32,6 1,8 M7 TRBV7-9 2,6 33,8 2 CMV TRBV7-9 3,1 2,3 2,3

[0454] Table 5: Chimeric HA-1 TCR. The mean fluorescence intensity (MFI) of the HA-1 pMHC tetramer is shown.

[0455] Since the expression of HA-1-TCR could not be enhanced by modifying specific sequences of the HA-1-TCR β-chain, other strategies to improve the cell surface expression of TCRs for gene transfer were explored. We investigated whether TCR codon optimization or the inclusion of cysteine residues (7) in the constant domains of the HA-1-TCR α and β chains resulted in functional HA-1-specific T cells after gene transfer. We analyzed the cell surface expression (5) of HA-1-TCR after transferring different constructs into virus-specific T cells known to have an endogenous TCR with weak competition for cell surface expression (weak competitor; pp50 VTE-specific T cells, Figure 3 ) and virus-specific T cells known to have an endogenous TCR with strong competition for cell surface expression (strong competitor; EBNA3A FLR-specific T cells, Figure 3 ). As Figure 3 shown in A, transferring the unmodified HA-1-TCR complex into weak competitor T cells resulted in 40% HA-1 tetramer-positive T cells, while after transferring the unmodified HA-1-TCR complex into strong competitor T cells, no clear expression of HA-1-TCR could be measured using tetramers after transferring the unmodified HA-1-TCR complex. Inclusion of cysteine residues in both HA-1-TCR chains increased the expression of HA-1-TCR, particularly in strong competitor virus-specific T cells. As expected, having cysteine residues in only one of the two HA-1-TCR chains significantly reduced the expression of HA-1-TCR. In addition, codon optimization increased the expression of HA-1-TCR in both weak and strong competitor virus-specific T cells. However, the increased HA-1-TCR expression did not seem to be due to increased expression of the HA1-TCR β-chain, but rather due to increased expression of the HA1-TCR α-chain, since T cells transferred with a combination of codon-optimized HA-1-TCR α-chain and wild-type HA-1-TCR β-chain showed a similar increase in the percentage of HA-1-tetramer-positive T cells compared to T cells transferred with codon-optimized HA-1-TCR α and β chains. In virus-specific T cells of both weak and strong competitors, the combination of codon optimization and cysteine-modified HA-1-TCR α-chain with cysteine-modified HA-1-TCR β-chain most significantly increased the expression of HA-1-TCR ( Figure 3 A).

[0456] To test whether the improved HA-1-TCR expression led to enhanced HA-1-specific function, HA-1-TCR td weak and strong competitor virus-specific T cells were tested against target cells loaded with HA-1 peptide as well as target cells endogenously expressing the HA-1 antigen ( Figure 3 B). Among weakly competitive sub-viral specific T cells, the combination of codon-optimized and cysteine-modified HA-1-TCR α-chain and cysteine-modified HA-1-TCR β-chain (Combination #8) showed the highest IFN-γ production against peptide-loaded target cells as well as against target cells presenting endogenously processed HA-1 antigen. Most notably, among strongly competitive T cells, this TCR combination was the only one capable of eliciting a significant HA-1-specific reactivity. In summary, the combination of cysteine modification of HA-1-TCR chains and codon optimization of HA-1-TCR α-chain led to efficient HA-1-TCR expression after gene transfer, even in strongly competitive T cells, and resulted in robust HA-1-specific functions.

[0457] To confirm the generality of these data, polyclonal peripheral CD8 + T cells and other weakly and strongly competitive T cells were transduced with a single retroviral vector encoding unmodified or codon-optimized and cysteine-modified HA-1-TCR α- and β-chains linked to a picornavirus-derived self-cleaving 2A sequence, and HA-1-TCR cell surface expression was tested ( Figure 4 A). Although we did not observe an increase in cell surface expression of the codon-optimized HA-1-TCR β-chain, the HA-1-TCR β-chain was also codon-optimized to ensure that the mRNA stability of the TCR β-chain would not have a negative impact on the expression of the TCR α-chain. Accordingly, transduction with the modified HA-1-TCR led to the most efficient cell surface expression in both weakly and strongly competitive T cells. Polyclonal CD8 + T cells showed significant surface expression of HA-1-TCR similar to that of strongly competitive T cells after transfer of the modified HA-1-TCR ( Figure 4 A).

[0458] To investigate whether this increased HA-1-TCR cell surface expression was consistent with clinically relevant HA-1-specific functions, the HA-1-specific cytotoxic activities ( Figure 4 B) and IFN-γ production ( Figure 4 C) of weakly and strongly competitive phenotype T cells transduced with unmodified or codon-optimized and cysteine-modified HA-1-TCR were analyzed. Weakly competitive T cells transduced with unmodified HA-1-TCR produced HA-1-specific cytotoxic responses and IFN-γ production against AML and ALL, while the introduction of the modified TCR enhanced the HA-1-specific responses (see Figure 4 B and C, respectively). In addition, strongly competitive T cells transduced with the modified HA-1-TCR were able to exhibit significant cytotoxic activities and IFN-γ production against HA-1 + malignant cells (see Figure 4 B and 4C). Collectively, these results confirm the generality of enhanced HA-1-TCR expression upon introduction of the modified HA-1-TCR into weak and strong competitor phenotype T cells and polyclonal CD8 + T cells, thus demonstrating that we can generate effective redirected HA-1-specific T cells.

[0459] For use in clinical therapy, the introduced TCR must be encoded by a retroviral construct without potentially immunogenic marker genes. Thus, we constructed an MP71 vector without marker genes that encodes the modified HA-1-TCR α and β chains and analyzed the weak ( Figure 5 ) and strong competitor T cells ( Figure 5 ) transduced with this clinically useful vector for a similar enhanced anti-leukemic reactivity (8). One week after transduction, IFN-γ ELISA ( Figure 5 ) was used to analyze the HA-1-specific reactivity of weak and strong competitor T cells against malignant target cells. The transduction efficiencies of the pLZRS and MP71 vectors were based on NGF-R or HA-1-tetramer staining and were demonstrated to be 15% and 2%, respectively. Although weak competitor T cells transduced with unmodified or modified HA-1-TCR recognized malignant cells equally well ( Figure 5 ) compared to T cells transduced with unmodified HA-1-TCR, strong competitor T cells transduced with the modified HA-1-TCR showed significantly enhanced IFNγ production against AML and ALL in all target cells. Collectively, TCR transfer with codon-optimized and cysteine-modified HA-1-TCR led to effective expression of the introduced HA-1-TCR and strong HA-1-specific function against clinically relevant target cells, both in weak and strong competitor T cells.

[0460] Based on previous results, we investigated whether this procedure could be scaled up for clinical purposes to rapidly engineer a therapeutically relevant number of virally specific T cells transduced with HA-1-TCR. To obtain therapeutic cell numbers after the entire procedure, donor leukocytes were incubated with one or two streptavidins consisting of relevant CMV and EBV peptide-HLA complexes, with a large T cell population present in the donor. For this, we performed 4 trial procedures ( 9 ) using 1 x 10 Figure 6 PBMCs isolated from leukocyte products of 4 healthy individuals (donors JBC, UPB, UHO, and UBQ). The leukocytes were incubated with the relevant streptavidin and purified using CliniMACS ( Figure 6 A-D). After isolation, the T cells were immediately incubated with D-biotin and the purity was analyzed by flow cytometry. As Figure 6 As shown in A - D, all positive fractions contained 60% virus - specific T cells, even when the starting material contained a low frequency of virus - specific T cells( Figure 6 D). For all 4 experimental procedures, the recovery rate of virus - specific T cells present in the starting material of the positive fraction was close to 60%. After CliniMACS separation, the positive fraction was cultured in T - cell medium containing irradiated autologous feeders (at a 1:5 ratio) and cytokines. Some streptavidin - rich cell lines were not transduced, while the majority of cell lines were transduced 2 - 3 days after separation with GMP - grade retroviral supernatant encoding HA - 1 - TCR (produced by Eufets, Germany). After an additional culture period of 8 - 12 days, the transduction efficiency and purity of the transduced T cells were analyzed using HA - 1 and viral tetramers. As determined by viral tetramers, the purity of all 4 streptavidin - rich cell lines that were not transduced was 97%( Figure 6 E - H). The transduction efficiency of the 4 HA - 1 - TCR - transduced streptavidin - rich cell lines was between 22.5% and 54.2%( Figure 6 E - H). Among the HA - 1 - TCR - transduced virus - specific T cells that stained positive with HA - 1 tetramer, the T cells mainly expressed HA - 1 - TCR and expressed a reduced level of viral TCR due to cell - surface expression competition. At the end of the culture period (day 14 after separation), all T - cell products were collected and viable cells were counted. The experimental procedures of JBC, UPB, and UHO produced high - purity antigen - specific T cells of ≥15x10 6 . The experimental procedure UBQ, which had a low frequency of virus - specific T cells in the starting material, produced 2x10 6 antigen - specific T cells at the end of the culture period. In summary, these results indicate that using a GMP - grade separation method, virus - specific T cells can be enriched from thawed PBMC material with a high recovery rate and be effectively transduced.

