NL2031118B1

NL2031118B1 - T cell receptors directed against transcription factor wt1 and uses thereof

Info

Publication number: NL2031118B1
Application number: NL2031118A
Authority: NL
Inventors: H M Heemskerk Mirjam; H Frederik Falkenburg J
Original assignee: Academisch Ziekenhuis Leiden
Priority date: 2022-03-01
Filing date: 2022-03-01
Publication date: 2023-09-07
Also published as: WO2023167583A1

Abstract

Novel nucleic acid compositions, vector systems, modified cells, and pharmaceutical compositions that encode or express T cell receptor components directed against transcription factor Vlﬁlms’ tumor gene 1 (VVT1) are provided herein. These novel components may be used to enhance an immune response in a subject diagnosed with a WT1 associated disease or condition, such as a hematological malignancy or a solid tumor. Associated methods for treating such subjects are also provided herein.

Description

T CELL RECEPTORS DIRECTED AGAINST TRANSCRIPTION FACTOR WT1 AND USES

THEREOF

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 (WT1) are provided herein. These novel components may be used to enhance an immune response in a subject diagnosed with a WT1 associated disease or condition, such as a hematological malignancy or a solid tumor. Associated methods for treating such subjects are also provided herein.

Background

Transcription factor Wilms’ tumor gene 1 (WT1) is expressed in a broad range of cancers, especially in ovarian carcinomas (OVCA), mesotheliomas, and acute myeloid leukemia (AML).[1, 2] These tumor types have low five-year survival rates of 49%, 12% and 29%, respectively, and there is a strong need for additional treatment options.[3, 4] In OVCA and AML patients, WT1 expression is associated with poor prognosis.[5, 6] This corresponds to the promoting role of WT1 in cancer progression through the induction of tumor angiogenesis and metastasis formation.[7]

WT1 is expressed in a wide range of hematological malignancies and solid tumors with only low- level expression in normal tissues.

Currently, most clinical studies targeting WT1 use peptide- or dendritic cell-based vaccines.

Mainly hematological malignancies are included in these trials, but also several solid tumor types such as gynecological malignancies and sarcomas are treated.[9-11] More recently, WT1 has been targeted by T-cell-based therapies as well. HLA class | restricted WT 1-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).[14-16] In these clinical studies anti-leukemic reactivity and persistence of WT1-specific T cells have been reported. However, there are still many issues to be solved to improve avidity and increase killing potential of tumor cells by vaccine activated T cells or infused WT 1-specific

T-cell products.[17]

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 WT 1-specific T cells present in the T-cell repertoire can be activated by WT1 peptide vaccines. In addition, the WT 1-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 WT 1-specific TCRs.[22]

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-HLA-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.[13, 26] Besides the relative low affinity of WT1-specific TCRs used in these clinical studies, the selection of WT1- specific peptides used for peptide vaccination studies and identification of TCRs can most likely also be optimized. WT1 peptides were thus far selected based on peptide binding prediction algorithms, and although these peptides efficiently bind to HLA class | it is unknown whether these peptides are efficiently processed and presented in HLA class | 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.

There is a need for improved means for treating a WT 1 associated disease or condition, such as hematological malignancy or a solid tumor.

Brief summary of the disclosure

In this study, WT1 peptides were identified from the HLA class | 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. Using broad panels of malignant cells and healthy cell subsets, 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. These 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.

Accordingly, 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 B chain variable (VB) domain, the composition comprising: (i) 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; and a nucleic acid sequence that encodes a TCR VB domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:8, or a functional fragment thereof, wherein the CDR3 sequences together specifically bind to WT1; or (il) 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 VB 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; or (iii) 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 VB 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 WT1; or (iv) 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 VB 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 WT1; or

(v) 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 VB 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.

Suitably: (i) the CDRS of the Va domain may comprise or consist of the amino acid sequence of SEQ ID

NO: 3, and the CDR3 of the VB domain may comprise or consist of the amino acid sequence of

SEQ ID NO:6; or (ii) 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 VB domain may comprise or consist of the amino acid sequence of

SEQ ID NO:20; or (iii) the CDRS of the Va domain may comprise or consist of the amino acid sequence of SEQ ID

NO: 31, and the CDR3 of the VB domain may comprise or consist of the amino acid sequence of

SEQ ID NO:34; or (iv) 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 VB domain may comprise or consist of the amino acid sequence of

SEQ ID NO:48; or (v) the CDR3 of the Va domain may comprise or consist of the amino acid sequence of SEQ ID

NO: 59, and the CDR3 of the VB domain may comprise or consist of the amino acid sequence of

SEQ ID NO:82.

Suitably: (i) 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 VB domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 9; (ii) 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 VB domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 23; or (iii) 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 VB domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 37; or

(iv) 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 V domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 51; or 5 {v) 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 VB domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 65.

Suitably, 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).

Suitably, 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.

Suitably, the nucleic acid sequence may be codon optimised for expression in a host cell, optionally wherein the host cell is a human cell.

Suitably, the nucleic acid composition may comprise a TCR a chain constant domain and/or a

TCR B chain constant domain.

Suitably, the encoded binding protein may comprise a TCR, an antigen binding fragment of a

TCR, a chimeric antigen receptor (CAR), or an ImmTAC.

Suitably, 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.

The invention also provides a vector system comprising a nucleic acid composition according to the invention.

Suitably, 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.

Suitably, 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.

Suitably, the modified cell may be a human cell.

The invention also provides 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 HLA- restricted WT1 antigen.

Suitably, the human subject may have at least one tumor.

Suitably, the subject may have been diagnosed with a WT1 associated disease or condition.

Suitably, the WT1 associated disease or condition may be a hematological malignancy or a solid tumor.

Suitably, 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.

Suitably, the hematological malignancy may be AML.

Suitably, 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.

Suitably, 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.

Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Various aspects of the invention are described in further detail below.

Brief description of the Figures

Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

Figure 1 shows MS data validation of the 8 WT 1-derived peptides. The 8 WT1 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;

Figure 2 shows high WT1 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.

WT1 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 WT 1-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-Y 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 4 shows recognition patterns of the 5 most promising WT1-specific T-cell clones.

Recognition patterns based on IFN-Y production (ng/mL) after overnight co-culture assays with 3 screening panels (E:T = 1:6 for cell lines and 1:16 for AML samples). All cell lines in panel A and

C 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. Dark grey bars depict high WT 1+ 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. Blast percentage of the

AML samples was on average 83% (range 40% — 99%), as determined by FACS expression (CD13, CD33 and CD34);

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-Y 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-y 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. (G-I) Killing percentages of the primary AML panel, OVCA cell line panel, and control panel included in the TCR-T cell screenings, depicted in Figure 9;

Figure 6 shows limited WT 1-specific reactivity of the RMF-specific T-cell clones. Comparison of

T-cell clones recognizing the RMFPNAPYL {SEQ ID NO: 88) (RMF) and VLDFAPPGA (SEQ ID

NO: 72) (VLD) peptide, both presented in HLA-A*02:01. (A) IFN-y production (ng/mL) of T-cell clones recognizing Raji pulsed with RMF/A2 or VLD/A2 peptide (1 uM) identified in 5 healthy donors. 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. (C)

T-cell clones stimulated with tumor cell lines (E:T = 1:6). All cell lines express the HLA-A*02:01, either wildtype (wt) or introduced by transduction (+A2). Percentage relative WT 1 expression is depicted, as determined by RT-qPCR. (D) T-cell clones stimulated with primary acute myeloid leukemia (AML) patient samples (E:T = 1:16). (x = Recognition of AML-5905 and AML-2467 is not shown for clone 5.1G11, since these samples are HLA-B*35:01 positive and the 5.1G11 clone demonstrated allo- HLA reactivity against this allele in an EBV-LCL panel);

Figure 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 WT 1-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. (A) Flow cytometry plots of purified TCR-T cells and their parental T-cell clones stained with the specific and a control pMHC-multimer. (B) IFN-y production (ng/ml) of T cells stimulated with Raji cells (transduced with HLA-A*01:01 or -B*35:01) or T2 cells (wildtype HLA-A*02:01 positive) pulsed with titrated peptide concentrations;

Figure 8 shows WT1-specific TCR-T cells recognize tumor cell lines and primary tumor samples, without recognition of healthy cell subsets. IFN-y production (ng/mL) of purified TCR-T cells co- cultured overnight with various targets. 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). (B) TCR-T cells stimulated with primary AML samples (E:T = 1:16). (C) TCR-T cells stimulated with primary OVCA samples (E:T = 1:6). (D) TCR-T cells stimulated with several healthy cell subsets (E:T = 1:4 for keratinocytes, fibroblasts and CD14+, 1:6 for

CD19+ cells, and 1:12 for CD34+ cells). The cell subsets were isolated from either an HLA-

A*01:01 + and HLA-B*35:01 + donor, or an HLA-A*02:01 + donor; and

Figure 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. The negative control, CD8+ cells transduced with the

CMV TCR, induced limited killing (max 9.6%) and was per target and E:T ratio subtracted from the tested TCR-T cells. (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.

The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

Various aspects of the invention are described in further detail below.

Detailed Description

Nucleic acid compositions that encode binding protein components

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 B chain variable (VB) domain is provided herein, the composition comprising: (a) a nucleic acid sequence that encodes a TCR Va domain comprising a CDR3 amino acid sequence; and (b) a nucleic acid sequence that encodes a TCR VB domain comprising a CDR3 amino acid sequence, wherein the CDR3 sequences together specifically bind to WT1.

As would be clear to a person of skill in the art, the CDR3 amino acid sequences described herein specifically bind to their target (in this case a WT1 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. These complexes are described in more detail elsewhere herein.

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. 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).

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 VB 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. In other words, 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.

Alternatively, 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. For example, 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). As another example, a nucleic acid sequence that encodes a TCR VB domain with the specified features described herein may be part of a larger nucleic acid sequence that encodes a functional TCR B chain (including the constant domain). As a further example, 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 B chain (including the constant domain), optionally wherein the sequence encoding the functional TCR a chain is separated from the sequence encoding the functional TCR

B 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 VB 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 B 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, 2008). 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).

In one example, 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. In one example, 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.

In certain examples, a TCR constant domain is modified to enhance pairing of desired TCR chains. For example, enhanced pairing between a heterologous TCR a chain and a heterologous

TCR B 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., Govers 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 B chain.

A binding protein that is encoded by the nucleic acid compositions described herein is specific for a WT1 antigen and comprises WT1 antigen specific-TCR components. However, 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. For example, 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. A non-limiting example of 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 B 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. InmTACs are therefore bispecific, combining WT1- recognizing TCR components with immune activating complexes.

In certain examples, 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. In other examples, 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). In further examples, 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. "Chimeric antigen receptor” (CAR) refers to a 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)) (see, e.g., Sadelain et al,

Cancer Discov., 3(4):388 (2013); see also Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2018), and Stone et al, Cancer Immunol. Immunother., 83(11): 1163 (2014)).

Methods for producing engineered TCRs are described in, for example, Bowerman et al, Mol.

Immunol, 5(15):3000 (2009). Methods for making CARs are well known in the art and are described, for example, in U.S. Patent No. 6,410,319; U.S. Patent No. 7,446,191; U.S. Patent

Publication No. 2010/065818; U.S. Patent No. 8,822,647; PCT Publication No. WO 2014/031687;

U.S. Patent No. 7,514,537; and Brentjens ef al, 2007, Clin. Cancer Res. 73:5426.

