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WO2023167583A1 - Récepteurs de cellules t dirigés contre le facteur de transcription wt1 et leurs utilisations - Google Patents

Récepteurs de cellules t dirigés contre le facteur de transcription wt1 et leurs utilisations Download PDF

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WO2023167583A1
WO2023167583A1 PCT/NL2023/050096 NL2023050096W WO2023167583A1 WO 2023167583 A1 WO2023167583 A1 WO 2023167583A1 NL 2023050096 W NL2023050096 W NL 2023050096W WO 2023167583 A1 WO2023167583 A1 WO 2023167583A1
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seq
acid sequence
amino acid
tcr
domain
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J.H. Frederik Falkenburg
Mirjam H.M. Heemskerk
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Leids Universitair Medisch Centrum LUMC
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Leids Universitair Medisch Centrum LUMC
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C07K16/3038—Kidney, bladder
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • Novel nucleic acid compositions, vector systems, modified cells, and pharmaceutical compositions that encode or express T cell receptor components directed against transcription factor Wilms’ tumor gene 1 are provided herein. These novel components may be used to enhance an immune response in a subject diagnosed with a WT 1 associated disease or condition, such as a hematological malignancy or a solid tumor. Associated methods for treating such subjects are also provided herein.
  • WT1 Transcription factor Wilms’ tumor gene 1
  • OVCA ovarian carcinomas
  • AML acute myeloid leukemia
  • WT1 has been targeted by T-cell-based therapies as well.
  • HLA class I restricted WT1-specific T cells were previously identified[12, 13], and in clinical trials AML and myelodysplastic syndrome (MDS) patients were treated with WT1 -reactive CD8+ T cells or WT1-TCR engineered T cells (TCR-T cells).
  • WT1 is expressed during embryogenesis and involved in the embryonic development of kidneys, gonads, and several organs lined by the mesothelium. In adults, WT1 is involved in homeostasis processes for tissue maintenance and recovery, resulting in expression in renal podocytes, epicardial cells, Sertoli cells, granulosa cells and hematopoietic stem cells. [18, 19] The expression levels in adults are expected to be sufficient to induce negative selection of high- affinity WT1-specific T cells during T-cell development, as a mechanism to centrally delete self- reactive T cells. As a consequence, only the remaining low-affinity WT1 -specific T cells present in the T-cell repertoire can be activated by WT 1 peptide vaccines.
  • WT1 -specific T cells and subsequently WT1 -specific TCR-T cells transferred into patients were of low avidity.
  • Strategies to circumvent self-tolerance to WT1 have been developed.
  • Increased reactivity was demonstrated by stimulating T cells with synthetic analog peptides derived from the WT1 protein[20, 21] or by using affinity enhanced WT1-specific TCRs.
  • Another strategy to circumvent self-tolerance is by searching for WT1 -specific T cells in the allogeneic-HLA (allo-HLA) repertoire. Since allo-HLA reactivity of T cells is not subjected to negative selection, high-affinity allo-H LA-restricted TCRs recognizing self antigens are present in the TCR repertoire. This approach previously allowed the identification of high affinity TCRs specific for several B-cell restricted antigens. [23-25] Also for WT1 , T cells recognizing HLA A*02:01 restricted peptides were successfully isolated using this approach.
  • WT 1 peptides were thus far selected based on peptide binding prediction algorithms, and although these peptides efficiently bind to HLA class I it is unknown whether these peptides are efficiently processed and presented in HLA class I at the cell surface of tumor cells. Furthermore, the thus far selected WT1 peptides were presented only in either HLA-A*02:01 or HLA-A*24:02, whereas more HLA alleles are necessary to cover a larger part of the patient population.
  • WT1 peptides were identified from the HLA class I ligandome of primary leukemia and OVCA patient samples, and a large-scale search was performed to identify high-affinity WT1- specific CD8+ T cells targeting these peptides from the allo HLA T-cell repertoire of healthy donors.
  • the inventors selected potent and safe WT1-specific T-cell clones.
  • the TCR sequences of these T-cell clones were analyzed and TCR gene transfer into CD8+ T cells installed antitumor reactivity against primary AML and OVCA patient samples. Five particularly advantageous TCRs were identified, which are described in detail herein.
  • TCRs have several advantages over the TCRs previously identified. See for example Figure 6, which compares a newly identified VLD-A2 specific T cell clone with a RMF-A2 specific T cell clone of the prior art.
  • the data presented therein demonstrates that both T cell clones exhibit high affinity towards their specific peptide ( Figure 6B), however only the VLD-A2 T cell clones are able to recognize and kill WT1 expressing (primary) tumor cells ( Figure 6C and 6D).
  • the inventors have therefore been able to identify high affinity T cell clones (and corresponding TCRs) that are both potent and safe.
  • the invention provides an isolated nucleic acid composition that encodes a transcription factor Wilms’ tumor 1 (WT1) antigen-specific binding protein having a TCR a chain variable (Va) domain and a TCR p chain variable (Vp) domain, the composition comprising:
  • nucleic acid sequence that encodes a TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ I D NO:3, or a functional fragment thereof; and a nucleic acid sequence that encodes a TCR Vp domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ I D NO:6, or a functional fragment thereof, wherein the CDR3 sequences together specifically bind to WT 1 ; or
  • nucleic acid sequence that encodes a TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:45, or a functional fragment thereof; and a nucleic acid sequence that encodes a TCR Vp domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:48, or a functional fragment thereof, wherein the CDR3 sequences together specifically bind to WT 1 ; or
  • a nucleic acid sequence that encodes a TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:59, or a functional fragment thereof; and a nucleic acid sequence that encodes a TCR Vp domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 62, or a functional fragment thereof, wherein the CDR3 sequences together specifically bind to WT1.
  • the CDR3 of the Va domain may comprise or consist of the amino acid sequence of SEQ ID NO: 3
  • the CDR3 of the Vp domain may comprise or consist of the amino acid sequence of SEQ ID NO:6;
  • the CDR3 of the Va domain may comprise or consist of the amino acid sequence of SEQ ID NO: 17, and the CDR3 of the p domain may comprise or consist of the amino acid sequence of SEQ ID NQ:20; or
  • the CDR3 of the Va domain may comprise or consist of the amino acid sequence of SEQ ID NO: 31
  • the CDR3 of the Vp domain may comprise or consist of the amino acid sequence of SEQ ID NO:34;
  • the CDR3 of the Va domain may comprise or consist of the amino acid sequence of SEQ ID NO: 45, and the CDR3 of the Vp domain may comprise or consist of the amino acid sequence of SEQ ID NO:48; or
  • the CDR3 of the Va domain may comprise or consist of the amino acid sequence of SEQ ID NO: 59
  • the CDR3 of the Vp domain may comprise or consist of the amino acid sequence of SEQ ID NO:62.
  • the Va domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 7; and the Vp domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 9;
  • the Va domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 21 ; and the Vp domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 23; or
  • the Va domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 35; and the Vp domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 37; or
  • the Va domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 49; and the Vp domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 51 ; or
  • the Va domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 63; and the Vp domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 65.
  • the WT1 antigen may comprise an amino acid sequence selected from the group consisting of: ALLPAVPSL (SEQ ID NO: 71), VLDFAPPGA (SEQ ID NO: 72), TPYSSDNLY (SEQ ID NO: 73) and VLDFAPPGASAY (SEQ ID NO: 74).
  • the encoded binding protein may be capable of specifically binding to a peptide:HLA complex selected from the group consisting of: a ALLPAVPSL:HLA-A*02:01 complex; a VLDFAPPGA:HLA-A*02:01 complex; a TPYSSDNLY:HLA-B*35:01 complex; and a VLDFAPPGASAY:HLA-A*01 :01 complex.
  • a peptide:HLA complex selected from the group consisting of: a ALLPAVPSL:HLA-A*02:01 complex; a VLDFAPPGA:HLA-A*02:01 complex; a TPYSSDNLY:HLA-B*35:01 complex; and a VLDFAPPGASAY:HLA-A*01 :01 complex.
  • the nucleic acid sequence may be codon optimised for expression in a host cell, optionally wherein the host cell is a human cell.
  • the nucleic acid composition may comprise a TCR a chain constant domain and/or a TCR chain constant domain.
  • the encoded binding protein may comprise a TCR, an antigen binding fragment of a TCR, a chimeric antigen receptor (CAR), or an ImmTAC.
  • the antigen binding fragment of a TCR may be a single chain TCR (scTCR) or a chimeric TCR dimer in which the antigen binding fragment of the TCR is linked to an alternative transmembrane and intracellular signalling domain.
  • scTCR single chain TCR
  • chimeric TCR dimer in which the antigen binding fragment of the TCR is linked to an alternative transmembrane and intracellular signalling domain.
  • the invention also provides a vector system comprising a nucleic acid composition according to the invention.
  • the vector may be a plasmid, a viral vector, or a cosmid, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
  • the invention also provides a modified cell comprising a nucleic acid composition according to the invention, or a vector system according to the invention.
  • the modified cell may be selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NK-T cell, a gamma-delta T cell, a hematopoietic stem cell, an inducible pluripotent stem cell, a progenitor cell, a T cell line and a NK-92 cell line.
  • the modified cell may be a human cell.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a nucleic acid composition according to the invention, a vector system according to the invention, or a modified cell according to the invention, and a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
  • the invention also provides a pharmaceutical composition according to the invention for use in inducing or enhancing an immune response in human subject diagnosed with a WT1 associated disease or condition.
  • the invention also provides a pharmaceutical composition according to the invention for use in stimulating a cell mediated immune response to a target cell population or tissue in a human subject.
  • the invention also provides a pharmaceutical composition according to the invention for use in providing anti-tumor immunity to a human subject.
  • the invention also provides a pharmaceutical composition according to the invention for use in treating an human subject having a disease or condition associated with an elevated level of H LA- restricted WT1 antigen.
  • the human subject may have at least one tumor.
  • the subject may have been diagnosed with a WT1 associated disease or condition.
  • the WT1 associated disease or condition may be a hematological malignancy or a solid tumor.
  • the hematological malignancy may be selected from the group consisting of: acute myeloid leukemia (AML), multiple myeloma, plasma cell leukemia, Acute lymphoblastoid leukemia (ALL) and B cell lymphoma, optionally wherein the B cell lymphoma is selected from the group consisting of: Diffuse large B cell lymphoma (DLBCL), High grade B cell lymphoma, Mantel cell lymphoma (MCL), Follicular lymphoma (FL), Hairy cell leukemia (HCL), and Burkitt Lymphoma.
  • AML acute myeloid leukemia
  • ALL Acute lymphoblastoid leukemia
  • B cell lymphoma optionally wherein the B cell lymphoma is selected from the group consisting of: Diffuse large B cell lymphoma (DLBCL), High grade B cell lymphoma, Man
  • the hematological malignancy may be AML.
  • the solid tumor may be selected from the group consisting of: ovarian carcinoma, mesothelioma, uterine carcinoma, testicular tumors, pancreatic carcinoma, lung carcinoma, kidney carcinoma, thymoma, sarcoma, prostate carcinoma, colorectal carcinoma, breast carcinoma, cervical carcinoma, stomach carcinoma, melanoma, bladder carcinoma, and kidney carcinoma.
  • the invention also provides a method of generating a binding protein that is capable of specifically binding to a peptide containing a WT 1 antigen and does not bind to a peptide that does not contain the WT1 antigen, comprising contacting a nucleic acid composition according to the invention with a cell under conditions in which the nucleic acid composition is incorporated and expressed by the cell.
  • the method may be ex vivo.
  • the invention also provides an isolated nucleic acid sequence comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs: 8, 10, 12, 14, 22, 24, 26, 28, 36, 38, 40, 42, 50, 52, 54, 56, 64, 66, 68, or 70.
  • the invention also provides an isolated nucleic acid sequence comprising or consisting of the nucleotide sequence of any one of SEQ ID NOs: 8, 10, 12, 14, 22, 24, 26, 28, 36, 38, 40, 42, 50, 52, 54, 56, 64, 66, 68, or 70 for use in therapy.
  • Figure 1 shows MS data validation of the 8 WT1 -derived peptides.
  • the 8 WT 1 peptides identified in our HLA ligandome analyses were validated by comparing tandem mass spectra of eluted peptides and synthetic peptides.
  • a - H For each peptide the tandem mass spectra of the eluted and synthetic peptides are shown, including the source of the eluted peptide;
  • FIG. 2 shows high WT 1 expression in primary OVCA, ALL, and AML samples, low or absent expression in various healthy cell subsets.
  • WT1 mRNA expression was measured by RT-qPCR.
  • WT 1 expression is shown as percentage relative to the three housekeeping genes GUSB, VPS29 and PSMB4, which was set at 100%. The minimum gene expression is set at 0.01%.
  • Dots illustrate in (A) primary tumor samples, or healthy cell subsets, and in (B) tumor cell lines. The vertical bars indicate mean with standard deviation.
  • OVCA primary ovarian carcinoma
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • iDCs and mDCs immature and mature dendritic cells
  • PTECs proximal tubular epithelial cells
  • PBECs primary bronchus epithelial cells
  • APL acute promyelocytic leukemia
  • Figure 3 shows screening approach to select specific and potent WT1-specific T-cell clones.
  • Recognition patterns by 7/19 T-cell clones recognizing the VLDFAPPGA (SEQ ID NO: 72) peptide presented in HLA-A*02:01. Clone 20.3D10 and 23.2G9 were selected as the most specific and potent T-cell clones. Excluded T-cell clones based on recognition in a panel are blurred. Recognition is based on IFN-/ production (ng/mL) after overnight co-culture assays (E:T 1 :6 for cell lines and 1 :16 for AML samples).
  • All cell lines in panel A and C were HLA-A*02:01 positive, either wildtype (wt) or the HLA-allele was introduced by transduction (+A2). Percentage relative WT1 expression is depicted, as determined by RT-qPCR. Dark grey bars depict high WT1 + targets and light grey bars the WT1- targets.
  • A Panel with WT1+ and WT- tumor cell lines.
  • B Panel with 25 EBV-LCLs, expressing all frequent HLA alleles (expression above 99%) in the Caucasian population. The HLA-allele is depicted if only one HLA-allele is recognized by the T- cell clone, meeting the requirement that all EBV-LCLs with this HLA-allele are recognized.
  • C Panel with primary acute myeloid leukemia (AML) and ovarian carcinoma (OVCA) patient samples;
  • Figure 5 shows reactivities by allo-HLA reactive T-cell clones in the included screening panels. Reactivities of the positive control T-cell clones that are reactive against a housekeeping gene presented in HLA-A*02:01 , HLA-A*01 :01 or HLA-B*35:01.
  • A-B IFN-/ production against the tumor cell line panel and primary AML panel included in T-cell clone screenings, depicted in Figure 3 and Figure 4.
  • C-F IFN-/ production against the tumor cell line panel, primary AML panel, primary OVCA panel, and healthy cell subsets panel included in TCR-T cell screenings, depicted in Figure 8.
  • T-cell clones also recognizing Raji transduced with WT1 (WT1 gene) on a similar level (>50%), are depicted with colored dots.
  • B The 4 most potent T-cell clones stimulated with Raji pulsed with titrated peptide. EC50 values represent the peptide concentrations needed to induce 50% of the maximum cytokine production, values were calculated based on sigmoidal curves.
  • FIG. 7 shows comparable pMHC-multimer staining and peptide sensitivity of TCR-T cells and their parental T-cell clones.
  • T-cell receptors (TCRs) of the 4 most promising WT1-specific T-cell clones were constructed and introduced in CD8+ cells via retroviral transduction. Shown are representative results of 2 independent experiments and 2 CD8+ donors, at day 10 post isolation.
  • FIG. 8 shows WT1 -specific TCR-T cells recognize tumor cell lines and primary tumor samples, without recognition of healthy cell subsets.
  • IFN- production ng/mL
  • All targets express the HLA-allele that presents the targeted peptide, either wildtype (wt) or the HLA-allele was introduced by transduction (+A2, +A1 , +B35).
  • Percentage relative WT1 expression is depicted, as determined by RT-qPCR. The error bars represent mean and SD and are based on one or two CD8+ donors with duplicate wells. A comparable experiment showed similar results (data not shown).
  • (A) TCR-T cells stimulated with tumor cell lines (E:T 1 :6).
  • FIG. 9 shows WT1-specific TCR-T cells efficiently kill primary AML samples and OVCA cell lines.
  • Purified TCR-T cells tested for cytotoxic capacity in a 6-hour 51Cr-release assay on primary AML patient samples and OVCA cell lines.
  • Plots illustrate percentage of killed cells on y-axis and E:T ratios on x-axis.
  • All targets express the HLA-allele that presents the targeted peptide, either wildtype (wt) or the HLA-allele was introduced by transduction (+A2, +A1 , +B35). Percentage relative WT1 expression is depicted, as determined by RT-qPCR. The error bars represent mean and SD and are based on triplicate wells.
  • A TCR-T cells tested against primary AML samples.
  • B TCR-T cells tested against OVCA cell lines.
  • C TCR-T cells tested against Raji only or Raji pulsed with WT 1 peptide.
  • WT1 Wilms’ tumor 1 (WT1) antigen-specific binding protein having a TCR a chain variable (Va) domain and a TCR p chain variable (VP) domain
  • WT1 Wilms’ tumor 1
  • Va chain variable
  • VP TCR p chain variable
  • the CDR3 amino acid sequences described herein specifically bind to their target (in this case a WT 1 peptide), when the target (i.e. the appropriate WT1 peptide) is presented in the context of HLA.
  • the binding proteins (and CDR3 sequences specifically described herein) are therefore capable of specifically binding to a WT1 peptide:HLA complex.
  • the invention provides an isolated nucleic acid composition that encodes a binding protein comprising T cell receptor (TCR) components that specifically bind a WT1 antigen.
  • TCR T cell receptor
  • the encoded binding protein is therefore capable of specifically binding to a peptide containing a WT1 antigen (e.g. a WT1 antigen comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74) and does not bind to a peptide that does not contain a WT1 antigen (e.g. it does not bind to a peptide that does not contain a WT1 antigen comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74).
  • a WT1 antigen e.g. a WT1 antigen comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74
  • the nucleic acid composition comprises (a) a nucleic acid sequence that encodes a TCR Va domain with the specified features described herein and (b) a nucleic acid sequence that encodes a TCR Vp domain with the specified features described herein.
  • the encoded TCR components form a WT1 antigen-specific binding protein.
  • the nucleic acid sequences of (a) and (b) above may be distinct nucleic acid sequences within the nucleic acid composition.
  • the TCR components of the binding protein may therefore be encoded by two (or more) nucleic acid sequences (with distinct nucleotide sequences) which, together, encode all of the TCR components of the binding protein.
  • some of the TCR components may be encoded by one nucleic acid sequence in the nucleic acid composition, and others may be encoded by another (distinct) nucleic acid sequence within the nucleic acid composition.
  • the nucleic acid sequences of (a) and (b) may be part of a single nucleic acid sequence.
  • the TCR components of the binding protein may therefore all be encoded by a single nucleic acid sequence (for example with a single open reading frame, or with multiple (e.g. 2 or more, three or more etc.) open reading frames).
  • Nucleic acid sequences described herein may form part of a larger nucleic acid sequence that encodes a larger component part of a functioning binding protein.
  • a nucleic acid sequence that encodes a TCR Va domain with the specified features described herein may be part of a larger nucleic acid sequence that encodes a functional TCR a chain (including the constant domain).
  • a nucleic acid sequence that encodes a TCR Vp domain with the specified features described herein may be part of a larger nucleic acid sequence that encodes a functional TCR chain (including the constant domain).
  • both nucleic acid sequences (a) and (b) above may be part of a larger nucleic acid sequence that encodes a combination of a functional TCR a chain (including the constant domain) and a functional TCR p chain (including the constant domain), optionally wherein the sequence encoding the functional TCR a chain is separated from the sequence encoding the functional TCR P chain by a linker sequence that enables coordinate expression of two proteins or polypeptides in the same nucleic acid sequence. More details on this are provided below.
  • the nucleic acid sequences described herein may alternatively encode a small component of a T cell receptor e.g. a TCR Va domain, or a TCR Vp domain, only.
  • the nucleic acid sequences may be considered as “building blocks” that provide essential components for peptide binding specificity.
  • the nucleic acid sequences described herein may be incorporated into a distinct nucleic acid sequence (e.g. a vector) that encodes the other elements of a functional binding protein such as a TCR, such that when the nucleic acid sequence described herein is incorporated, a new nucleic acid sequence is generated that encodes e.g.
  • a TCR a chain and/or a TCR p chain that specifically binds to a WT1 antigen e.g. wherein the WT1 antigen comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74.
  • the nucleic acid sequences described herein therefore have utility as essential components that confer binding specificity for a WT1 antigen, and thus can be used to generate a larger nucleic acid sequence encoding a binding protein with the required antigen binding activity and specificity.
  • the nucleic acid sequences described herein may be codon optimised for expression in a host cell, for example they may be codon optimised for expression in a human cell, such as a cell of the immune system, a inducible pluripotent stem cell (iPSC), a hematopoietic stem cell, a T cell, a primary T cell, a T cell line, a NK cell, or a natural killer T cell (Scholten et al, Clin. Immunol. 119: 135, 2006).
  • the T cell can be a CD4+ or a CD8+ T cell.
  • Codon optimisation is a well-known method in the art for maximizing expression of a nucleic acid sequence in a particular host cell. For instance, one or more cysteine residues may also be introduced into the encoded TCR alpha and beta chain components (e.g. to reduce the risk of mispairing with endogenous TCR chains).