[0461] HA - 1 - specific function was tested in 3 HA - 1 - TCR - transduced virus - specific T cells (JBC, UHO, UBQ) in a peptide titration assay, and IFN - γ production was measured. All 3 transduced virus - specific T - cell lines showed HA - 1 - specific dose - dependent IFN - γ production comparable to that of HA - 1 - specific control T - cell clones. In addition, no non - tdT cells produced HA - 1 - specific IFN - γ. To investigate whether HA - 1 - TCR - transduced virus - specific T cells could recognize malignant primary leukemia cells presenting endogenously processed HA1 H antigen, HA - 1 - TCR - transduced virus - specific T cells were used against HLA - A2 pos primary ALL cells (HA1 H pos or HA1 H neg ) were tested. As Figure 7 shown in Panel B, all HA-1-TCR transduced virus-specific T cell lines (but except for non-transduced virus-specific T cells) were able to produce IFN-γ after stimulation with HA-1 pos primary ALL cells, while no IFN-γ was produced after stimulation with HA-1 neg primary ALL cells. HA-1-TCR transduced virus-specific T cell lines and non-transduced virus-specific T cells produced IFN-γ after stimulation with virus peptide-pulsed T2 cells. In addition, all 4 HA-1-TCR transduced virus-specific T cells were tested for their HA-1 specific cytotoxic responses ( pos Panel C) against virus or HA-1 peptide-pulsed T2 cells or against HLA-A2 H pos or HA1 H neg ) on primary ALL and AML samples. The results showed that HA-1-TCR transduced virus-specific T cells could effectively lyse HLA-A2 Figure 7 HA1 pos HA1 H 1 pos primary ALL and AML samples. In addition, they showed comparable cytotoxic responses to virus peptide-pulsed T2 cells as non-transduced virus-specific T cells. These results indicate that it is feasible to reproducibly produce HA-1-TCR modified T cells with strong anti-leukemia reactivity using GMP-grade production methods.

[0462] Recently, it has been shown that patients treated with CD19CAR modified T cells from the patient after allogeneic stem cell transplantation do not induce GvHD. Therefore, we tested whether polyclonal CD8+ T cells could be transduced and efficiently express HA1-TCR on the cell surface. For this purpose, we isolated CD8+ T cells from healthy individuals by MACS and transduced the T cells 2 days after PHA-specific stimulation in IL-2 medium supplemented with GMP-grade retroviral supernatant encoding HA1-TCR (codon-optimized and cysteine-modified). Transduction of polyclonal CD8+ T cells yielded 50% HA1-TCR positive T cells, and the modified T cells very efficiently recognized HA1 H positive target cells, as demonstrated by high IFN-γ production after stimulation with various different HLA-A*0201+ target cells (EBV-MRJ, U266, AML3) expressing HA1 H , while not expressing immunogenic HA1 H The HLA-A2*0201+ target cells were not recognized (EBV-IZA, EBV-JY, AML2)( Figure 8 ).

[0463] In addition, these HA-1 TCR-engineered CD8+ T cells mediated effective anti-leukemia responses in a multiple myeloma xenograft model ( Figure 9 ), indicating that these HA-1 TCR-engineered T cells have a high anti-tumor reactivity in vitro and in vivo. Therefore, these data suggest that patients with relapsed or refractory hematological malignancies can be effectively treated with these effective HA1-TCR-modified T cells.

[0464] Figure 10 and 11 Analysis settings:

[0465] CD8+ T cells from two healthy HLA-A*02:01+ donors (which are homozygous for the HA-1R variant) were transduced with lentivirus using two different HA1-H-specific TCRs (TCR LUMC and TCR FHCRC of WO2018058002A1). The TRBV-CDR3-TRBJ, TRAV-CDR3-TRBJ, and murine α and β C regions of FHCRC and LUMC were cloned into the pES.12-6 vector (see SEQ ID NO:89 and SEQ ID NO:90 below).

[0466] Subsequently, the transduced cells were sorted for murine Cβ+ and CD8+ cells and amplified (rapid amplification protocol).

[0467] To analyze functional avidity, T2 cells were loaded with graded concentrations of HA-1H peptide (VLHDDLLEA (SEQ ID NO:10), 10 -12 M - 10 -5 M), incubated at 37 °C for 1 - 2 hours, washed with PBS and resuspended in medium. The transduced T cells were co-cultured with the peptide-loaded T2 cells at an effector / target ratio of 2:1 (20,000 effectors / 96 wells). After 20 hours of co-culture, IFN-γ in the supernatant was measured by ELISA.

[0468] To analyze non-specific recognition of the HA-1R peptide, the same effector cell preparation as above was co-cultured with graded concentrations of HA-1R peptide (VLRDDLLEA (SEQ ID NO:79), 10 -8 M - 10 -5 M). Peptide loading, co-culture, and reading were performed as described above.

[0469] Nucleic acid and amino acid sequences of interest:

[0470] SEQ ID NO:1 (HA-1 H Amino acid sequence of CDR3 in the Vα domain of TCR M7): CAGNTGGFKTIF

[0471] SEQ ID NO:2 (HA-1 H Amino acid sequence of CDR3 in the Vα domain of TCR M2): CAARNSGAGSYQLTF

[0472] SEQ ID NO:3 (HA-1 H Amino acid sequence of CDR3 in the Vα domain of TCR FK47.83): CAASNLVF

[0473] SEQ ID NO:4 (HA-1 H Amino acid sequence of CDR3 in the Vβ domain of TCR M7): CASSLLGNQPQHF

[0474] SEQ ID NO:5 (HA-1 H Amino acid sequence of CDR3 in the Vβ domain of TCR M2): CASLTVQNTEAFF

[0475] SEQ ID NO:6 (HA-1 H Amino acid sequence of CDR3 in the Vβ domain of TCR FK47.83): CASSLVVVDEQFF

[0476] SEQ ID NO:7 (HA-1 H TCR M7, HA-1 H TCR M2 or HA-1 H Amino acid sequence of CDR1 in the Vβ domain of TCR FK47.83): SEHNRL

[0477] SEQ ID NO:8 (HA-1 H TCR M2 or HA-1 H Amino acid sequence of CDR1 in the Vα domain of TCR FK47.83): DSASNY

[0478] SEQ ID NO:9 (HA-1 H TCR M2 or HA-1 H Amino acid sequence of CDR1 in the Vα domain of TCR FK47.83): GACAGTGCCTCAAACTAC

[0479] SEQ ID NO:10 (HA-1 H (Amino acid sequence of the antigen): VLHDDLLEA

[0480] SEQ ID NO:11 (HA-1 H (Nucleic acid sequence of CDR3 of the Vα domain of TCR M7): TGTGCAGGCAATACTGGAGGCTTCAAAACTATCTTT

[0481] SEQ ID NO:12 (HA-1 H (Codon-optimized nucleic acid sequence of CDR3 of the Vα domain of TCR M7): TGTGCCGGCAATACCGGCGGCTTCAAGACCATCTTC

[0482] SEQ ID NO:13 (HA-1 H (Nucleic acid sequence of CDR3 of the Vβ domain of TCR M7): TGTGCCAGCAGCTTATTGGGTAATCAGCCCCAGCATTTT

[0483] SEQ ID NO:14 (HA-1 H (Codon-optimized nucleic acid sequence of CDR3 of the Vβ domain of TCR M7): TGCGCCAGCTCCCTGCTGGGCAACCAGCCCCAGCACTTC

[0484] SEQ ID NO:15 (HA-1 H TCR M7, HA-1 H TCR M2 or HA-1 H (Nucleic acid sequence of CDR1 of the Vβ domain of TCR FK47.83): TCTGAACACAACCGCCTT

[0485] SEQ ID NO:16 (HA-1 H TCR M7, HA-1 H TCR M2 or HA-1 H (Codon-optimized nucleic acid sequence of CDR1 of the Vβ domain of TCR FK47.83): AGCGAGCACAACCGGCTG

[0486] SEQ ID NO:17 (HA-1 H (Amino acid sequence of the Vα (VJ) domain of TCR M7):

[0487] MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGNTGGFKTIFGAGTRLFVKA

[0488] SEQ ID NO:18 (HA-1 H (Amino acid sequence of the Vβ (VDJ) domain of TCR M7):

[0489] MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLLGNQPQHFGDGTRLSIL

[0490] SEQ ID NO:19 (HA-1 H (Nucleic acid sequence of the Vα (VJ) domain of TCR M7):

[0491] ATGCTACTCATCACATCAATGTTGGTCTTATGGATGCAATTGTCACAGGTGAATGGACAACAGGTAATGCAAATTCCTCAGTACCAGCATGTACAAGAAGGAGAAGACTTCACCACGTACTGCAATTCCTCAACTACTTTAAGCAATATACAGTGGTATAAGCAAAGGCCTGGTGGACATCCCGTTTTTTTGATACAGTTAGTGAAGAGTGGAGAAGTGAAGAAGCAGAAAAGACTGACATTTCAGTTTGGAGAAGCAAAAAAGAACAGCTCCCTGCACATCACAGCCACCCAGACTACAGATGTAGGAACCTACTTCTGTGCAGGCAATACTGGAGGCTTCAAAACTATCTTTGGAGCAGGAACAAGACTATTTGTTAAAGCA

[0492] SEQ ID NO:20 (HA-1 H (Codon-optimized nucleic acid sequence of the Vα (VJ) domain of TCR M7):

[0493] ATGCTGCTGATCACCTCCATGCTGGTGCTGTGGATGCAGCTGTCCCAGGTGAACGGCCAGCAGGTGATGCAGATCCCCCAGTACCAGCACGTGCAGGAGGGCGAGGATTTCACCACCTACTGTAACAGCAGCACCACCCTGAGCAACATCCAGTGGTACAAGCAGAGACCTGGCGGCCACCCCGTGTTCCTGATCCAGCTGGTGAAGAGCGGCGAGGTGAAGAAGCAGAAGCGGCTGACCTTCCAGTTCGGCGAGGCCAAGAAGAATAGCAGCCTGCACATCACCGCCACCCAGACCACCGATGTGGGCACCTACTTCTGTGCCGGCAATACCGGCGGCTTCAAGACCATCTTCGGAGCCGGCACCAGACTGTTCGTGAAGGCC

[0494] SEQ ID NO:21 (HA-1 H Nucleic acid sequence of the Vβ (VDJ) domain of TCR M7:

[0495] ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAGGACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGCAGCTTATTGGGTAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTA

[0496] SEQ ID NO:22 (HA-1 H Codon-optimized nucleic acid sequence of the Vβ (VDJ) domain of TCR M7:

[0497] ATGGGCACCAGCCTGCTGTGCTGGATGGCCCTGTGCCTGCTGGGCGCTGACCATGCTGATACCGGCGTGAGCCAGGACCCCCGGCACAAGATCACCAAGCGGGGCCAGAACGTGACCTTCAGATGCGACCCCATCAGCGAGCACAACCGGCTGTACTGGTACAGACAGACCCTGGGCCAGGGCCCCGAGTTCCTGACCTACTTCCAGAACGAGGCCCAGCTGGAAAAGAGCCGGCTGCTGTCCGACCGGTTCAGCGCCGAGCGGCCCAAGGGCAGCTTCAGCACCCTGGAAATCCAGCGGACCGAGCAGGGCGACAGCGCCATGTACCTGTGCGCCAGCTCCCTGCTGGGCAACCAGCCCCAGCACTTCGGCGACGGCACCAGACTGAGCATCCTG

[0498] SEQ ID NO:23 (HA-1 H (Nucleic acid sequence of CDR3 of Vα domain of TCR M2): TGTGCAGCAAGGAACTCTGGGGCTGGGAGTTACCAACTCACTTTC

[0499] SEQ ID NO:24 (HA-1 H (Codon-optimized nucleic acid sequence of CDR3 of Vα domain of TCR M2): TGCGCCGCCCGGAACAGCGGCGCCGGCAGCTACCAGCTGACCTTC

[0500] SEQ ID NO:25 (HA-1 H (Nucleic acid sequence of CDR3 of Vβ domain of TCR M2): TGTGCCAGCTTGACGGTACAGAACACTGAAGCTTTCTTT

[0501] SEQ ID NO:26 (HA-1 H (Codon-optimized nucleic acid sequence of CDR3 of Vβ domain of TCR M2): TGCGCCAGCCTGACCGTGCAGAACACCGAGGCCTTCTTC

[0502] SEQ ID NO: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: GACAGCGCCAGCAACTAC

[0503] SEQ ID NO:28 (HA-1 H TCR M2 or HA-1 H Amino acid sequence of CDR2 of the Vα domain of TCR FK47.83: IRSNVGE

[0504] SEQ ID NO:29 (HA-1 H Amino acid sequence of the Vα (VJ) domain of TCR M2:

[0505] MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAARNSGAGSYQLTFGKGTKLSVIP

[0506] SEQ ID NO:30 (HA-1 H Amino acid sequence of the Vβ (VDJ) domain of TCR M2:

[0507] MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASLTVQNTEAFFGQGTRLTVV

[0508] SEQ ID NO:31 (HA-1 H Nucleic acid sequence of the Vα (VJ) domain of TCR M2:

[0509] ATGACATCCATTCGAGCTGTATTTATATTCCTGTGGCTGCAGCTGGACTTGGTGAATGGAGAGAATGTGGAGCAGCATCCTTCAACCCTGAGTGTCCAGGAGGGAGACAGCGCTGTTATCAAGTGTACTTATTCAGACAGTGCCTCAAACTACTTCCCTTGGTATAAGCAAGAACTTGGAAAAAGACCTCAGCTTATTATAGACATTCGTTCAAATGTGGGCGAAAAGAAAGACCAACGAATTGCTGTTACATTGAACAAGACAGCCAAACATTTCTCCCTGCACATCACAGAGACCCAACCTGAAGACTCGGCTGTCTACTTCTGTGCAGCAAGGAACTCTGGGGCTGGGAGTTACCAACTCACTTTCGGGAAGGGGACCAAACTCTCGGTCATACCA

[0510] SEQ ID NO:32 (HA-1 H Nucleic acid sequence with codon optimization of the Vα (VJ) domain of TCR M2):

[0511] ATGACCAGCATCCGGGCCGTGTTCATCTTCCTGTGGCTGCAGCTGGACCTGGTGAACGGCGAGAACGTGGAGCAGCACCCCAGCACCCTGAGCGTGCAGGAGGGCGACAGCGCCGTGATCAAGTGCACCTACAGCGACAGCGCCAGCAACTACTTCCCCTGGTACAAGCAGGAGCTGGGCAAGCGGCCCCAGCTGATCATCGACATCCGGAGCAACGTGGGCGAGAAGAAGGACCAGCGGATCGCCGTGACCCTGAACAAGACCGCCAAGCACTTCAGCCTGCACATCACCGAGACCCAGCCCGAGGACAGCGCCGTGTACTTCTGCGCCGCCCGGAACAGCGGCGCCGGCAGCTACCAGCTGACCTTCGGCAAGGGCACCAAGCTGAGCGTGATCCCC

[0512] SEQ ID NO:33 (HA-1 H Nucleic acid sequence of the Vβ (VDJ) domain of TCR M2):

[0513] ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAGAACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGCTTGACGGTACAGAACACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTA

[0514] SEQ ID NO:34 (HA-1 H (Codon-optimized nucleic acid sequence of the Vβ (VDJ) domain of TCR M2):

[0515] ATGGGCACCAGCCTGCTGTGCTGGATGGCCCTGTGCCTGCTGGGCGCCGACCACGCCGACACCGGCGTGAGCCAGAACCCCCGGCACAAGATCACCAAGCGGGGCCAGAACGTGACCTTCCGGTGCGACCCCATCAGCGAGCACAACCGGCTGTACTGGTACCGGCAGACCCTGGGCCAGGGCCCCGAGTTCCTGACCTACTTCCAGAACGAGGCCCAGCTGGAGAAGAGCCGGCTGCTGAGCGACCGGTTCAGCGCCGAGCGGCCCAAGGGCAGCTTCAGCACCCTGGAGATCCAGCGGACCGAGCAGGGCGACAGCGCCATGTACCTGTGCGCCAGCCTGACCGTGCAGAACACCGAGGCCTTCTTCGGCCAGGGCACCCGGCTGACCGTGGTG

[0516] SEQ ID NO:35 (HA-1 H Nucleic acid sequence of CDR3 of the Vα domain of TCR FK47.83: TGTGCAGCAAGTAATCTGGTCTTT

[0517] SEQ ID NO:36 (HA-1 H Codon-optimized nucleic acid sequence of CDR3 of the Vα domain of TCR FK47.83: TGCGCCGCCAGCAACCTGGTGTTC

[0518] SEQ ID NO:37 (HA-1 H Nucleic acid sequence of CDR3 of the Vβ domain of TCR FK47.83: TGTGCCAGCAGCTTAGTCGTTGTGGATGAGCAGTTCTTC

[0519] SEQ ID NO:38 (HA-1 H Codon-optimized nucleic acid sequence of CDR3 of the Vβ domain of TCR FK47.83: TGCGCCAGCAGCCTGGTGGTGGTGGACGAGCAGTTCTTC

[0520] SEQ ID NO:39 (HA-1 H TCR M2 or HA-1 H Nucleic acid sequence of CDR2 of the Vα domain of TCR FK47.83: ATTCGTTCAAATGTGGGCGAA

[0521] SEQ ID NO:40 (HA-1 H TCR M2 or HA-1 H Codon-optimized nucleic acid sequence of CDR2 of the Vα domain of TCR FK47.83: ATCCGGAGCAACGTGGGCGAG

[0522] SEQ ID NO:41 (HA-1 H Amino acid sequence of the Vα (VJ) domain of TCR FK47.83: MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAASNLVFGAGTILRVKS

[0523] SEQ ID NO:42 (HA-1 H Amino acid sequence of the Vβ (VDJ) domain of TCR FK47.83

[0524] MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLVVVDEQFFGPGTRLTVL

[0525] SEQ ID NO:43 (HA-1 H Nucleic acid sequence of the Vα (VJ) domain of TCR FK47.83

[0526] ATGACATCCATTCGAGCTGTATTTATATTCCTGTGGCTGCAGCTGGACTTGGTGAATGGAGAGAATGTGGAGCAGCATCCTTCAACCCTGAGTGTCCAGGAGGGAGACAGCGCTGTTATCAAGTGTACTTATTCAGACAGTGCCTCAAACTACTTCCCTTGGTATAAGCAAGAACTTGGAAAAAGACCTCAGCTTATTATAGACATTCGTTCAAATGTGGGCGAAAAGAAAGACCAACGAATTGCTGTTACATTGAACAAGACAGCCAAACATTTCTCCCTGCACATCACAGAGACCCAACCTGAAGACTCGGCTGTCTACTTCTGTGCAGCAAGTAATCTGGTCTTTGGCGCAGGAACCATTCTGAGAGTCAAGTCC

[0527] SEQ ID NO:44 (HA-1 H Codon-optimized nucleic acid sequence of the Vα (VJ) domain of TCR FK47.83

[0528] ATGACCAGCATCCGGGCCGTGTTCATCTTCCTGTGGCTGCAGCTGGACCTGGTGAACGGCGAGAACGTGGAGCAGCACCCCAGCACCCTGAGCGTGCAGGAGGGCGACAGCGCCGTGATCAAGTGCACCTACAGCGACAGCGCCAGCAACTACTTCCCCTGGTACAAGCAGGAGCTGGGCAAGCGGCCCCAGCTGATCATCGACATCCGGAGCAACGTGGGCGAGAAGAAGGACCAGCGGATCGCCGTGACCCTGAACAAGACCGCCAAGCACTTCAGCCTGCACATCACCGAGACCCAGCCCGAGGACAGCGCCGTGTACTTCTGCGCCGCCAGCAACCTGGTGTTCGGCGCCGGCACCATCCTGCGGGTGAAGAGC

[0529] SEQ ID NO:45 (HA-1 H Nucleic acid sequence of the Vβ (VDJ) domain of TCR FK47.83):

[0530] ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAGAACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGCAGCTTAGTCGTTGTGGATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTA

[0531] SEQ ID NO:46 (HA-1 H Codon-optimized nucleic acid sequence of the Vβ (VDJ) domain of TCR FK47.83):