The 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 B-chain, a-chain or both, or any combination thereof.

A T cell receptor (TCR) is a molecule found on the surface of T cells (T lymphocytes) that is responsible for recognising a peptide that is bound to (presented by) a major histocompatibility complex (MHC) molecule on a target cell. 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}. In an example, 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.

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”. Similarly, 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. Similarly, 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.

As described herein, 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.

Accordingly, 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: 71to 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.

Accordingly, in one example, 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.

In one example, 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.

In a further example, the WT1 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 TCR is composed of two different polypeptide chains. In humans, 95% of TCRs consist of an alpha (a) chain and a beta (B) chain (encoded by TRA and TRB respectively). When the TCR 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.

An isolated nucleic acid composition that encodes a WT1 antigen-specific binding protein is provided herein having a TCR a chain variable (Va) domain and a TCR B chain variable (VB) domain. In one example the nucleic acid composition described herein may comprise a TCR a chain constant domain and/or a TCR chain constant domain.

The variable domain of each chain has three hypervariable regions (also called complementarity determining regions (CDRs)). Accordingly, 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. Similarly, the TCR beta variable domain (referred to herein as a TCR VB domain, TCR V beta domain, VB domain or V beta domain, beta variable domain etc) also comprises a (different) CDR1, CDR2, and CDR3 region. 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.

As will be clear to a person of skill in the art, the phrase “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.

As will be clear to a person of skill in the art, the phrase “TCR B 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.

TCR Components

The isolated nucleic acid composition described herein encodes a WT 1 antigen-specific binding protein. As discussed herein, 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). (i) TCR components that interact with ALLPAVPSL (SEQ ID NO: 71)

As provided elsewhere herein, the inventors identified TCR clone 22.1H1 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 22.1H1 are SEQ ID NO:s 1 to 14.

In one embodiment, 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 (VB) domain is provided, 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:3, or a functional fragment thereof; and a nucleic acid sequence that encodes a TCR VB domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:6, or a functional fragment thereof, wherein the CDR3 sequences together specifically bind to WT1.

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. As would be clear to a person of skill in the art, 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 WT1 antigen (e.g. to the peptide ALLPAVPSL (SEQ ID NO: 71)) when the CDR3 is part of TCR Va domain).

Such functional variants are therefore encompassed herein.

For example, 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:3). In other words, 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.

As stated above, functional variants of SEQ ID NO:3 retain their ability to confer specific binding to a WT1 antigen (e.g. the peptide shown in SEQ ID NO:71) when the CDRS is part of TCR Va domain.

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 WT1antigen (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.

In one example, the CDR3 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 3. In examples where the TCR Va domain CDR3 has 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 ID 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. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

For example, 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:1). In other words, 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. As stated previously, 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. As stated above, functional variants of SEQ

ID NO: 1 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 Va domain).

In one example, the CDR1 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO:1. In examples where the TCR Va domain CDR1 has 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.

For example, 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:2). In other words, 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. As stated previously, 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). As stated above, a functional variant of SEQ

ID NO: 2 retains the ability to specifically bind to HLA-A*02:01.

In one example, the CDR2 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 2. In examples where the TCR Va domain CDR2 has 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 WT1 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: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 NQ: 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. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

In one example, 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). In other words, a functional TCR Va domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO:7 is also encompassed. As stated previously, 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). In other words, 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 7).

As an example, 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. In this example, the TCR Va domain

CDR1 may have an amino acid sequence of SEQ ID NO: 1 and the TCR Va domain CDR2 may have an amino acid sequence of SEQ ID NO: 2.

As another example, 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. In this example, the TCR Va domain CDR1 may have an amino acid sequence of SEQ ID NO: 1 and the TCR Va domain CDR2 may have an amino acid sequence of

SEQ ID NO: 2.

In examples where the TCR Va domain has the amino acid sequence of SEQ ID NO:7, 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 phrase “genetically degenerate sequence thereof” is used interchangeably with “derivative thereof” herein.

For the avoidance of doubt, 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 B chain) is encoded in the MP71-TCR-flex retroviral vector. However, 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.

By way of example only, 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. Recently, 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).

Moreover, naked (synthetic) DNA/RNA can also be used to introduce the TCR. As an example, a pMSGV retroviral vector with pre-cloned TCR-Ca and Cb genes as described in LV Coren et al.,

BioTechniques 2015 may be used to provide an appropriate constant domain. Alternatively, single stranded or double stranded DNA or RNA can be inserted by homologous directed repair into the

TCR locus (see Roth ef a/ 2018 Nature vol 559; page 405). As a further option, non — homologous end joining is possible.

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). In other words, a functional TCR a chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 11 is also encompassed. As stated previously, 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). In other words, 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 11).

As an example, 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. In this example, 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.

In examples where the TCR a chain has the amino acid sequence of SEQ ID NO:11, 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 22.1H1.

In one example, 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.

In another example, 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.

In another example, 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.

As provided above, the inventors identified TCR clone 22.1H1 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 22.1H1 are SEQ ID NO:s 1 to 14.

Accordingly, an example of an appropriate TCR VB 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:6. As would be clear to a person of skill in the art, 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 VB domain). Such functional variants are therefore encompassed herein.

For example, appropriate (functional) VB domain CDR3 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 8, 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g.

SEQ ID NO: 6). In other words, appropriate (functional) VB 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.

As stated above, functional variants of SEQ ID NO: 6 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 VB domain.

Functional variants may be naturally occurring, synthetic, or synthetically improved functional variants of SEQ ID NO: 8. 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 CDRS.

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.

In one example, the CDR3 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 6. In examples where the TCR VB domain CDR3 has the amino acid sequence of

SEQ ID NO:6, the CDR3 may be encoded by Any appropriate nucleic acid sequence.

The encoded TCR VB 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 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: 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. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

For example, appropriate functional VB 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 4). In other words, appropriate (functional) VB domain CDR1 amino acid sequences may vary from the sequence shown in SEQ

ID NO:4 by one or several amino acids. As stated previously, 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). As stated above, 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 VB domain).

In one example, the CDR1 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 4. In examples where the TCR Va domain CDR1 has the amino acid sequence of

SEQ ID NO:4, the CDR1 may be encoded by any appropriate nucleic acid sequence.

The encoded TCR VB 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.

For example, appropriate functional VB 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 5). In other words, appropriate (functional) VB domain CDR2 amino acid sequences may vary from the sequence shown in SEQ ID NO: 5 by one or several amino acids. As stated previously, 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). As stated above, a functional variant of SEQ

ID NO: 5 retains the ability to specifically bind to HLA-A*02:01.

In one example, the CDR2 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 5. In examples where the TCR VB domain CDR2 has the amino acid sequence of

SEQ ID NO:5, the CDR2 may be encoded by any appropriate nucleic acid sequence.

The encoded TCR VB 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 VB domain may have an amino acid sequence of SEQ ID NO: 9, or a functional variant thereof (i.e. wherein the variant TCR VB domain retains the 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). 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. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

In one example, the encoded TCR VB 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). In other words, a functional TCR VB domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 9 is also encompassed. As stated previously, 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 VB 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). In other words, 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 9).

As an example, the encoded TCR VB 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 VB domain comprises a

CDRS having an amino acid sequence of SEQ ID NO: 6. In this example, the TCR VB domain

CDR1 may have an amino acid sequence of SEQ ID NO:4 and the TCR VB domain CDR2 may have an amino acid sequence of SEQ ID NO: 5.

In examples where the TCR VB domain has the amino acid sequence of SEQ ID NO:9, the TCR

VB 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).

For the avoidance of doubt, the nucleic acid sequence encoding the TCR VB domain may also encode a TCR B chain constant domain. Examples of suitable constant domains are generally discussed above.

An example of a specific TCR B chain amino acid sequence that includes a TCR VB 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 B 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). In other words, a functional TCR B chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 13 is also encompassed. As stated previously, 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 B chain that do not form CDRs (i.e. the variant may have the CDRs of SEQ

ID NO: 8, SEQ ID NO: 4 and/or SEQ ID NO: 5, and still have 25% (or less) sequence variability compared to SEQ ID NO:13. In other words, 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 13).

As an example, the encoded TCR B 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 B chain comprises a

CDR3 having an amino acid sequence of SEQ ID NO: 6. In this example, the TCR B chain CDR1 may have an amino acid sequence of SEQ ID NO: 4 and the TCR B chain CDR2 may have an amino acid sequence of SEQ ID NO: 5.

In examples where the TCR B chain has the amino acid sequence of SEQ ID NO:13, the TCR B 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 B chain of clone 22.1H1.

In an example, the nucleic acid composition provided herein comprises a nucleic acid sequence that encodes a TCR VB domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:6, or a functional fragment thereof.

In another example, the CDR3 of the VB domain of a nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO:6.

In a further example, the VB 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.

The TCR VB domain sequences derived from TCR clone 22.1H1 discussed above are particularly compatible with the TCR Va domain sequences derived from TCR clone 22.1H1 discussed elsewhere herein.

Accordingly, in one example, 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 VB domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:6, or a functional fragment thereof.

In a particular example, a nucleic acid composition described herein encodes a WT 1antigen- 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 VB domain with a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:6. In addition, the WT1 antigen may comprise or consist of the sequence shown in SEQ ID NO: 71. Furthermore, the

TCR Va domain may be part of a TCR a chain having a constant domain and the TCR VB domain may be part of a TCR B chain having a constant domain.

In this particular example, 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 VB domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 9. In one example, the Va domain comprises the amino acid sequence of SEQ ID NO: 7 and the VB domain comprises the amino acid sequence of SEQ ID NO: 9. In such cases, the Va domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 8; and the VB domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 10.

In this particular example, 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. Furthermore, the TCR VB 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.

For the avoidance of doubt, this particular example encompasses components of TCR clone 22.1H1 exemplified herein. The different components of TCR clone 22.1H1 and their respective

SEQ ID Nos are summarised in Table 5 below.

As stated in more detail elsewhere herein, the nucleic acid composition described herein encodes both a TCR Va domain and a TCR VB domain, which form the binding protein that is capable of specifically binding to a WT1 antigen. In examples where the TCR Va domain and the TCR VB domain are encoded by the same nucleic acid sequence, the TCR Va domain and TCR VB domain may be joined together via a linker, e.g. a linker that enables expression of two proteins or polypeptides from the same vector. By way of example, 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. Any other appropriate linker may also be used. As a further example, the nucleic acid sequence encading the TCR Va domain and nucleic acid sequence encoding the TCR VB domain may be cloned into a vector with dual internal promoters (see e.g. 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 VB domain is well within the routine capabilities of a person of skill in the art.

Additional appropriate polypeptide domains may also be encoded by the nucleic acid sequences that encode the TCR Va domain and/or the TCR VB domain. By way of example only, 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 WO2018/071758.

In one example, the nucleic acid composition described herein may encode a soluble TCR. For example, 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). 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. W02004/106380; U.S.

Patent Application Publication No. 2004/0202657; U.S. Pat. No. 6,750,325). Immune mobilising

MTCR Against Cancer (ImmTAC; Immunocore Limited, Milton Partk, Abington, Oxon, United

Kingdom) are bifunctional proteins that combine affinity monoclonal T cell receptor (mTCR) targeting with a therapeutic mechanism of action (i.e., an anti-CD3 scFv). In another example, 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.