  • the nucleic acid sequences described herein are codon optimised for expression in a suitable host cell, and/or are modified to introduce codons encoding one or more cysteine amino acids (e.g. into the constant domain of the encoded TCR alpha chain and/or the encoded TCR beta chain) to reduce the risk of mispairing with endogenous TCR chains.
  • the nucleic acid sequences described herein are codon optimised for expression in a suitable host cell, optionally wherein the host cell is a human cell.
  • a TCR constant domain is modified to enhance pairing of desired TCR chains.
  • enhanced pairing between a heterologous TCR a chain and a heterologous TCR p chain due to a modification may result in the preferential assembly of a TCR comprising two heterologous chains over an undesired mispairing of a heterologous TCR chain with an endogenous TCR chain (see, e.g., Covers et al, Trends Mol. Med. 16(2): 11 (2010)).
  • Exemplary modifications to enhance pairing of heterologous TCR chains include the introduction of complementary cysteine residues in each of the heterologous TCR a chain and chain.
  • a binding protein that is encoded by the nucleic acid compositions described herein is specific for a WT 1 antigen and comprises WT 1 antigen specific-TCR components.
  • the encoded binding protein is not limited to being a TCR.
  • Other appropriate binding proteins that comprise the specified WT1 antigen specific -TCR components are also encompassed.
  • the encoded binding protein may comprise a TCR, an antigen binding fragment of a TCR, a chimeric antigen receptor (CAR), or an immTAC.
  • TCRs, antigen binding fragments thereof, CARs and ImmTACs are well defined in the art.
  • an antigen binding fragment of a TCR is a single chain TCR (scTCR) or a chimeric dimer composed of the antigen binding fragments of the TCR a and TCR p chain linked to transmembrane and intracellular domains of a dimeric complex so that the complex is a chimeric dimer TCR (cdTCR).
  • An ImmTAC comprises a TCR connected to an anti-CD3 antibody. ImmTACs are therefore bispecific, combining WT1- recognizing TCR components with immune activating complexes.
  • an antigen-binding fragment of a TCR comprises a single chain TCR (scTCR), which comprises both the TCR Va and TCR Vp domains, but only a single TCR constant domain.
  • an antigen-binding fragment of a TCR comprises a chimeric TCR dimer in which the antigen binding fragment of the TCR is linked to an alternative transmembrane and intracellular signalling domain (where the alternative transmembrane and intracellular signalling domain are not naturally found in TCRs).
  • an antigen-binding fragment of a TCR or a chimeric antigen receptor is chimeric (e.g., comprises amino acid residues or motifs from more than one donor or species), humanized (e.g., comprises residues from a non- human organism that are altered or substituted so as to reduce the risk of immunogenicity in a human), or human.
  • CAR Chimeric antigen receptor
  • fusion protein that is engineered to contain two or more naturally-occurring amino acid sequences linked together in a way that does not occur naturally or does not occur naturally in a host cell, which fusion protein can function as a receptor when present on a surface of a cell.
  • CARs described herein include an extracellular portion comprising an antigen binding domain (i.e., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as an scFv derived from an antibody or TCR specific for an antigen (e.g.
  • a cancer antigen etc or an antigen binding domain derived or obtained from a killer immunoreceptor from an NK cell linked to a transmembrane domain and one or more intracellular signalling domains (optionally containing co-stimulatory domain(s))
  • intracellular signalling domains optionally containing co-stimulatory domain(s)
  • binding proteins described herein may also be expressed as part of a transgene construct that encodes additional accessory proteins, such as a safety switch protein, a tag, a selection marker, a CD8 co-receptor p-chain, a-chain or both, or any combination thereof.
  • additional accessory proteins such as a safety switch protein, a tag, a selection marker, a CD8 co-receptor p-chain, a-chain or both, or any combination thereof.
  • TCR T cell receptor
  • T lymphocytes T cells
  • MHC major histocompatibility complex
  • the invention is directed to nucleic acid compositions that encode binding proteins comprising TCR components that interact with a particular peptide in the context of the appropriate serotype of MHC, i.e. a WT1 antigen in the context of HLA- A*01 :01 , HLA-A*02:01 , and/or HLA-B*35:01 (in other words, the encoded binding protein is capable of specifically binding to a WT1 antigen: specific HLA complex).
  • the invention is directed to nucleic acid compositions that encode binding proteins comprising TCR components that interact with a particular peptide in the context of the appropriate serotype of MHC, i.e. ALLPAVPSL (SEQ ID NO: 71) in the context of HLA-A*02:01; VLDFAPPGA (SEQ ID NO: 72) in the context of HLA-A*02:01; TPYSSDNLY (SEQ ID NO: 73) in the context of HLA- B*35:01 ; or VLDFAPPGASAY (SEQ ID NO: 74) in the context of HLA-A*01 : 01.
  • ALLPAVPSL SEQ ID NO: 71
  • VLDFAPPGA SEQ ID NO: 72
  • TPYSSDNLY SEQ ID NO: 73
  • HLA- B*35:01 a particular serotype of MHC
  • HLA-A*02:01 is a globally common human leukocyte antigen serotype within the HLA-A serotype group. Peptides that are presented by HLA-A*02:01 to TCRs are described as being “HLA- A*02:01 restricted”.
  • HLA-A*01 :01 , and HLA-B*35:01 are also common human leukocyte antigen serotypes within the HLA-A and HLA-B serotype groups.
  • Peptides that are presented by HLA-A*01 :01 to TCRs are described as being “HLA-A*01 :01 restricted”.
  • peptides that are presented by HLA- B*35:01 to TCRs are described as being “HLA-B*35:01 restricted”.
  • HLA-A*01 :01 is also referred to herein as HLA-A1.
  • HLA-A*02:01 is also referred to herein as HLA-A2; and
  • HLA-B*35:01 is also referred to herein as HLA-B35.
  • the inventors have identified several WT1 derived peptides presented on malignant cells in HLA-A*01 :01 (HLA-A1), HLA-A*02:01 (HLA-A2), and HLA-B*35:01 (HLA-B35). Specifically, the inventors identified the WT1 derived peptides SEQ ID NO: 71 to 74.
  • the WT1 antigen specifically bound by a binding protein described herein may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74.
  • the antigen may be an antigenic fragment (i.e. a portion) of an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74, it may consist of an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74, or it may comprise (i.e. include within a longer sequence) an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74.
  • the inventors identified that the WT1 derived peptides ALLPAVPSL (SEQ ID NO: 71) and VLDFAPPGA (SEQ ID NO: 72) are capable of being presented by HLA-A*02:01 ; that the WT1 derived peptide TPYSSDNLY (SEQ ID NO: 73) is capable of being presented by HLA-B*35:01 ; and that the WT1 derived peptide VLDFAPPGASAY (SEQ ID NO: 74) is capable of being presented by HLA-A*01 :01.
  • the encoded binding protein is capable of specifically binding to a peptide:HLA complex selected from the group consisting of: a ALLPAVPSL:HLA-A*02:01 complex; a VLDFAPPGA:HLA-A*02:01 complex; a TPYSSDNLY:HLA-B*35:01 complex; and a VLDFAPPGASAY:HLA-A*01 :01 complex.
  • a peptide:HLA complex selected from the group consisting of: a ALLPAVPSL:HLA-A*02:01 complex; a VLDFAPPGA:HLA-A*02:01 complex; a TPYSSDNLY:HLA-B*35:01 complex; and a VLDFAPPGASAY:HLA-A*01 :01 complex.
  • the WT1 derived peptide of the peptide:HLA complex comprises an antigenic fragment of an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74.
  • the WT 1 derived peptide of the peptide:HLA complex comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO:71 to 74.
  • the TOR is composed of two different polypeptide chains. In humans, 95% of TCRs consist of an alpha (a) chain and a beta (P) chain (encoded by TRA and TRB respectively).
  • a chain a chain
  • P chain a chain
  • the TOR engages with peptide in the context of HLA (e.g. in the context of HLA-A*01 :01 , HLA-A*02:01 , and/or HLA-B*35:01 , as appropriate)
  • the T cell is activated through signal transduction.
  • the alpha and beta chains of the TCR are highly variable in sequence. Each chain is composed of two extracellular domains, a variable domain (V) and a constant domain (C).
  • the constant domain is proximal to the T cell membrane followed by a transmembrane region and a short cytoplasmic tail while the variable domain binds to the peptide/HLA complex.
  • nucleic acid composition that encodes a WT1 antigen-specific binding protein having a TCR a chain variable (Va) domain and a TCR chain variable (VP) domain.
  • the nucleic acid composition described herein may comprise a TCR a chain constant domain and/or a TCR p chain constant domain.
  • the variable domain of each chain has three hypervariable regions (also called complementarity determining regions (CDRs)).
  • the TCR alpha variable domain (referred to herein as a TCR Va domain, TCR V alpha domain, Va domain or V alpha domain, alpha variable domain etc) comprises a CDR1 , a CDR2 and CDR3 region.
  • the TCR beta variable domain (referred to herein as a TCR Vp domain, TCR V beta domain, Vp domain or V beta domain, beta variable domain etc) also comprises a (different) CDR1 , CDR2, and CDR3 region.
  • CDR3 In each of the alpha and beta variable domains it is CDR3 that is mainly responsible for recognizing the peptide being presented by the HLA molecules.
  • TCR a chain variable domain refers to the variable (V) domain (extracellular domain) of a TCR alpha chain, and thus includes three hypervariable regions (CDR1 , CDR2 and the specified CDR3), as well as the intervening sequences, but does not include the constant (C) domain of the alpha chain, which does not form part of the variable domain.
  • TCR p chain variable domain refers to the variable (V) domain (extracellular domain) of a TCR beta chain, and thus includes three hypervariable regions (CDR1 , CDR2 and the specified CDR3), as well as the intervening sequences, but does not include the constant (C) domain of the beta chain, which does not form part of the variable domain.
  • the isolated nucleic acid composition described herein encodes a WT1 antigen-specific binding protein.
  • the inventors have identified several TCRs that specifically bind to a WT1 antigen selected from ALLPAVPSL (SEQ ID NO: 71), VLDFAPPGA (SEQ ID NO: 72), TPYSSDNLY (SEQ ID NO: 73) and VLDFAPPGASAY (SEQ ID NO: 74).
  • TCR clone 22.1 H1 which interacts with ALLPAVPSL (SEQ ID NO: 71) in the context of HLA-A*02:01.
  • ALLPAVPSL SEQ ID NO: 71
  • sequences provided herein that correspond to TCR clone 22.1 H1 are SEQ ID NO:s 1 to 14.
  • an isolated nucleic acid composition that encodes a Wilms’ tumor 1 (WT1) antigen-specific binding protein having a TCR a chain variable (Va) domain and a TCR chain variable (VP) domain
  • the composition comprising: a nucleic acid sequence that encodes a TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ I D NO:3, or a functional fragment thereof; and a nucleic acid sequence that encodes a TCR Vp domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ I D NO:6, or a functional fragment thereof, wherein the CDR3 sequences together specifically bind to WT1.
  • TCR Va domain CDR3 amino acid sequence that confers specific binding to a WT1 antigen e.g. to ALLPAVPSL (SEQ ID NO: 71)
  • SEQ ID NO:3 An example of an appropriate TCR Va domain CDR3 amino acid sequence that confers specific binding to a WT1 antigen (e.g. to ALLPAVPSL (SEQ ID NO: 71)) is shown in SEQ ID NO:3.
  • variants of the amino acid sequence shown in SEQ ID NO:3 may also be functional (i.e. retain their ability to confer specific binding to a WT 1 antigen (e.g. to the peptide ALLPAVPSL (SEQ ID NO: 71)) when the CDR3 is part of TCR Va domain).
  • WT 1 antigen e.g. to the peptide ALLPAVPSL (SEQ ID NO: 71)
  • appropriate (functional) Va domain CDR3 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 3, i.e. they may have at least 80%, at least 86%, at least 93%, or 100% sequence identity to SEQ ID NO: 3.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:3).
  • appropriate (functional) Va domain CDR3 amino acid sequences may vary from the sequence shown in SEQ ID NO:3 by one or several (e.g. two etc) amino acids.
  • SEQ ID NO:3 retain their ability to confer specific binding to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO:71) when the CDR3 is part of TCR Va domain.
  • WT 1 antigen e.g. the peptide shown in SEQ ID NO:71
  • Functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:3.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one, two or more amino acids of SEQ ID NO:3, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the CDR3.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 3 that do not specifically bind to a WTIantigen (e.g. the peptide shown in SEQ ID NO:71).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:3 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • the CDR3 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 3.
  • the CDR3 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Va domain may comprise, in addition to the specified CDR3, a CDR1 comprising an amino acid sequence of SEQ ID NO: 1 , or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to the WT1 antigen (e.g. the peptide shown in SEQ ID NO:71)).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:1.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ I D NO: 1 , or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 1 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO:71).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:1 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • appropriate functional Va domain CDR1 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 1 , i.e. it may have at least 80%, at least 85%, or 100% sequence identity to SEQ ID NO: 1.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:1).
  • appropriate functional Va domain CDR1 amino acid sequences may vary from the sequence shown in SEQ ID NO: 1 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO:1.
  • functional variants of SEQ ID NO: 1 retain the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO:71) when the CDR1 is part of TCR Va domain).
  • the CDR1 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO:1.
  • the CDR1 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Va domain may also comprise, in addition to the specified CDR3 (and optionally the specified CDR1 above), a CDR2 comprising an amino acid sequence of SEQ ID NO:2, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to HLA-A*02:01).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:2.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:2, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 2 that do not specifically bind to HLA-A*02:01. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 2 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • appropriate functional Va domain CDR2 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 2, i.e. it may have at least 80%, at least 87%, or 100% sequence identity to SEQ ID NO: 2.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:2).
  • appropriate (functional) Va domain CDR2 amino acid sequences may vary from the sequence shown in SEQ ID NO:2 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO:2).
  • a functional variant of SEQ ID NO: 2 retains the ability to specifically bind to HLA-A*02:01.
  • the CDR2 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 2.
  • the CDR2 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Va domain may therefore comprise the CDRs mentioned in detail above (by SEQ ID specifically i.e. SEQ ID NO:3, SEQ ID NO: 1 and SEQ ID NO: 2, or functional variants thereof), with appropriate intervening sequences between the CDRs.
  • the encoded TCR Va domain may comprise an amino acid sequence of SEQ ID NO:7, or a functional variant thereof (i.e. wherein the variant TCR Va domain retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO:71) when part of a binding protein described herein).
  • a functional variant may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:7.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:7, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 7 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO:71).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:7 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • the encoded TCR Va domain may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 7, whilst retaining the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO:71).
  • a functional TCR Va domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO:7 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO:7 may all be in regions of the TCR Va domain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 3, SEQ ID NO: 1 and/or SEQ ID NO: 2, and still have 25% (or less) sequence variability compared to SEQ ID NO:7).
  • the sequence of the CDRs of SEQ ID NO: 7 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 7).
  • the encoded TCR Va domain may 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: 7, wherein the TCR Va domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 3.
  • the TCR Va domain CDR1 may have an amino acid sequence of SEQ ID NO: 1
  • the TCR Va domain CDR2 may have an amino acid sequence of SEQ ID NO: 2.
  • the encoded TCR Va domain may comprise an amino acid sequence having at the amino acid sequence of SEQ ID NO: 7, with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions), wherein the TCR Va domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 3.
  • the TCR Va domain CDR1 may have an amino acid sequence of SEQ ID NO: 1
  • the TCR Va domain CDR2 may have an amino acid sequence of SEQ ID NO: 2.
  • the TCR Va domain may be encoded by the nucleic acid sequence of SEQ ID NO:8, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
  • the nucleic acid sequence encoding the TCR Va domain may also encode a TCR a chain constant domain.
  • An example of a suitable constant domain (for either a TCR a chain or a TCR p chain) is encoded in the MP71-TCR-flex retroviral vector.
  • the invention is not limited to this specific constant domain, and encompasses any appropriate TCR a chain constant domain.
  • the constant domain may be murine derived, human derived or humanised. Methods for identifying or generating appropriate constant domains are well known to a person of skill in the art and are well within their routine capabilities.
  • the constant domain may be encoded by or derived from a vector, such as a lentiviral, retroviral or plasmid vector but also adenovirus, adeno-associated virus, vaccinia virus, canary poxvirus or herpes virus vectors in which murine or human constant domains are pre-cloned.
  • a vector such as a lentiviral, retroviral or plasmid vector but also adenovirus, adeno-associated virus, vaccinia virus, canary poxvirus or herpes virus vectors in which murine or human constant domains are pre-cloned.
  • minicircles have also been described for TCR gene transfer (non-viral Sleeping Beauty transposition from minicircle vectors as published by R Monjezi, et al., 2017).
  • naked (synthetic) DNA/RNA can also be used to introduce the TCR.
  • a pMSGV retroviral vector with pre-cloned TCR-Ca and Cb genes as described in LV Coren et al., BioT echniques 2015 may be used to provide an appropriate constant domain.
  • single stranded or double stranded DNA or RNA can be inserted by homologous directed repair into the TCR locus (see Roth eta/2018 Nature vol 559; page 405).
  • non - homologous end joining is possible.
  • SEQ ID NO: 11 An example of a specific TCR a chain amino acid sequence that includes a TCR Va domain described herein with an appropriate constant domain is shown in SEQ ID NO: 11.
  • Appropriate functional variants of SEQ ID NO:11 are also encompassed (e.g. variants having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 11 , wherein the variant TCR a chain amino acid sequence retains its ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO:71) when part of a binding protein described herein).
  • a WT1 antigen e.g. the peptide shown in SEQ ID NO:71
  • a functional TCR a chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO:11 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO:11 may all be in regions of the TCR a chain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 3, SEQ ID NO: 1 and/or SEQ ID NO: 2, and still have 25% (or less) sequence variability compared to SEQ ID NO:11).
  • the sequence of the CDRs of SEQ ID NO: 11 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 11).
  • the encoded TCR a chain may 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: 11 , wherein the TCR a chain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 3.
  • the TCR a chain CDR1 may have an amino acid sequence of SEQ ID NO:1 and the TCR a chain CDR2 may have an amino acid sequence of SEQ ID NO: 2.
  • the TCR a chain may be encoded by the nucleic acid sequence of SEQ ID NO:12, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 12 is the nucleic acid sequence for TCR a chain of clone 22.1 H1.
  • the nucleic acid composition provided herein comprises a nucleic acid sequence that encodes a TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:3, or a functional fragment thereof.
  • the CDR3 of the Va domain of a nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO: 3.
  • the Va domain of a nucleic acid composition provided herein comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 7.
  • the inventors identified TCR clone 22.1 H1 which interacts with ALLPAVPSL (SEQ ID NO: 71) in the context of HLA-A*02:01.
  • the sequences provided herein that correspond to TCR clone 22.1 H1 are SEQ ID NO:s 1 to 14.
  • an example of an appropriate TCR p domain CDR3 amino acid sequence that confers specific binding to a WT1 antigen is shown in SEQ I D NO:6.
  • a WT1 antigen e.g. to ALLPAVPSL (SEQ ID NO: 71)
  • SEQ I D NO:6 an example of an appropriate TCR p domain CDR3 amino acid sequence that confers specific binding to a WT1 antigen (e.g. to ALLPAVPSL (SEQ ID NO: 71)
  • variants of the amino acid sequence shown in SEQ ID NO:6 may also be functional (i.e. retain their ability to confer specific binding to a WT1 antigen (e.g. the peptide shown in SEQ ID NO:71) when the CDR3 is part of TCR p domain). Such functional variants are therefore encompassed herein.
  • appropriate (functional) p domain CDR3 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 6, i.e. they may have at least 80%, at least 81 %, at least 87%, at least 93%, or 100% sequence identity to SEQ ID NO: 6.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 6).
  • appropriate (functional) p domain CDR3 amino acid sequences may vary from the sequence shown in SEQ ID NO: 6 by one or several (e.g. two) amino acids.
  • functional variants of SEQ ID NO: 6 retain their ability to confer specific binding to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 71) when the CDR3 is part of TCR p domain.
  • Functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 6.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 6, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the CDR3.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 6 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 71). Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 6 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • the CDR3 of the Vp domain comprises or consists of the amino acid sequence of SEQ ID NO: 6.
  • the CDR3 may be encoded by Any appropriate nucleic acid sequence.
  • the encoded TOR Vp domain may comprise, in addition to the specified CDR3, a CDR1 comprising an amino acid sequence of SEQ ID NO: 4, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 71)).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 4.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 4, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 4 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 71).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 4 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • appropriate functional Vp domain CDR1 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 4, i.e. it may have at least 80%, or 100% sequence identity to SEQ ID NO: 4.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 4).
  • appropriate (functional) Vp domain CDR1 amino acid sequences may vary from the sequence shown in SEQ ID NO:4 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO:4).
  • functional variants of SEQ ID NO: 4 retain the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 71) when the CDR1 is part of TCR Vp domain).
  • the CDR1 of the Vp domain comprises or consists of the amino acid sequence of SEQ ID NO: 4.
  • the CDR1 may be encoded by any appropriate nucleic acid sequence. 1
  • the encoded TCR Vp domain may also comprise, in addition to the specified CDR3 (and optionally the specified CDR1 above), a CDR2 having an amino acid sequence of SEQ ID NO: 5, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to HLA-A*02:01).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 5.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 5, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 5 that do not specifically bind to HLA-A*02:01. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 5 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • appropriate functional Vp domain CDR2 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 5, i.e. it may have at least 80%, at least 83%, or 100% sequence identity to SEQ ID NO: 5.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 5).