[0532] ATGGGCACCAGCCTGCTGTGCTGGATGGCCCTGTGCCTGCTGGGCGCCGACCACGCCGACACCGGCGTGAGCCAGAACCCCCGGCACAAGATCACCAAGCGGGGCCAGAACGTGACCTTCCGGTGCGACCCCATCAGCGAGCACAACCGGCTGTACTGGTACCGGCAGACCCTGGGCCAGGGCCCCGAGTTCCTGACCTACTTCCAGAACGAGGCCCAGCTGGAGAAGAGCCGGCTGCTGAGCGACCGGTTCAGCGCCGAGCGGCCCAAGGGCAGCTTCAGCACCCTGGAGATCCAGCGGACCGAGCAGGGCGACAGCGCCATGTACCTGTGCGCCAGCAGCCTGGTGGTGGTGGACGAGCAGTTCTTCGGCCCCGGCACCCGGCTGACCGTGCTG

[0533] SEQ ID NO:47 (HA-1 H Amino acid sequence of the Vα (VJ) domain and constant region domain of TCR M7:

[0534] MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGNTGGFKTIFGAGTRLFVKANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*

[0535] SEQ ID NO:48 (HA-1 H Amino acid sequence of the Vα (VJ) domain and constant region domain (mouse) of TCR M7:

[0536] MKSLRVLLVILWLQLSWVWSQGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGNTGGFKTIFGAGTRLFVKADIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS*

[0537] SEQ ID NO:49 (HA-1 H Nucleic acid sequences of the Vα (VJ) domain and constant domain of TCR M7):

[0538] ATGCTACTCATCACATCAATGTTGGTCTTATGGATGCAATTGTCACAGGTGAATGGACAACAGGTAATGCAAATTCCTCAGTACCAGCATGTACAAGAAGGAGAAGACTTCACCACGTACTGCAATTCCTCAACTACTTTAAGCAATATACAGTGGTATAAGCAAAGGCCTGGTGGACATCCCGTTTTTTTGATACAGTTAGTGAAGAGTGGAGAAGTGAAGAAGCAGAAAAGACTGACATTTCAGTTTGGAGAAGCAAAAAAGAACAGCTCCCTGCACATCACAGCCACCCAGACTACAGATGTAGGAACCTACTTCTGTGCAGGCAATACTGGAGGCTTCAAAACTATCTTTGGAGCAGGAACAAGACTATTTGTTAAAGCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA

[0539] SEQ ID NO:50 (HA-1 H (Nucleic acid sequence with codon optimization of the Vα (VJ) domain and constant region domain of TCR M7):

[0540] ATGCTGCTGATCACCTCCATGCTGGTGCTGTGGATGCAGCTGTCCCAGGTGAACGGCCAGCAGGTGATGCAGATCCCCCAGTACCAGCACGTGCAGGAGGGCGAGGATTTCACCACCTACTGTAACAGCAGCACCACCCTGAGCAACATCCAGTGGTACAAGCAGAGACCTGGCGGCCACCCCGTGTTCCTGATCCAGCTGGTGAAGAGCGGCGAGGTGAAGAAGCAGAAGCGGCTGACCTTCCAGTTCGGCGAGGCCAAGAAGAATAGCAGCCTGCACATCACCGCCACCCAGACCACCGATGTGGGCACCTACTTCTGTGCCGGCAATACCGGCGGCTTCAAGACCATCTTCGGAGCCGGCACCAGACTGTTCGTGAAGGCCAACATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGGGACAGCAAGAGCAGCGACAAGAGCGTGTGTCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGTGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTTTTCCCCAGCCCTGAGAGCAGCTGTGACGTGAAACTGGTGGAGAAGAGCTTCGAGACCGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCAGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGACTGTGGTCCAGCTGA

[0541] SEQ ID NO:51 (HA-1 H Codon-optimized nucleic acid sequence of the Vα (VJ) domain and constant region domain of TCR M7 (mouse):

[0542] ATGAAGAGCCTGCGCGTGCTGCTGGTCATCCTGTGGCTGCAATTGTCGTGGGTCTGGAGCCAAATGCTGCTGATCACCTCCATGCTGGTGCTGTGGATGCAGCTGTCCCAGGTGAACGGCCAGCAGGTGATGCAGATCCCCCAGTACCAGCACGTGCAGGAGGGCGAGGATTTCACCACCTACTGTAACAGCAGCACCACCCTGAGCAACATCCAGTGGTACAAGCAGAGACCTGGCGGCCACCCCGTGTTCCTGATCCAGCTGGTGAAGAGCGGCGAGGTGAAGAAGCAGAAGCGGCTGACCTTCCAGTTCGGCGAGGCCAAGAAGAATAGCAGCCTGCACATCACCGCCACCCAGACCACCGATGTGGGCACCTACTTCTGTGCCGGCAATACCGGCGGCTTCAAGACCATCTTCGGAGCCGGCACCAGACTGTTCGTGAAGGCCGACATTCAGAACCCGGAACCGGCTGTATACCAGCTGAAGGACCCCCGATCTCAGGATAGTACTCTGTGCCTGTTCACCGACTTTGATAGTCAGATCAATGTGCCTAAAACCATGGAATCCGGAACTTTTATTACCGACAAGTGCGTGCTGGATATGAAAGCCATGGACAGTAAGTCAAACGGCGCCATCGCTTGGAGCAATCAGACATCCTTCACTTGCCAGGATATCTTCAAGGAGACCAACGCAACATACCCATCCTCTGACGTGCCCTGTGATGCCACCCTGACAGAGAAGTCTTTCGAAACAGACATGAACCTGAATTTTCAGAATCTGAGCGTGATGGGCCTGAGAATCCTGCTGCTGAAGGTCGCTGGGTTTAATCTGCTGATGACACTGCGGCTGTGGTCCTCATGA

[0543] SEQ ID NO:52 (HA-1 H Amino acid sequences of the Vβ (VDJ) domain and the constant region domain of TCR M7):

[0544] MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLLGNQPQHFGDGTRLSILEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF*

[0545] SEQ ID NO:53 (HA-1 H Amino acid sequence of the Vβ (VDJ) domain and constant domain of TCR M7 (mouse):

[0546] MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLLGNQPQHFGDGTRLSILEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS*

[0547] SEQ ID NO:54 (HA-1 H Nucleic acid sequence of the Vβ (VDJ) domain and constant domain of TCR M7:

[0548] ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAGGACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGCAGCTTATTGGGTAATCAGCCCCAGCATTTTGGTGATGGGACTCGACTCTCCATCCTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTCTGA

[0549] SEQ ID NO:55 (HA-1 H Codon-optimized nucleic acid sequences of the Vβ (VDJ) domain and constant region domain of TCR M7

[0550] ATGGGCACCAGCCTGCTGTGCTGGATGGCCCTGTGCCTGCTGGGCGCTGACCATGCTGATACCGGCGTGAGCCAGGACCCCCGGCACAAGATCACCAAGCGGGGCCAGAACGTGACCTTCAGATGCGACCCCATCAGCGAGCACAACCGGCTGTACTGGTACAGACAGACCCTGGGCCAGGGCCCCGAGTTCCTGACCTACTTCCAGAACGAGGCCCAGCTGGAAAAGAGCCGGCTGCTGTCCGACCGGTTCAGCGCCGAGCGGCCCAAGGGCAGCTTCAGCACCCTGGAAATCCAGCGGACCGAGCAGGGCGACAGCGCCATGTACCTGTGCGCCAGCTCCCTGCTGGGCAACCAGCCCCAGCACTTCGGCGACGGCACCAGACTGAGCATCCTGGAAGATCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTGGTGTGCCTGGCCACCGGCTTTTTCCCCGACCACGTGGAGCTGTCTTGGTGGGTGAACGGCAAAGAGGTGCACAGCGGCGTCAGCACCGACCCCCAGCCCCTGAACGACAGCCGGTACTGCCTGTCTAGCCGGCTGCGGGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACAGAGCCAAGCCCGTGACCCAGATCGTGTCCGCCGAGGCCTGGGGCAGAGCCGACTGCGGCTTCACCAGCGTGTCCTACCAGCAGGGCGTGCTGTCTGCCACCATCCTGTACGAGATCCTGCTGGGGAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTGAAGCGGAAGGACTTCTGA

[0551] SEQ ID NO:56 (HA-1 H Codon-optimized nucleic acid sequence of the Vβ (VDJ) and constant domain of the TCR M7 domain (mouse):

[0552] ATGGGCACCAGCCTGCTGTGCTGGATGGCCCTGTGCCTGCTGGGCGCTGACCATGCTGATACCGGCGTGAGCCAGGACCCCCGGCACAAGATCACCAAGCGGGGCCAGAACGTGACCTTCAGATGCGACCCCATCAGCGAGCACAACCGGCTGTACTGGTACAGACAGACCCTGGGCCAGGGCCCCGAGTTCCTGACCTACTTCCAGAACGAGGCCCAGCTGGAAAAGAGCCGGCTGCTGTCCGACCGGTTCAGCGCCGAGCGGCCCAAGGGCAGCTTCAGCACCCTGGAAATCCAGCGGACCGAGCAGGGCGACAGCGCCATGTACCTGTGCGCCAGCTCCCTGCTGGGCAACCAGCCCCAGCACTTCGGCGACGGCACCAGACTGAGCATCCTGGAAGATCTACGTAACGTGACACCACCCAAAGTCTCACTGTTTGAGCCTAGCAAGGCAGAAATTGCCAACAAGCAGAAGGCCACCCTGGTGTGCCTGGCAAGAGGGTTCTTTCCAGATCACGTGGAGCTGTCCTGGTGGGTCAACGGCAAAGAAGTGCATTCTGGGGTCTGCACCGACCCCCAGGCTTACAAGGAGAGTAATTACTCATATTGTCTGTCAAGCCGGCTGAGAGTGTCCGCCACATTCTGGCACAACCCTAGGAATCATTTCCGCTGCCAGGTCCAGTTTCACGGCCTGAGTGAGGAAGATAAATGGCCAGAGGGGTCACCTAAGCCAGTGACACAGAACATCAGCGCAGAAGCCTGGGGACGAGCAGACTGTGGCATTACTAGCGCCTCCTATCATCAGGGCGTGCTGAGCGCCACTATCCTGTACGAGATTCTGCTGGGAAAGGCCACCCTGTATGCTGTGCTGGTCTCCGGCCTGGTGCTGATGGCCATGGTCAAGAAAAAGAACTCTTGA