In one example, 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. In this example, the linker is non-cleavable. In an alternative embodiment, 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. (ii) TCR components that interact with VLDFAPPGA (SEQ ID NO: 72)

As provided elsewhere herein, 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 20.2D10 are SEQ ID NO:s 15 to 28.

In one embodiment, 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 (VB) domain is provided, 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 VB 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.

Accordingly, 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. As would be clear to a person of skill in the art, 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). Such functional variants are therefore encompassed herein.

For example, 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 17).

In other words, 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.

As stated above, functional variants of 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.

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 CDRS.

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. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

In one example, the CDR3 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 17. In examples where the TCR Va domain CDR3 has 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 WT1 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. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

For example, 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:15). In other words, 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. As stated previously, 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. As stated above, 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).

In one example, the CDR1 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 15. In examples where the TCR Va domain CDR1 has the amino acid sequence of SEQ ID NO:15, the CDR1 may be encoded by any appropriate nucleic acid sequence.

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:186, 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:18 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.

For example, appropriate functional Va domain CDR2 amino acid sequences may have at least 80% sequence identity to SEQ ID NO:18, i.e. it may have at least 80%, at least 87%, or 100% sequence identity to SEQ ID NO:16. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NQ:16). In other words, 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. As stated previously, 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). As stated above, a functional variant of SEQ

ID NO:16 retains the ability to specifically bind to HLA-A*02:01.

In one example, the CDR2 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 16. In examples where the TCR Va domain CDR2 has the amino acid sequence of SEQ ID NO:18, the CDR2 may be encoded by any appropriate nucleic acid sequence.

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 WT1 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. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

In one example, 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). In other words, a functional TCR Va domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO:21 is also encompassed. As stated previously, 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:186, and still have 25% (or less) sequence variability compared to SEQ ID NO:21). In other words, 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 21).

As an example, 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: 21, wherein the TCR Va domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 17. In this example, 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.

As another example, 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. In this example, 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.

In examples where the TCR Va domain has the amino acid sequence of SEQ ID NO:21, 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).

For the avoidance of doubt, 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.

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). In other words, a functional TCR a chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO:25 is also encompassed. As stated previously, 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

ID NO: 17, SEQ ID NO: 15 and/or SEQ ID NO: 18, and still have 25% (or less) sequence variability compared to SEQ ID NO: 11). In other words, 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 25).

As an example, 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. In this example, 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.

In examples where the TCR a chain has the amino acid sequence of SEQ ID NO:25, the TCR a 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:26 is the nucleic acid sequence for TCR a chain of clone 20.3D10.

In one example, 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.

In another example, 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.

ID NO: 21.

As provided elsewhere herein, 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.

An example of an appropriate TCR VB 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:20. As would be clear to a person of skill in the art, variants of the amino acid sequence shown in SEQ

ID NO:20 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: 72) when the CDR3 is part of TCR VB domain). Such functional variants are therefore encompassed herein.

For example, appropriate (functional) VB 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g.

SEQ ID NO: 20). In other words, appropriate (functional) VB 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.

As stated above, functional variants of SEQ ID NO: 20 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 VB 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 CDRS.

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.

In one example, the CDR3 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 20. In examples where the TCR VB domain CDR3 has the amino acid sequence of SEQ ID NO:20, the CDR3 may be encoded by any appropriate nucleic acid sequence.

The encoded TCR VB 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 WT1 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. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

For example, appropriate functional VB 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 18). In other words, appropriate (functional) VB domain CDR1 amino acid sequences may vary from the sequence shown in SEQ

ID NO: 18 by one or several amino acids. As stated previously, 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). As stated above, 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 V domain).

In one example, the CDR1 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 18. In examples where the TCR Va domain CDR1 has the amino acid sequence of SEQ ID NO:18, the CDR1 may be encoded by any appropriate nucleic acid sequence.

The encoded TCR VB 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.

For example, appropriate functional VB 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 19). In other words, appropriate (functional) VB domain CDR2 amino acid sequences may vary from the sequence shown in SEQ ID NO: 19 by one or several amino acids. As stated previously, 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). As stated above, a functional variant of

SEQ ID NO: 19 retains the ability to specifically bind to HLA-A*02:01.

In one example, the CDR2 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 19. In examples where the TCR VB domain CDR2 has the amino acid sequence of SEQ ID NO: 19, the CDR2 may be encoded by any appropriate nucleic acid sequence.

SEQ ID specifically i.e. SEQ ID NO:20, SEQ ID NO: 18 and SEQ ID NO: 19, or functional variants thereof), with appropriate intervening sequences between the CDRs.

The encoded TCR VB domain may have an amino acid sequence of SEQ ID NO: 23, or a functional variant thereof (i.e. wherein the variant TCR VB domain retains the 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). 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. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

In one example, the encoded TCR VB 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). In other words, a functional TCR VB domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 23 is also encompassed. As stated previously, 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 VB 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). In other words, 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 23).

As an example, the encoded TCR VB 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 VB domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 20. In this example, the TCR VB domain

CDR1 may have an amino acid sequence of SEQ ID NO:18 and the TCR VB domain CDR2 may have an amino acid sequence of SEQ ID NO: 19.

In examples where the TCR V domain has the amino acid sequence of SEQ ID NO:23, the TCR

VB 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).

An example of a specific TCR B chain amino acid sequence that includes a TCR VB 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 B 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). In other words, a functional TCR B chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 27 is also encompassed. As stated previously, 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 B chain 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:27. In other words, 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 27).

As an example, the encoded TCR B 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 B chain comprises a

CDR3 having an amino acid sequence of SEQ ID NO: 20. In this example, the TCR B chain CDR1 may have an amino acid sequence of SEQ ID NO: 18 and the TCR B chain CDR2 may have an amino acid sequence of SEQ ID NO: 19.

In examples where the TCR B chain has the amino acid sequence of SEQ ID NO:27, the TCR B 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 B chain of clone 20.3D10.

In an example, the nucleic acid composition provided herein comprises a nucleic acid sequence that encodes a TCR VB domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:20, or a functional fragment thereof.

In another example, the CDR3 of the VB domain of a nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO:20.

ID NO: 23.

The TCR VB 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.

Accordingly, in one example, 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 VB domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:20, or a functional fragment thereof.

In a particular example, a nucleic acid composition described herein encodes a WT1 antigen- 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: 17; and a TCR VB domain with a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:20. In addition, the WT1 antigen may comprise or consist of the sequence shown in SEQ ID NO: 72. Furthermore, the

In this particular example, 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 VB domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 23. In one example, the Va domain comprises the amino acid sequence of SEQ ID NO: 21 and the VB domain comprises the amino acid sequence of SEQ ID NO: 23. In such cases, the Va domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 22; and the VB domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 24.

In this particular example, 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. Furthermore, the TCR VB 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.

For the avoidance of doubt, 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.

As stated in more detail elsewhere herein, the nucleic acid composition described herein encodes both a TCR Va domain and a TCR VB domain, which form the binding protein that is capable of specifically binding to a WT1 antigen. In examples where the TCR Va domain and the TCR VB domain are encoded by the same nucleic acid sequence, the TCR Va domain and TCR VB 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 VB domain are also discussed generally elsewhere herein.

In one example, 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. These are discussed generally in more detail elsewhere herein. (iii) alternative TCR components that interact with VLDFAPPGA (SEQ ID NO: 72)

As provided elsewhere herein, the inventors have also 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.

In one embodiment, 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 (VB) domain is provided, 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 VB 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.

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. As would be clear to a person of skill in the art, 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 WT1 antigen (e.g. to the peptide VLDFAPPGA (SEQ ID NO: 72)) when the CDR3 is part of TCR Va domain).

Such functional variants are therefore encompassed herein.

For example, 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:31).

In other words, 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.

As stated above, functional variants of SEQ ID NO: 31 retain their ability to confer specific binding to a WT1 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. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

In one example, the CDR3 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 31. In examples where the TCR Va domain CDR3 has 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 WT1 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. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

For example, 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 29). In other words, 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. As stated previously, 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. As stated above, 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).

In one example, the CDR1 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 29. In examples where the TCR Va domain CDR1 has the amino acid sequence of SEQ ID NO: 29, the CDR1 may be encoded by any appropriate nucleic acid sequence.

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.

For example, 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO:30). In other words, appropriate (functional) Va domain CDR2 amino acid sequences may vary from the sequence shown in SEQ ID NO:30 by one or several amino acids. As stated previously, 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). As stated above, a functional variant of

SEQ ID NO: 30 retains the ability to specifically bind to HLA-A*02:01.

In one example, the CDR2 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 30. In examples where the TCR Va domain CDR2 has the amino acid sequence of SEQ ID NO:30, the CDR2 may be encoded by any appropriate nucleic acid sequence.

SEQ ID specifically i.e. SEQ ID NO: 31, SEQ ID NO: 22 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 WT1 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: 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. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

In one example, 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). In other words, a functional TCR Va domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 35 is also encompassed. As stated previously, 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). In other words, 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 35).

As an example, 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: 35, wherein the TCR Va domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 31. In this example, 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.

As another example, 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. In this example, 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.

In examples where the TCR Va domain has the amino acid sequence of SEQ ID NO: 35, 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).

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). In other words, a functional TCR a chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 39 is also encompassed. As stated previously, 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 TCR a chain that do not form CDRs (i.e. the variant may have the CDRs of SEQ

ID NO: 31, SEQ ID NO: 28 and/or SEQ ID NO: 30, and still have 25% (or less) sequence variability compared to SEQ ID NO: 39). In other words, 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 39).

As an example, 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. In this example, the TCR a chain CDR1 may have an amino acid sequence of SEQ ID NO: 28 and the TCR a chain CDR2 may have an amino acid sequence of SEQ ID NO: 30.

In examples where the TCR a chain has the amino acid sequence of SEQ ID NO: 39, 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 NO:40 is the nucleic acid sequence for TCR a chain of clone 23.2G9.

In one example, 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.

In another example, 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.

ID NO: 35.

As provided above, 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.

An example of an appropriate TCR VB 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:34. As would be clear to a person of skill in the art, 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 WT1 antigen (i.e. the peptide shown in SEQ ID NO: 72) when the CDR3 is part of TCR VB domain). Such functional variants are therefore encompassed herein.

For example, appropriate (functional) VB 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 34).

In other words, appropriate (functional) VB 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. As stated above, functional variants of SEQ ID NO: 34 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 VB 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.

In one example, the CDR3 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 34. In examples where the TCR VB domain CDR3 has the amino acid sequence of SEQ ID NO:34, the CDR3 may be encoded by any appropriate nucleic acid sequence.

The encoded TCR VB 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 WT1 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. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

For example, appropriate functional VB 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 32). In other words, appropriate (functional) VB domain CDR1 amino acid sequences may vary from the sequence shown in SEQ

ID NO:32 by one or several amino acids. As stated previously, 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). As stated above, 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 VB domain).

In one example, the CDR1 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 32. In examples where the TCR Va domain CDR1 has the amino acid sequence of SEQ ID NO:32, the CDR1 may be encoded by any appropriate nucleic acid sequence.

The encoded TCR VB 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.

For example, appropriate functional VB 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 33). In other words, appropriate (functional) VB domain CDR2 amino acid sequences may vary from the sequence shown in SEQ ID NO: 33 by one or several amino acids. As stated previously, 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). As stated above, a functional variant of

SEQ ID NO: 33 retains the ability to specifically bind to HLA-A*02:01.