  • appropriate (functional) Vp domain CDR2 amino acid sequences may vary from the sequence shown in SEQ ID NO: 5 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO: 5).
  • a functional variant of SEQ ID NO: 5 retains the ability to specifically bind to HLA-A*02:01.
  • the CDR2 of the Vp domain comprises or consists of the amino acid sequence of SEQ ID NO: 5.
  • the CDR2 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Vp domain may therefore comprise the CDRs mentioned in detail above (by SEQ ID specifically i.e. SEQ ID NO:6, SEQ ID NO: 4 and SEQ ID NO: 5, or functional variants thereof), with appropriate intervening sequences between the CDRs.
  • the encoded TCR Vp domain may have an amino acid sequence of SEQ ID NO: 9, or a functional variant thereof (i.e. wherein the variant TCR Vp domain retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 71) when part of a binding protein described herein).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 9.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 9, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 9 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 71).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:9 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • the encoded TCR Vp domain may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 9, whilst retaining the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 71).
  • a functional TCR Vp domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 9 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO:9 may all be in regions of the TCR Vp domain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 6, SEQ ID NO: 4 and/or SEQ ID NO: 5, and still have 25% (or less) sequence variability compared to SEQ ID NO: 9).
  • the sequence of the CDRs of SEQ ID NO: 9 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 9).
  • the encoded TCR Vp domain may 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: 9, wherein the TCR Vp domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 6.
  • the TCR Vp domain CDR1 may have an amino acid sequence of SEQ ID NO:4 and the TCR p domain CDR2 may have an amino acid sequence of SEQ ID NO: 5.
  • the TCR VP domain may be encoded by the nucleic acid sequence of SEQ ID NO: 10, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
  • the nucleic acid sequence encoding the TCR p domain may also encode a TCR p chain constant domain. Examples of suitable constant domains are generally discussed above.
  • SEQ ID NO: 13 An example of a specific TCR p chain amino acid sequence that includes a TCR p domain described herein and an appropriate constant domain is shown in SEQ ID NO: 13.
  • Appropriate functional variants of SEQ ID NO: 13 are also encompassed (e.g. variants having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 13, wherein the variant TCR p chain amino acid sequence retains its ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 71) when part of a binding protein described herein).
  • a WT1 antigen e.g. the peptide shown in SEQ ID NO: 71
  • a functional TCR p chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 13 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO: 13 may all be in regions of the TCR p chain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 6, SEQ ID NO: 4 and/or SEQ ID NO: 5, and still have 25% (or less) sequence variability compared to SEQ ID NO:13.
  • the sequence of the CDRs of SEQ ID NO: 13 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 13).
  • the encoded TCR p chain may 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: 13, wherein the TCR p chain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 6.
  • the TCR p chain CDR1 may have an amino acid sequence of SEQ ID NO: 4 and the TCR p chain CDR2 may have an amino acid sequence of SEQ ID NO: 5.
  • the TCR chain may be encoded by the nucleic acid sequence of SEQ ID NO:14, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO: 14 is the nucleic acid sequence for TCR p chain of clone 22.1 H1.
  • the nucleic acid composition provided herein comprises a nucleic acid sequence that encodes a TCR Vp domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:6, or a functional fragment thereof.
  • the CDR3 of the Vp domain of a nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO:6.
  • the Vp domain of a nucleic acid composition provided herein comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 9.
  • TCR Vp domain sequences derived from TCR clone 22.1 H1 discussed above are particularly compatible with the TCR Va domain sequences derived from TCR clone 22.1 H1 discussed elsewhere herein.
  • a nucleic acid composition described herein encodes a WT1 antigen-specific binding protein having TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:3, or a functional fragment thereof; and a nucleic acid sequence that encodes a TCR p domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:6, or a functional fragment thereof.
  • a nucleic acid composition described herein encodes a WTIantigen- specific binding protein having a TCR Va domain with a CDR3 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and a TCR Vp domain with a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:6.
  • the WT1 antigen may comprise or consist of the sequence shown in SEQ ID NO: 71.
  • the TCR Va domain may be part of a TCR a chain having a constant domain and the TCR Vp domain may be part of a TCR p chain having a constant domain.
  • the Va domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 7; and the Vp domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 9.
  • the Va domain comprises the amino acid sequence of SEQ ID NO: 7 and the p domain comprises the amino acid sequence of SEQ ID NO: 9.
  • the Va domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 8; and the Vp domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ I D NO: 10.
  • the TCR Va domain may include a CDR1 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO:1 and a CDR2 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO:2.
  • the TCR Vp domain may include a CDR1 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO:4 and a CDR2 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 5.
  • this particular example encompasses components of TCR clone 22.1 H1 exemplified herein.
  • the different components of TCR clone 22.1 H1 and their respective SEQ ID Nos are summarised in Table 5 below.
  • the nucleic acid composition described herein encodes both a TCR Va domain and a TCR Vp domain, which form the binding protein that is capable of specifically binding to a WT 1 antigen.
  • the TCR Va domain and the TCR Vp domain may be joined together via a linker, e.g. a linker that enables expression of two proteins or polypeptides from the same vector.
  • a linker comprising a porcine teschovirus-1 2A (P2A) sequence may be used, such as 2A sequences from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A) or Thosea asigna virus (T2A) as published by A.L. Szymczak et al., Nature Biotechnology 22, 589 - 594 (2004) or 2A-like sequences.
  • 2A and 2A-like sequences are linkers that are cleavable once the nucleic acid molecule has been transcribed and translated.
  • Another example of a linker is an internal ribosomal entry sites (IRES) which enables translation of two proteins or polypeptides from the same transcript.
  • IRS internal ribosomal entry sites
  • any other appropriate linker may also be used.
  • the nucleic acid sequence encoding the TCR Va domain and nucleic acid sequence encoding the TCR Vp domain may be cloned into a vector with dual internal promoters (see e.g. S Jones et al., Human Gene Ther 2009).
  • S Jones et al., Human Gene Ther 2009 The identification of appropriate linkers and vectors that enable expression of both the TCR Va domain and the TCR Vp domain is well within the routine capabilities of a person of skill in the art.
  • nucleic acid sequences that encode the TCR Va domain and/or the TCR p domain may also be encoded by the nucleic acid sequences that encode the TCR Va domain and/or the TCR p domain.
  • the nucleic acid sequence may comprise a membrane targeting sequence that provides for transport of the encoded polypeptide to the cell surface membrane of the modified cell.
  • Other appropriate additional domains are well known and are described, for example, in WO2016/071758.
  • the nucleic acid composition described herein may encode a soluble TCR.
  • the nucleic acid composition may encode the variable domain of the TCR alpha and beta chains respectively together with an immune-modulator molecule such as a CD3 agonist (e.g. an anti-CD3 scFv).
  • 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 signal transduction of the TCR.
  • Antibodies specific for the human CD3 antigen are well known. One such antibody is the murine monoclonal antibody OKT3, which is the first monoclonal antibody approved by the FDA. Other antibodies specific for CD3 have also been reported (see e.g.
  • Immune mobilising mTCR Against Cancer are bifunctional proteins that combine affinity monoclonal T cell receptor (mTCR) targeting with a therapeutic mechanism of action (i.e. , an anti-CD3 scFv).
  • mTCR monoclonal T cell receptor
  • a soluble TCR of the invention may be combined with a radioisotope or a toxic drug. Appropriate radioisotopes and/or toxic drugs are well known in the art and are readily identifiable by a person of ordinary skill in the art.
  • the nucleic acid composition may encode a chimeric single chain TCR wherein the TCR alpha chain variable domain is linked to the TCR beta chain variable domain and a constant domain which is e.g. fused to the CD3 zeta signalling domain.
  • the linker is non-cleavable.
  • the nucleic acid composition may encode a chimeric two chain TCR in which the TCR alpha chain variable domain and the TCR beta chain variable domain are each linked to a CD3 zeta signalling domain or other transmembrane and intracellular domains. Methods for preparing such single chain TCRs and two chain TCRs are well known in the art; see for example RA Willemsen et al, Gene Therapy 2000.
  • TCR components that interact with VLDFAPPGA SEQ ID NO: 72
  • TCR clone 20.3D10 which interacts with VLDFAPPGA (SEQ ID NO: 72) in the context of HLA-A*02:01.
  • sequences provided herein that correspond to TCR clone 20.2D10 are SEQ ID NO:s 15 to 28.
  • an isolated nucleic acid composition that encodes a Wilms’ tumor 1 (WT1) antigen-specific binding protein having a TCR a chain variable (Va) domain and a TCR p chain variable (VP) domain
  • the composition comprising: a nucleic acid sequence that encodes a TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 17, or a functional fragment thereof; and a nucleic acid sequence that encodes a TCR Vp domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 20, or a functional fragment thereof, wherein the CDR3 sequences together specifically bind to WT1.
  • TCR Va domain CDR3 amino acid sequence that confers specific binding to a WT1 antigen e.g. to VLDFAPPGA (SEQ ID NO: 72)
  • SEQ ID NO: 17 another example of an appropriate TCR Va domain CDR3 amino acid sequence that confers specific binding to a WT1 antigen (e.g. to VLDFAPPGA (SEQ ID NO: 72)) is shown in SEQ ID NO: 17.
  • variants of the amino acid sequence shown in SEQ ID NO:17 may also be functional (i.e. retain their ability to confer specific binding to a WT1 antigen (e.g. to the peptide VLDFAPPGA (SEQ ID NO: 72)) when the CDR3 is part of TCR Va domain).
  • WT1 antigen e.g. to the peptide VLDFAPPGA (SEQ ID NO: 72)
  • appropriate (functional) Va domain CDR3 amino acid sequences may have at least 80% sequence identity to SEQ ID NO:17, i.e. they may have at least 80%, at least 84%, at least 92%, or 100% sequence identity to SEQ ID NO: 17.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:17).
  • appropriate (functional) Va domain CDR3 amino acid sequences may vary from the sequence shown in SEQ ID NO: 17 by one or several (e.g. two etc) amino acids.
  • SEQ ID NO: 17 retain their ability to confer specific binding to a WT1 antigen (e.g. the peptide shown in SEQ ID NO:72) when the CDR3 is part of TCR Va domain.
  • a WT1 antigen e.g. the peptide shown in SEQ ID NO:72
  • Functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 17.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one, two or more amino acids of SEQ ID NO:17, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the CDR3.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 17 that do not specifically bind to a WT1 antigen (e.g. to the peptide shown in SEQ ID NO72).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 17 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • the CDR3 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 17.
  • the CDR3 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Va domain may comprise, in addition to the specified CDR3, a CDR1 comprising an amino acid sequence of SEQ ID NO: 15, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO:72)).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 15.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 15, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 15 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO:72).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 15 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • appropriate functional Va domain CDR1 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 15, i.e. it may have at least 80%, at least 83%, or 100% sequence identity to SEQ ID NO: 15.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:15).
  • appropriate functional Va domain CDR1 amino acid sequences may vary from the sequence shown in SEQ ID NO: 15 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO: 15.
  • functional variants of SEQ ID NO: 15 retain the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO:72) when the CDR1 is part of TCR Va domain).
  • the CDR1 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 15.
  • the CDR1 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Va domain may also comprise, in addition to the specified CDR3 (and optionally the specified CDR1 above), a CDR2 comprising an amino acid sequence of SEQ ID NO: 16, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to HLA-A*02:01).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 16.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:16, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO:16 that do not specifically bind to HLA-A*02:01. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 16 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • appropriate functional Va domain CDR2 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 16, i.e. it may have at least 80%, at least 87%, or 100% sequence identity to SEQ ID NO: 16.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:16).
  • appropriate (functional) Va domain CDR2 amino acid sequences may vary from the sequence shown in SEQ ID NO:16 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO: 16).
  • the CDR2 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 16.
  • the CDR2 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TOR Va domain may therefore comprise the CDRs mentioned in detail above (by SEQ ID specifically i.e. SEQ ID NO:17, SEQ ID NO:15 and SEQ ID NO: 16, or functional variants thereof), with appropriate intervening sequences between the CDRs.
  • the encoded TCR Va domain may comprise an amino acid sequence of SEQ ID NO:21 , or a functional variant thereof (i.e. wherein the variant TCR Va domain retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO:72) when part of a binding protein described herein).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO:21.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO:21 , or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO:21 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO:72).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:21 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • the encoded TCR Va domain may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO:21 , whilst retaining the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO:72).
  • a functional TCR Va domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO:21 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO:21 may all be in regions of the TCR Va domain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 17, SEQ ID NO:15 and/or SEQ ID NO:16, and still have 25% (or less) sequence variability compared to SEQ ID NO:21).
  • the sequence of the CDRs of SEQ ID NO: 21 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 21).
  • the encoded TOR Va domain may 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: 21 , wherein the TCR Va domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the TCR Va domain CDR1 may have an amino acid sequence of SEQ ID NO: 15 and the TCR Va domain CDR2 may have an amino acid sequence of SEQ ID NO: 16.
  • the encoded TCR Va domain may comprise an amino acid sequence having at the amino acid sequence of SEQ ID NO: 21 , with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions), wherein the TCR Va domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the TCR Va domain CDR1 may have an amino acid sequence of SEQ ID NO: 15 and the TCR Va domain CDR2 may have an amino acid sequence of SEQ ID NO: 16.
  • the TCR Va domain may be encoded by the nucleic acid sequence of SEQ ID NO: 22, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
  • the nucleic acid sequence encoding the TCR Va domain may also encode a TCR a chain constant domain. Examples of suitable constant domains are generally discussed above.
  • SEQ ID NO: 25 An example of a specific TCR a chain amino acid sequence that includes a TCR Va domain described herein with an appropriate constant domain is shown in SEQ ID NO: 25.
  • Appropriate functional variants of SEQ ID NO: 25 are also encompassed (e.g. variants having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 25, wherein the variant TCR a chain amino acid sequence retains its ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO:72) when part of a binding protein described herein).
  • a WT1 antigen e.g. the peptide shown in SEQ ID NO:72
  • a functional TCR a chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO:25 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO:25 may all be in regions of the TCR a chain that do not form CDRs (i.e. the variant may have the CDRs of SEQ I D NO: 17, SEQ I D NO: 15 and/or SEQ I D NO: 16, and still have 25% (or less) sequence variability compared to SEQ ID NO:25).
  • the sequence of the CDRs of SEQ ID NO: 25 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 25).
  • the encoded TCR a chain may 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: 25, wherein the TCR a chain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 17.
  • the TCR a chain CDR1 may have an amino acid sequence of SEQ ID NO: 15 and the TCR a chain CDR2 may have an amino acid sequence of SEQ ID NO: 16.
  • the TCR a chain may be encoded by the nucleic acid sequence of SEQ ID NO:26, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO:26 is the nucleic acid sequence for TCR a chain of clone 20.3D10.
  • the nucleic acid composition provided herein comprises a nucleic acid sequence that encodes a TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 17, or a functional fragment thereof.
  • the CDR3 of the Va domain of a nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO: 17.
  • Va domain of a nucleic acid composition provided herein comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 21.
  • the inventors have also identified TCR clone 20.3D10 which interacts with VLDFAPPGA (SEQ ID NO: 72) in the context of HLA-A*02:01.
  • the sequences provided herein that correspond to TCR clone 20.3D10 are SEQ ID NO:s 15 to 28.
  • TCR p domain CDR3 amino acid sequence that confers specific binding to a WT 1 antigen e.g. to VLDFAPPGA (SEQ ID NO: 72)
  • SEQ ID NQ:20 An example of an appropriate TCR p domain CDR3 amino acid sequence that confers specific binding to a WT 1 antigen (e.g. to VLDFAPPGA (SEQ ID NO: 72)) is shown in SEQ ID NQ:20.
  • variants of the amino acid sequence shown in SEQ ID NQ:20 may also be functional (i.e. retain their ability to confer specific binding to a WT 1 antigen (i.e.. the peptide shown in SEQ ID NO: 72) when the CDR3 is part of TCR p domain). Such functional variants are therefore encompassed herein.
  • appropriate (functional) p domain CDR3 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 20, i.e. they may have at least 80%, at least 81 %, at least 87%, at least 93%, or 100% sequence identity to SEQ ID NO: 20.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 20).
  • appropriate (functional) p domain CDR3 amino acid sequences may vary from the sequence shown in SEQ ID NO: 20 by one or several (e.g. two) amino acids.
  • functional variants of SEQ ID NO: 20 retain their ability to confer specific binding to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 72) when the CDR3 is part of TCR p domain.
  • Functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 20.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 20, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the CDR3.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 20 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 20 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • the CDR3 of the Vp domain comprises or consists of the amino acid sequence of SEQ ID NO: 20.
  • the CDR3 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TOR Vp domain may comprise, in addition to the specified CDR3, a CDR1 comprising an amino acid sequence of SEQ ID NO: 18, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 72)).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 18.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 18, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 18 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 18 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • appropriate functional Vp domain CDR1 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 18, i.e. it may have at least 80%, or 100% sequence identity to SEQ ID NO: 18.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 18).
  • appropriate (functional) Vp domain CDR1 amino acid sequences may vary from the sequence shown in SEQ ID NO: 18 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO: 18).
  • functional variants of SEQ ID NO: 18 retain the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72) when the CDR1 is part of TCR Vp domain).
  • the CDR1 of the Vp domain comprises or consists of the amino acid sequence of SEQ ID NO: 18.
  • the CDR1 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Vp domain may also comprise, in addition to the specified CDR3 (and optionally the specified CDR1 above), a CDR2 having an amino acid sequence of SEQ ID NO: 19, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to HLA-A*02:01).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 19.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 19, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 19 that do not specifically bind to HLA-A*02:01. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 19 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • appropriate functional Vp domain CDR2 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 19, i.e. it may have at least 80%, at least 83%, or 100% sequence identity to SEQ ID NO: 19.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 19).
  • appropriate (functional) Vp domain CDR2 amino acid sequences may vary from the sequence shown in SEQ ID NO: 19 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO: 19).
  • a functional variant of SEQ ID NO: 19 retains the ability to specifically bind to HLA-A*02:01.
  • the CDR2 of the Vp domain comprises or consists of the amino acid sequence of SEQ ID NO: 19.
  • the CDR2 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Vp domain may therefore comprise the CDRs mentioned in detail above (by SEQ ID specifically i.e. SEQ ID NQ:20, SEQ ID NO: 18 and SEQ ID NO: 19, or functional variants thereof), with appropriate intervening sequences between the CDRs.
  • the encoded TCR Vp domain may have an amino acid sequence of SEQ ID NO: 23, or a functional variant thereof (i.e. wherein the variant TCR Vp domain retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 72) when part of a binding protein described herein).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 23.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 23, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 23 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:23 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • the encoded TCR Vp domain may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 23, whilst retaining the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72).
  • a functional TCR Vp domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 23 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO:23 may all be in regions of the TCR Vp domain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 20, SEQ ID NO: 18 and/or SEQ ID NO: 19, and still have 25% (or less) sequence variability compared to SEQ ID NO: 23).
  • the sequence of the CDRs of SEQ ID NO: 23 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 23).
  • the encoded TCR Vp domain may 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: 23, wherein the TCR Vp domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 20.
  • the TCR Vp domain CDR1 may have an amino acid sequence of SEQ ID NO:18 and the TCR p domain CDR2 may have an amino acid sequence of SEQ ID NO: 19.
  • the TCR VP domain may be encoded by the nucleic acid sequence of SEQ ID NO:24, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
  • the nucleic acid sequence encoding the TCR p domain may also encode a TCR p chain constant domain. Examples of suitable constant domains are generally discussed above.
  • SEQ ID NO: 27 An example of a specific TCR p chain amino acid sequence that includes a TCR p domain described herein and an appropriate constant domain is shown in SEQ ID NO: 27.
  • Appropriate functional variants of SEQ ID NO: 27 are also encompassed (e.g. variants having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 27, wherein the variant TCR p chain amino acid sequence retains its ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72) when part of a binding protein described herein).
  • a WT1 antigen e.g. the peptide shown in SEQ ID NO: 72
  • a functional TCR p chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 27 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO:27 may all be in regions of the TCR p chain that do not form CDRs (i.e. the variant may have the CDRs of SEQ I D NO: 20, SEQ I D NO: 18 and/or SEQ I D NO: 19, and still have 25% (or less) sequence variability compared to SEQ ID NO:27.
  • the sequence of the CDRs of SEQ ID NO: 27 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 27).
  • the encoded TCR p chain may 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: 27, wherein the TCR p chain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 20.
  • the TCR p chain CDR1 may have an amino acid sequence of SEQ ID NO: 18 and the TCR p chain CDR2 may have an amino acid sequence of SEQ ID NO: 19.
  • the TCR chain may be encoded by the nucleic acid sequence of SEQ ID NO:28, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO:28 is the nucleic acid sequence for TCR p chain of clone 20.3D10.
  • the nucleic acid composition provided herein comprises a nucleic acid sequence that encodes a TCR Vp domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NQ:20, or a functional fragment thereof.
  • the CDR3 of the Vp domain of a nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NQ:20.
  • the Vp domain of a nucleic acid composition provided herein comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 23.
  • TCR Vp domain sequences derived from TCR clone 20.3D10 discussed above are particularly compatible with the TCR Va domain sequences derived from TCR clone 20.3D10 discussed elsewhere herein.