[0553] SEQ ID NO:57 (HA-1 H Amino acid sequences of the Vα (VJ) domain and constant region domain of TCR M2:

[0554] MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAARNSGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*

[0555] SEQ ID NO:58 (HA-1 H Amino acid sequences of the Vα (VJ) domain and constant region domain of TCR M2 (mouse):

[0556] MKSLRVLLVILWLQLSWVWSQGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAARNSGAGSYQLTFGKGTKLSVIPDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS*

[0557] SEQ ID NO:59 (HA-1 H Nucleic acid sequences of the Vα (VJ) domain and constant region domain of TCR M2:

[0558] ATGACATCCATTCGAGCTGTATTTATATTCCTGTGGCTGCAGCTGGACTTGGTGAATGGAGAGAATGTGGAGCAGCATCCTTCAACCCTGAGTGTCCAGGAGGGAGACAGCGCTGTTATCAAGTGTACTTATTCAGACAGTGCCTCAAACTACTTCCCTTGGTATAAGCAAGAACTTGGAAAAAGACCTCAGCTTATTATAGACATTCGTTCAAATGTGGGCGAAAAGAAAGACCAACGAATTGCTGTTACATTGAACAAGACAGCCAAACATTTCTCCCTGCACATCACAGAGACCCAACCTGAAGACTCGGCTGTCTACTTCTGTGCAGCAAGGAACTCTGGGGCTGGGAGTTACCAACTCACTTTCGGGAAGGGGACCAAACTCTCGGTCATACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGTTGTGGTCCAGCTGA

[0559] SEQ ID NO:60 (HA-1 H Nucleic acid sequence with codon optimization for the Vα (VJ) domain and constant region domain of TCR M2):

[0560] ATGACCAGCATCCGGGCCGTGTTCATCTTCCTGTGGCTGCAGCTGGACCTGGTGAACGGCGAGAACGTGGAGCAGCACCCCAGCACCCTGAGCGTGCAGGAGGGCGACAGCGCCGTGATCAAGTGCACCTACAGCGACAGCGCCAGCAACTACTTCCCCTGGTACAAGCAGGAGCTGGGCAAGCGGCCCCAGCTGATCATCGACATCCGGAGCAACGTGGGCGAGAAGAAGGACCAGCGGATCGCCGTGACCCTGAACAAGACCGCCAAGCACTTCAGCCTGCACATCACCGAGACCCAGCCCGAGGACAGCGCCGTGTACTTCTGCGCCGCCCGGAACAGCGGCGCCGGCAGCTACCAGCTGACCTTCGGCAAGGGCACCAAGCTGAGCGTGATCCCCAACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAGAAGAGCTTCGAGACCGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGAGCAGCTGA

[0561] SEQ ID NO:61 (HA-1 H Codon-optimized nucleic acid sequence of the Vα (VJ) domain and constant domain of TCR M2 (mouse):

[0562] ATGAAGAGCCTGCGCGTGCTGCTGGTCATCCTGTGGCTGCAATTGTCGTGGGTCTGGAGCCAAGGCGAGAACGTGGAGCAGCACCCCAGCACCCTGAGCGTGCAGGAGGGCGACAGCGCCGTGATCAAGTGCACCTACAGCGACAGCGCCAGCAACTACTTCCCCTGGTACAAGCAGGAGCTGGGCAAGCGGCCCCAGCTGATCATCGACATCCGGAGCAACGTGGGCGAGAAGAAGGACCAGCGGATCGCCGTGACCCTGAACAAGACCGCCAAGCACTTCAGCCTGCACATCACCGAGACCCAGCCCGAGGACAGCGCCGTGTACTTCTGCGCCGCCCGGAACAGCGGCGCCGGCAGCTACCAGCTGACCTTCGGCAAGGGCACCAAGCTGAGCGTGATCCCCGACATTCAGAACCCGGAACCGGCTGTATACCAGCTGAAGGACCCCCGATCTCAGGATAGTACTCTGTGCCTGTTCACCGACTTTGATAGTCAGATCAATGTGCCTAAAACCATGGAATCCGGAACTTTTATTACCGACAAGTGCGTGCTGGATATGAAAGCCATGGACAGTAAGTCAAACGGCGCCATCGCTTGGAGCAATCAGACATCCTTCACTTGCCAGGATATCTTCAAGGAGACCAACGCAACATACCCATCCTCTGACGTGCCCTGTGATGCCACCCTGACAGAGAAGTCTTTCGAAACAGACATGAACCTGAATTTTCAGAATCTGAGCGTGATGGGCCTGAGAATCCTGCTGCTGAAGGTCGCTGGGTTTAATCTGCTGATGACACTGCGGCTGTGGTCCTCATGA

[0563] SEQ ID NO:62 (HA-1 H Amino acid sequence of the Vβ (VDJ) domain and constant region domain of TCR M2):

[0564] MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASLTVQNTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF*

[0565] SEQ ID NO:63 (HA-1 H Amino acid sequence of the Vβ (VDJ) domain and constant region domain (mouse) of TCR M2:

[0566] MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASLTVQNTEAFFGQGTRLTVVEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS*

[0567] SEQ ID NO:64 (HA-1 H Nucleic acid sequence of the Vβ (VDJ) domain and constant region domain of TCR M2:

[0568] ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAGAACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGCTTGACGGTACAGAACACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACAGTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTTCCCTGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTCTGA

[0569] SEQ ID NO:65 (HA-1 H Codon-optimized nucleic acid sequences of the Vβ (VDJ) domain and constant region domain of TCR M2

[0570] ATGGGCACCAGCCTGCTGTGCTGGATGGCCCTGTGCCTGCTGGGCGCCGACCACGCCGACACCGGCGTGAGCCAGAACCCCCGGCACAAGATCACCAAGCGGGGCCAGAACGTGACCTTCCGGTGCGACCCCATCAGCGAGCACAACCGGCTGTACTGGTACCGGCAGACCCTGGGCCAGGGCCCCGAGTTCCTGACCTACTTCCAGAACGAGGCCCAGCTGGAGAAGAGCCGGCTGCTGAGCGACCGGTTCAGCGCCGAGCGGCCCAAGGGCAGCTTCAGCACCCTGGAGATCCAGCGGACCGAGCAGGGCGACAGCGCCATGTACCTGTGCGCCAGCCTGACCGTGCAGAACACCGAGGCCTTCTTCGGCCAGGGCACCCGGCTGACCGTGGTGGAGGACCTGAACAAGGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGAGCTGCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGCCGGGCCGACTGCGGCTTCACCAGCGTGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGAGCGCCCTGGTGCTGATGGCCATGGTGAAGCGGAAGGACTTCTGA

[0571] SEQ ID NO:66 (HA-1 H Codon-optimized nucleic acid sequences of the Vβ (VDJ) domain and the constant region domain (mouse) of TCR M2

[0572] ATGGGCACCAGCCTGCTGTGCTGGATGGCCCTGTGCCTGCTGGGCGCCGACCACGCCGACACCGGCGTGAGCCAGAACCCCCGGCACAAGATCACCAAGCGGGGCCAGAACGTGACCTTCCGGTGCGACCCCATCAGCGAGCACAACCGGCTGTACTGGTACCGGCAGACCCTGGGCCAGGGCCCCGAGTTCCTGACCTACTTCCAGAACGAGGCCCAGCTGGAGAAGAGCCGGCTGCTGAGCGACCGGTTCAGCGCCGAGCGGCCCAAGGGCAGCTTCAGCACCCTGGAGATCCAGCGGACCGAGCAGGGCGACAGCGCCATGTACCTGTGCGCCAGCCTGACCGTGCAGAACACCGAGGCCTTCTTCGGCCAGGGCACCCGGCTGACCGTGGTGGAAGATCTACGTAACGTGACACCACCCAAAGTCTCACTGTTTGAGCCTAGCAAGGCAGAAATTGCCAACAAGCAGAAGGCCACCCTGGTGTGCCTGGCAAGAGGGTTCTTTCCAGATCACGTGGAGCTGTCCTGGTGGGTCAACGGCAAAGAAGTGCATTCTGGGGTCTGCACCGACCCCCAGGCTTACAAGGAGAGTAATTACTCATATTGTCTGTCAAGCCGGCTGAGAGTGTCCGCCACATTCTGGCACAACCCTAGGAATCATTTCCGCTGCCAGGTCCAGTTTCACGGCCTGAGTGAGGAAGATAAATGGCCAGAGGGGTCACCTAAGCCAGTGACACAGAACATCAGCGCAGAAGCCTGGGGACGAGCAGACTGTGGCATTACTAGCGCCTCCTATCATCAGGGCGTGCTGAGCGCCACTATCCTGTACGAGATTCTGCTGGGAAAGGCCACCCTGTATGCTGTGCTGGTCTCCGGCCTGGTGCTGATGGCCATGGTCAAGAAAAAGAACTCTTGA

[0573] SEQ ID NO:67 (HA-1 H Amino acid sequences of the Vα (VJ) domain and constant region domain of TCR FK47.83:

[0574] MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAASNLVFGAGTILRVKSYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*

[0575] SEQ ID NO:68 (HA-1 H Amino acid sequences of the Vα (VJ) domain and constant region domain (mouse) of TCR FK47.83:

[0576] MKSLRVLLVILWLQLSWVWSQGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAASNLVFGAGTILRVKSDIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS*