In one example, the CDR2 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 33. In examples where the TCR VB domain CDR2 has the amino acid sequence of SEQ ID NO:33, the CDR2 may be encoded by any appropriate nucleic acid sequence.

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 VB domain may have an amino acid sequence of SEQ ID NO: 37, or a functional variant thereof (i.e. wherein the variant TCR VB domain retains the 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). 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. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

In one example, the encoded TCR VB 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). In other words, a functional TCR VB domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 37 is also encompassed. As stated previously, 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 VB 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). In other words, 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 37).

As an example, the encoded TCR VB 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 VB domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 34. In this example, the TCR VB domain

CDR1 may have an amino acid sequence of SEQ ID NO:32 and the TCR VB domain CDR2 may have an amino acid sequence of SEQ ID NO: 33.

In examples where the TCR VB domain has the amino acid sequence of SEQ ID NO:37, the TCR

VB 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).

An example of a specific TCR B chain amino acid sequence that includes a TCR VB 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 B 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). In other words, a functional TCR B chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 41 is also encompassed. As stated previously, 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 B 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. In other words, 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 41).

As an example, the encoded TCR B 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 B chain comprises a

CDR3 having an amino acid sequence of SEQ ID NO: 34. In this example, the TCR B chain CDR1 may have an amino acid sequence of SEQ ID NO: 32 and the TCR B chain CDR2 may have an amino acid sequence of SEQ ID NO: 33.

In examples where the TCR B chain has the amino acid sequence of SEQ ID NO:41, the TCR B 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 B chain of clone 23.2G9.

In an example, the nucleic acid composition provided herein comprises a nucleic acid sequence that encodes a TCR VB domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:34, or a functional fragment thereof.

In another example, the CDR3 of the VB domain of a nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO:34.

ID NO: 37.

The TCR VB 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.

Accordingly, in one example, 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 VB domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:34, or a functional fragment thereof.

In a particular example, a nucleic acid composition described herein encodes a WT1 antigen- 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: 31; and a TCR VB domain with a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:34. In addition, the WT1 antigen may comprise or consist of the sequence shown in SEQ ID NO: 100. Furthermore, the

In this particular example, 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 VB domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 37. In one example, the Va domain comprises the amino acid sequence of SEQ ID NO: 35 and the VB domain comprises the amino acid sequence of SEQ ID NO: 37. In such cases, the Va domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 36; and the VB domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 38.

In this particular example, 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 NO:30. Furthermore, the TCR VB 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.

For the avoidance of doubt, 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.

In one example, 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. These are discussed generally in more detail elsewhere herein. (iv) TCR components that interact with TPYSSDNLY (SEQ ID NO: 73)

As provided elsewhere herein, the inventors have also 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.

In one embodiment, 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 B chain variable (VB) domain is provided, 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 VB 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 WT1.

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.

As would be clear to a person of skill in the art, 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 WT1 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.

For example, 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 45).

In other words, 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.

As stated above, functional variants of SEQ ID 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.

In one example, the CDR3 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 45. In examples where the TCR Va domain CDR3 has 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 WT1 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.

For example, 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 43). In other words, 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. As stated previously, 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. As stated above, 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).

In one example, the CDR1 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 43. In examples where the TCR Va domain CDR1 has the amino acid sequence of SEQ ID NO: 43, the CDR1 may be encoded by any appropriate nucleic acid sequence.

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.

For example, 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 44). In other words, 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. As stated previously, 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). As stated above, a functional variant of

SEQ ID NO: 44 retains the ability to specifically bind to HLA-B*35:01.

In one example, the CDR2 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 44. In examples where the TCR Va domain CDR2 has the amino acid sequence of SEQ ID NO: 44, the CDR2 may be encoded by any appropriate nucleic acid sequence.

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 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: 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.

In one example, 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). In other words, a functional TCR Va domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 49 is also encompassed. As stated previously, 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). In other words, 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 49).

As an example, 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: 49, wherein the TCR Va domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 45. In this example, 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.

As another example, 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 O 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. In this example, 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.

In examples where the TCR Va domain has the amino acid sequence of SEQ ID NO: 49, 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).

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). In other words, a functional TCR a chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO:53 is also encompassed. As stated previously, 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

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: 53). In other words, the 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 53).

As an example, 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. In this example, 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.

In examples where the TCR a chain has the amino acid sequence of SEQ ID NO:53, 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.

In one example, 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.

In another example, 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.

ID NO: 48.

As provided above, 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.

An example of an appropriate TCR VB 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. As would be clear to a person of skill in the art, 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 WT1 antigen (i.e. the peptide shown in SEQ ID NO: 73) when the CDR3 is part of TCR VB domain). Such functional variants are therefore encompassed herein.

For example, appropriate (functional) VB 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 48).

In other words, appropriate (functional) VB 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. As stated above, functional variants of SEQ ID NO: 48 retain their ability to confer specific binding to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 73) when the CDR3 is part of TCR VB 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.

In one example, the CDR3 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 48. In examples where the TCR VB domain CDR3 has the amino acid sequence of SEQ ID NO:48, the CDR3 may be encoded by Any appropriate nucleic acid sequence.

The encoded TCR VB 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 WT1 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.

For example, appropriate functional VB 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: 48. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 48). In other words, appropriate

(functional) VB domain CDR1 amino acid sequences may vary from the sequence shown in SEQ

ID NO:46 by one or several amino acids. As stated previously, 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). As stated above, 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 VB domain).

In one example, the CDR1 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 46. In examples where the TCR Va domain CDR1 has the amino acid sequence of SEQ ID NO:48, the CDR1 may be encoded by any appropriate nucleic acid sequence.

The encoded TCR VB 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.

For example, appropriate functional VB 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 47). In other words, appropriate (functional) VB domain CDR2 amino acid sequences may vary from the sequence shown in SEQ ID NO: 47 by one or several amino acids. As stated previously, 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). As stated above, a functional variant of

SEQ ID NO: 47 retains the ability to specifically bind to HLA-B*35:01.

In one example, the CDR2 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 47. In examples where the TCR VB domain CDR2 has the amino acid sequence of SEQ ID NO:47, the CDR2 may be encoded by any appropriate nucleic acid sequence.

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 VB domain may have an amino acid sequence of SEQ ID NO: 51, or a functional variant thereof (i.e. wherein the variant TCR VB 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. 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: 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.

In one example, the encoded TCR VB 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). In other words, a functional TCR VB domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 51 is also encompassed. As stated previously, 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 VB 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). In other words, 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 51).

As an example, the encoded TCR VB 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 VB domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 48. In this example, the TCR VB domain

CDR1 may have an amino acid sequence of SEQ ID NO:46 and the TCR VB domain CDR2 may have an amino acid sequence of SEQ ID NO: 47.

In examples where the TCR VB domain has the amino acid sequence of SEQ ID NO:51, the TCR

VB 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).

An example of a specific TCR B chain amino acid sequence that includes a TCR VB 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 B 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). In other words, a functional TCR 8 chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 55 is also encompassed. As stated previously, 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 B chain 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:55. In other words, the 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 55).

As an example, the encoded TCR B 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 B chain comprises a

CDR3 having an amino acid sequence of SEQ ID NO: 48. In this example, the TCR B chain CDR1 may have an amino acid sequence of SEQ ID NO: 46 and the TCR B chain CDR2 may have an amino acid sequence of SEQ ID NO: 47.

In examples where the TCR B chain has the amino acid sequence of SEQ ID NO:55, the TCR B 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 B chain of clone 17.2G4.

In an example, the nucleic acid composition provided herein comprises a nucleic acid sequence that encodes a TCR VB domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:48, or a functional fragment thereof.

In another example, the CDR3 of the VB domain of a nucleic acid composition provided herein comprises or consists of the amino acid sequence of SEQ ID NO:48.

ID NO: 51.

The TCR VB 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.

Accordingly, in one example, 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 VB domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:48, or a functional fragment thereof.

In a particular example, a nucleic acid composition described herein encodes a WT1 antigen- 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: 45; and a TCR VB domain with a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:48. In addition, the WT1 antigen may comprise or consist of the sequence shown in SEQ ID NO: 101. Furthermore, the

In this particular example, 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 VB domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 51. In one example, the Va domain comprises the amino acid sequence of SEQ ID NO: 49 and the VB domain comprises the amino acid sequence of SEQ ID NO: 51. In such cases, the Va domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 50; and the VB domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 52.

In this particular example, 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. Furthermore, the TCR VB 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.

For the avoidance of doubt, 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.

In one example, 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. These are discussed generally in more detail elsewhere herein. (v) TCR components that interact with VLDFAPPGASAY (SEQ ID NO: 74)

As provided elsewhere herein, the inventors have also 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.

In one embodiment, an isolated nucleic acid composition that encodes a Wilms’ tumor 1 (WT 1) antigen-specific binding protein having a TCR a chain variable (Va) domain and a TCR B chain variable (VB) domain is provided, 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 VB 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.

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. As would be clear to a person of skill in the art, 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 WT1 antigen (e.g. to the peptide VLDFAPPGASAY (SEQ ID NO: 74)) when the CDR3 is part of TCR

Va domain). Such functional variants are therefore encompassed herein.

For example, 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 59).

In other words, 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.

As stated above, functional variants of SEQ ID 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.

In one example, the CDR3 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 59. In examples where the TCR Va domain CDR3 has 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 WT1 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.

For example, 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 57). In other words, 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. As stated previously, 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. As stated above, 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).

In one example, the CDR1 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 57. In examples where the TCR Va domain CDR1 has the amino acid sequence of SEQ ID NO: 57, the CDR1 may be encoded by any appropriate nucleic acid sequence.

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.

For example, 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. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 58). In other words, 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. As stated previously, 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). As stated above, a functional variant of

SEQ ID NO: 58 retains the ability to specifically bind to HLA-A*01:01.

In one example, the CDR2 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 58. In examples where the TCR Va domain CDR2 has the amino acid sequence of SEQ ID NO: 58, the CDR2 may be encoded by any appropriate nucleic acid sequence.

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 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: 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: 83 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.

In one example, 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). In other words, a functional TCR Va domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 63 is also encompassed. As stated previously, 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). In other words, the sequence of the CDRs of

SEQ ID NO: 83 may be retained whilst the rest of the sequence is varied, as appropriate within the “at least 75% identity” parameters specified above. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 63).

As an example, 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: 63, wherein the TCR Va domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 59. In this example, 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.

As another example, 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. In this example, 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.

In examples where the TCR Va domain has the amino acid sequence of SEQ ID NO: 63, 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).

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). In other words, a functional TCR a chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO:67 is also encompassed. As stated previously, 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). In other words, the 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 87).

As an example, 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. In this example, 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.

In examples where the TCR a chain has the amino acid sequence of SEQ ID NO: 67, 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.

In one example, 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.

In another example, 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.

ID NO: 63.

As provided above, 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.

An example of an appropriate TCR VB 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:62.

SEQ ID NO:82 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 VB domain).

Such functional variants are therefore encompassed herein.

For example, appropriate (functional) VB domain CDR3 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 82, i.e. they may have at least 80%, at least 87%, or 100% sequence identity to SEQ ID NO: 62. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 62). In other words, appropriate (functional) VB 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. As stated above, functional variants of SEQ ID NO: 62 retain their ability to confer specific binding to a WT1 antigen (e.g. the peptide shown in SEQ ID NO: 74) when the CDR3 is part of TCR VB 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.