  • a nucleic acid composition described herein encodes a WT1 antigen-specific binding protein having TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 17, or a functional fragment thereof; and a nucleic acid sequence that encodes a TCR p domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NQ:20, or a functional fragment thereof.
  • a nucleic acid composition described herein encodes a WT1 antigenspecific binding protein having a TCR Va domain with a CDR3 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 17; and a TCR Vp domain with a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NQ:20.
  • the WT1 antigen may comprise or consist of the sequence shown in SEQ ID NO: 72.
  • the TCR Va domain may be part of a TCR a chain having a constant domain and the TCR Vp domain may be part of a TCR p chain having a constant domain.
  • the Va domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 21 ; and the Vp domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 23.
  • the Va domain comprises the amino acid sequence of SEQ ID NO: 21 and the p domain comprises the amino acid sequence of SEQ ID NO: 23.
  • the Va domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 22; and the Vp domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 24.
  • the TCR Va domain may include a CDR1 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO:15 and a CDR2 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 16.
  • the TCR Vp domain may include a CDR1 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO:18 and a CDR2 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 19.
  • this particular example encompasses components of TCR clone 20.3D10 exemplified herein.
  • the different components of TCR clone 20.3D10 and their respective SEQ ID Nos are summarised in Table 6 below.
  • the nucleic acid composition described herein encodes both a TCR Va domain and a TCR Vp domain, which form the binding protein that is capable of specifically binding to a WT 1 antigen.
  • the TCR Va domain and the TCR Vp domain may be joined together via a linker. Suitable linkers are discussed generally elsewhere herein. Additional appropriate polypeptide domains that may also be encoded by the nucleic acid sequences that encode the TCR Va domain and/or the TCR Vp domain are also discussed generally elsewhere herein.
  • the nucleic acid composition described herein may encode a soluble TCR or a chimeric single chain TCR wherein the TCR alpha chain variable domain is linked to the TCR beta chain variable domain and a constant domain which is e.g. fused to the CD3 zeta signalling domain.
  • TCR clone 23.2G9 which interacts with VLDFAPPGA (SEQ ID NO: 72) in the context of HLA-A*02:01.
  • sequences provided herein that correspond to TCR clone 23.2G9 are SEQ ID NO:s 29 to 42.
  • an isolated nucleic acid composition that encodes a Wilms’ tumor 1 (WT1) antigen-specific binding protein having a TCR a chain variable (Va) domain and a TCR p chain variable (VP) domain
  • the composition comprising: a nucleic acid sequence that encodes a TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:31 , or a functional fragment thereof; and a nucleic acid sequence that encodes a TCR Vp domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:34, or a functional fragment thereof, wherein the CDR3 sequences together specifically bind to WT.
  • TCR Va domain CDR3 amino acid sequence that confers specific binding to a WT1 antigen e.g. to VLDFAPPGA (SEQ ID NO: 72)
  • SEQ ID NO:31 An example of an appropriate TCR Va domain CDR3 amino acid sequence that confers specific binding to a WT1 antigen (e.g. to VLDFAPPGA (SEQ ID NO: 72)) is shown in SEQ ID NO:31.
  • variants of the amino acid sequence shown in SEQ ID NO:31 may also be functional (i.e. retain their ability to confer specific binding to a WT 1 antigen (e.g. to the peptide VLDFAPPGA (SEQ ID NO: 72)) when the CDR3 is part of TCR Va domain).
  • WT 1 antigen e.g. to the peptide VLDFAPPGA (SEQ ID NO: 72)
  • appropriate (functional) Va domain CDR3 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 31 , i.e. they may have at least 80%, at least 84%, at least 92%, or 100% sequence identity to SEQ ID NO: 31.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:31).
  • appropriate (functional) Va domain CDR3 amino acid sequences may vary from the sequence shown in SEQ ID NO:31 by one or several (e.g. two etc) amino acids.
  • SEQ ID NO: 31 retain their ability to confer specific binding to a WT 1 antigen (i.e.. the peptide shown in SEQ ID NO: 72) when the CDR3 is part of TCR Va domain.
  • Functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 31.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one, two or more amino acids of SEQ ID NO: 31 , or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the CDR3.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 31 that do not specifically bind to a WT1 antigen (i.e. the peptide shown in SEQ ID NO: 72).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 31 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • the CDR3 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 31.
  • the CDR3 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Va domain may comprise, in addition to the specified CDR3, a CDR1 comprising an amino acid sequence of SEQ ID NO: 29, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 72)).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 29.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 29, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 29 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 29 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • appropriate functional Va domain CDR1 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 29, i.e. it may have at least 80%, at least 83%, or 100% sequence identity to SEQ ID NO: 29.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 29).
  • appropriate functional Va domain CDR1 amino acid sequences may vary from the sequence shown in SEQ ID NO: 29 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO: 29.
  • functional variants of SEQ ID NO: 29 retain the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72) when the CDR1 is part of TCR Va domain).
  • the CDR1 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 29.
  • the CDR1 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Va domain may also comprise, in addition to the specified CDR3 (and optionally the specified CDR1 above), a CDR2 comprising an amino acid sequence of SEQ ID NO: 30, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to HLA-A*02:01).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 30.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 30, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 30 that do not specifically bind to HLA-A*02:01. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 30 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • appropriate functional Va domain CDR2 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 30, i.e. it may have at least 80%, at least 83%, or 100% sequence identity to SEQ ID NO: 30.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NQ:30).
  • appropriate (functional) Va domain CDR2 amino acid sequences may vary from the sequence shown in SEQ ID NQ:30 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO: 30).
  • the CDR2 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 30.
  • the CDR2 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TOR Va domain may therefore comprise the CDRs mentioned in detail above (by SEQ ID specifically i.e. SEQ ID NO: 31 , SEQ ID NO: 29 and SEQ ID NO: 30, or functional variants thereof), with appropriate intervening sequences between the CDRs.
  • the encoded TCR Va domain may comprise an amino acid sequence of SEQ ID NO: 35, or a functional variant thereof (i.e. wherein the variant TCR Va domain retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 72) when part of a binding protein described herein).
  • a functional variant may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 35.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 35, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 35 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 35 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • the encoded TCR Va domain may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 35, whilst retaining the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72).
  • a functional TCR Va domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 35 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO: 35 may all be in regions of the TCR Va domain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 31 , SEQ ID NO: 29 and/or SEQ ID NO: 30, and still have 25% (or less) sequence variability compared to SEQ ID NO: 35).
  • the sequence of the CDRs of SEQ ID NO: 35 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 35).
  • the encoded TOR Va domain may 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: 35, wherein the TCR Va domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 31 .
  • the TCR Va domain CDR1 may have an amino acid sequence of SEQ ID NO: 29 and the TCR Va domain CDR2 may have an amino acid sequence of SEQ ID NO: 30.
  • the encoded TCR Va domain may comprise an amino acid sequence having at the amino acid sequence of SEQ ID NO: 35, with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions), wherein the TCR Va domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 31.
  • the TCR Va domain CDR1 may have an amino acid sequence of SEQ ID NO: 29 and the TCR Va domain CDR2 may have an amino acid sequence of SEQ ID NO: 30.
  • the TCR Va domain may be encoded by the nucleic acid sequence of SEQ ID NO: 36, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
  • the nucleic acid sequence encoding the TCR Va domain may also encode a TCR a chain constant domain. Examples of suitable constant domains are generally discussed above.
  • SEQ ID NO: 39 An example of a specific TCR a chain amino acid sequence that includes a TCR Va domain described herein with an appropriate constant domain is shown in SEQ ID NO: 39.
  • Appropriate functional variants of SEQ ID NO: 39 are also encompassed (e.g. variants having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 39, wherein the variant TCR a chain amino acid sequence retains its ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72) when part of a binding protein described herein).
  • a WT1 antigen e.g. the peptide shown in SEQ ID NO: 72
  • a functional TCR a chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 39 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO: 39 may all be in regions of the TOR a chain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 31 , SEQ ID NO: 29 and/or SEQ ID NO: 30, and still have 25% (or less) sequence variability compared to SEQ ID NO: 39).
  • the sequence of the CDRs of SEQ ID NO: 39 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 39).
  • the encoded TCR a chain may 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: 39, wherein the TCR a chain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 31.
  • the TCR a chain CDR1 may have an amino acid sequence of SEQ ID NO: 29 and the TCR a chain CDR2 may have an amino acid sequence of SEQ ID NO: 30.
  • the TCR a chain may be encoded by the nucleic acid sequence of SEQ ID NO: 40, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NQ:40 is the nucleic acid sequence for TCR a chain of clone 23.2G9.
  • the nucleic acid composition provided herein comprises a nucleic acid sequence that encodes a TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:31 , or a functional fragment thereof.
  • the CDR3 of the Va domain of a nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO: 31.
  • the Va domain of a nucleic acid composition provided herein comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 35.
  • the inventors identified TCR clone 23.2G9 which interacts with VLDFAPPGA (SEQ ID NO: 72) in the context of HLA-A*02:01.
  • the sequences provided herein that correspond to TCR clone 23.2G9 are SEQ ID NO:s 29 to 42.
  • TCR p domain CDR3 amino acid sequence that confers specific binding to a WT 1 antigen e.g. to VLDFAPPGA (SEQ ID NO: 72)
  • SEQ ID NO:34 An example of an appropriate TCR p domain CDR3 amino acid sequence that confers specific binding to a WT 1 antigen (e.g. to VLDFAPPGA (SEQ ID NO: 72)) is shown in SEQ ID NO:34.
  • variants of the amino acid sequence shown in SEQ ID NO:34 may also be functional (i.e. retain their ability to confer specific binding to a WT 1 antigen (i.e. the peptide shown in SEQ ID NO: 72) when the CDR3 is part of TCR p domain). Such functional variants are therefore encompassed herein.
  • appropriate (functional) p domain CDR3 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 34, i.e. they may have at least 80%, at least 86%, at least 93%, or 100% sequence identity to SEQ ID NO: 34.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 34).
  • appropriate (functional) p domain CDR3 amino acid sequences may vary from the sequence shown in SEQ ID NO: 34 by one or several (e.g. two) amino acids.
  • functional variants of SEQ ID NO: 34 retain their ability to confer specific binding to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 72) when the CDR3 is part of TCR p domain.
  • Functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 34.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 34, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the CDR3.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 34 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 34 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • the CDR3 of the Vp domain comprises or consists of the amino acid sequence of SEQ ID NO: 34.
  • the CDR3 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TOR Vp domain may comprise, in addition to the specified CDR3, a CDR1 comprising an amino acid sequence of SEQ ID NO: 32, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 72)).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 32.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 32, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 32 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 32 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • appropriate functional Vp domain CDR1 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 32, i.e. it may have at least 80%, or 100% sequence identity to SEQ ID NO: 32.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 32).
  • appropriate (functional) Vp domain CDR1 amino acid sequences may vary from the sequence shown in SEQ ID NO:32 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO:32).
  • functional variants of SEQ ID NO: 32 retain the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72) when the CDR1 is part of TCR Vp domain).
  • the CDR1 of the Vp domain comprises or consists of the amino acid sequence of SEQ ID NO: 32.
  • the CDR1 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Vp domain may also comprise, in addition to the specified CDR3 (and optionally the specified CDR1 above), a CDR2 having an amino acid sequence of SEQ ID NO: 33, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to HLA-A*02:01).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 33.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 33, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 33 that do not specifically bind to HLA-A*02:01. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 33 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • appropriate functional Vp domain CDR2 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 33, i.e. it may have at least 80%, at least 83%, or 100% sequence identity to SEQ ID NO: 33.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 33).
  • appropriate (functional) Vp domain CDR2 amino acid sequences may vary from the sequence shown in SEQ ID NO: 33 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO: 33).
  • a functional variant of SEQ ID NO: 33 retains the ability to specifically bind to HLA-A*02:01.
  • the CDR2 of the Vp domain comprises or consists of the amino acid sequence of SEQ ID NO: 33.
  • the CDR2 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Vp domain may therefore comprise the CDRs mentioned in detail above (by SEQ ID specifically i.e. SEQ ID NO:34, SEQ ID NO: 32 and SEQ ID NO: 33, or functional variants thereof), with appropriate intervening sequences between the CDRs.
  • the encoded TCR Vp domain may have an amino acid sequence of SEQ ID NO: 37, or a functional variant thereof (i.e. wherein the variant TCR Vp domain retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 72) when part of a binding protein described herein).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 37.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 37, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 37 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:37 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • the encoded TCR Vp domain may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 37, whilst retaining the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72).
  • a functional TCR Vp domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 37 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO:37 may all be in regions of the TCR Vp domain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 34, SEQ ID NO: 32 and/or SEQ ID NO: 33, and still have 25% (or less) sequence variability compared to SEQ ID NO: 37).
  • the sequence of the CDRs of SEQ ID NO: 37 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 37).
  • the encoded TCR Vp domain may 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: 37, wherein the TCR Vp domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 34.
  • the TCR Vp domain CDR1 may have an amino acid sequence of SEQ ID NO:32 and the TCR p domain CDR2 may have an amino acid sequence of SEQ ID NO: 33.
  • the TCR VP domain may be encoded by the nucleic acid sequence of SEQ ID NO:38, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
  • the nucleic acid sequence encoding the TCR p domain may also encode a TCR p chain constant domain. Examples of suitable constant domains are generally discussed above.
  • SEQ ID NO: 41 An example of a specific TCR p chain amino acid sequence that includes a TCR p domain described herein and an appropriate constant domain is shown in SEQ ID NO: 41.
  • Appropriate functional variants of SEQ ID NO: 41 are also encompassed (e.g. variants having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 41 , wherein the variant TCR p chain amino acid sequence retains its ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 72) when part of a binding protein described herein).
  • a WT1 antigen e.g. the peptide shown in SEQ ID NO: 72
  • a functional TCR p chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 41 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO:41 may all be in regions of the TCR p chain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 34, SEQ ID NO: 32 and/or SEQ ID NO: 33, and still have 25% (or less) sequence variability compared to SEQ ID NO:41 .
  • the sequence of the CDRs of SEQ ID NO: 41 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 41).
  • the encoded TCR p chain may comprise an amino acid sequence having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 41 , wherein the TCR p chain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 34.
  • the TCR p chain CDR1 may have an amino acid sequence of SEQ ID NO: 32 and the TCR p chain CDR2 may have an amino acid sequence of SEQ ID NO: 33.
  • the TCR chain may be encoded by the nucleic acid sequence of SEQ ID NO: 42, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO:42 is the nucleic acid sequence for TCR p chain of clone 23.2G9.
  • the nucleic acid composition provided herein comprises a nucleic acid sequence that encodes a TCR Vp domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:34, or a functional fragment thereof.
  • the CDR3 of the Vp domain of a nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO:34.
  • the Vp domain of a nucleic acid composition provided herein comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 37.
  • TCR Vp domain sequences derived from TCR clone 23.2G9 discussed above are particularly compatible with the TCR Va domain sequences derived from TCR clone 23.2G9 discussed elsewhere herein.
  • a nucleic acid composition described herein encodes a WT1 antigen-specific binding protein having TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:31 , or a functional fragment thereof; and a nucleic acid sequence that encodes a TCR p domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:34, or a functional fragment thereof.
  • a nucleic acid composition described herein encodes a WT1 antigenspecific binding protein having a TCR Va domain with a CDR3 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 31 ; and a TCR Vp domain with a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:34.
  • the WT1 antigen may comprise or consist of the sequence shown in SEQ ID NO: 72.
  • the TCR Va domain may be part of a TCR a chain having a constant domain and the TCR Vp domain may be part of a TCR p chain having a constant domain.
  • the Va domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 35; and the Vp domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 37.
  • the Va domain comprises the amino acid sequence of SEQ ID NO: 35 and the p domain comprises the amino acid sequence of SEQ ID NO: 37.
  • the Va domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 36; and the Vp domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 38.
  • the TCR Va domain may include a CDR1 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 29 and a CDR2 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NQ:30.
  • the TCR Vp domain may include a CDR1 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO:32 and a CDR2 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 33.
  • this particular example encompasses components of TCR clone 23.2G9 exemplified herein.
  • the different components of TCR clone 23.2G9 and their respective SEQ ID Nos are summarised in Table 7 below.
  • the nucleic acid composition described herein encodes both a TCR Va domain and a TCR Vp domain, which form the binding protein that is capable of specifically binding to a WT 1 antigen.
  • the TCR Va domain and the TCR Vp domain may be joined together via a linker. Suitable linkers are discussed generally elsewhere herein. Additional appropriate polypeptide domains that may also be encoded by the nucleic acid sequences that encode the TCR Va domain and/or the TCR Vp domain are also discussed generally elsewhere herein.
  • the nucleic acid composition described herein may encode a soluble TCR or a chimeric single chain TCR wherein the TCR alpha chain variable domain is linked to the TCR beta chain variable domain and a constant domain which is e.g. fused to the CD3 zeta signalling domain.
  • TCR components that interact with TPYSSDNLY SEQ ID NO: 73
  • TCR clone 17.2G4 which interacts with TPYSSDNLY (SEQ ID NO: 73) in the context of HLA-B*35:01.
  • sequences provided herein that correspond to TCR clone 17.2G4 are SEQ ID NO:s 43 to 56.
  • an isolated nucleic acid composition that encodes a Wilms’ tumor 1 (WT1) antigen-specific binding protein having a TCR a chain variable (Va) domain and a TCR p chain variable (VP) domain
  • the composition comprising: a nucleic acid sequence that encodes a TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:45, or a functional fragment thereof; and a nucleic acid sequence that encodes a TCR Vp domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:48, or a functional fragment thereof, wherein the CDR3 sequences together specifically bind to WT 1 .
  • TCR Va domain CDR3 amino acid sequence that confers specific binding to a WT1 antigen e.g. to TPYSSDNLY (SEQ ID NO: 73)
  • SEQ ID NO: 45 An example of an appropriate TCR Va domain CDR3 amino acid sequence that confers specific binding to a WT1 antigen (e.g. to TPYSSDNLY (SEQ ID NO: 73)) is shown in SEQ ID NO: 45.
  • variants of the amino acid sequence shown in SEQ ID NO: 45 may also be functional (i.e. retain their ability to confer specific binding to a WT 1 antigen (e.g. to the peptide TPYSSDNLY (SEQ ID NO: 73)) when the CDR3 is part of TCR Va domain). Such functional variants are therefore encompassed herein.
  • appropriate (functional) Va domain CDR3 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 45, i.e. they may have at least 80%, at least 84%, at least 92%, or 100% sequence identity to SEQ ID NO: 45.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 45).
  • appropriate (functional) Va domain CDR3 amino acid sequences may vary from the sequence shown in SEQ ID NO: 45 by one or several (e.g. two etc) amino acids.
  • SEQ I D NO: 45 retain their ability to confer specific binding to a WT1 antigen (i.e.. the peptide shown in SEQ ID NO: 73) when the CDR3 is part of TCR Va domain.
  • Functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 45.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one, two or more amino acids of SEQ ID NO: 45, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the CDR3.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 45 that do not specifically bind to a WT1 antigen (i.e. the peptide shown in SEQ ID NO: 73). Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 45 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • the CDR3 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 45.
  • the CDR3 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Va domain may comprise, in addition to the specified CDR3, a CDR1 comprising an amino acid sequence of SEQ ID NO: 43, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 73)).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 43.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 43, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 43 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 73). Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 43 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • appropriate functional Va domain CDR1 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 43, i.e. it may have at least 80%, at least 83%, or 100% sequence identity to SEQ ID NO: 43.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 43).
  • appropriate functional Va domain CDR1 amino acid sequences may vary from the sequence shown in SEQ ID NO: 43 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO: 43.
  • functional variants of SEQ ID NO: 43 retain the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 73) when the CDR1 is part of TCR Va domain).
  • the CDR1 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 43.
  • the CDR1 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Va domain may also comprise, in addition to the specified CDR3 (and optionally the specified CDR1 above), a CDR2 comprising an amino acid sequence of SEQ ID NO: 44, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to HLA-B*35:01).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 44.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 44, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 44 that do not specifically bind to HLA-B*35:01. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 44 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • appropriate functional Va domain CDR2 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 44, i.e. it may have at least 80%, at least 85%, or 100% sequence identity to SEQ ID NO: 44.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 44).
  • appropriate (functional) Va domain CDR2 amino acid sequences may vary from the sequence shown in SEQ ID NO: 44 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO: 44).
  • the CDR2 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 44.
  • the CDR2 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TOR Va domain may therefore comprise the CDRs mentioned in detail above (by SEQ ID specifically i.e. SEQ ID NO:45, SEQ ID NO: 43 and SEQ ID NO: 44, or functional variants thereof), with appropriate intervening sequences between the CDRs.
  • the encoded TCR Va domain may comprise an amino acid sequence of SEQ ID NO: 49, or a functional variant thereof (i.e. wherein the variant TCR Va domain retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 73) when part of a binding protein described herein).
  • a functional variant may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 49.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 49, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 49 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 73). Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 49 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • the encoded TCR Va domain may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 49, whilst retaining the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 73).
  • a functional TCR Va domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 49 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO: 49 may all be in regions of the TCR Va domain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 45, SEQ ID NO: 43 and/or SEQ ID NO: 44, and still have 25% (or less) sequence variability compared to SEQ ID NO:49).
  • the sequence of the CDRs of SEQ ID NO: 49 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 49).
  • the encoded TOR Va domain may 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: 49, wherein the TCR Va domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 45.