[0577] SEQ ID NO:69 (HA-1 H Nucleic acid sequences of the Vα (VJ) domain and constant region domain of TCR FK47.83:

[0578] ATGACATCCATTCGAGCTGTATTTATATTCCTGTGGCTGCAGCTGGACTTGGTGAATGGAGAGAATGTGGAGCAGCATCCTTCAACCCTGAGTGTCCAGGAGGGAGACAGCGCTGTTATCAAGTGTACTTATTCAGACAGTGCCTCAAACTACTTCCCTTGGTATAAGCAAGAACTTGGAAAAAGACCTCAGCTTATTATAGACATTCGTTCAAATGTGGGCGAAAAGAAAGACCAACGAATTGCTGTTACATTGAACAAGACAGCCAAACATTTCTCCCTGCACATCACAGAGACCCAACCTGAAGACTCGGCTGTCTACTTCTGTGCAGCAAGTAATCTGGTCTTTGGCGCAGGAACCATTCTGAGAGTCAAGTCCTATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGTTGTGGTCCAGCTGA

[0579] SEQ ID NO:70 (HA-1 H (Nucleic acid sequence with codon optimization of the Vα (VJ) domain and constant region domain of TCR FK47.83):

[0580] ATGACCAGCATCCGGGCCGTGTTCATCTTCCTGTGGCTGCAGCTGGACCTGGTGAACGGCGAGAACGTGGAGCAGCACCCCAGCACCCTGAGCGTGCAGGAGGGCGACAGCGCCGTGATCAAGTGCACCTACAGCGACAGCGCCAGCAACTACTTCCCCTGGTACAAGCAGGAGCTGGGCAAGCGGCCCCAGCTGATCATCGACATCCGGAGCAACGTGGGCGAGAAGAAGGACCAGCGGATCGCCGTGACCCTGAACAAGACCGCCAAGCACTTCAGCCTGCACATCACCGAGACCCAGCCCGAGGACAGCGCCGTGTACTTCTGCGCCGCCAGCAACCTGGTGTTCGGCGCCGGCACCATCCTGCGGGTGAAGAGCTACATCCAGAACCCCGACCCCGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGAGCCAGAGCAAGGACAGCGACGTGTACATCACCGACAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCCAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAGAAGAGCTTCGAGACCGACACCAACCTGAACTTCCAGAACCTGAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGAGCAGCTGA

[0581] SEQ ID NO:71 (HA-1 H (Nucleic acid sequence with codon optimization for the Vα (VJ) domain and constant region domain (mouse) of TCR FK47.83):

[0582] ATGAAGAGCCTGCGCGTGCTGCTGGTCATCCTGTGGCTGCAATTGTCGTGGGTCTGGAGCCAAGGCGAGAACGTGGAGCAGCACCCCAGCACCCTGAGCGTGCAGGAGGGCGACAGCGCCGTGATCAAGTGCACCTACAGCGACAGCGCCAGCAACTACTTCCCCTGGTACAAGCAGGAGCTGGGCAAGCGGCCCCAGCTGATCATCGACATCCGGAGCAACGTGGGCGAGAAGAAGGACCAGCGGATCGCCGTGACCCTGAACAAGACCGCCAAGCACTTCAGCCTGCACATCACCGAGACCCAGCCCGAGGACAGCGCCGTGTACTTCTGCGCCGCCAGCAACCTGGTGTTCGGCGCCGGCACCATCCTGCGGGTGAAGAGCGACATTCAGAACCCGGAACCGGCTGTATACCAGCTGAAGGACCCCCGATCTCAGGATAGTACTCTGTGCCTGTTCACCGACTTTGATAGTCAGATCAATGTGCCTAAAACCATGGAATCCGGAACTTTTATTACCGACAAGTGCGTGCTGGATATGAAAGCCATGGACAGTAAGTCAAACGGCGCCATCGCTTGGAGCAATCAGACATCCTTCACTTGCCAGGATATCTTCAAGGAGACCAACGCAACATACCCATCCTCTGACGTGCCCTGTGATGCCACCCTGACAGAGAAGTCTTTCGAAACAGACATGAACCTGAATTTTCAGAATCTGAGCGTGATGGGCCTGAGAATCCTGCTGCTGAAGGTCGCTGGGTTTAATCTGCTGATGACACTGCGGCTGTGGTCCTCATGA

[0583] SEQ ID NO:72 (HA-1 H Amino acid sequence of the Vβ (VDJ) domain and constant region domain of TCR FK47.83):

[0584] MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLVVVDEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG*

[0585] SEQ ID NO:73 (HA-1 H Amino acid sequence of the Vβ (VDJ) domain and constant domain of TCR FK47.83 (mouse):

[0586] MGTSLLCWMALCLLGADHADTGVSQNPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLVVVDEQFFGPGTRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS*

[0587] SEQ ID NO:74 (HA-1 H Nucleic acid sequence of the Vβ (VDJ) domain and constant domain of TCR FK47.83):

[0588] ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAGAACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGCAGCTTAGTCGTTGTGGATGAGCAGTTCTTCGGGCCAGGGACACGGCTCACCGTGCTAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTATGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG

[0589] SEQ ID NO:75 (HA-1 H Codon-optimized nucleic acid sequences of the Vβ (VDJ) domain and the constant domain of TCR FK47.83

[0590] ATGGGCACCAGCCTGCTGTGCTGGATGGCCCTGTGCCTGCTGGGCGCCGACCACGCCGACACCGGCGTGAGCCAGAACCCCCGGCACAAGATCACCAAGCGGGGCCAGAACGTGACCTTCCGGTGCGACCCCATCAGCGAGCACAACCGGCTGTACTGGTACCGGCAGACCCTGGGCCAGGGCCCCGAGTTCCTGACCTACTTCCAGAACGAGGCCCAGCTGGAGAAGAGCCGGCTGCTGAGCGACCGGTTCAGCGCCGAGCGGCCCAAGGGCAGCTTCAGCACCCTGGAGATCCAGCGGACCGAGCAGGGCGACAGCGCCATGTACCTGTGCGCCAGCAGCCTGGTGGTGGTGGACGAGCAGTTCTTCGGCCCCGGCACCCGGCTGACCGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCCAGCGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCCACCGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGTGCACCGACCCCCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCCGGTACTGCCTGAGCAGCCGGCTGCGGGTGAGCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCCGGTGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGAGCGCCGAGGCCTGGGGCCGGGCCGACTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGAGCGCCCTGGTGCTGATGGCCATGGTGAAGCGGAAGGACAGCCGGGGCTGA

[0591] SEQ ID NO:76 (HA-1 H Codon-optimized nucleic acid sequence of the Vβ (VDJ) domain and constant region domain (mouse) of TCR FK47.83:

[0592] ATGGGCACCAGCCTGCTGTGCTGGATGGCCCTGTGCCTGCTGGGCGCCGACCACGCCGACACCGGCGTGAGCCAGAACCCCCGGCACAAGATCACCAAGCGGGGCCAGAACGTGACCTTCCGGTGCGACCCCATCAGCGAGCACAACCGGCTGTACTGGTACCGGCAGACCCTGGGCCAGGGCCCCGAGTTCCTGACCTACTTCCAGAACGAGGCCCAGCTGGAGAAGAGCCGGCTGCTGAGCGACCGGTTCAGCGCCGAGCGGCCCAAGGGCAGCTTCAGCACCCTGGAGATCCAGCGGACCGAGCAGGGCGACAGCGCCATGTACCTGTGCGCCAGCAGCCTGGTGGTGGTGGACGAGCAGTTCTTCGGCCCCGGCACCCGGCTGACCGTGCTGGAAGATCTACGTAACGTGACACCACCCAAAGTCTCACTGTTTGAGCCTAGCAAGGCAGAAATTGCCAACAAGCAGAAGGCCACCCTGGTGTGCCTGGCAAGAGGGTTCTTTCCAGATCACGTGGAGCTGTCCTGGTGGGTCAACGGCAAAGAAGTGCATTCTGGGGTCTGCACCGACCCCCAGGCTTACAAGGAGAGTAATTACTCATATTGTCTGTCAAGCCGGCTGAGAGTGTCCGCCACATTCTGGCACAACCCTAGGAATCATTTCCGCTGCCAGGTCCAGTTTCACGGCCTGAGTGAGGAAGATAAATGGCCAGAGGGGTCACCTAAGCCAGTGACACAGAACATCAGCGCAGAAGCCTGGGGACGAGCAGACTGTGGCATTACTAGCGCCTCCTATCATCAGGGCGTGCTGAGCGCCACTATCCTGTACGAGATTCTGCTGGGAAAGGCCACCCTGTATGCTGTGCTGGTCTCCGGCCTGGTGCTGATGGCCATGGTCAAGAAAAAGAACTCTTGA

[0593] SEQ ID NO:77 (Nucleotide sequence encoding amino acids 1 to 80 of HA-1 TCR BV7-9( Figure 2 A)):

[0594] AGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTC

[0595] SEQ ID NO:78 (Nucleotide sequence encoding amino acids 1 to 80 of HA-2 TCR BV7-8( Figure 2 A)):

[0596] AGGTGTGATCCAATTTCGGGTCATGTATCCCTTTTTTGGTACCAACAGGCCCTGGGGCAGGGGCCAGAGTTTCTGACTTATTTCCAGAATGAAGCTCAACTAGACAAATCGGGGCTGCCC

[0597] SEQ ID NO:79 (Amino acid sequence of HA-1R): VLRDDLLEA

[0598] SEQ ID NO:80 (HA-1 H Amino acid sequence of CDR1 of Vα domain of TCR M7): TTLSN

[0599] SEQ ID NO:81 (HA-1 H Nucleic acid sequence of CDR1 of Vα domain of TCR M7): ACTACTTTAAGCAAT

[0600] SEQ ID NO:82 (HA-1 H Codon-optimized nucleic acid sequence of CDR1 of Vα domain of TCR M7): ACCACCCTGAGCAAC