In one example, the CDR3 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 62. In examples where the TCR VB domain CDR3 has the amino acid sequence of SEQ ID NO:82, the CDR3 may be encoded by any appropriate nucleic acid sequence.

The encoded TCR VB 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 WT1 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: 80, 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.

For example, appropriate functional VB domain CDR1 amino acid sequences may have at least 80% sequence identity to SEQ ID NO: 80, i.e. it may have at least 80%, or 100% sequence identity to SEQ ID NO: 60. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 60). In other words, appropriate (functional) VB domain CDR1 amino acid sequences may vary from the sequence shown in SEQ

ID NO:60 by one or several amino acids. As stated previously, the variant may comprise an amino acid substitution such as a conservative amino acid substitution compared to the sequence shown in SEQ ID NO:60). As stated above, 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: 101) when the CDR1 is part of TCR VB domain).

In one example, the CDR1 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 80. In examples where the TCR Va domain CDR1 has the amino acid sequence of SEQ ID NO:60, the CDR1 may be encoded by any appropriate nucleic acid sequence.

The encoded TCR VB 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. 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: 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: 81 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.

For example, appropriate functional VB 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: 81. Suitably, percent identity is calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 61). In other words, appropriate (functional) VB domain CDR2 amino acid sequences may vary from the sequence shown in SEQ ID NO: 61 by one or several amino acids. As stated previously, 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). As stated above, a functional variant of SEQ ID NO: 61 retains the ability to specifically bind to HLA-A*01:01.

In one example, the CDR2 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 61. In examples where the TCR VB domain CDR2 has the amino acid sequence of SEQ ID NO:81, the CDR2 may be encoded by any appropriate nucleic acid sequence.

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 VB domain may have an amino acid sequence of SEQ ID NO: 65, or a functional variant thereof (i.e. wherein the variant TCR VB 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 mere 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. Methods for identifying functional and non-functional variants are well known to a person of ordinary skill in the art.

In one example, the encoded TCR VB 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). In other words, a functional TCR VB domain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 85 is also encompassed. As stated previously, 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 VB 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). In other words, 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 65).

As an example, the encoded TCR VB 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 VB domain comprises a CDR3 having an amino acid sequence of SEQ ID NO: 82. In this example, the TCR VB domain

CDR1 may have an amino acid sequence of SEQ ID NO:80 and the TCR VB domain CDR2 may have an amino acid sequence of SEQ ID NO: 81.

In examples where the TCR VB domain has the amino acid sequence of SEQ ID NO:65, the TCR

VB 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).

An example of a specific TCR B chain amino acid sequence that includes a TCR VB 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 B 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). In other words, a functional TCR B chain with one or several amino acid substitutions compared to the sequence of SEQ ID NO: 69 is also encompassed. As stated previously, 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 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. In other words, the 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. Suitably, percent identity can be calculated as the percentage of identity to the entire length of the reference sequence (e.g. SEQ ID NO: 69).

As an example, the encoded TCR 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 B chain comprises a

CDR3 having an amino acid sequence of SEQ ID NO: 62. In this example, the TCR B chain CDR1 may have an amino acid sequence of SEQ ID NO: 60 and the TCR B chain CDR2 may have an amino acid sequence of SEQ ID NO: 61.

In examples where the TCR B chain has the amino acid sequence of SEQ ID NO:69, the TCR B 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 NO:70 is the nucleic acid sequence for TCR B chain of clone 12.5H9.

In an example, the nucleic acid composition provided herein comprises a nucleic acid sequence that encodes a TCR VB domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:62, or a functional fragment thereof.

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

ID NO: 65.

The TCR VB 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.

Accordingly, in one example, 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 VB domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO:62, or a functional fragment thereof.

In a particular example, a nucleic acid composition described herein encodes a WT1 antigen- 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: 59; and a TCR VB domain with a CDR3 comprising or consisting of the amino acid sequence of SEQ ID NO:82. In addition, the WT1 antigen may comprise or consist of the sequence shown in SEQ ID NO: 101. Furthermore, the

In this particular example, 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 VB domain may comprise an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of, SEQ ID NO: 65. In one example, the Va domain comprises the amino acid sequence of SEQ ID NO: 83 and the VB domain comprises the amino acid sequence of SEQ ID NO: 65. In such cases, the Va domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 84; and the VB domain may be encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 66.

In this particular example, 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. Furthermore, the TCR VB domain may include a CDR1 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO:60 and a CDR2 amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 61.

For the avoidance of doubt, 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.

As stated in more detail elsewhere herein, the nucleic acid composition described herein encodes both a TCR Va domain and a TCR VB domain, which form the binding protein that is capable of specifically binding to a WT1 antigen. In examples where the TCR Va domain and the TCR VB domain are encoded by the same nucleic acid sequence, the TCR Va domain and TCR Vf 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 VB domain are also discussed generally elsewhere herein.

In one example, 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. These are discussed generally in more detail elsewhere herein.

Vector systems

A vector system is also provided which includes a nucleic acid composition described herein. The vector system may have one or more vectors. As discussed previously, 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. In examples where all of the binding protein components are encoded by a single nucleic acid sequence, the nucleic acid sequence may be present within a single vector (and thus the vector system described herein may comprise of one vector only). In examples where 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. In this example, the vector system will comprise a plurality of distinct vectors (i.e. vectors with different nucleotide sequences).

Accordingly, in one example, a vector system is provided, comprising a nucleic acid composition described herein.

Any appropriate vector can be used. By way of example only, 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). Alternatively, single stranded or double stranded DNA or RNA can be used to transfect lymphocytes with a TCR of interest (see Roth et al 2018 Nature vol 559; page 405).

In one example, 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.

As used herein, 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. Preferably the vector is suitable for expression in a cell (i.e. the vector is an “expression vector”). Preferably, the vector is suitable for expression in a human T cell such as a CD8" T cell or CD4* T cell, or stem cell, iPS cell, or NK cell. In certain aspects, the vector is a viral vector, such as a retroviral vector, a lentiviral vector or an adeno-associated vector.

Optionally, the vector is selected from the group consisting of an adenovirus, vaccinia virus, canary poxvirus, herpes virus, minicircle vector and synthetic DNA or synthetic RNA.

Preferably 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” as used herein, 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 for transcription factors, transcriptional terminators, polyadenylation sites etc. Optionally, 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.

Optionally, the vector comprises the nucleic acid sequence of interest operably linked to a promoter. "Promoter", as used herein, 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. “Transcriptional terminator” as used herein, refers to a DNA element, which terminates the function of RNA polymerases responsible for transcribing DNA into RNA. Preferred transcriptional terminators are characterized by arun of T residues preceded by a GC rich dyad symmetrical region.

The vector may comprise a translational control element. “Translational control element”, as used herein, refers to DNA or RNA elements that control the translation of mRNA. Preferred translational control elements are ribosome binding sites. Preferably, 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" as used herein, refers to a motif on the DNA recognized by a restriction enzyme.

The vector may comprise a selectable marker. "Selectable marker" as used herein, 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.

Optionally, the vector may also comprise a suicide gene. “Suicide gene” as used herein, refers to proteins that induce death of the modified cell upon treatment with specific drugs. By way of example, suicide can be induced of 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 Caspase (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.

Preferably 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.

A person of skill in the art will be well aware of the molecular techniques available for the preparation of (expression) vectors and how the (expression) vectors may be transduced or transfected into an appropriate host cell (thereby generating a modified cell described further below). The (expression) vector system described herein can be introduced into cells by conventional techniques such as transformation, transfection or transduction. “Transformation”, “transfection” and “transduction” 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. Appropriate methods for introducing nucleic acid sequences and vectors into host cells such as human cells are well known in the art; see for example

Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory,

Cold Spring Harbor, N.Y; Ausubel et al (1987) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY; Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110; Luchansky et al (1988) Mol. Microbiol. 2, 637-646. Further conventional methods that are suitable for preparing expression vectors and introducing them into appropriate host cells are described in detail in

WO2016/07 1758 for example.

It is understood that it some examples, 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 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. For example, 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.

The term "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). In the context of the invention, the exogenous sequence comprises at least one of the T cell receptor component parts described herein for any of clones 22.1H1, 20.3D10, 23.2G9, 17.2G4 or 12.5H9 (e.g. the sequences etc that encode the CDR3 sequences that are specific for a WT1 antigen (e.g. the peptide of SEQ ID NO: 71, 72, 73 or 74)).

The term “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.

In one example, 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 NK cell). The host cell (and thus the modified cell) may be an autologous or allogeneic cell (e.g. 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 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.

Accordingly, in an example, the modified cell is a human cell.

The host cell (and thus the modified cell) may be any cell that is able to confer anti-tumour immunity after TCR gene transfer. Non limiting examples of appropriate cells include autologous or allogeneic a CD8 T cell, a CD4 T cell, 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.

Accordingly, in one example 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.

In the context of the methods of treatment described herein, the host cell {and thus the modified cell) is typically for administration to a HLA-A*01:01, HLA-A*02:01, and/or HLA-B*35:01 positive human subject. In view of this, 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).

In the context of the methods of treatment described herein, the host cell (and thus the modified cell} that is to be administered to the subject can either be autologous or allogeneic.

Advantageously, 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

WT1 antigen, and thus can be used to treat or prevent WT1 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.

Pharmaceutical compositions

A nucleic acid composition, vector system, modified cell, or isolated nucleic acid sequence described herein may be provided as part of a pharmaceutical composition. Advantageously, 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.

As used herein, "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. By way of example, 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. The term “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.

Treatment of a subject

Pharmaceutical 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).

Typically, the subject in need of treatment has a disease or condition that is associated with an elevated level of HLA-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. In one example, the

WT1 associated disease or condition may be a hyperproliferative disease or condition.

In one example, the WT1 associated disease or condition may be a hematological malignancy.

In other words, it may be a hematological malignancy with an elevated level of HLA-restricted

WT1 antigens (i.e. WT1 antigens that are presented at the cell surface in the context of an HLA).

Examples of appropriate hematological malignancies are well known in the art, and 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.

In one example, the WT 1 associated disease or condition is AML.

In an alternative example, the WT1 associated disease or condition may be a solid tumor. In other words, it may be a solid tumor with an elevated level of HLA-restricted WT1 antigens (i.e. WT1 antigens that are presented at the cell surface in the context of an HLA). Examples of appropriate solid tumours are well known in the art, and 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. For example, the WT1 associated disease or condition may be a HLA-restricted WT1 antigen expressing tumor or cancer. In other words, the WT1 associated disease or condition may be a

WT1 positive tumor or cancer.

In an example, the pharmaceutical composition provided herein is for use in inducing or enhancing an immune response in human subject diagnosed with a WT 1 associated disease or condition.

As would be clear to a person skilled in the art, an appropriate therapy for subject in need thereof (e.g. an appropriate pharmaceutical composition described herein) may be selected based on the

HLA serotype of the subject.

In one example, if the subject in need thereof is HLA-A*02:01 positive, an appropriate therapy (e.g. an appropriate pharmaceutical composition described herein) may comprise (i) components of TCR clone 22.1H1 exemplified herein; (ii) components of TCR clone 20.3D10 exemplified herein; or (iii) components of TCR clone 23.2G9 exemplified herein. Accordingly, TCRs comprising components of (i) TCR clone 22.1H1 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.