  • the TCR Va domain CDR1 may have an amino acid sequence of SEQ ID NO: 43 and the TCR Va domain CDR2 may have an amino acid sequence of SEQ ID NO: 44.
  • the encoded TCR Va domain may comprise an amino acid sequence having at the amino acid sequence of SEQ ID NO: 49, with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions), wherein the TCR Va domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 45.
  • the TCR Va domain CDR1 may have an amino acid sequence of SEQ ID NO: 43 and the TCR Va domain CDR2 may have an amino acid sequence of SEQ ID NO: 44.
  • the TCR Va domain may be encoded by the nucleic acid sequence of SEQ ID NO: 50, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
  • the nucleic acid sequence encoding the TCR Va domain may also encode a TCR a chain constant domain. Examples of suitable constant domains are generally discussed above.
  • SEQ ID NO: 53 An example of a specific TCR a chain amino acid sequence that includes a TCR Va domain described herein with an appropriate constant domain is shown in SEQ ID NO: 53.
  • Appropriate functional variants of SEQ ID NO:53 are also encompassed (e.g. variants having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 53, wherein the variant TCR a chain amino acid sequence retains its ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 73) when part of a binding protein described herein).
  • a WT1 antigen e.g. the peptide shown in SEQ ID NO: 73
  • a functional TCR a chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO:53 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO:53 may all be in regions of the TCR a chain that do not form CDRs (i.e. the variant may have the CDRs of SEQ I D NO: 45, SEQ I D NO: 43 and/or SEQ I D NO: 44, and still have 25% (or less) sequence variability compared to SEQ ID NO: 53).
  • sequence of the CDRs of SEQ ID NO: 53 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 53).
  • the encoded TCR a chain may 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: 53, wherein the TCR a chain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 45.
  • the TCR a chain CDR1 may have an amino acid sequence of SEQ ID NO:43 and the TCR a chain CDR2 may have an amino acid sequence of SEQ ID NO: 44.
  • the TCR a chain may be encoded by the nucleic acid sequence of SEQ ID NO:54, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO:54 is the nucleic acid sequence for TCR a chain of clone 17.2G4.
  • the nucleic acid composition provided herein comprises a nucleic acid sequence that encodes a TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:45, or a functional fragment thereof.
  • the CDR3 of the Va domain of a nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO: 45.
  • the Va domain of a nucleic acid composition provided herein comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 49.
  • the inventors identified TCR clone 17.2G4 which interacts with TPYSSDNLY (SEQ ID NO: 73) in the context of HLA-B*35:01.
  • the sequences provided herein that correspond to TCR clone 17.2G4 are SEQ ID NO:s 43 to 56.
  • TCR Vp domain CDR3 amino acid sequence that confers specific binding to a WT1 antigen e.g. to TPYSSDNLY (SEQ ID NO: 73)
  • SEQ ID NO:48 An example of an appropriate TCR Vp domain CDR3 amino acid sequence that confers specific binding to a WT1 antigen (e.g. to TPYSSDNLY (SEQ ID NO: 73)) is shown in SEQ ID NO:48.
  • variants of the amino acid sequence shown in SEQ ID NO:48 may also be functional (i.e. retain their ability to confer specific binding to a WT 1 antigen (i.e.. the peptide shown in SEQ ID NO: 73) when the CDR3 is part of TCR Vp domain). Such functional variants are therefore encompassed herein.
  • appropriate (functional) Vp domain CDR3 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 48, i.e. they may have at least 80%, at least 84%, at least 92%, or 100% sequence identity to SEQ ID NO: 48.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 48).
  • appropriate (functional) Vp domain CDR3 amino acid sequences may vary from the sequence shown in SEQ ID NO: 48 by one or several (e.g. two) amino acids.
  • functional variants of SEQ ID NO: 48 retain their ability to confer specific binding to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 73) when the CDR3 is part of TCR Vp domain.
  • Functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 48.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 48, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the CDR3.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 48 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 73). Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 48 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • the CDR3 of the Vp domain comprises or consists of the amino acid sequence of SEQ ID NO: 48.
  • the CDR3 may be encoded by Any appropriate nucleic acid sequence.
  • the encoded TOR Vp domain may comprise, in addition to the specified CDR3, a CDR1 comprising an amino acid sequence of SEQ ID NO: 46, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 73)).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 46.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 46, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 46 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 73). Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 46 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • appropriate functional Vp domain CDR1 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 46, i.e. it may have at least 80%, or 100% sequence identity to SEQ ID NO: 46.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 46).
  • appropriate (functional) Vp domain CDR1 amino acid sequences may vary from the sequence shown in SEQ ID NO:46 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO:46).
  • functional variants of SEQ ID NO: 46 retain the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 73) when the CDR1 is part of TCR Vp domain).
  • the CDR1 of the Vp domain comprises or consists of the amino acid sequence of SEQ ID NO: 46.
  • the CDR1 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Vp domain may also comprise, in addition to the specified CDR3 (and optionally the specified CDR1 above), a CDR2 having an amino acid sequence of SEQ ID NO: 47, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to HLA-B*35:01).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 47.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 47, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 47 that do not specifically bind to HLA-B*35:01. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 47 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • appropriate functional Vp domain CDR2 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 47, i.e. it may have at least 80%, at least 83%, or 100% sequence identity to SEQ ID NO: 47.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 47).
  • appropriate (functional) Vp domain CDR2 amino acid sequences may vary from the sequence shown in SEQ ID NO: 47 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO: 47).
  • a functional variant of SEQ ID NO: 47 retains the ability to specifically bind to HLA-B*35:01.
  • the CDR2 of the Vp domain comprises or consists of the amino acid sequence of SEQ ID NO: 47.
  • the CDR2 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Vp domain may therefore comprise the CDRs mentioned in detail above (by SEQ ID specifically i.e. SEQ ID NO:48, SEQ ID NO: 46 and SEQ ID NO: 47, or functional variants thereof), with appropriate intervening sequences between the CDRs.
  • the encoded TCR Vp domain may have an amino acid sequence of SEQ ID NO: 51 , or a functional variant thereof (i.e. wherein the variant TCR Vp domain retains the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 73) when part of a binding protein described herein).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 51.
  • variant also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 51 , or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 51 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 73). Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:51 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • the encoded TCR Vp domain may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 51 , whilst retaining the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 73).
  • a functional TCR Vp domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 51 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO:51 may all be in regions of the TCR Vp domain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 48, SEQ ID NO: 46 and/or SEQ ID NO: 47, and still have 25% (or less) sequence variability compared to SEQ ID NO: 51).
  • the sequence of the CDRs of SEQ ID NO: 51 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 51).
  • the encoded TCR Vp domain may 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: 51 , wherein the TCR Vp domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 48.
  • the TCR Vp domain CDR1 may have an amino acid sequence of SEQ ID NO:46 and the TCR p domain CDR2 may have an amino acid sequence of SEQ ID NO: 47.
  • the TCR VP domain may be encoded by the nucleic acid sequence of SEQ ID NO:52, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
  • the nucleic acid sequence encoding the TCR p domain may also encode a TCR p chain constant domain. Examples of suitable constant domains are generally discussed above.
  • SEQ ID NO: 55 An example of a specific TCR p chain amino acid sequence that includes a TCR p domain described herein and an appropriate constant domain is shown in SEQ ID NO: 55.
  • Appropriate functional variants of SEQ ID NO: 55 are also encompassed (e.g. variants having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 55, wherein the variant TCR p chain amino acid sequence retains its ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 73) when part of a binding protein described herein).
  • a WT1 antigen e.g. the peptide shown in SEQ ID NO: 73
  • a functional TCR p chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 55 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO:55 may all be in regions of the TCR p chain that do not form CDRs (i.e. the variant may have the CDRs of SEQ I D NO: 48, SEQ I D NO: 46 and/or SEQ I D NO: 47, and still have 25% (or less) sequence variability compared to SEQ ID NO:55.
  • sequence of the CDRs of SEQ ID NO: 55 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 55).
  • the encoded TCR p chain may 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: 55, wherein the TCR p chain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 48.
  • the TCR p chain CDR1 may have an amino acid sequence of SEQ ID NO: 46 and the TCR p chain CDR2 may have an amino acid sequence of SEQ ID NO: 47.
  • the TCR chain may be encoded by the nucleic acid sequence of SEQ ID NO:56, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO:56 is the nucleic acid sequence for TCR p chain of clone 17.2G4.
  • the nucleic acid composition provided herein comprises a nucleic acid sequence that encodes a TCR Vp domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:48, or a functional fragment thereof.
  • the CDR3 of the Vp domain of a nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO:48.
  • the Vp domain of a nucleic acid composition provided herein comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 51.
  • TCR Vp domain sequences derived from TCR clone 17.2G4 discussed above are particularly compatible with the TCR Va domain sequences derived from TCR clone 17.2G4 discussed elsewhere herein.
  • a nucleic acid composition described herein encodes a WT1 antigen-specific binding protein having TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:45, or a functional fragment thereof; and a nucleic acid sequence that encodes a TCR p domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:48, or a functional fragment thereof.
  • a nucleic acid composition described herein encodes a WT1 antigenspecific binding protein having a TCR Va domain with a CDR3 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 45; and a TCR Vp domain with a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:48.
  • the WT1 antigen may comprise or consist of the sequence shown in SEQ ID NO: 73.
  • the TCR Va domain may be part of a TCR a chain having a constant domain and the TCR Vp domain may be part of a TCR p chain having a constant domain.
  • the Va domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 49; and the Vp domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 51.
  • the Va domain comprises the amino acid sequence of SEQ ID NO: 49 and the p domain comprises the amino acid sequence of SEQ ID NO: 51.
  • the Va domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 50; and the Vp domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 52.
  • the TCR Va domain may include a CDR1 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 43 and a CDR2 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO:44.
  • the TCR Vp domain may include a CDR1 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO:46 and a CDR2 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 47.
  • this particular example encompasses components of TCR clone 17.2G4 exemplified herein.
  • the different components of TCR clone 17.2G4 and their respective SEQ ID Nos are summarised in Table 8 below.
  • the nucleic acid composition described herein encodes both a TCR Va domain and a TCR Vp domain, which form the binding protein that is capable of specifically binding to a WT 1 antigen.
  • the TCR Va domain and the TCR Vp domain may be joined together via a linker. Suitable linkers are discussed generally elsewhere herein. Additional appropriate polypeptide domains that may also be encoded by the nucleic acid sequences that encode the TCR Va domain and/or the TCR Vp domain are also discussed generally elsewhere herein.
  • the nucleic acid composition described herein may encode a soluble TCR or a chimeric single chain TCR wherein the TCR alpha chain variable domain is linked to the TCR beta chain variable domain and a constant domain which is e.g. fused to the CD3 zeta signalling domain.
  • TCR components that interact with VLDFAPPGASAY SEQ ID NO: 74
  • TCR clone 12.5H9 which interacts with VLDFAPPGASAY (SEQ ID NO: 74) in the context of HLA-A*01 :01.
  • the sequences provided herein that correspond to TCR clone 12.5H9 are SEQ ID NO:s 57 to 70.
  • an isolated nucleic acid composition that encodes a Wilms’ tumor 1 (WT1) antigen-specific binding protein having a TCR a chain variable (Va) domain and a TCR p chain variable (VP) domain
  • the composition comprising: a nucleic acid sequence that encodes a TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:59, or a functional fragment thereof; and a nucleic acid sequence that encodes a TCR Vp domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 62, or a functional fragment thereof, wherein the CDR3 sequences together specifically bind to WT 1 .
  • TCR Va domain CDR3 amino acid sequence that confers specific binding to a WT1 antigen e.g. to VLDFAPPGASAY (SEQ ID NO: 74)
  • SEQ ID NO: 59 An example of an appropriate TCR Va domain CDR3 amino acid sequence that confers specific binding to a WT1 antigen (e.g. to VLDFAPPGASAY (SEQ ID NO: 74)) is shown in SEQ ID NO: 59.
  • variants of the amino acid sequence shown in SEQ ID NO: 59 may also be functional (i.e. retain their ability to confer specific binding to a WT 1 antigen (e.g. to the peptide VLDFAPPGASAY (SEQ ID NO: 74)) when the CDR3 is part of TCR Va domain).
  • WT 1 antigen e.g. to the peptide VLDFAPPGASAY (SEQ ID NO: 74)
  • appropriate (functional) Va domain CDR3 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 59, i.e. they may have at least 80%, at least 81 %, at least 90%, or 100% sequence identity to SEQ ID NO: 59.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 59).
  • appropriate (functional) Va domain CDR3 amino acid sequences may vary from the sequence shown in SEQ ID NO: 59 by one or several (e.g. two etc) amino acids.
  • SEQ I D NO: 59 retain their ability to confer specific binding to a WT1 antigen (i.e. the peptide shown in SEQ ID NO: 74) when the CDR3 is part of TCR Va domain.
  • Functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 59.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one, two or more amino acids of SEQ ID NO: 59, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the CDR3.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 59 that do not specifically bind to a WT1 antigen (i.e. the peptide shown in SEQ ID NO: 74). Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 59 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • the CDR3 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 59.
  • the CDR3 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Va domain may comprise, in addition to the specified CDR3, a CDR1 comprising an amino acid sequence of SEQ ID NO: 57, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 74)).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 57.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 57, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 57 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 74). Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 57 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • appropriate functional Va domain CDR1 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 57, i.e. it may have at least 80%, at least 85%, or 100% sequence identity to SEQ ID NO: 57.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 57).
  • appropriate functional Va domain CDR1 amino acid sequences may vary from the sequence shown in SEQ ID NO: 57 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO: 57.
  • functional variants of SEQ ID NO: 57 retain the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 74) when the CDR1 is part of TCR Va domain).
  • the CDR1 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 57.
  • the CDR1 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Va domain may also comprise, in addition to the specified CDR3 (and optionally the specified CDR1 above), a CDR2 comprising an amino acid sequence of SEQ ID NO: 58, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to HLA-A*01 :01).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 58.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 58, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 58 that do not specifically bind to HLA-A*01 :01. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 58 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • appropriate functional Va domain CDR2 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 58, i.e. it may have at least 80%, at least 85%, or 100% sequence identity to SEQ ID NO: 58.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 58).
  • appropriate (functional) Va domain CDR2 amino acid sequences may vary from the sequence shown in SEQ ID NO: 58 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO: 58).
  • the CDR2 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 58.
  • the CDR2 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TOR Va domain may therefore comprise the CDRs mentioned in detail above (by SEQ ID specifically i.e. SEQ ID NO:59, SEQ ID NO: 57 and SEQ ID NO: 58, or functional variants thereof), with appropriate intervening sequences between the CDRs.
  • the encoded TCR Va domain may comprise an amino acid sequence of SEQ ID NO: 63, or a functional variant thereof (i.e. wherein the variant TCR Va domain retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 74) when part of a binding protein described herein).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 63.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 63, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 63 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 74). Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 63 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • the encoded TCR Va domain may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 63, whilst retaining the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 74).
  • a functional TCR Va domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 63 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO: 63 may all be in regions of the TCR Va domain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 59, SEQ ID NO: 57 and/or SEQ ID NO: 58, and still have 25% (or less) sequence variability compared to SEQ ID NO:7).
  • the sequence of the CDRs of SEQ ID NO: 63 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 63).
  • the encoded TOR Va domain may 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: 63, wherein the TCR Va domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 59.
  • the TCR Va domain CDR1 may have an amino acid sequence of SEQ ID NO: 57 and the TCR Va domain CDR2 may have an amino acid sequence of SEQ ID NO: 58.
  • the encoded TCR Va domain may comprise an amino acid sequence having at the amino acid sequence of SEQ ID NO: 63, with 0 to 10 (or 0 to 5) amino acid substitutions, insertions or deletions), wherein the TCR Va domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 59.
  • the TCR Va domain CDR1 may have an amino acid sequence of SEQ ID NO: 57 and the TCR Va domain CDR2 may have an amino acid sequence of SEQ ID NO: 58.
  • the TCR Va domain may be encoded by the nucleic acid sequence of SEQ ID NO: 64, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
  • the nucleic acid sequence encoding the TCR Va domain may also encode a TCR a chain constant domain. Examples of suitable constant domains are generally discussed above.
  • SEQ ID NO: 67 An example of a specific TCR a chain amino acid sequence that includes a TCR Va domain described herein with an appropriate constant domain is shown in SEQ ID NO: 67.
  • Appropriate functional variants of SEQ ID NO:67 are also encompassed (e.g. variants having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 67, wherein the variant TCR a chain amino acid sequence retains its ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 74) when part of a binding protein described herein).
  • a WT1 antigen e.g. the peptide shown in SEQ ID NO: 74
  • a functional TCR a chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO:67 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO:67 may all be in regions of the TCR a chain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 59, SEQ ID NO: 57 and/or SEQ ID NO: 58, and still have 25% (or less) sequence variability compared to SEQ ID NO: 67).
  • sequence of the CDRs of SEQ ID NO: 67 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 67).
  • the encoded TCR a chain may 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, wherein the TCR a chain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 59.
  • the TCR a chain CDR1 may have an amino acid sequence of SEQ ID NO:57 and the TCR a chain CDR2 may have an amino acid sequence of SEQ ID NO: 58.
  • the TCR a chain may be encoded by the nucleic acid sequence of SEQ ID NO: 68, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NO:68 is the nucleic acid sequence for TCR a chain of clone 12.5H9.
  • the nucleic acid composition provided herein comprises a nucleic acid sequence that encodes a TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:59, or a functional fragment thereof.
  • the CDR3 of the Va domain of a nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO: 59.
  • the Va domain of a nucleic acid composition provided herein comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 63.
  • the inventors identified TCR clone 12.5H9 which interacts with VLDFAPPGASAY (SEQ ID NO: 74) in the context of HLA-A*01 :01.
  • the sequences provided herein that correspond to TCR clone 12.5H9 are SEQ ID NO:s 57 to 70.
  • TCR p domain CDR3 amino acid sequence that confers specific binding to a WT 1 antigen e.g. to VLDFAPPGASAY (SEQ ID NO: 74)
  • SEQ ID NO:62 An example of an appropriate TCR p domain CDR3 amino acid sequence that confers specific binding to a WT 1 antigen (e.g. to VLDFAPPGASAY (SEQ ID NO: 74)) is shown in SEQ ID NO:62.
  • variants of the amino acid sequence shown in SEQ ID NO:62 may also be functional (i.e. retain their ability to confer specific binding to a WT1 antigen (i.e. the peptide shown in SEQ ID NO: 74) when the CDR3 is part of TCR p domain). Such functional variants are therefore encompassed herein.
  • appropriate (functional) p domain CDR3 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 62, i.e. they may have at least 80%, at least 87%, or 100% sequence identity to SEQ ID NO: 62.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 62).
  • appropriate (functional) p domain CDR3 amino acid sequences may vary from the sequence shown in SEQ ID NO: 62 by one or several (e.g. two) amino acids.
  • functional variants of SEQ ID NO: 62 retain their ability to confer specific binding to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 74) when the CDR3 is part of TCR p domain.
  • Functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 62.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 62, or substitution, deletion or insertion of non-critical amino acids in non- critical regions of the CDR3.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 62 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 74). Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 62 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • the CDR3 of the Vp domain comprises or consists of the amino acid sequence of SEQ ID NO: 62.
  • the CDR3 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TOR Vp domain may comprise, in addition to the specified CDR3, a CDR1 comprising an amino acid sequence of SEQ ID NO: 60, or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to a WT 1 antigen (e.g. the peptide shown in SEQ ID NO: 74)).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 60.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 60, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 60 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 74). Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 60 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • appropriate functional Vp domain CDR1 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 60, i.e. it may have at least 80%, or 100% sequence identity to SEQ ID NO: 60.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 60).
  • appropriate (functional) Vp domain CDR1 amino acid sequences may vary from the sequence shown in SEQ ID NQ:60 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NQ:60).
  • functional variants of SEQ ID NO: 60 retain the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 74) when the CDR1 is part of TCR Vp domain).
  • the CDR1 of the Vp domain comprises or consists of the amino acid sequence of SEQ ID NO: 60.
  • the CDR1 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Vp domain may also comprise, in addition to the specified CDR3 (and optionally the specified CDR1 above), a CDR2 having an amino acid sequence of SEQ ID NO: 61 , or a functional variant thereof (i.e. wherein the variant retains the ability to specifically bind to HLA-A*01 :01).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 61.
  • variant also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 61 , or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 61 that do not specifically bind to HLA-A*01 :01. Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 61 or a substitution, insertion or deletion in critical amino acids or critical regions. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.
  • appropriate functional Vp domain CDR2 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 61 , i.e. it may have at least 80%, at least 83, or 100% sequence identity to SEQ ID NO: 61.
  • percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 61).
  • appropriate (functional) Vp domain CDR2 amino acid sequences may vary from the sequence shown in SEQ ID NO: 61 by one or several amino acids.
  • the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO: 61).
  • a functional variant of SEQ ID NO: 61 retains the ability to specifically bind to HLA-A*01 :01 .
  • the CDR2 of the Vp domain comprises or consists of the amino acid sequence of SEQ ID NO: 61.
  • the CDR2 may be encoded by any appropriate nucleic acid sequence.
  • the encoded TCR Vp domain may therefore comprise the CDRs mentioned in detail above (by SEQ ID specifically i.e. SEQ ID NO:62, SEQ ID NO: 60 and SEQ ID NO: 61 , or functional variants thereof), with appropriate intervening sequences between the CDRs.