[0601] SEQ ID NO:83 (HA-1 H Amino acid sequence of CDR2 of Vα domain of TCR M7): LVKSGEV

[0602] SEQ ID NO:84 (HA-1 H Nucleic acid sequence of CDR2 of Vα domain of TCR M7): TTAGTGAAGAGTGGAGAAGTG

[0603] SEQ ID NO:85 (HA-1 H (Codon-optimized nucleic acid sequence of CDR2 of the Vα domain of TCR M7): CTGGTGAAGAGCGGCGAGGTG

[0604] SEQ ID NO:86 (HA-1 H TCR M7, HA-1 H TCR M2 or HA-1 H (Amino acid sequence of CDR2 of the Vβ domain of TCR FK47.83): FQNEAQ

[0605] SEQ ID NO:87 (HA-1 H TCR M7, HA-1 H TCR M2 or HA-1 H (Nucleic acid sequence of CDR2 of the Vβ domain of TCR FK47.83): TTCCAGAATGAAGCTCAA

[0606] SEQ ID NO:88 (HA-1 H TCR M7, HA-1 H TCR M2 or HA-1 H (Codon-optimized nucleic acid sequence of CDR2 of the Vβ domain of TCR FK47.83): TTCCAGAACGAGGCCCAG

[0607] SEQ ID NO:89 (Mouse C-β region)

[0608] gagcaaggccgagatcgccaacaagcagaaagccaccctcgtgtgcctggccagaggcttcttccccgaccatgtggaactgtcttggtgggtcaacggcaaagaggtgcacagcggagtgtccaccgaccctcaggcctacaaagagagcaactacagctactgcctgagcagcagactgcgggtgtccgccaccttctggcacaacccccggaaccacttcagatgccaggtgcagtttcacggcctgagcgaagaggacaagtggcccgaaggctcccccaagcccgtgacccagaatatctctgccgaggcctggggcagagccgactgtggaattaccagcgccagctaccaccagggcgtgctgtctgccaccatcctgtacgagatcctgctgggcaaggccaccctgtacgccgtgctggtgtctggcctggt

[0609] SEQ ID NO:90 (Mouse C–α region)

[0610] caggacagcaccctgtgcctgttcaccgacttcgacagccagatcaacgtgcccaagaccatggaaagcggcaccttcatcaccgacaagacagtgctggacatgaaggccatggacagcaagtccaacggcgcaatcgcctggtccaaccagaccagcttcacatgccaggacatcttcaaagagacaaacgccacataccccagcagcgacgtgccctgtgatgccaccctgacagagaagtccttcgagacagacatgaacctgaacttccagaatctgtccgtgatgggcctgagaatcctgctgctgaaggtggccggcttcaatctgctgatgaccctgcggctgtggtccagctga

[0611] SEQ ID NO:91 (Seattle TCR2, Vβ domain from WO2018 / 058002)

[0612] MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSLVKGEKLFFGSGTQLSVL

[0613] SEQ ID NO:92 (Seattle TCR2, Vα domain from WO2018 / 058002)

[0614] METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVIGLGGTYKYIFGTGTRLKVLAN

[0615] References

[0616] 1. Marijt WA, Heemskerk MH, Kloosterboer FM, Goulmy E, Kester MG, van der Hoorn MA, et al. Hematopoiesis-restricted minor histocompatibility antigens HA-1- or HA-2-specific T cells can induce complete remissions of relapsed leukemia. Proc Natl Acad Sci U S A 2003; 100:2742-7.

[0617] 2. den Haan JM, Sherman NE, Blokland E, Huczko E, Koning F, Drijfhout JW, et al. Identification of a graft versus host disease-associated human minor histocompatibility antigen. Science 1995; 268:1476-80.

[0618] 3. Mommaas B, van Halteren AG, Pool J, van der Veken L, Wieles B, Heemskerk MH, et al. Adult and cord blood T cells can acquire HA-1 specificity through HA-1 T-cell receptor gene transfer. Haematologica 2005;90:1415-21.

[0619] 4. Verdijk RM, Mutis T, Wilke M, Pool J, Schrama E, Brand A, et al. Exclusive TCRVbeta chain usage of ex vivo generated minor Histocompatibility antigen HA-1 specific cytotoxic T cells: implications for monitoring of immunotherapy of leukemia by TCRBV spectratyping. Hematol J 2002;3:271-5.

[0620] 5. Heemskerk MH, Hagedoorn RS, van der Hoorn MA, van der Veken LT, Hoogeboom M, Kester MG, et al. Efficiency of T cell receptor expression in dual specific T cells is controlled by the intrinsic qualities of the TCR chains within the TCR-CD3 complex. Blood 2006.

[0621] 6. van Loenen, M.M., de Boer, R., Hagedoorn, R.S., van Egmond, E.H., Falkenburg, J.H. & Heemskerk, M.H. (2011) Optimization of the HA-1-specific T-cell receptor for gene therapy of hematologic malignancies, Haematologica. 96, 477-481

[0622] 7. van Loenen MM, de Boer R, Amir AL, Hagedoorn RS, Volbeda GL, Willemze R, et al. Mixed T cell receptor dimers harbor potentially harmful neoreactivity. Proc Natl Acad Sci U S A 2010;107:10972-7.

[0623] 8. van Loenen, M.M., de Boer, R., van Liempt, E., Meij, P., Jedema, I., Falkenburg, J.H. & Heemskerk, M.H. (2014) A Good Manufacturing Practice procedure to engineer donor virus-specific T cells into potent anti-leukemic effector cells, Haematologica. 99, 759-768.

[0624] 9. Meij P, Jedema I, van der Hoorn MA, Bongaerts R, Cox L, Wafelman AR, et al. Generation and administration of HA-1-specific T-cell lines for the treatment of patients with relapsed leukemia after allogeneic stem cell transplantation: a pilot study. Haematologica. 2012;97(8):1205-8.

[0625] Lefranc M.-P. "Unique database numbering system for immunogenetic analysis" Immunology Today, 18:509(1997).

[0626] Lefranc M.-P. "The IMGT unique numbering for immunoglobulins, T cell Receptors and Ig-like domains" The immunologist, 7, 132 - 136(1999).

[0627] 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).