In one example, if the subject in need thereof is HLA-B*35.01 positive, an appropriate therapy (e.g. an appropriate pharmaceutical composition described herein) may comprise components of

TCR clone 17.2G4 exemplified herein. Accordingly, 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.

In one example, if the subject in need thereof is HLA-A*01:01 positive, an appropriate therapy (e.g. an appropriate pharmaceutical composition described herein) may comprise components of

TCR clone 12.5H9 exemplified herein. Accordingly, 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.

The phrase “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).

In another example, 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.

In another example, 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. In such an example,

the target cell population or tissue may be a HLA-restricted WT1 antigen expressing target cell population or tissue. Typically, it is a HLA-restricted WT 1 antigen expressing target cell population or tissue. For example, it may be a target cell population or tissue comprising a HLA-restricted

WT1 antigen expressing tumor or cancer.

The pharmaceutical composition may also be for use in providing anti-tumor immunity to a human subject.

In another example, the pharmaceutical composition may be for use in treating a human subject having a disease or condition associated with an elevated level of HLA-restricted WT1 antigen.

A person of skill in the art will be fully aware of WT1 associated diseases or conditions that may be treated in accordance with the invention. Appropriate examples of such diseases or conditions are discussed elsewhere herein.

As would be clear to a person skilled in the art, the WT1 associated diseases or conditions may comprise at least one tumor (particularly, at least one HLA-restricted WT1 antigen expressing tumor).

As used herein, the terms “treat”, “treating” and "treatment" are taken to include an intervention performed with the intention of preventing the development or altering the pathology of a condition, disorder or symptom (e.g. a WT1 associated disease or condition). Accordingly, "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.

As used herein 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.

The 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.

The compositions described herein may be in any form suitable for the above modes of administration. For example, compositions comprising modified cells may in any form suitable for infusion. As further examples, suitable forms for parenteral injection (including, subcutaneous, intramuscular, intravascular or infusion) include a sterile solution, suspension or emulsion.

Alternatively, 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.

Advantageously, the 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. Using TCR gene transfer, modified cells suitable for infusion may be generated within a few days.

The 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. For example, 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.

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

Methods of generating 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 is also provided, 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.

In the context of the binding proteins described herein, the WT1 antigen comprises or consists of a sequence comprising an amino acid sequence 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. Alternatively, 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 sequence is incorporated and expressed by the cell to generate the binding protein. In one example, the method is not a method of treatment of the human or animal body.

Appropriate in vivo, in vitro and ex vivo methods for contacting a nucleic acid sequence (or vector systems) with a cell under conditions in which the nucleic acid sequence (or vector) is incorporated and expressed by the cell are well known, as described elsewhere herein.

As stated elsewhere herein, 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. Furthermore, 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.

General definitions

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. In one example, the nucleotide sequence lacks introns. In other words, it is an intronless nucleic acid sequence. For example, the nucleotide sequence may be a DNA sequence that does not comprise intron sequences.

As used herein, “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. In other words, 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. Such a 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. The term "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). For example, 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. In this context, such promoters may be considered exogenous promoters. Examples of appropriate promoters are described elsewhere.

As used herein "specifically binds" or "specific for" refers to an association or union of a binding protein (e.g., TCR receptor) or a binding domain (or fusion protein thereof) to a target molecule with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10°M™* (which equals the ratio of the on-rate [kon] to the off-rate [Kog] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample. Binding proteins or binding domains (or fusion proteins thereof) 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 Ka of at least 107 Mt, at least 103 M7, at least 10°M™', at least 1019 M*, at least 10" Mt, at least 1072 M7, or at least 1013 M. Low affinity" binding proteins or binding domains refer to those binding proteins or binding domains having a Ka of up to 107 M™* up to 10°

Mt, up to 105M". Alternatively, affinity can be defined as an equilibrium dissociation constant (Ka) of a particular binding interaction with units of M (e.g., 10° Mto 1073 M).

In certain embodiments, 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. For example, enhanced affinity may be due to a Ka (equilibrium association constant) for the target antigen that is higher than the wild type binding domain, due to a Ky (dissociation constant) for the target antigen that is less than that of the wild type binding domain, due to an off-rate (kor) for the target antigen that is less than that of the wild type binding domain, or a combination thereof. In certain embodiments, 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.

As used herein, the terms “WT 1", “Wilms’ tumor 1”, “transcription factor Wilms’ tumor 1” and “WT1 protein” refer to the transcription factor that is encoded by Wilms’ tumor gene 1 (the WT1 gene) on chromosome 11p . The WT1 protein is a transcription factor that plays an important role in cell growth and differentiation. The WT1 gene is highly expressed in leukemia and various types of solid tumors, whereas WT1 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, WT1 transcription factor, and WT-1. In humans it can be uniquely identified by Uniprot identifiers: P19544 and Q6PI38.

As used herein, the term "WT1 antigen" or "WT1 peptide antigen" or "WT1-containing peptide antigen" refers to a naturally or synthetically produced peptide portion of a WT1 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 WT1 peptide:MHC (e.g., HLA) complex can specifically bind to such as complex. Typically, for the purposes of this disclosure, the WT1 peptide antigen comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74. Additionally, for the purposes of this disclosure, 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.

The term "WT 1-specific binding protein," as used herein, refers to a protein or polypeptide, such as a TCR 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. Typically, for the purposes of this disclosure, the WT1 peptide antigen comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74, and 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.

In certain embodiments, 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% M, less than about 10° M, less than about 10° M, less than about 107! M, less than about 1072 M, or less than about 10713 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. In certain embodiments, a WT1-specific binding protein comprises a WT1-specific immunoglobulin superfamily binding protein or binding portion thereof. Typically, for the purposes of this disclosure, the WT1 peptide antigen comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74, and 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. In other words, in certain embodiments, 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 HLA or is in an equivalent structural formation as when it is being presented by the specific HLA. As discussed elsewhere herein, the inventors identified that 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 WT1 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.

Accordingly, in certain examples, 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. In another example, 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. In another example, 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.

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 WT 1-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. an amino acid sequence selected from the group consisting of: SEQ ID NO: 71 to 74) and does not bind in a significant amount to other polypeptides. Thus 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. This binding may also be determined indirectly in the context of a modified T cell that expresses a WT1 TCR. In assays such as, for example, an assay discussed herein, the modified T cell is specifically reactive against a OVCA cell line or an AML cell line. Thus, 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.

A “non-essential” (or “non-critical”) 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. For example, 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). Thus, 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. Alternatively, in another embodiment, mutations can be introduced randomly, and the resultant mutants can be screened for activity to identify mutants that retain activity.

Calculations of sequence homology or identity (the terms are used interchangeably herein) between sequences are performed as follows.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the 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). In a preferred embodiment, 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. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein 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. In a preferred embodiment, 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 BLOSUM 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. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at hitp://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 (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Alternatively, the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et al. (1989) CAB/OS 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 NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-410). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Altschul et al. (1997, Nucl.

Acids Res. 25:3389-3402). When using BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See <http://www.ncbi.nlm.nih.gov>.

The 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. In other words, amino acid sequences or nucleic acid sequences having one or several (e.g. two, three, four etc) 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). In such examples, the one or several (e.g. two, three, four etc) amino acid or nucleic acid substitutions may be conservative substitutions. For example, 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).

[3] Lefranc M.-P. et al. "IMGT unique numbering for immunoglobulin and Tcell receptor variable domains and Ig superfamily V-like domains" Dev. Comp. Immunol., 27, 55-77 (2003).

[4] Lefranc M.-P. et al. "IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains" Dev. Comp. Immunol., 2005, 29, 185-203 PMID: 15572068.

As used herein, the term “ex vivo” refers to “outside” the body. The term “in vitro” can be used to encompass “ex vivo’ components, compositions and methods.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. For example, Singleton and Sainsbury, Dictionary of Microbiology and Molecular

Biology, 2d Ed., John Wiley and Sons, NY (1994); and Hale and Marham, The Harper

Collins Dictionary of Biology, Harper Perennial, NY (1991) provide those of skill in the art with a general dictionary of many of the terms used in the invention. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present invention, the preferred methods and materials are described herein. Accordingly, the terms defined immediately below are more fully described by reference to the Specification as a whole. Also, as used herein, the singular terms "a", "an," and "the" include the plural reference unless the context clearly indicates otherwise. Unless otherwise indicated, 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.

Aspects of the invention are demonstrated by the following non-limiting examples.

EXAMPLES

Transcription factor Wilms’ tumor gene 1 (WT1) 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. In clinical trials, WT1 is targeted using peptide- or dendritic cell-based vaccines and

T-cell receptor (TCR)-based therapies. Antitumor reactivities were reported, but T-cell reactivity is hampered by self-tolerance to WT1 and limited number of WT1 peptides, which were thus far selected based on peptide prediction algorithms. In this study, 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 WT1 peptides derived from the HLA class | associated ligandome of primary leukemia and ovarian carcinoma (OVCA) samples.

Using broad panels of malignant cells and healthy cell subsets, T-cell clones were selected that demonstrated potent and safe anti-WT1 T-cell reactivity against 5 of these 8 WT1 peptides.

Notably no T- cell clones with antitumor reactivity were identified for WT1 peptides previously used in clinical trials, suggesting limited processing and presentation of these peptides. 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. 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.

Materials and Methods

WT1 expression by Real-Time Quantitative Polymerase Chain Reaction

WT1 expression was quantified by Real-Time Quantitative Polymerase Chain Reaction (RT- gPCR). Total RNA was isolated using the RNAqueous-Micro Kit (Ambion) or ReliaPrep RNA Cell

Miniprep System (Promega). First strand cDNA synthesis was performed with Moloney murine leukemia virus reverse transcriptase and Oligo (dT) primers (Invitrogen by Thermo Fisher

Scientific). RT-gPCR was performed using Fast Start TagDNA Polymerase (Roche) and

EvaGreen (Biotium), and gene expression was measured on the Lightcycler 480 (Roche).

Expression was calculated as percentage relative to the average of housekeeping genes GUSB,

VPS29 and PSMB4, which was set at 100%. All samples and genes were run in triplicate with 10 ng cDNA per reaction. The following primers were used:

WT1: forward: AGACCCACACCAGGACTCAT (SEQ ID NO: 75); reverse:

GATGCATGTTGTGATGGCGG (SEQ ID NO: 76);

GUSB: forward: ACTGAACAGTCACCGACGAG (SEQ ID NO: 77); reverse:

GGAACGCTGCACTTTTTGGT (SEQ ID NO: 78);

PSMB4: forward: GTTTCCGCAACATCTCTCGC (SEQ ID NO: 79); reverse:

CATCAATCACCATCTGGCCG (SEQ ID NO: 80);

VPS29: forward: TGAGAGGAGACTTCGATGAGAATC (SEQ ID NO: 81); reverse:

TCTGCAACAGGGCTAAGCTG (SEQ ID NO: 82).

Sample collection for peptide elution

To identify T-cell epitopes derived from the WT1 gene, in total 37 tumor samples were collected.

Cell pellets (2*10%9 — 610*10"9 cells) were made of 11 ALL samples, 15 AML samples, 1 HCL sample and 2 OVCA cell lines. Also 7 solid primary OVCA samples (2.3 gram — 30 gram) were included and 1 ascites primary OVCA sample (6*10%9 cells). The OVCA samples were residual material and collected anonymously. Solid OVCA tumors were sliced into small pieces and dead, clotted or non-tumor material was removed. 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 IU/ml to remove DNA/RNA complexes during lysis.