  • the encoded TCR Vp domain may have an amino acid sequence of SEQ ID NO: 65, or a functional variant thereof (i.e. wherein the variant TCR Vp domain retains the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 74) when part of a binding protein described herein).
  • Such functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 65.
  • the term “variant” also encompasses homologues and fragments. Functional variants will typically contain only conservative substitutions of one or more amino acids of SEQ ID NO: 65, or substitution, deletion or insertion of non-critical amino acids in non-critical regions of the protein.
  • Non-functional variants are amino acid sequence variants of SEQ ID NO: 65 that do not specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 74).
  • Non-functional variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:65 or a substitution, insertion or deletion in critical amino acids or critical regions.
  • the encoded TCR Vp domain may have an amino acid sequence having at least 75%, at least 80%, at least 85% or at least 90% (or at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 65, whilst retaining the ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 74).
  • a functional TCR Vp domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 65 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO:65 may all be in regions of the TCR Vp domain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 62, SEQ ID NO: 60 and/or SEQ ID NO: 61 , and still have 25% (or less) sequence variability compared to SEQ ID NO: 65).
  • the sequence of the CDRs of SEQ ID NO: 65 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 65).
  • the encoded TCR Vp domain may 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: 65, wherein the TCR Vp domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 62.
  • the TCR Vp domain CDR1 may have an amino acid sequence of SEQ ID NO:60 and the TCR p domain CDR2 may have an amino acid sequence of SEQ ID NO: 61.
  • the TCR VP domain may be encoded by the nucleic acid sequence of SEQ ID NO: 66, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code).
  • the nucleic acid sequence encoding the TCR p domain may also encode a TCR p chain constant domain. Examples of suitable constant domains are generally discussed above.
  • SEQ ID NO: 69 An example of a specific TCR p chain amino acid sequence that includes a TCR p domain described herein and an appropriate constant domain is shown in SEQ ID NO: 69.
  • Appropriate functional variants of SEQ ID NO: 69 are also encompassed (e.g. variants having at least 75% (e.g. at least 75%, at least 80%, at least 85%, at least 90%, at least 95% etc) sequence identity to the amino acid sequence of SEQ ID NO: 69, wherein the variant TCR p chain amino acid sequence retains its ability to specifically bind to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 74) when part of a binding protein described herein).
  • a WT1 antigen e.g. the peptide shown in SEQ ID NO: 74
  • a functional TCR p chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 69 is also encompassed.
  • the amino acid substitution may be a conservative amino acid substitution.
  • the variability in sequence compared to SEQ ID NO:69 may all be in regions of the TCR p chain that do not form CDRs (i.e. the variant may have the CDRs of SEQ ID NO: 62, SEQ ID NO: 60 and/or SEQ ID NO: 61 , and still have 25% (or less) sequence variability compared to SEQ ID NO:69.
  • sequence of the CDRs of SEQ ID NO: 69 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above.
  • percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 69).
  • the encoded TCR p chain may 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: 69, wherein the TCR p chain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 62.
  • the TCR p chain CDR1 may have an amino acid sequence of SEQ ID NO: 60 and the TCR p chain CDR2 may have an amino acid sequence of SEQ ID NO: 61.
  • the TCR chain may be encoded by the nucleic acid sequence of SEQ ID NO:70, or a genetically degenerate sequence thereof (i.e. other nucleic acid sequences that encode the same protein as a result of the degeneracy of the genetic code). It is noted that SEQ ID NQ:70 is the nucleic acid sequence for TCR p chain of clone 12.5H9.
  • the nucleic acid composition provided herein comprises a nucleic acid sequence that encodes a TCR Vp domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:62, or a functional fragment thereof.
  • the CDR3 of the Vp domain of a nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO:62.
  • the Vp domain of a nucleic acid composition provided herein comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 65.
  • TCR Vp domain sequences derived from TCR clone 12.5H9 discussed above are particularly compatible with the TCR Va domain sequences derived from TCR clone 12.5H9 discussed elsewhere herein.
  • a nucleic acid composition described herein encodes a WT1 antigen-specific binding protein having TCR Va domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:59, or a functional fragment thereof; and a nucleic acid sequence that encodes a TCR p domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:62, or a functional fragment thereof.
  • a nucleic acid composition described herein encodes a WT1 antigenspecific binding protein having a TCR Va domain with a CDR3 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 59; and a TCR Vp domain with a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:62.
  • the WT1 antigen may comprise or consist of the sequence shown in SEQ ID NO: 74.
  • the TCR Va domain may be part of a TCR a chain having a constant domain and the TCR Vp domain may be part of a TCR p chain having a constant domain.
  • the Va domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 63; and the Vp domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 65.
  • the Va domain comprises the amino acid sequence of SEQ ID NO: 63 and the p domain comprises the amino acid sequence of SEQ ID NO: 65.
  • the Va domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 64; and the Vp domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 66.
  • the TCR Va domain may include a CDR1 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO:57 and a CDR2 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO:58.
  • the TCR Vp domain may include a CDR1 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NQ:60 and a CDR2 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 61.
  • this particular example encompasses components of TCR clone 12.5H9 exemplified herein.
  • the different components of TCR clone 12.5H9 and their respective SEQ ID Nos are summarised in Table 9 below.
  • the nucleic acid composition described herein encodes both a TCR Va domain and a TCR Vp domain, which form the binding protein that is capable of specifically binding to a WT 1 antigen.
  • the TCR Va domain and the TCR Vp domain may be joined together via a linker. Suitable linkers are discussed generally elsewhere herein. Additional appropriate polypeptide domains that may also be encoded by the nucleic acid sequences that encode the TCR Va domain and/or the TCR Vp domain are also discussed generally elsewhere herein.
  • the nucleic acid composition described herein may encode a soluble TCR or a chimeric single chain TCR wherein the TCR alpha chain variable domain is linked to the TCR beta chain variable domain and a constant domain which is e.g. fused to the CD3 zeta signalling domain.
  • a vector system which includes a nucleic acid composition described herein.
  • the vector system may have one or more vectors.
  • the binding protein components that are encoded by the nucleic acid composition may be encoded by one or more nucleic acid sequences in the nucleic acid composition.
  • the nucleic acid sequence may be present within a single vector (and thus the vector system described herein may comprise of one vector only).
  • the binding protein components are encoded by two or more nucleic acid sequences (wherein the plurality of nucleic acid sequences, together, encode all of the components of the binding protein) these two or more nucleic acid sequences may be present within one vector (e.g. in different open reading frames of the vector), or may be distributed over two or more vectors.
  • the vector system will comprise a plurality of distinct vectors (i.e. vectors with different nucleotide sequences).
  • a vector system comprising a nucleic acid composition described herein.
  • the vector may be a plasmid, a cosmid, or a viral vector, such as a retroviral vector or a lentiviral vector.
  • Adenovirus, adeno- associated virus, vaccinia virus, canary poxvirus, herpes virus, minicircle vectors and naked (synthetic) DNA/RNA may also be used (for details on minicircle vectors, see for example non- viral Sleeping Beauty transposition from minicircle vectors as published by R Monjezi et al., Leukemia 2017).
  • single stranded or double stranded DNA or RNA can be used to transfect lymphocytes with a TCR of interest (see Roth et a/ 2018 Nature vol 559; page 405).
  • the vector is a plasmid, a viral vector, or a cosmid, optionally wherein the vector is selected from the group consisting of a retrovirus, lentivirus, adeno-associated virus, adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or RNA.
  • the term “vector” refers to a nucleic acid sequence capable of transporting another nucleic acid sequence to which it has been operably linked.
  • the vector can be capable of autonomous replication or it can integrate into a host DNA.
  • the vector may include restriction enzyme sites for insertion of recombinant DNA and may include one or more selectable markers or suicide genes.
  • the vector can be a nucleic acid sequence in the form of a plasmid, a bacteriophage or a cosmid.
  • the vector is suitable for expression in a cell (i.e. the vector is an “expression vector”).
  • the vector is suitable for expression in a human T cell such as a CD8 + T cell or CD4 + T cell, or stem cell, iPS cell, or NK cell.
  • the vector is a viral vector, such as a retroviral vector, a lentiviral vector or an adeno-associated vector.
  • the vector is selected from the group consisting of an adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or synthetic RNA.
  • the (expression) vector is capable of propagation in a host cell and is stably transmitted to future generations.
  • the vector may comprise regulatory sequences.
  • Regulatory sequences refers to, DNA or RNA elements that are capable of controlling gene expression. Examples of expression control sequences include promoters, enhancers, silencers, TATA- boxes, internal ribosomal entry sites (IRES), attachment sites fortranscription factors, transcriptional terminators, polyadenylation sites etc.
  • the vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. Regulatory sequences include those which direct constitutive expression, as well as tissue-specific regulatory and/or inducible sequences.
  • the vector comprises the nucleic acid sequence of interest operably linked to a promoter.
  • Promoter refers to the nucleotide sequences in DNA to which RNA polymerase binds to start transcription.
  • the promoter may be inducible or constitutively expressed. Alternatively, the promoter is under the control of a repressor or stimulatory protein.
  • the promoter may be one that is not naturally found in the host cell (e.g. it may be an exogenous promoter). The skilled person in the art is well aware of appropriate promoters for use in the expression of target proteins, wherein the selected promoter will depend on the host cell.
  • “Operably linked” refers to a single or a combination of the below-described control elements together with a coding sequence in a functional relationship with one another, for example, in a linked relationship so as to direct expression of the coding sequence.
  • the vector may comprise a transcriptional terminator.
  • T ranscriptional terminator refers to a DNA element, which terminates the function of RNA polymerases responsible for transcribing DNA into RNA. Preferred transcriptional terminators are characterized by a run of T residues preceded by a GC rich dyad symmetrical region.
  • the vector may comprise a translational control element.
  • Translational control element refers to DNA or RNA elements that control the translation of mRNA.
  • Preferred translational control elements are ribosome binding sites.
  • the translational control element is from a homologous system as the promoter, for example a promoter and its associated ribozyme binding site. Preferred ribosome binding sites are known, and will depend on the chosen host cell.
  • the vector may comprise restriction enzyme recognition sites.
  • Restriction enzyme recognition site refers to a motif on the DNA recognized by a restriction enzyme.
  • the vector may comprise a selectable marker.
  • Selectable marker refers to proteins that, when expressed in a host cell, confer a phenotype onto the cell which allows a selection of the cell expressing said selectable marker gene. Generally this may be a protein that confers a new beneficial property onto the host cell (e.g. antibiotic resistance) or a protein that is expressed on the cell surface and thus accessible for antibody binding. Appropriate selectable markers are well known in the art.
  • the vector may also comprise a suicide gene.
  • suicide gene encodes a protein that induce death of the modified cell upon treatment with specific drugs.
  • suicide can be induced in cells modified by the herpes simplex virus thymidine kinase gene upon treatment with specific nucleoside analogs including ganciclovir, cells modified by human CD20 upon treatment with anti-CD20 monoclonal antibody and cells modified with inducible Caspase9 (iCasp9) upon treatment with AP1903 (reviewed by BS Jones, LS Lamb, F Goldman, A Di Stasi; Improving the safety of cell therapy products by suicide gene transfer. Front Pharmacol. (2014) 5:254). Appropriate suicide genes are well known in the art.
  • the vector comprises those genetic elements which are necessary for expression of the binding proteins described herein by a host cell.
  • the elements required for transcription and translation in the host cell include a promoter, a coding region for the protein(s) of interest, and a transcriptional terminator.
  • the (expression) vector system described herein can be introduced into cells by conventional techniques such as transformation, transfection or transduction.
  • Transformation refer generally to techniques for introducing foreign (exogenous) nucleic acid sequences into a host cell, and therefore encompass methods such as electroporation, microinjection, gene gun delivery, transduction with retroviral, lentiviral or adeno- associated vectors, lipofection, superfection etc.
  • the specific method used typically depends on both the type of vector and the cell.
  • the host cell is contacted with the vector system (e.g. viral vector) in vitro, ex vivo, and in some examples, the host cell is contacted with the vector system (e.g. viral vector) in vivo.
  • the vector system e.g. viral vector
  • the term "host cell” includes any cell into which the nucleic acid composition or vector system described herein may be introduced. Once a nucleic acid molecule or vector system has been introduced into the cell, it may be referred to as a “modified cell” herein. Once the nucleic acid molecule or vector is introduced into the host cell, the resultant modified cell should be capable of expressing the encoded binding protein (and e.g. correctly localising the encoded binding protein for its intended function e.g. transporting the encoded binding protein to the cell surface).
  • the nucleic acid composition or vector system may be introduced into the cell using any conventional method known in the art.
  • the nucleic acid composition or vector system may be introduced using CRISPR technology. Insertion of the nucleic acid sequences at the endogenous TCR locus by engineering with CRISPR/Cas9 and homologous directed repair (HDR) or non-homologous end joining (NHEJ) is therefore encompassed. Other conventional methods such as transfection, transduction or transformation of the cell may also be used.
  • modified cell refers to a genetically altered (e.g. recombinant) cell.
  • the modified cell includes at least one exogenous nucleic acid sequence (i.e. a nucleic acid sequence that is not naturally found in the host cell).
  • the exogenous sequence comprises at least one of the T cell receptor component parts described herein for any of clones 22.1 H1 , 20.3D10, 23.2G9, 17.2G4 or 12.5H9 (e.g. the sequences etc that encode the CDR3 sequences that are specific for a WT 1 antigen (e.g. the peptide of SEQ ID NO: 71 , 72, 73 or 74)).
  • modified cell refers to the particular subject cell and also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a modified cell comprises a nucleic acid composition or a vector system provided herein.
  • the host cell (and thus the modified cell) is typically a eukaryotic cell, and particularly a human cell (e.g. a T cell such as a CD8 + T cell or a CD4 + T cell, or a mixture thereof, or a hematopoietic stem cell, an iPSC, or gamma-delta T cell, or a pluripotent stem cell, or a NK-T cell or NK cell).
  • the host cell (and thus the modified cell) may be an autologous or allogeneic cell (e.g.
  • a CD8 + T cell or a CD4 + T cell such as a CD8 + T cell or a CD4 + T cell, or a mixture thereof, or a hematopoietic stem cell, an iPSC, or gamma-delta T cell, or a pluripotent stem cell, or a NK-T cell or NK cell).
  • Allogeneic cell refers to a cell derived from a different individual to the individual to which it is later administered. In other words, the host cell (and thus the modified cell) may be an isolated cell from a distinct individual compared to the subject to be treated.
  • Autologous cell refers to a cell derived from the individual to which it is also later administered. In other words, the host cell (and thus the modified cell) may be an isolated cell from the subject that is to be treated.
  • the modified cell is a human cell.
  • the host cell may be any cell that is able to confer anti-tumour immunity after TCR gene transfer.
  • suitable cells include autologous or allogeneic CD8 T cells, CD4 T cells, Natural Killer (NK) cells, NKT cells, gamma-delta T cells, inducible pluripotent stem cells (iPSCs), hematopoietic stem cells or other progenitor cells and any other autologous or allogeneic cell or cell line (NK-92 for example or T cell lines) that is able to confer anti-tumor immunity after TCR gene transfer.
  • NK Natural Killer
  • iPSCs inducible pluripotent stem cells
  • hematopoietic stem cells or other progenitor cells and any other autologous or allogeneic cell or cell line (NK-92 for example or T cell lines) that is able to confer anti-tumor immunity after TCR gene transfer.
  • the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, an NK-T cell, a gamma-delta T cell, a hematopoietic stem cell, an inducible pluripotent stem cell, a progenitor cell, a T cell line and a NK-92 cell line.
  • the host cell and thus the modified cell
  • the host cell is typically for administration to a HLA-A*01 :01 , HLA-A*02:01 , and/or HLA-B*35:01 positive human subject.
  • the host cell (and thus the modified cell) is typically HLA-A*01 :01 , HLA-A*02:01 , and/or HLA-B*35:01 positive but needs to be WT1 antigen negative (i.e. modified cells can either be HLA-A*01 :01 , HLA-A*02:01 , and/or HLA-B*35:01 positive or negative).
  • the host cell (and thus the modified cell) that is to be administered to the subject can either be autologous or allogeneic.
  • the modified cell is capable of expressing the binding protein encoded by the nucleic acid composition or vector system described herein (i.e. the TCR component parts) such that the modified cell provides an immunotherapy that specifically targets cells that express a WT 1 antigen, and thus can be used to treat or prevent WT 1 associated diseases or conditions in a corresponding HLA-A*01:01, HLA-A*02:01 , and/or HLA-B*35:01 positive human subject. More details on this use are given below.
  • a nucleic acid composition, vector system, modified cell, or isolated nucleic acid sequence described herein may be provided as part of a pharmaceutical composition.
  • Such compositions may be administered to a human subject in need thereof (as described elsewhere herein).
  • a particularly suitable composition may be selected based on the HLA serotype of the human subject, as described in detail elsewhere herein.
  • a pharmaceutical composition may comprise a nucleic acid composition, vector system, modified cell, or isolated nucleic acid sequence described herein along with a pharmaceutically acceptable excipient, adjuvant, diluent and/or carrier.
  • compositions may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents or compounds.
  • pharmaceutically acceptable refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected nucleic acid composition, vector system, modified cell, or isolated nucleic acid sequence without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • Excipients are natural or synthetic substances formulated alongside an active ingredient (e.g. a nucleic acid sequence, a nucleic acid composition, vector or vector system, modified cell, or isolated nucleic acid as provided herein), included for the purpose of bulking-up the formulation or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as facilitating drug absorption or solubility.
  • Excipients can also be useful in the manufacturing process, to aid in the handling of the active substance concerned such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation over the expected shelf life.
  • Pharmaceutically acceptable excipients are well known in the art. A suitable excipient is therefore easily identifiable by one of ordinary skill in the art.
  • suitable pharmaceutically acceptable excipients include water, saline, aqueous dextrose, glycerol, ethanol, and the like.
  • Adjuvants are pharmacological and/or immunological agents that modify the effect of other agents in a formulation.
  • Pharmaceutically acceptable adjuvants are well known in the art. A suitable adjuvant is therefore easily identifiable by one of ordinary skill in the art.
  • Diluents are diluting agents. Pharmaceutically acceptable diluents are well known in the art. A suitable diluent is therefore easily identifiable by one of ordinary skill in the art.
  • Carriers are non-toxic to recipients at the dosages and concentrations employed and are compatible with other ingredients of the formulation.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • Pharmaceutically acceptable carriers are well known in the art. A suitable carrier is therefore easily identifiable by one of ordinary skill in the art.
  • compositions described herein may advantageously be administered to a HLA- A*01 :01, HLA-A*02:01 , and/or HLA-B*35:01 positive human subject in need thereof (where certain compositions are more suitable for certain human subjects, based on their HLA status, as described in more detail elsewhere herein).
  • the subject in need of treatment has a disease or condition that is associated with an elevated level of H LA-restricted WT1 antigens (i.e. WT1 antigens that are presented at the cell surface in the context of an HLA).
  • the disease or condition is typically a WT1 associated disease or condition.
  • the WT 1 associated disease or condition may be a hyperproliferative disease or condition.
  • the WT1 associated disease or condition may be a hematological malignancy.
  • it may be a hematological malignancy with an elevated level of H LA-restricted WT 1 antigens (i.e. WT 1 antigens that are presented at the cell surface in the context of an HLA).
  • hematological malignancies include, for example acute myeloid leukemia (AML), multiple myeloma, plasma cell leukemia, Acute lymphoblastoid leukemia (ALL) and B cell lymphoma, optionally wherein the B cell lymphoma is selected from the group consisting of: Diffuse large B cell lymphoma (DLBCL), High grade B cell lymphoma, Mantel cell lymphoma (MCL), Follicular lymphoma (FL), Hairy cell leukemia (HCL), and Burkitt Lymphoma.
  • AML acute myeloid leukemia
  • ALL Acute lymphoblastoid leukemia
  • B cell lymphoma optionally wherein the B cell lymphoma is selected from the group consisting of: Diffuse large B cell lymphoma (DLBCL), High grade B cell lymphoma, Mantel cell lymphoma (MCL), Follicular lymphoma (FL), Hairy cell leukemia (HCL), and Burkit
  • the WT1 associated disease or condition is AML.
  • the WT1 associated disease or condition may be a solid tumor.
  • it may be a solid tumor with an elevated level of H LA-restricted WT1 antigens (i.e. WT1 antigens that are presented at the cell surface in the context of an HLA).
  • WT1 antigens that are presented at the cell surface in the context of an HLA.
  • appropriate solid tumours include, for example ovarian carcinoma, mesothelioma, uterine carcinoma, testicular tumors, pancreatic carcinoma, lung carcinoma, kidney carcinoma, thymoma, sarcoma, prostate carcinoma, colorectal carcinoma, breast carcinoma, cervical carcinoma, stomach carcinoma, melanoma, bladder carcinoma, and kidney carcinoma.
  • the WT1 associated disease or condition may be a hyperproliferative disease or condition.
  • the WT1 associated disease or condition may be a H LA-restricted WT1 antigen expressing tumor or cancer.
  • the WT1 associated disease or condition may be a WT 1 positive tumor or cancer.
  • the pharmaceutical composition provided herein is for use in inducing or enhancing an immune response in human subject diagnosed with a WT1 associated disease or condition.
  • an appropriate therapy for subject in need thereof may be selected based on the HLA serotype of the subject.