[0628] 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 Sequence Listing <110> Leiden University Medical Center, Leiden University Medical Center <120> Binding Protein Specific for HA-1H and Its Use <130> P257143WO <150> NL 2021789 <151> 2018-10-10 <160> 92 <170> PatentIn version 3.5 <210> 1 <211> 12 <212> PRT <213> Homo sapiens <400> 1 Cys Ala Gly Asn Thr Gly Gly Phe Lys Thr Ile Phe 1 5 10 <210> 2 <211> 15 <212> PRT <213> Homo sapiens <400> 2 Cys Ala Ala Arg Asn Ser Gly Ala Gly Ser Tyr Gln Leu Thr Phe 1 5 10 15 <210> 3 <211> 8 <212> PRT <213> Homo sapiens <400> 3 Cys Ala Ala Ser Asn Leu Val Phe 1 5 <210> 4 <211> 13 <212> PRT <213> Homo sapiens <400> 4 Cys Ala Ser Ser Leu Leu Gly Asn Gln Pro Gln His Phe 1 5 10 <210> 5 <211> 13 <212> PRT <213> Homo sapiens <400> 5 Cys Ala Ser Leu Thr Val Gln Asn Thr Glu Ala Phe Phe 1 5 10 <210> 6 <211> 13 <212> PRT <213> Homo sapiens <400> 6 Cys Ala Ser Ser Leu Val Val Val Asp Glu Gln Phe Phe 1 5 10 <210> 7 <211> 6 <212> PRT <213> Homo sapiens <400> 7 Ser Glu His Asn Arg Leu 1 5 <210> 8 <211> 6 <212> PRT <213> Homo sapiens <400> 8 Asp Ser Ala Ser Asn Tyr 1 5 <210> 9 <211> 18 <212> DNA <213> Homo sapiens <400> 9 gacagtgcct caaactac 18 <210> 10 <211> 9 <212> PRT <213> Homo sapiens <400> 10 Val Leu His Asp Asp Leu Leu Glu Ala 1 5 <210> 11 <211> 36 <212> DNA <213> Homo sapiens <400> 11 tgtgcaggca atactggagg cttcaaaact atcttt 36 <210> 12 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized nucleic acid sequence of CDR3 of Va domain of HA-1H TCR M7 <400> 12 tgtgccggca ataccggcgg cttcaagacc atcttc 36 <210> 13 <211> 39 <212> DNA <213> Homo sapiens <400> 13 tgtgccagca gcttattggg taatcagccc cagcatttt 39 <210> 14 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized nucleic acid sequence of CDR3 of the Vb domain of HA-1H TCR M7 <400> 14 tgcgccagct ccctgctggg caaccagccc cagcacttc 39 <210> 15 <211> 18 <212> DNA <213> Homo sapiens <400> 15 tctgaacaca accgcctt 18 <210> 16 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized nucleic acid sequence of CDR1 of the Vb domain of HA-1H TCR M7, HA-1H TCR M2 or HA-1H TCR FK47.83 <400> 16 agcgagcaca accggctg 18 <210> 17 <211> 128 <212> PRT <213> Homo sapiens <400> 17 Met Leu Leu Ile Thr Ser Met Leu Val Leu Trp Met Gln Leu Ser Gln 1 5 10 15 Val Asn Gly Gln Gln Val Met Gln Ile Pro Gln Tyr Gln His Val Gln 20 25 30 Glu Gly Glu Asp Phe Thr Thr Tyr Cys Asn Ser Ser Thr Thr Leu Ser 35 40 45 Asn Ile Gln Trp Tyr Lys Gln Arg Pro Gly Gly His Pro Val Phe Leu 50 55 60 Ile Gln Leu Val Lys Ser Gly Glu Val Lys Lys Gln Lys Arg Leu Thr 65 70 75 80 Phe Gln Phe Gly Glu Ala Lys Lys Asn Ser Ser Leu His Ile Thr Ala 85 90 95 Thr Gln Thr Thr Asp Val Gly Thr Tyr Phe Cys Ala Gly Asn Thr Gly 100 105 110 Gly Phe Lys Thr Ile Phe Gly Ala Gly Thr Arg Leu Phe Val Lys Ala 115 120 125 <210> 18 <211> 132 <212> PRT <213> Homo sapiens <400> 18 Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala 1 5 10 15 Asp His Ala Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr 20 25 30 Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His 35 40 45 Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe 50 55 60 Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu 65 70 75 80 Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu 85 90 95 Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala 100 105 110 Ser Ser Leu Leu Gly Asn Gln Pro Gln His Phe Gly Asp Gly Thr Arg 115 120 125 Leu Ser Ile Leu 130 <210> 19 <211> 384 <212> DNA <213> Homo sapiens <400> 19 atgctactca tcacatcaat gttggtctta tggatgcaat tgtcacaggt gaatggacaa 60 caggtaatgc aaattcctca gtaccagcat gtacaagaag gagaagactt caccacgtac 120 tgcaattcct caactacttt aagcaatata cagtggtata agcaaaggcc tggtggacat 180 cccgtttttt tgatacagtt agtgaagagt ggagaagtga agaagcagaa aagactgaca 240 tttcagtttg gagaagcaaa aaagaacagc tccctgcaca tcacagccac ccagactaca 300 gatgtaggaa cctacttctg tgcaggcaat actggaggct tcaaaactat ctttggagca 360 ggaacaagac tatttgttaa agca 384 <210> 20 <211> 384 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized nucleic acid sequence of the Va (VJ) domain of HA-1H TCR M7 <400> 20 atgctgctga tcacctccat gctggtgctg tggatgcagc tgtcccaggt gaacggccag 60 caggtgatgc agatccccca gtaccagcac gtgcaggagg gcgaggattt caccacctac 120 tgtaacagca gcaccaccct gagcaacatc cagtggtaca agcagagacc tggcggccac 180 cccgtgttcc tgatccagct ggtgaagagc ggcgaggtga agaagcagaa gcggctgacc 240 ttccagttcg gcgaggccaa gaagaatagc agcctgcaca tcaccgccac ccagaccacc 300 gatgtgggca cctacttctg tgccggcaat accggcggct tcaagaccat cttcggagcc 360 ggcaccagac tgttcgtgaa ggcc 384 <210> 21 <211> 396 <212> DNA <213> Homo sapiens <400> 21 atgggcacca gcctcctctg ctggatggcc ctgtgtctcc tgggggcaga tcacgcagat 60 actggagtct cccaggaccc cagacacaag atcacaaaga ggggacagaa tgtaactttc 120 aggtgtgatc caatttctga acacaaccgc ctttattggt accgacagac cctggggcag 180 ggcccagagt ttctgactta cttccagaat gaagctcaac tagaaaaatc aaggctgctc 240 agtgatcggt tctctgcaga gaggcctaag ggatctttct ccaccttgga gatccagcgc 300 acagagcagg gggactcggc catgtatctc tgtgccagca gcttattggg taatcagccc 360 cagcattttg gtgatgggac tcgactctcc atccta 396 <210> 22 <211> 396 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized nucleic acid sequence of the Vb (VDJ) domain of HA-1H TCR M7 <400> 22 atgggcacca gcctgctgtg ctggatggcc ctgtgcctgc tgggcgctga ccatgctgat 60 accggcgtga gccaggaccc ccggcacaag atcaccaagc ggggccagaa cgtgaccttc 120 agatgcgacc ccatcagcga gcacaaccgg ctgtactggt acagacagac cctgggccag 180 ggccccgagt tcctgaccta cttccagaac gaggcccagc tggaaaagag ccggctgctg 240 tccgaccggt tcagcgccga gcggcccaag ggcagcttca gcaccctgga aatccagcgg 300 accgagcagg gcgacagcgc catgtacctg tgcgccagct ccctgctggg caaccagccc 360 cagcacttcg gcgacggcac cagactgagc atcctg 396 <210> 23 <211> 45 <212> DNA <213> Homo sapiens <400> 23 tgtgcagcaa ggaactctgg ggctgggagt taccaactca ctttc 45 <210> 24 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized nucleic acid sequence of CDR3 of the Va domain of HA-1H TCR M2 <400> 24 tgcgccgccc ggaacagcgg cgccggcagc taccagctga ccttc 45 <210> 25 <211> 39 <212> DNA <213> Homo sapiens <400> 25 tgtgccagct tgacggtaca gaacactgaa gctttcttt 39 <210> 26 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Codon-optimized nucleic acid sequence of CDR3 of Vb domain of HA-1H TCR M2 <400> 26 tgcgccagcc tgaccgtgca gaacaccgag gccttcttc 39 <210> 27 <211> 18 &...

Claims

1. An isolated nucleic acid composition encoding an HA-1 antigen-specific binding protein having a variable region of the TCRα chain (Vα) and a variable region of the TCRβ chain (Vβ), the composition comprising: H ​ (a) A nucleic acid sequence encoding a TCR Vα domain, wherein the TCR Vα domain comprises the CDR3 amino acid sequence of SEQ ID NO: 1, the CDR1 amino acid sequence of SEQ ID NO: 80, and the CDR2 amino acid sequence of SEQ ID NO: 83; and (b) A nucleic acid sequence encoding a TCR Vβ domain, the TCR Vβ domain having an amino acid sequence encoded by the TRBV7-9 gene, wherein the Vβ domain comprises the CDR3 amino acid sequence of SEQ ID NO: 4, the CDR1 amino acid sequence of SEQ ID NO: 7, and the CDR2 amino acid sequence of SEQ ID NO:

86.

2. The isolated nucleic acid composition according to claim 1, wherein the TRBV7-9 gene is TRBV7-9*01 or TRBV7-9*03.

3. The isolated nucleic acid composition according to claim 1 or 2, wherein said HA-1 H antigen comprises the amino acid sequence shown in SEQ ID NO:

10.

4. The isolated nucleic acid composition according to claim 1 or 2, wherein the encoded binding protein is capable of specifically binding to HA-1 H Antigen: HLA-A*0201 complex.

5. The isolated nucleic acid composition according to claim 1 or 2, wherein the nucleic acid sequence pair is codon-optimized for expression in a host cell.

6. The isolated nucleic acid composition according to claim 5, wherein the host cell is a human T cell.

7. The isolated nucleic acid composition according to claim 1, wherein: (i) the Vα domain comprises the amino acid sequence of SEQ ID NO: 17; or (ii) the Vβ domain comprises the amino acid sequence of SEQ ID NO:

18.

8. The isolated nucleic acid composition according to claim 1, wherein: (i) the Vα domain comprises the amino acid sequence of SEQ ID NO: 17; and (ii) the Vβ domain comprises the amino acid sequence of SEQ ID NO:

18.

9. The isolated nucleic acid composition according to claim 7 or 8, wherein: (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.

10. The isolated nucleic acid composition according to claim 1 or 2, further comprising a TCR α-chain constant region and / or a TCR β-chain constant region.

11. The isolated nucleic acid composition according to claim 1 or 2, wherein the encoded binding protein comprises a TCR, an antigen-binding fragment of a TCR, or a chimeric antigen receptor (CAR).

12. The isolated nucleic acid composition according to claim 11, wherein the antigen-binding fragment of the TCR is a single-chain TCR (scTCR).

13. A vector system comprising the nucleic acid composition according to any one of claims 1 to 12.

14. The vector system according to claim 13, wherein the vector is a plasmid, a viral vector, or a cosmid.

15. The vector system according to claim 14, wherein the vector is selected from the group consisting of: retrovirus, adeno-associated virus, adenovirus, vaccinia virus, canarypox virus, herpes virus, and minicircle vector.

16. The vector system according to claim 14, wherein the vector is selected from the group consisting of: lentivirus and synthetic DNA or RNA.

17. A modified cell transfected or transduced with the nucleic acid composition according to any one of claims 1 to 12 or the vector system according to any one of claims 13 to 16, wherein the modified cell is HLA-A*0201 negative and / or HA-1 H negative.

18. The modified cell according to claim 17, wherein the modified cell is selected from the group consisting of: CD8 T cells, CD4 T cells, NK cells, NK-T cells, γ-δ T cells, hematopoietic stem cells and progenitor cells.

19. The modified cell according to claim 17, wherein the modified cell is selected from the group consisting of: T cell line or NK-92 cell line.

20. The modified cell according to claim 17 or 18 or 19, wherein the modified cell is a human cell.

21. A pharmaceutical composition comprising the nucleic acid composition according to any one of claims 1 to 12, the vector system according to any one of claims 13 to 16 or the modified cell according to any one of claims 17 to 20, and a pharmaceutically acceptable excipient, adjuvant, diluent and / or carrier.

22. Use of the pharmaceutical composition according to claim 21 for the preparation of a medicament for treating or preventing recurrence of hematological malignancies expressing HA-1 antigen after allogeneic stem cell transplantation (allo-SCT) in HLA-A*0201-positive human subjects H ​ wherein the hematological malignancy is acute myeloid leukemia (AML) or multiple myeloma.

23. The use according to claim 22, wherein: the hematological malignancy is acute myeloid leukemia.

24. The use according to claim 22 or 23, wherein one or more of the modified cells in the composition of claim 21 are allogeneic to the subject.

25. A method for producing a binding protein that is capable of specifically binding to a peptide containing the HA-1 H antigen and not binding to a peptide that does not contain the HA-1 H antigen, the method comprising contacting a nucleic acid composition according to any one of claims 1 to 12 with a cell under conditions such that the nucleic acid composition is incorporated into the cell and expressed by the cell.

26. The method according to claim 25, wherein, The method is ex vivo.

Images