HLA typing was performed of all samples and WT1 expression was analyzed by RT-gPCR.

HLA class |-peptide elution procedure, fractionation and mass spectrometry

Peptide elution was performed as outlined previously.[38] In short, the cell pellets were lysed and subjected to an immunoaffinity column to collect bound peptide-HLA complexes, with either an

HLA class- | antibody (W8/32) or an HLA-A*02:01 antibody (BB7.2). To separate the peptides, 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).

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). All unique WT1- derived peptides with a length between 8 and 14 amino acids, a minimal Best Mascot lon (BMI) score of 20, a mass accuracy of 10 ppm and predicted to bind to a common HLA molecule (HLA-

A*01:01, -A*02:01, -A*03:01, -A*24:02, - B*07:02, -B*35:01, -C*07:01, -C*07:02) according to the netMHC peptide binding algorithm[27] were selected.

Peptide synthesis and pMHC-multimer production

Eight peptides met all the criteria and their synthetic peptides were in-house synthesized using standard Fmoc chemistry. By mass spectrometry the tandem mass spectra of the eluted peptides were validated with synthetic peptides (Figure 1). Of in total 12 WT1-derived peptides, pMHC- multimer complexes were generated with minor modifications.[40] In short, monomers consisting of the selected HLA allele heavy chain, human beta-2 microglobulin (B2M) light chain and selected peptide were purified by gel-filtration high-performance liquid chromatography and biotinylated. Subsequently, p-MHC multimers were generated by adding PE-conjugated streptavidin (Invitrogen, Thermo Fisher Scientific).

Cell Culture

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 IU/mL IL-2 (Novartis Pharma). Every 10-14 days, 0.2*10%6 T cells were (re)stimulated with 11046 irradiated (35 Gy) PBMCs, 0.1*1016 irradiated (55 Gy) EBV-LCLs and 0.8 pg/mL phytohemagglutinin (PHA) (Oxoid Microbiology Products, Thermo Fisher Scientific).

Most tumor cell lines were cultured in IMDM, 10% FBS, 1.5% L-glutamine and 1% Pen/Strep.

Ovarian tumor cell lines were cultured in Dulbecco's Modified Eagle Medium (DMEM, high glucose 4.5 g/L, NEAA) (Gibco), 8% FBS, 2% L-glutamine and 1% Pen/Strep. Using the

PlasmoTest Mycoplasma Detection Kit (InvivoGen), all cell lines were found to be mycoplasma negative. Of all cell lines, HLA typing was performed and WT1 expression was analyzed by RT- gPCR. If needed, the WT1 gene or an HLA allele was introduced by retroviral transduction. These genes were expressed in MP71 retroviral backbone vectors with marker genes nerve growth factor receptor (NGF-R), green fluorescent protein (GFP), CD34 or mouse CD19 (mCD19). Target cells were enriched for marker gene expression via MACS or FACS and purity was confirmed by

FACS.

Primary 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.

Healthy cell subset isolation

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 co- culturing 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 (DCs) were differentiated in vitro from isolated CD14+ cells.

Immature DCs were generated by culturing 0.1*10%6 cells/mL for 3 days in IMDM supplemented with 100 ng/mL GM-CSF (Sandoz Novartis Pharma), 500 IU/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 ug/ml PGE-2 (Sigma

Aldrich), 500 IU/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 1g/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 ug/mL bovine pituitary extract (BPE) and 2 ng/mL epithelial growth factor (EGF) (both Thermo Fisher Scientific 37000-015).

Antibodies and FACS analysis

Fluorescence activated cell sorting (FACS) was performed on an LSR Il 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 (Invitrogen/MHCDO0829), murine TCR-B (mTCR-B) 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),

CD88 PE (BD/555658). AML samples with: CD13 PE (BD/347406), CD33 FITC (BD/555626) and

CD34 APC (BD/555824).

Isolation of WT 1-specific T cells by pMHC-multimer enrichment

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 WT 1-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 lll cell sorter (BD Biosciences) in a 96 well round bottom plate containing 5*1074 irradiated PBMCs (35Gy) and 5*10%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 reactivity assays

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 uL TCM in 384-well flat- bottom plates (Greiner Bio-One). To upregulate HLA expression, adherent target cells were treated for 48 hours with 100 IU/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.

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 (1uM).

T-cell mediated cytotoxicity was measured in a 68-hour *'chromium release assay. Target cells were labeled with 100 pCi *'chromium (PerkinElmer) for 1 hour at 37°C, washed, and co-cultured with T cells at various E:T ratios in 100 ul TCM per well in 96-well U-bottom culture plates (Costar).

Spontaneous and maximum 5'Cr release for all targets were measured in separate plates with per well 100 uL TCM or 100 pL TCM with 1% Triton-X 100 (Sigma-Aldrich), respectively. After 6 hours of co-culture, 25 pL supernatant was harvested, transferred to 96-well LumaPlates (PerkinElmer) and *tchromium release was measured in counts per minute on a 2450 Microbeta? plate counter (PerkinElmer). The percentage of killed target cells was calculated with the following formula = ((experimental release — spontaneous release)/(maximum release — spontaneous release)) *100.

TCR identification and production of retroviral supernatants

TCR a and B chains of the selected T-cell clones were identified by sequencing with minor modifications, as previously described.[41] 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 B primers, a SA.rt anchor template-switching oligonucleotide (TSO), and SMARTScribe Reverse Transcriptase (Takara,

Clontech).[42] The TCR a and B 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 VB families were determined of the NGS data using the MiIXCR software and ImMunoGeneTics (IMGT) database.[43] 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 TCRaB constant domains and P2A sequence to link TCR chains, resulting in optimized TCR expression and increased preferential pairing.[44] Apart from the WT1- specific TCRs, 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.

TCR gene transfer to healthy donor CD8+ T cells

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. After removal of the retroviral supernatant, 0.3*10%6 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-B

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.

Study approval

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.

Results

Identification of WT 1-derived peptides in ovarian carcinoma and leukemia samples

To identify WT1-derived peptides that are efficiently expressed in HLA class | at the cell surface of tumors, patient samples were selected based on WT1 expression levels. By RT-qPCR, gene expression was measured in primary OVCA, acute lymphoblastic leukemia (ALL), and AML samples, and healthy cell subsets. WT1 expression (>5% relative expression compared to housekeeping genes) was observed in all OVCA samples (16/16), most ALL samples (3/4) and in more than half of the AML samples (18/30) (Figure 2A). In addition, a variety of tumor cell-lines were tested, of which OVCA, AML, and ALL cell-lines were most prominently positive for WT1 (Figure 2B). These results indicate that especially tumor samples of OVCA, ALL and AML patients could be used for the identification of naturally expressed WT1 peptides.

The HLA class | 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 lon (BMI) score of 20 and a mass accuracy of 10 ppm were selected. 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, -C*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).

1 WTEGQSN | 84 A*01:01 | 43 OVCA | 162% | Solid 8 A*“01: | B*08: | C*04:

HSTGY -G1 tumor gram | 01 01 01

A*11: | B*35: | C*07: 01 01 01 33 ALL- 9%* 62x1 | A*O1 B*18 C*04 1833 09 A*03 B*35 c*07 41 AML- | 13% 110x | A*01: | B*08: | C*07: 4443 10%9 | 01 01 01 42 AML- | 27% 550x | A*01: | B*08: | C*07: 10197 10%9 | 01 01 04

A*02: | B*44: | C*16: 01 03 01 2 | VLDFAPPG | 74 A*01:01 | 36 AML- | 13% 110x | A*01: | B*08: | C*07:

ASAY 4443 10%9 | 01 01 01 3 | ALLPAVPS | 71 A*02:01 | 31 OVCA | 57% Ascites | 7x10 | A*02: | B*38: | C*05:

L -L23 “9 01 01, 01

A*26: B“44: | C“12: 01 02 03 4 | VLDFAPPG | 72 A*02:01 | 26 OVCA | 57% Ascites | 7x10 | A*02: | B*38: | C*05:

A -L23 A9 01 01, 01

A*26: | B*44: | C12: 01 02 03

FGPPPPSQ | 85 A*02:01 | 42 OVCA | 57% Ascites | 7x10 | A*02: | B*38: | C*05:

A -L23 AQ 01 01, 01

A*28: | B*44: | C*12: 01 02 03

AML- | 27% 550x | A*01: | B*08: | C*07: 10197 10%9 | 01 01 01

A*02: | B*44: | C*16: 01 03 01

AQFPNHSF A*03:01 | 20 Cell 9% Cell 2x10 | A*03: | B*40: | C*03:

K line- line AQ 01 01 04

COV3 62.4 7 | HAAQFPNH | 87 B*35:01 | 36 HCL- Spleen | 100x | A*02 B*35 C*04

SF 4512 1000 | A*29

TPYSSDNL | 73 B*35.01 | 41 ALL- | 9% 610x | A*11: | B*35: | C*02:

Y 2184 10%9 | 01 01 02

B*40: | C*04: 02 01

Table 1: Overview of the characteristics of materials included in the HLA ligandome analyses from which the WT1 peptides were identified. The materials are indicated per WT1 peptide, including their predicted HLA binding molecule based on NetMHC and best Mascot ion score (BMI). WT1 expression was determined.by RT-qPCR (*ALL-1833 by Illumina HT-12.0 microarray). OVCA: primary ovarian carcinoma, ALL: acute lymphoblastic leukemia, AML: acute myeloid leukemia,

HCL: hairy cell leukemia, n.d. = not determined.

Eight peptides were identified and the sequences of these peptides were validated by comparing mass spectra of eluted peptides with the mass spectra of synthetic peptides (Figure 1). WT1 peptides frequently studied and used in clinical trials; RMFPNAPYL (SEQ ID NO: 88) (HLA-

A*02:01 restricted)[16], CMTWNQMNL (SEQ ID NO: 89) (HLA-A*02:01 and HLA-A*24:02 restricted)[14, 26] and RWPSCQKKF (SEQ ID NO: 90) (HLA-A*24:02 restricted)[28] were not identified in our large HLA-ligandome database, whereas 20 and 9 of the eluted tumor samples expressed HLA-A*02:01 or HLA-A*24:02, respectively. Considering the frequent use of these peptides, we added them to our final set of WT1 peptides, and in total 12 pMHC-multimer complexes were generated (Table 2).

Nr Peptide HLA Sample/cell line source Best

BMI

WTEGQSNHSTGY | A*01:01 | OVCA-G1, ALL-1833, | 43 84

AML-4443, AML-10197 2 | VLDFAPPGASAY A*01:01 | AML-4443

HAAQFPNHSF B*35:01 | HCL-4512

B | TPYSSDNLY B*35:01 | ALL-2184

Table 2: Overview of the 12 WT 1 peptides included in this study. For the 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.

Large-scale WT 1-reactive T-cell search in the allogeneic T-cell repertoire of 28 healthy donors

With the pMHC-multimers WT1-reactive T cells were searched for, with the ultimate goal to identify high- affinity WT 1-specific TCRs. PBMCs of 28 healthy HLA typed donors were incubated with pMHC-multimers, pMHC-multimer positive CD8+ T cells were enriched by MACS, and single- cell sorted (Table 3).

T-cell clones

Number of T-cell clones reactive

Screened T- reactive .