  • an appropriate therapy e.g. an appropriate pharmaceutical composition described herein
  • an appropriate therapy may comprise (i) components of TCR clone 22.1 H1 exemplified herein; (ii) components of TCR clone 20.3D10 exemplified herein; or (iii) components of TCR clone 23.2G9 exemplified herein.
  • TCRs comprising components of (i) TCR clone 22.1 H1 exemplified herein; (ii) TCR clone 20.3D10 exemplified herein; or (iii) TCR clone 23.2G9 exemplified herein are particularly suitable for administration, or treating, stimulating, providing appropriate immunity (e.g. anti-tumor immunity etc) in HLA-A*02:01 positive human subjects.
  • appropriate immunity e.g. anti-tumor immunity etc
  • an appropriate therapy e.g. an appropriate pharmaceutical composition described herein
  • TCRs comprising components of TCR clone 17.2G4 exemplified herein are particularly suitable for administration, or treating, stimulating, providing appropriate immunity (e.g. anti-tumor immunity etc) in HLA-B*35:01 positive human subjects.
  • an appropriate therapy e.g. an appropriate pharmaceutical composition described herein
  • TCRs comprising components of TCR clone 12.5H9 exemplified herein are particularly suitable for administration, or treating, stimulating, providing appropriate immunity (e.g. anti-tumor immunity etc) in HLA-A*01 :01 positive human subjects.
  • induced or enhanced immune response refers to an increase in the immune response (e.g. a cell mediated immune response such as a T cell mediated immune response) of the subject during or after treatment compared to their immune response prior to treatment.
  • An “induced or enhanced” immune response therefore encompasses any measurable increase in the immune response that is directly or indirectly targeted to the disease or condition being treated (or prevented).
  • the pharmaceutical composition may be for use in stimulating a cell mediated immune response to a target cell population or tissue in a human subject.
  • the pharmaceutical composition may be for use in stimulating a cell mediated immune response to a target cell population or tissue in a human subject.
  • the target cell population or tissue may be a H LA-restricted WT1 antigen expressing target cell population or tissue.
  • it is a H LA-restricted WT1 antigen expressing target cell population or tissue.
  • it may be a target cell population or tissue comprising a H LA-restricted WT 1 antigen expressing tumor or cancer.
  • the pharmaceutical composition may also be for use in providing anti-tumor immunity to a human subject.
  • the pharmaceutical composition may be for use in treating a human subject having a disease or condition associated with an elevated level of H LA-restricted WT1 antigen.
  • WT 1 associated diseases or conditions that may be treated in accordance with the invention. Appropriate examples of such diseases or conditions are discussed elsewhere herein.
  • the WT1 associated diseases or conditions may comprise at least one tumor (particularly, at least one H LA-restricted WT1 antigen expressing tumor).
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted condition, disorder or symptom.
  • Treatment therefore encompasses a reduction, slowing or inhibition of the amount or concentration of target cells, for example as measured in a sample obtained from the subject, of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% when compared to the amount or concentration of target cells before treatment.
  • Methods of measuring the amount or concentration of target cells include, for example, qRT-PCR, and quantification of disease specific biomarkers in a sample obtained from the subject.
  • the term “subject” refers to an individual, e.g., a human, having or at risk of having a specified condition, disorder or symptom.
  • the subject may be a patient i.e. a subject in need of treatment in accordance with the invention.
  • the subject may have received treatment for the condition, disorder or symptom. Alternatively, the subject has not been treated prior to treatment in accordance with the present invention.
  • compositions described herein can be administered to the subject by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be by infusion or by intramuscular, intravascular, intracavity, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, percutaneous administration.
  • compositions described herein may be in any form suitable for the above modes of administration.
  • compositions comprising modified cells may in any form suitable for infusion.
  • suitable forms for parenteral injection include a sterile solution, suspension or emulsion.
  • the route of administration may be by direct injection into the target area, or by regional delivery or by local delivery. The identification of suitable dosages of the compositions of the invention is well within the routine capabilities of a person of skill in the art.
  • compositions described herein may be formulated for use in T cell receptor (TCR) gene transfer, an approach that is rapid, reliable and capable of generating large quantities of T cells with specificity for the WT1 antigenic peptides (e.g. the peptides shown in any one of SEQ ID NO:71 to 74), regardless of the patient’s pre-existing immune repertoire.
  • TCR T cell receptor
  • modified cells suitable for infusion may be generated within a few days.
  • compositions described herein are for administration in an effective amount.
  • An “effective amount” is an amount that alone, or together with further doses, produces the desired (therapeutic or non-therapeutic) response.
  • the effective amount to be used will depend, for example, upon the therapeutic (or non-therapeutic) objectives, the route of administration, and the condition of the patient/subject.
  • the suitable dosage of the composition of the invention for a given patient/subject will be determined by the attending physician (or person administering the composition), taking into consideration various factors known to modify the action of the composition of the invention for example severity and type of haematological malignancy, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors.
  • the dosages and schedules may be varied according to the particular condition, disorder or symptom the overall condition of the patient/subject. Effective dosages may be determined by either in vitro or in vivo methods.
  • compositions described herein are advantageously presented in unit dosage form.
  • binding proteins e.g. TCRs
  • a method of generating a binding protein that is capable of specifically binding to a peptide containing a WT1 antigen and does not bind to a peptide that does not contain the WT1 antigen comprising contacting a nucleic acid composition (or vector system) described herein with a cell under conditions in which the nucleic acid composition is incorporated and expressed by the cell.
  • the WT 1 antigen comprises or consists of a seguence comprising an amino acid seguence selected from the group consisting of: SEQ ID NO:71 to 74, or a functional fragment or variant thereof.
  • the method may be carried out on the (host) cell ex vivo or in vitro.
  • the method may be performed in vivo, wherein the nucleic acid composition (or vector system) is administered to the subject and is contacted with the cell in vivo, under conditions in which the nucleic acid seguence is incorporated and expressed by the cell to generate the binding protein.
  • the method is not a method of treatment of the human or animal body.
  • nucleic acid seguence or vector systems
  • the binding protein comprise a TCR, an antigen binding fragment of a TCR, a ImmTAC or a chimeric antigen receptor (CAR). Further details are provided elsewhere herein.
  • the binding proteins described herein may be used therapeutically, as described elsewhere herein.
  • the binding proteins may be used in a diagnostic setting, e.g. to detect the presence of WT1 presented in the context of an appropriate HLA at the cell surface of diseased/malignant tissues.
  • nucleic acid sequence As used herein “nucleic acid sequence”, “polynucleotide”, “nucleic acid” and “nucleic acid molecule” are used interchangeably to refer to an oligonucleotide sequence or polynucleotide sequence.
  • the nucleotide sequence may be of genomic, synthetic or recombinant origin, and may 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 analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs.
  • the nucleotide sequence lacks introns. In other words, it is an intronless nucleic acid sequence.
  • the nucleotide sequence may be a DNA sequence that does not comprise intron sequences.
  • isolated nucleic acid sequence or “isolated nucleic acid composition” refers to a nucleic acid sequence that is not in its natural environment when it is linked to its naturally associated sequence(s) that is/are also in its/their natural environment.
  • an isolated nucleic acid sequence/composition is not a native nucleotide sequence/composition, wherein "native nucleotide sequence/composition” means an entire nucleotide sequence that is in its native environment and when operatively linked to an entire promoter with which it is naturally associated, which promoter is also in its native environment.
  • nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition ⁇ e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide.
  • gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region ("leader and trailer") as well as intervening sequences (introns) between individual coding segments (exons).
  • the nucleic acid sequences of the invention may be a non-naturally occurring nucleic acid sequence (e.g. it may be that the entire sequence does not occur in its entirety in nature).
  • the nucleic acid sequence of the invention may be operably linked to a promoter, wherein the promoter is not naturally associated with equivalent human nucleic acid sequences in nature (e.g. human TCR sequences or fragments thereof); i.e. it is not the entire promoter that is naturally associated with the nucleic acid in its natural environment.
  • such promoters may be considered exogenous promoters. Examples of appropriate promoters are described elsewhere.
  • binding protein e.g., TCR receptor
  • binding domain or fusion protein thereof
  • K a an affinity or K a (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10 5 M -1 (which equals the ratio of the on-rate [k on ] to the off-rate [k O ff] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample.
  • Binding proteins or binding domains may be classified as “high affinity” binding proteins or binding domains (or fusion proteins thereof) or as “low affinity” binding proteins or binding domains (or fusion proteins thereof).
  • "High affinity” binding proteins or binding domains refer to those binding proteins or binding domains having 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 1 ° M -1 , at least 10 11 M’ 1 , at least 10 12 M -1 , or at least 10 13 M -1 .
  • binding proteins or binding domains refer to those binding proteins or binding domains having a K a of up to 10 7 M’ 1 ’ up to 10 6 M’ 1 , up to 10 5 M -1 .
  • affinity can be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10' 5 M to 10' 13 M).
  • a receptor or binding domain may have "enhanced affinity," which refers to selected or engineered receptors or binding domains with stronger binding to a target antigen than a wild type (or parent) binding domain.
  • enhanced affinity may be due to a K a (equilibrium association constant) for the target antigen that is higher than the wild type binding domain, due to a Kd (dissociation constant) for the target antigen that is less than that of the wild type binding domain, due to an off-rate (k O ff) for the target antigen that is less than that of the wild type binding domain, or a combination thereof.
  • enhanced affinity TCRs can be codon optimized to enhance expression in a particular host cell, such as a cell of the immune system, a inducible pluripotent stem cell (iPSC), a hematopoietic stem cell, a T cell, a primary T cell, a T cell line, a NK cell, or a natural killer T cell (Scholten et al, Clin. Immunol. 119: 135, 2006).
  • the T cell can be a CD4+ or a CD8+ T cell, or gamma-delta T cell.
  • WT1 Wilms’ tumor 1
  • transcription factor Wilms’ tumor 1 WT 1 protein
  • WT 1 protein refers to the transcription factor that is encoded by Wilms’ tumor gene 1 (the WT1 gene) on chromosome 11 p .
  • the WT 1 protein is a transcription factor that plays an important role in cell growth and differentiation.
  • the WT 1 gene is highly expressed in leukemia and various types of solid tumors, whereas WT 1 is a tumor marker convenient for the detection of minimal residual disease of leukemia.
  • WT1 is also referred to as WT1 , AEWS-GUD, NPHS4, WAGR, WIT-2, WT33, Wilms tumor 1 , WT 1 transcription factor, and WT-1 .
  • WT1 In humans it can be uniquely identified by Uniprot identifiers: P19544 and Q6PI38.
  • WT1 antigen or “WT1 peptide antigen” or “WT1 -containing peptide antigen” refers to a naturally or synthetically produced peptide portion of a WT 1 protein ranging in length from about 7 amino acids, about 8 amino acids, about 9 amino acids, about 10 amino acids, up to about 20 amino acids, which can form a complex with a MHC (e.g., HLA) molecule, and a binding protein of this disclosure specific for a WT 1 peptide: MHC (e.g., HLA) complex can specifically bind to such as complex.
  • MHC e.g., HLA
  • the WT1 peptide antigen comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74.
  • the WT1 peptide antigen:HLA complex typically comprises a peptide:HLA complex selected from the group consisting of: a ALLPAVPSL:HLA-A*02:01 complex; a VLDFAPPGA:HLA-A*02:01 complex; a TPYSSDNLY:HLA-B*35:01 complex; and a VLDFAPPGASAY:HLA-A*01 :01 complex.
  • WT1-specific binding protein refers to a protein or polypeptide, such as a TOR or CAR, that specifically binds to a WT1 peptide antigen (or to a WT1 peptide antigen:HLA complex, e.g., on a cell surface), and does not bind a peptide sequence that does not include the WT1 peptide antigen.
  • the WT1 peptide antigen comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74
  • the WT1 peptide antigen:HLA complex comprises a peptide:HLA complex selected from the group consisting of: a ALLPAVPSL:HLA-A*02:01 complex; a VLDFAPPGA:HLA-A*02:01 complex; a TPYSSDNLY:HLA-B*35:01 complex; and a VLDFAPPGASAY:HLA-A*01 :01 complex, as appropriate.
  • a WT1-specific binding protein specifically binds to a WT1 peptide antigen (or a WT1 peptide antigen:HLA complex) with a Kd of less than about 10' 8 M, less than about 10' 9 M, less than about 10' 1 ° M, less than about 10' 11 M, less than about 10' 12 M, or less than about 10' 13 M, or with an affinity that is about the same as, at least about the same as, or is greater than at or about the affinity exhibited by an exemplary WT1 -specific binding protein provided herein, such as any of the WT1-specific TCRs provided herein, for example, as measured by the same assay.
  • a WT1 -specific binding protein comprises a WT1-specific immunoglobulin superfamily binding protein or binding portion thereof.
  • the WT1 peptide antigen comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74
  • the WT1 peptide antigen:HLA complex comprises a peptide:HLA complex selected from the group consisting of: a ALLPAVPSL:HLA-A*02:01 complex; a VLDFAPPGA:HLA-A*02:01 complex; a TPYSSDNLY:HLA-B*35:01 complex; and a VLDFAPPGASAY:HLA-A*01:01 complex, as appropriate.
  • the selective binding may be in the context of WT1 antigen presentation by HLA-A*01 :01, HLA- A*02:01 , and/or HLA-B*35:01.
  • a binding protein that “specifically binds to a WT1 antigen” may only do so when it is being presented (i.e. it is bound by) by a specific H LA or is in an equivalent structural formation as when it is being presented by the specific HLA.
  • WT1 derived peptides according to SEQ ID NO:71 and SEQ ID NO: 72 are capable of being presented by HLA- A*02:01 ; that the WT 1 derived peptide of SEQ ID NO: 73 is capable of being presented by HLA- B*35:01 ; and that the WT1 derived peptide of SEQ ID NO: 74 is capable of being presented by HLA-A*01 :01.
  • a binding protein that “specifically binds to a WT1 antigen”, in particular the peptide of SEQ ID NO:71 or SEQ ID NO:72, may only do so when it is being presented (i.e. it is bound by) HLA-A*02:01 or is in an equivalent structural formation as when it is being presented by HLA-A*02:01.
  • a binding protein that “specifically binds to a WT1 antigen”, in particular the peptide of SEQ ID NO: 73 may only do so when it is being presented (i.e. it is bound by) HLA-B*35:01 , or is in an equivalent structural formation as when it is being presented by HLA-B*35:01.
  • a binding protein that “specifically binds to a WT1 antigen”, in particular a peptide of SEQ ID NO:74 may only do so when it is being presented (i.e. it is bound by) HLA-A*01 :01 or is in an equivalent structural formation as when it is being presented by HLA-A*01 :01.
  • T cell receptor By “specifically bind(s) to” as it relates to a T cell receptor, or as it refers to a recombinant T cell receptor, nucleic acid fragment, variant, or analog, or a modified cell, such as, for example, the WT1 T cell receptors, and WT1 -expressing modified cells herein, is meant that the T cell receptor, or fragment thereof, recognizes, or binds selectively to a WT1 antigen (e.g. wherein the WT1 antigen comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74). Under certain conditions, for example, in an immunoassay, for example an immunoassay discussed herein, the T cell receptor binds to WT1 (e.g.
  • the T cell receptor may bind to WT1 (e.g. an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74) with at least 10, 100, or 1000, fold more affinity than to a control antigenic polypeptide.
  • WT1 e.g. an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74
  • This binding may also be determined indirectly in the context of a modified T cell that expresses a WT1 TOR.
  • the modified T cell is specifically reactive against a OVCA cell line or an AML cell line.
  • the modified WT1 -expressing T cell may bind to a OVCA cell line or an AML cell line with at least 10, 100, or 1000, fold more reactivity when compared to its reactivity against a control cell line that is not a OVCA cell line or an AML cell line.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of (e.g., the sequence identified by SEQ ID NO herein) without abolishing or, more preferably, without substantially altering a biological activity, whereas an “essential” (or “critical”) amino acid residue results in such a change.
  • amino acid residues that are conserved are predicted to be particularly non-amenable to alteration, except that amino acid residues within the hydrophobic core of domains can generally be replaced by other residues having approximately equivalent hydrophobicity without significantly altering activity.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced 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 with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • a nonessential (or non-critical) amino acid residue in a protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly, and the resultant mutants can be screened for activity to identify mutants that retain activity.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a 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%, and 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.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”.
  • the percent identity between the 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 need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman et al. (1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a BLOSLIM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • 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 a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6.
  • a particularly preferred set of parameters are a BLOSLIM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
  • the percent identity between two amino acid or nucleotide sequences can 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.
  • the nucleic acid and protein sequences described herein can be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the N BLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-410).
  • gapped BLAST can be utilized as described in Altschul et al. (1997, Nucl. Acids Res. 25:3389-3402).
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • polypeptides and nucleic acid molecules described herein can have amino acid sequences or nucleic acid sequences sufficiently or substantially identical to the sequences identified 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 minimum number of identical or equivalent (e.g. with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain or common functional activity.
  • amino acid sequences or nucleic acid sequences having one or several e.g.
  • amino acid or nucleic acid substitutions compared to the corresponding sequences identified by SEQ ID NO may be sufficiently or substantially identical to the sequences identified by SEQ ID NO (provided that they retain the requisite functionality).
  • the one or several (e.g. two, three, four etc) amino acid or nucleic acid substitutions may be conservative substitutions.
  • amino acid or nucleotide sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently or substantially identical.
  • TCR sequences are defined according to IMGT. See the LeFranc references herein for further details i.e. [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).
  • ex vivo refers to “outside” the body.
  • in vitro can be used to encompass “ex vivo” components, compositions and methods.
  • nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art.
  • WT 1 Transcription factor Wilms’ tumor gene 1 (WT 1) is an ideal tumor target based on its expression in a wide range of tumors, low-level expression in normal tissues and promoting role in cancer progression.
  • WT1 is targeted using peptide- or dendritic cell-based vaccines and T-cell receptor (TCR)-based therapies.
  • TCR T-cell receptor
  • the inventors have overcome both limitations by searching in the allogeneic T-cell repertoire of healthy donors for high-avidity WT1- specific T cells, specific for WT 1 peptides derived from the HLA class I associated ligandome of primary leukemia and ovarian carcinoma (OVCA) samples.
  • WT1-specific T cells specific for WT 1 peptides derived from the HLA class I associated ligandome of primary leukemia and ovarian carcinoma (OVCA) samples.
  • OVCA primary leukemia and ovarian carcinoma
  • the TCR sequences of 5 T-cell clones were analyzed and TCR gene transfer into CD8+ T cells installed antitumor reactivity against acute myeloid leukemia (AML) and OVCA patient samples.
  • AML acute myeloid leukemia
  • the 23.2G9 clone is only shown in Figure 3, but the TCR sequence of this T cell clone was also analyzed and transferred into CD8+ T cells, results demonstrated comparable results as 20.3D10 TCR, therefore these data were not added into the results section.
  • WT1 forward: AGACCCACACCAGGACTCAT (SEQ ID NO: 75); reverse:
  • GUSB forward: ACTGAACAGTCACCGACGAG (SEQ ID NO: 77); reverse:
  • PSMB4 forward: GTTTCCGCAACATCTCTCGC (SEQ ID NO: 79); reverse:
  • CATCAATCACCATCTGGCCG (SEQ ID NO: 80);
  • VPS29 forward: TGAGAGGAGACTTCGATGAGAATC (SEQ ID NO: 81); reverse:
  • the small tumor-pieces were added to a C-tube (Miltenyi Biotec) with ice cold buffer without detergent and complete Protease Inhibitor (Sigma- Aldrich), to prevent protein degradation. Using a gentleMACS (Miltenyi Biotec) procedure the small tumor-pieces were dissociated until an almost homogenous cell solution. Benzonase (Merck) was added in a concentration of 125 lU/ml to remove DNA/RNA complexes during lysis. HLA typing was performed of all samples and WT 1 expression was analyzed by RT-qPCR.
  • Peptide elution was performed as outlined previously.
  • the cell pellets were lysed and subjected to an immunoaffinity column to collect bound peptide-HLA complexes, with either an HLA class- I antibody (W6/32) or an HLA-A*02:01 antibody (BB7.2).
  • bound peptide-HLA complexes were dissociated with 10% acetic acid and filtrated using a 10 kDa membrane.
  • Eluted peptide pools were either fractionated by strong cation exchange chromatography (SCX)[38] or by high pH reversed phase fractionation (High pH-RP)[39], SCX and high pH-RP peptide fractions were lyophilized, dissolved in 95/3/0.1 water/ acetonitrile/formic acid v/v/v and subsequently analyzed by data-dependent MS/MS on either an LTQ FT Ultra equipped with a nanoflow liquid chromatography 1100 HPLC system (Agilent Technologies) or a Q Exactive mass spectrometer equipped with an easy-nLC 1000 (Thermo Fisher Scientific).
  • SCX strong cation exchange chromatography
  • High pH-RP high pH reversed phase fractionation
  • Proteome Discoverer version 2.1 (Thermo Fisher Scientific) was used for peptide and protein identification, using the mascot search node for identification (mascot version 2.2.04) and the UniProt Homo Sapiens database (UP000005640; Jan 2015; 67,911 entries).
  • BMI Best Mascot Ion
  • T cells were cultured in T-cell medium (TCM) composed of Iscove’s Modified Dulbecco’s Medium (IMDM) (Lonza), 5% heat-inactivated Fetal Bovine Serum (FBS) (Gibco, Thermo Fisher Scientific), 5% human serum (Sanquin Reagents), 1.5% L-glutamine (Lonza), 1% Pen/Strep (Lonza) and 100 IIJ/mL IL-2 (Novartis Pharma).
  • TCM T-cell medium
  • IMDM Modified Dulbecco’s Medium
  • FBS heat-inactivated Fetal Bovine Serum
  • 0.2*10 A 6 T cells were (re)stimulated with 1*10 A 6 irradiated (35 Gy) PBMCs, 0.1*10 A 6 irradiated (55 Gy) EBV-LCLs and 0.8 pg/mL phytohemagglutinin (PHA) (Oxoid Microbiology Products, Thermo Fisher Scientific).
  • PHA phytohemagglutinin
  • NGF-R nerve growth factor receptor
  • GFP green fluorescent protein
  • CD34 mouse CD19
  • mCD19 mouse CD19
  • AML samples were thawed one day before being used as target cells in screening experiments.
  • Cells were cultured overnight at 37 degrees Celsius and 5% CO2 in IMDM containing 10% human serum and if needed, live cells were isolated using Ficoll gradient separation. Blast percentage of the AML samples was on average 83% (range 40% - 99%), as determined by FACS expression (CD13, CD33 and CD34).
  • Primary OVCA cells were thawed 3 days before being used as target cells in screening experiments. Cells were cultured in IMDM, 10% FBS, 1.5% L-glutamine and 1 % Pen/Strep, on FBS pre-coated plates.
  • Hematopoietic healthy cell subsets were isolated from PBMCs of healthy donors, either HLA- A*01 :01 and HLA-B*35:01 positive or HLA-A*02:01 positive.
  • CD14, CD19 and CD34 positive cells were enriched by magnetic associated cell sorting (MACS) using anti-CD14 MicroBeads (Miltenyi Biotec/200-070-118), anti- CD19 MicroBeads (Miltenyi Biotec/130-060-301) or anti-CD34 MicroBeads (Miltenyi Biotec/130-046-702).
  • Activated CD19+ B-cells were generated by coculturing CD19+ cells on CD40L-transduced irradiated (70 Gy) mouse fibroblasts for 7 days in IMDM supplemented with 2 ng/mL IL-4 (Schering-Plough) and 10% human serum.
  • Immature and mature CD14-derived dendritic cells were differentiated in vitro from isolated CD14+ cells. Immature DCs were generated by culturing 0.1*10 A 6 cells/mL for 3 days in IMDM supplemented with 100 ng/mL GM-CSF (Sandoz Novartis Pharma), 500 IIJ/mL IL-4 (Schering-Plough), and 10% human serum.
  • Mature DCs were generated by culturing immature DCs for an additional 3 days in IMDM supplemented with 100 ng/mL GM-CSF, 10 ng/ml TNFalpha (CellGenix), 10 ng/mL IL- 1b (Bioscource Invitrogen), 10 ng/mL IL-6 (Sandoz Novartis Pharma), 1 pg/ml PGE-2 (Sigma Aldrich), 500 IIJ/mL IFN-y (Boehringer Ingelheim), and 10% human serum.
  • Isolated CD34+ hematopoietic precursor cells were directly used after isolation. Purity of the isolated and generated cells was assessed using FACS analysis.
  • Fibroblasts from skin biopsies were cultured in DMEM with 1 g/L glucose (Lonza BE12-708F) and 10% FBS. Keratinocytes from skin biopsies were cultured in keratinocyte serum free medium (Thermo Fisher Scientific 17005-059) supplemented with 30 pg/mL bovine pituitary extract (BPE) and 2 ng/mL epithelial growth factor (EGF) (both Thermo Fisher Scientific 37000-015).
  • BPE bovine pituitary extract
  • EGF epithelial growth factor
  • Fluorescence activated cell sorting was performed on an LSR II flow cytometer (BD Biosciences) and data was analyzed using FlowJo software (TreeStar). T cells were stained with the following conjugated antibodies: CD4 FITC (BD/555346), CD14 FITC (BD/555397), CD19 FITC (BD/555412), CD8 AF700 (lnvitrogen/MHCD0829), murine TCR- (mTCR-P) APC (BD/553174) and pMHC-multimers PE.
  • Target cells with transduced WT1 or HLA-alleles were stained with: NGFR/CD271 APC (Sanbio/CL10013APC), CD34 APC (BD/555824), murine CD19 PE (BD/557399), HLA-A2 PE (BD/558570).
  • Non-malignant hematopoietic subsets with: CD14 FITC (BD/555397), CD19 FITC (BD/555412), CD34 APC (BD/555824), CD80 PE (BD/557227), CD86 PE (BD/555658).
  • AML samples with: CD13 PE (BD/347406), CD33 FITC (BD/555626) and CD34 APC (BD/555824).
  • Buffy coats of healthy donors negative for HLA-A*01 :01 , HLA-A*02:01 , HLA-A*03:01 , HLA- A*24:02, or HLA-B*35:01 were collected after informed consent (Sanquin).
  • PBMCs were isolated using Ficoll gradient separation and incubated with a selection of WT1-specific pMHC-multimers for 1 hour at 4°C or 15 minutes at 37°C. pMHC-multimers were only included if the healthy donor was negative for the restricted HLA allele.
  • pMHC-multimer bound cells were MACS enriched using anti-PE MicroBeads (Miltenyi Biotec/130-048-801).
  • the positive fraction was stained with AF700-conjugated antibody against CD8 and FITC-conjugated antibodies against CD4, CD14 and CD19.
  • pMHC-multimer and CD8 positive cells were single-cell sorted using an Aria III cell sorter (BD Biosciences) in a 96 well round bottom plate containing 5*10 A 4 irradiated PBMCs (35Gy) and 5*10 A 3 EBV-JY cells (55Gy) in 100ul T-cell medium with 0.8 pg/ml PHA.
  • T-cell recognition was assessed 10 - 14 days after stimulation, followed by restimulation or storage of the selected T-cell clones.
  • T-cell recognition was measured by an IFN-y ELISA (Sanquin). 5,000 T cells were co-cultured overnight with target cells in various effector-to-target (E:T) ratios in 60 pL TCM in 384-well flatbottom plates (Greiner Bio-One). To upregulate HLA expression, adherent target cells were treated for 48 hours with 100 lll/ml IFN-y (Boehringer Ingelheim) before co-culture. All T cells and target cells were washed thoroughly before co-culture to remove expansion-related cytokines.
  • E:T effector-to-target
  • Supernatants were transferred during the ELISA procedure using the Hamilton Microlab STAR Liquid Handling System (Hamilton company) and diluted 1 :5, 1 :25 and/or 1 :125 to quantify IFN-y production levels within the area of the standard curve.
  • the Hamilton System was also used to split 96-well T-cell cultures into 4 wells of 384-well flat bottom plates during our large-scale T-cell search in 28 healthy donors, making it feasible to screen the T cells against different combinations of Raji cells loaded with and without peptides (1 pM).
  • T-cell mediated cytotoxicity was measured in a 6-hour 51 chromium release assay.
  • Target cells were labeled with 100 pCi 51 chromium (PerkinElmer) for 1 hour at 37°C, washed, and co-cultured with T cells at various E:T ratios in 100 pl TCM per well in 96-well U-bottom culture plates (Costar). Spontaneous and maximum 51 Cr release for all targets were measured in separate plates with per well 100 pL TCM or 100 pL TCM with 1% Triton-X 100 (Sigma-Aldrich), respectively.
  • TCR a and p chains of the selected T-cell clones were identified by sequencing with minor modifications, as previously described.
  • mRNA was isolated by the Dynabeads mRNA DIRECT Kit (Invitrogen) or total RNA was isolated by the ReliaPrep RNA cell Miniprep System (Promega).
  • TCR cDNA was generated using TCR constant a and primers, a SA.rt anchor template-switching oligonucleotide (TSO), and SMARTScribe Reverse Transcriptase (Takara, Clontech).[42]
  • TCR a and p products were generated in a first PCR using Phusion Flash (Thermo Fisher Scientific), followed by a second PCR that was used to include 2 sided barcode sequences for the different T-cell clones. Barcoded TCR PCR products were pooled and TCR sequences were identified by HiSeq or NovaSeq (GenomeScan).
  • the Va and Vp families were determined of the NGS data using the MiXCR software and ImMunoGeneTics (IMGT) database.
  • the TCR chains were codon optimized, synthesized and cloned in MP71-TCR-flex retroviral vectors by Baseclear.
  • the MP71-TCR-flex vector contains codon-optimized and cysteine-modified murine TCRa constant domains and P2A sequence to link TCR chains, resulting in optimized TCR expression and increased preferential pairing.
  • a murinized CMV-specific TCR (NLVPMVATV (SEQ ID NO: 83) peptide presented in HLA-A*02:01) was included as a negative control.
  • Phoenix-AMPHO (ATCC) cells were transiently transfected with the created constructs and after 48 hours retroviral supernatants were harvested and stored at -80°C.
  • CD8+ T cells were isolated from PBMCs of different healthy individuals by MACS using anti-CD8 MicroBeads (Miltenyi Biotech/130-045-201). CD8+ T cells were stimulated with irradiated autologous feeders (40 Gy) and 0.8 pg/mL PHA in 24-well flat-bottom culture plates (Costar). Two days after stimulation, CD8+ T cells were transferred to 24-well flat-bottom suspension culture plates (Greiner Bio- One) for retroviral transduction. These plates were first coated with 30 pg/ml retronectin (Takara, Clontech) and blocked with 2% human serum albumin. Retroviral supernatants were added, and plates were centrifuged at 3000 g for 20 minutes at 4°C.
  • CD8+ T cells were transferred per well. After O/N incubation, CD8+ T cells were transferred to 24-well flat- bottom culture plates (Costar). Seven days after stimulation, CD8+ T cells were MACS enriched for the murine TCR, using mTCR-p APC antibody (BD/553174) and anti-APC MicroBeads (Miltenyi Biotec/130-090- 855). Ten days after stimulation, CD8+ T cells were functionally tested and purity was checked by FACS.
  • AML patient samples were used from the Leiden University Medical Center Biobank for Hematological Diseases. This study was approved by the Institutional Review Board of the Leiden University Medical Center (IRB LUMC approval number B16.039). Materials from patients and healthy individuals were collected after written informed consent according to the Declaration of Helsinki.
  • the HLA class I associated ligandome was determined of 11 ALL patients, 15 AML patients, 1 hairy cell leukemia (HCL) patient, and 8 OVCA patients, as well as 2 OVCA cell lines. All WT1 peptides with a length between 8 and 14 amino acids, a minimal Best Mascot Ion (BMI) score of 20 and a mass accuracy of 10 ppm were selected.
  • BMI Best Mascot Ion
  • Peptides were synthesized if they were predicted to bind common HLA molecules (HLA-A*01 :01, -A*02:01 , -A*03:01 , -A*24:02, -B*07:02, -B*35:01 , -0*07:01 , -C*07:02) according to netMHC peptide binding algorithm[27], and matched the HLA typing of the material from which the peptides originated (Table 1).
  • Table 2 Overview of the 12 WT1 peptides included in this study. Forthe 8 WT1 peptides identified in our HLA ligandome analyses, the source and best Mascot ion score (BMI) is listed. The 4 peptides not identified in this study, but previously described in literature, were added at the end of the table.
  • OVCA primary ovarian carcinoma
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • HCL hairy cell leukemia.
  • Table 3 Overview of the number of T-cell clones screened per healthy donor and T-cell clones reactive against WT1 peptide and/or transduced WT1.
  • H LA class I typing For each healthy donor, H LA class I typing, number of PBMCs, and expanded single-cell sorted T-cell clones are listed.
  • 461 of 7916 screened T-cell clones were reactive against Raji pulsed with the WT1 peptide pool (1 pM), and 71 clones were also reactive against Raji transduced with WT1. The reactivity is based on IFN- production (ng/mL) after an overnight co-culture stimulation assay.
  • pMHC-multimers were only included if the donor was negative for the HLA allele, enabling searching within the allo-HLA T-cell repertoire, and thereby circumventing self-tolerance.
  • 650*10 A 6 PBMCs per donor were used and between 20 and 658 pMHC-multimer positive CD8+ T-cell clones were expanded after single-cell sorting (Table 3).
  • T-cell clones were WT 1 peptide specific, recognizing WT 1 peptide pool pulsed Raji cells, whereas non-pulsed Raji cells were not recognized.
  • the other T-cell clones were either reactive against all stimulator cells, nonreactive or reactive against one specific HLA- allele, independent of added peptides.
  • the peptide-specific T-cell clones were subsequently tested against the individual peptides and screened for recognition of endogenously processed and presented WT1. 71 of the 461 T-cell clones (17%) recognized WT1 transduced Raji cells.
  • the peptide specificity of these T-cell clones is summarized in Table 4, T-cell clones were identified for 9 out of 12 WT 1 peptides.
  • Table 4 Overview of T-ce l clones reactive against transduced WT1 and those TCRs further analyzed. Number of healthy donors included in the T-cell search is listed. In total 71 of the isolated T-cell clones were reactive against both Raji pulsed with the WT1 peptide pool and Raji transduced with WT 1. The TCRs of 4 T-cell clones were seguenced and further analyzed.
  • T-cell clones were analyzed in additional screenings. This selection approach is illustrated in Figure 3 for 7 T-cell clones recognizing the VLDFAPPGA (SEQ ID NO: 72) peptide in HLA-A*02:01. T cells were tested against a tumor cell line panel composed of WT 1 positive and negative tumor cell lines that were positive or transduced with the corresponding HLA restriction molecule ( Figure 3A). The T-cell clones recognizing at least 2 WT 1 positive tumor cell lines, combined with no recognition of WT 1 negative tumor cell lines were selected (28/71 T-cell clones).
  • T-cell clones were additionally screened against a panel of 25 Epstein-Barr virus transformed lymphoblastoid cell lines (EBV-LCL), expressing all HLA alleles with an allele freguency > 1 % present in the Caucasian population (Figure 3B).
  • EBV-LCL Epstein-Barr virus transformed lymphoblastoid cell lines
  • Eight T-cell clones were excluded that showed HLA cross-reactivity against one or several prevalent HLA alleles, exemplified by clone 23.2E4.
  • Those T-cell clones that recognize only one non-prevalent HLA allele were not excluded, exemplified by clone 20.3D10.
  • the antitumor reactivity against primary AML and OVCA patient samples expressing variable levels of WT1 was analyzed ( Figure 3C).
  • ALLPAVPSL SEQ ID NO: 71
  • HLA-A*02:01 ALL/A2
  • clone 12.5H9 is specific for
  • VLDFAPPGASAY (SEQ ID NO: 74) in HLA-A*01 :01 (VLD/A1), clone 17.2G4 is specific for
  • TPYSSDNLY SEQ ID NO: 73 in HLA- B*35:01 (TPY/B35), and clone 17.2D6 is specific for
  • HAAQFPNHSF SEQ ID NO: 87
  • HLA-B*35:01 HLA/B35
  • All 5 WT1 -specific clones demonstrated antitumor reactivity against WT 1 positive tumor cell lines. Only those tumor cell lines with a WT1 expression below 15% were more variable recognized ( Figure 4A). As depicted in Figure 4B, 2 of the 5 T-cell clones showed HLA cross-reactivity against one non-prevalent HLA allele.
  • clone 12.5H9 VLD/A1 showed the highest recognition of AML-4443 (13% relative WT1 expression), from which the VLD/A1 peptide was eluted (Table 2), whereas the other AML samples with WT 1 expression between 23-27% were less well recognized.
  • clone 20.3D10 VLD/A2 and the other VLD/A2 reactive T-cell clones ( Figure 3) did not recognize AML-6588 (10%) and AML-4716 (3%), while the 22.1 H1 ALL/A2 clone recognized both. This also accounts for some of the HLA- B*35:01 positive primary AML patient samples.
  • T-cell clones were specific for the peptides that were previously identified based on peptide prediction algorithms, including the most freguently used RMFPNAPYL (SEQ ID NO: 88) peptide presented in HLA-A*02:01 (RMF/A2).
  • RMF/A2 the most freguently used RMFPNAPYL
  • RMF/A2 the most freguently used RMFPNAPYL
  • TCRs of the selected T-cell clones were analyzed in more detail. 4 of the 5 T-cell clones: 20.3D10 VLD/A2 , 22.1 H 1 ALL/A2 , 12.5H9 VLD/A1 , and 17.2G4 TPY/B35 based on potency were continued with.
  • the TCR a and p chains were identified by seguencing and transferred using retroviral vectors into CD8+ T cells of multiple donors.
  • TCR-engineered T cells were enriched based on murine TCR (mTCR) expression and pMHC multimer staining demonstrated that the TCR-T cells efficiently express the TCR at the cell surface ( Figure 7A). Functional reactivity of the TCR-T cells measured by peptide titration experiments was guite comparable to their parental T-cell clones ( Figure 7B).
  • TCR-T cells To investigate the antitumor potential of the TCR-T cells, they were screened against multiple tumor panels and assessed for IFN- production.
  • the 4 TCRs demonstrated effective antitumor reactivity, recognizing WT1 positive tumor cell lines, whereas WT1 negative cell lines were not recognized ( Figure 8A). Similar as observed for the T-cell clones, those tumor cell lines with a WT1 expression below 15% were more variable recognized.
  • the included WT1 positive primary AML and OVCA patient samples were recognized ( Figure 8B-C).
  • TCR-T 12.5H9 VLD/A1 The level of recognition of these primary tumor samples correlated with the level of WT1 expression, except for TCR-T 12.5H9 VLD/A1 that only recognized the AML-4443 sample in which the VLD/A1 peptide was initially eluted (Table 2).
  • the engineered T cells were tested against a variety of different healthy cell subsets. Keratinocytes, fibroblasts, and several hematopoietic cell subsets, including CD34+ hematopoietic precursor cells, CD14+ derived immature and mature dendritic cells, and B cells were tested for recognition by the TCR-T cells.
  • WT1-specific TCR-T cells efficiently kill primary AML samples and OVCA cell lines
  • TCR-T 20.3D10 VLD/A2 , 22.1 H1 ALL/A2 , and 17.2G4 TPY/B35 results in efficient killing of primary AML patient samples and OVCA cell lines.
  • these TCRs are considered as promising candidates for TCR gene transfer strategies for the treatment of AM L and OVCA patients, and other solid tumors.
  • TCR 12.5H9 VLD/A1 is considered as a potential candidate for TCR gene transfer strategies for patients with WT1 expressing solid tumors, including ovarian cancers.
  • the HLA class I restriction molecules for TCR 20.3D10 VLD/A2 , TCR 22.1 H1 ALL/A2 , TCR 12.5H9 VLD/A1 , and TCR 17.2G4 TPY/B35 are common, 39% of the global population expresses HLA-A*02:01 , 17% expresses HLA- A*01 :01 and 8.5% expresses HLA-B*35:01.[29] WT1-TCR-T cells induced potent killing of WT1 positive primary AML and OVCA patient samples and/or OVCA cell lines, without reactivity against healthy cell subsets and WT 1 negative tumor cells.
  • RMF/A2 peptide is not a suitable target for T-cell based tumor immunotherapies, since a high avidity RMF/A2-specific TCR was not reactive against naturally WT1 expressing tumor cells.
  • RMF/A2-specific T-cells were easily found in the autologous-HLA (auto-HLA) T-cell repertoire of most healthy individuals, but no antitumor reactivity was observed.
  • auto-HLA autologous-HLA
  • WT1-TCRs demonstrated potent antitumor reactivity against AML and OVCA tumors.
  • RT- qPCR WT1 expression in primary OVCA AML as well as ALL patient samples was confirmed.
  • WT1 is additionally expressed in a broad variety of other tumors[34], indicating that also other solid tumors can be treated with WT1-TCR therapy.
  • WT 1 promotes cancer progression through the induction of tumor angiogenesis and metastasis formation.
  • WT 1 is a strong predictor of leukemia relapse and is used as marker for minimal residual disease (MRD).[35] Also in MDS patients, overexpression of WT1 is associated with a higher risk for disease progression and AML transformation. [36] Finally, in solid tumors WT1 expression is also associated with poor prognosis, this is among others related to increased epithelial-to- mesenchymal transition. [37]
  • the 20.3D10 VLD/A2 , 22.1 H 1 ALL/A2 , 12.5H9 VLD/A1 , and 17.2G4 TPY/B35 TCRs were identified in a large- scale search for potent and safe WT1 -specific TCRs present in the allo-HLA repertoire. These TCRs are expected to be a more potent option than the currently used WT1-TCRs from the auto-HLA repertoire. Also the naturally expressed WT 1 peptides identified from the HLA class I associated ligandome of primary leukemia and OVCA patients, are expected to improve future vaccine and TCR gene therapy studies for WT1 -positive tumors.
  • SEQ ID NO: 80 CATCAATCACCATCTGGCCG - PSMB reverse;

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Abstract

L'invention concerne de nouvelles compositions d'acide nucléique, des systèmes de vecteur, des cellules modifiées et des compositions pharmaceutiques qui codent ou expriment des composants de récepteur de cellule dirigés contre le facteur de transcription WT1 ("Wilms tumor gene 1"). Ces nouveaux composants peuvent être utilisés pour améliorer une réponse immunitaire chez un sujet diagnostiqué avec une maladie ou un état associé à WT1, telle qu'une hémopathie maligne ou une tumeur solide. L'invention concerne également des méthodes associées pour traiter de tels sujets.
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WO2025238364A1 (fr) * 2024-05-16 2025-11-20 Oxford University Innovation Limited Récepteurs de lymphocytes t spécifiques du peptide wt1 restreint par hla-e et leurs utilisations

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