HLA class | type PBMCs cell clones against WT4 against (x106) gainst transduced peptide WT on EF] [ea [+ unknown

A01, A31, B51,

A01, A03, B13, mel EEE] | ww |e

A24, A29, B15, aaa) ge | ee un MEF ne unknown

AO3, A31, BO7, oo) EEE oe oan) EERE | om | ew | ow a ga | wm | a | 0 own] EEE | we | we |e oe al BEE | wm | ow | + | oo

DE | wo | ow |e | 1 one] gia | we | ow one] BES | we | we ona] GE] | ww |e asa20 | zate | ae | 7

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. For each healthy donor, HLA class | typing, number of PBMCs, and expanded single-cell sorted T-cell clones are listed. In total 461 of 7916 screened T-cell clones were reactive against Raji pulsed with the WT1 peptide pool (1 uM), and 71 clones were also reactive against Raji transduced with WT 1. The reactivity is based on IFN-y 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. On average 650106 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). Two weeks after clonal expansion, a peptide specificity screening was conducted on 7916 isolated T-cell clones.

Burkitt lymphoma cell line Raji, negative for WT1, transduced with HLA-alleles of interest, were ulsed with or without the WT1 peptide pool, and used as stimulator cells. In total 461 T-cell p pep p clones (6%) were WT1 peptide specific, recognizing WT1 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 WT1 peptides.

Peptide HLA Donors WT1- used for | reactive

T-cell T-cell Ivzed isolation clones analyze

WiEGASNHSTGY | e4 [aormt] 2 | [ 5 | FoPPPPSOA | & [ao] 1 | 7 6 | AQPPNRSFK | 8 [Awa] 5 | 1 7 | AAQFPNHSF | 37 [80] qe | B 3 | TPYSSONLY | 75 [eso] 19 | 3 | 17264 3 | RWEPNAPYL | 8 [at] 7 | B © | OMTWNQUNL | 85 [aat] 7 | 2

A | RWPSCORKF | © [aa] 1 “fo | CWTWNQUNL | 85 [eo] 4

DLL [ 4

Table 4: Overview of T-cell 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 WT1. The TCRs of 4 T-cell clones were sequenced and further analyzed.

Identification of 5 safe and potent WT 1-reactive T-cell clones

To select the most safe and potent WT 1-reactive T-cell clones, the 71 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 WT1 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 WT1 positive tumor cell lines, combined with no recognition of WT1 negative tumor cell lines were selected (28/71 T-cell clones). To avoid allo-HLA cross-reactivity, selected 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 frequency > 1% present in the Caucasian population (Figure 3B). 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. Finally, to investigate clinical potential of the remaining 20/71 T-cell clones, the antitumor reactivity against primary AML and OVCA patient samples expressing variable levels of WT1 was analyzed (Figure 3C).

Using this selection strategy we identified 5/20 promising WT1-specific T-cell clones, that demonstrated potent antitumor reactivity against WT1 positive primary AML and/or OVCA patient samples. The recognition patterns of these T-cell clones, recognizing 5 different WT1 peptides in the context of 3 different HLA class | molecules, are shown in Figure 4. Clone 20.3D10 is specific for VLDFAPPGA (SEQ ID NO: 72) in HLA-A*02:01 (VLD/A2), clone 22.1H1 is specific for

ALLPAVPSL (SEQ ID NO: 71) also in 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) also in HLA-B*35:01 (HAA/B35). All 5 WT1-specific clones demonstrated antitumor reactivity against WT1 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. The global frequencies of these HLA-alleles are low (HLA-A*33:01: 1.77% and HLA-

A*02:05: 1.17%), implicating that when one of these TCRs would be used for clinical practice only a small group of patients would not be suitable candidates for this particular TCR-based therapy[29]. Apart from the recognized BV-LCLs, no other target cells used in the assays expressed these HLA-alleles. Furthermore, primary AML and OVCA patient samples with WT1 expression above 10% were recognized by the different WT 1-specific T-cell clones (Figure 4C).

Interestingly, not all recognition patterns correlated with WT1 expression levels. For example clone 12.5H9 v:2/At 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 WT1 expression between 23-27% were less well recognized. In addition, clone 20.3D10V:P/%? and the other VLD/A2 reactive T-cell clones (Figure 3) did not recognize AML-6588 (10%) and AML-4716 (3%), while the 22.1H1AYA2 clone recognized both. This also accounts for some of the HLA-

B*35:01 positive primary AML patient samples. Clone 17.2G4 7835 recognized AML-5905 (44%), whereas clone 17.2D6HAA/B35 did not, while both recognized the other 2 primary AML patient samples. The most likely explanation is a difference in processing and presentation of these WT1 peptides between different primary AML patient samples, since similar reactivity by allo-HLA reactive T-cell clones directed against these different AML samples was observed (Figure 5A-B).

Notably none of the selected T-cell clones were specific for the peptides that were previously identified based on peptide prediction algorithms, including the most frequently used

RMFPNAPYL (SEQ ID NO: 88) peptide presented in HLA-A*02:01 (RMF/A2). Although high numbers of T-cell clones specific for RMF/A2 peptide were identified, of which a limited number were reactive against overexpressed WT1 transduced Raji cells (546%), all these T-cell clones were not reactive against WT1 positive tumor cell lines and primary AML patient samples. To illustrate the limited reactivity, RMF/A2 and VLD/A2 reactive T-cell clones isolated from 5 donors were compared. In Figure 6A it is shown that 12% of the RMF/A2-specific T-cell clones were reactive against WT1 transduced cells, whereas over 33% of the VLD/A2-specific T-cell clones were reactive against WT1 transduced cells. The most potent T-cell clones for both peptides were selected and no difference in peptide sensitivity was observed in a peptide titration (Figure 6B).

However, reactivity against WT1 positive tumor cell lines was very limited by all RMF/A2 clones (Figure 6C), and none of the primary AML patient samples were recognized (Figure 6D). In contrast, VLD/A2-specific T-cell clones were highly reactive against the WT 1 positive tumor cell lines as well as primary AML patient samples.

TCR gene transfer in CD8+ T cells installs potent WT 1-specific reactivity without on-target off- tumor toxicity

Next, to test whether the TCRs of the selected T-cell clones can be used for TCR gene therapeutic strategies, their TCRs were analyzed in more detail. 4 of the 5 T-cell clones: 20.3D10VLP/AZ, 22 AH1AAZ 12.5H9VED/A and 17.2G4 PYB35 based on potency were continued with. The TCR a and B chains were identified by sequencing and transferred using retroviral vectors into CD8+ T cells of multiple donors. TCR-engineered T cells (TCR-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 quite comparable to their parental T-cell clones (Figure 7B).

To investigate the antitumor potential of the TCR-T cells, they were screened against multiple tumor panels and assessed for IFN-y 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

WT 1 expression below 15% were more variable recognized. The included WT1 positive primary

AML and OVCA patient samples were recognized (Figure 8B-C). The level of recognition of these primary tumor samples correlated with the level of WT1 expression, except for TCR-T 12.5H9V-D/At that only recognized the AML-4443 sample in which the VLD/A1 peptide was initially eluted (Table 2).

To investigate the safety of the TCR-T cells, 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. No WT1 expression was detected in these healthy cell subsets, only in CD34+ hematopoietic precursor cells limited

WT1 expression (<0.5%) was observed (Figure 2A). The TCR-T cells demonstrated no reactivity against the different healthy cell subsets, even CD34+ hematopoietic precursor cells were not or limited recognized, indicating that these TCR-T cells do not exhibit on-target off-tumor reactivity (Figure 8D). Similar reactivities against all targets in Figure 8 were observed by the allo-HLA reactive T-cell clones (Figure 5C-F). In conclusion, TCR gene transfer of WT1-TCRs into CD8+

T cells installed high WT 1-specific reactivity against WT1 positive tumor cells, without indications of on-target off-tumor reactivity.

WT1-specific TCR-T cells efficiently kill primary AML samples and OVCA cell lines

Finally, to investigate clinical potential of the 4 selected TCR-T cells, cytotoxic capacity against primary AML patient samples and OVCA cell lines was measured in a 86-hour chromium release assay (Figure 9A-B). Raji pulsed with and without WT1 peptide were included as control (Figure 9C). Both 20.3D10"PA2 22 1H1ALA2 and 17.2G4 'PYB35 TCR-T cells killed AML samples and

OVCA cell lines. The percentage of killed tumor cells correlates with the level of WT1 expression and expected maximum killing by the allo-HLA reactive T-cell clones (Figure 5G-I). Interestingly, even the 9% WT1 expression in cell line COV362.4 seems sufficient to induce killing. Killing by the 12.5H9VE2'M TCR was restricted to the 2 WT1 positive OVCA cell lines.

Overall, these results demonstrate that TCR-T 20.3D10V:PA2 22 1H1AMA2 and 17.2G4 TPYB35 results in efficient killing of primary AML patient samples and OVCA cell lines. To conclude, these

TCRs are considered as promising candidates for TCR gene transfer strategies for the treatment of AML and OVCA patients, and other solid tumors. TCR 12.5H9V:P/A js considered as a potential candidate for TCR gene transfer strategies for patients with WT1 expressing solid tumors, including ovarian cancers.

Discussion

In this study, there is described 8 WT1 peptides that were identified from the HLA class associated ligandome of primary leukemia and OVCA patient samples. In a large-scale search for WT 1-specific TCRs present in the allo-HLA repertoire, T-cell clones directed against 5 different

WT1 peptides presented in 3 different HLA class | molecules were identified. No WT 1-specific T- cell clones reactive against tumor cells could be identified that recognized those peptides previously identified based on peptide prediction algorithms. By gene transfer of 4 of the 5 WT1-

TCRs into CD8+ T cells, the antitumor potential was analyzed as well as the safety of the TCR-T cells, and the results demonstrate that these WT1-TCRs can be considered as promising candidates for TCR gene transfer strategies in AML and/or OVCA patients.

The HLA class | restriction molecules for TCR 20.3D10VPA2 TCR 22.1H1AA2 TCR 12.5H9VEDAt and TCR 17.2G4 'PY®35 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 WT1 negative tumor cells. These results indicate that

WT1-TCR-T cells do not exhibit off- target or on-target off-tumor toxicity, even not against CD34+ hematopoietic precursor cells expressing low levels of WT1 (<0.5%). TCR 20.3D10V:2/*2 and TCR 22. 1H1AYA2 demonstrated some HLA cross- reactivity against HLA-A*33:01 and HLA-A*02:05, respectively. The frequencies of these HLA-alleles are however below 2%, implicating that only a small group of patients will not be suitable candidates for this particular TCR-based therapy.[29]

These results demonstrate the relevance of establishing the HLA class | associated ligandome of tumors, since only WT1-specific T-cell clones reactive against naturally WT1 expressing tumor cells were isolated for those peptides that were identified in the HLA class | associated ligandome.

The most commonly used WT1 peptide is the RMF peptide presented in HLA-A*02:01. To our knowledge this peptide has never been found in peptide-elution databases and since we were also not able to elute this peptide in our large set of WT1 expressing HLA-A*02:01 tumor samples, we question whether this peptide is efficiently processed and presented in WT1 positive tumors.

Although high numbers of T-cell clones recognizing the RMF/A2 peptide were identified, the recognition of naturally WT1 expressing tumor cell lines and primary AML patient samples was absent (Figure 6). Only WT1 transduced Raji cells (546% relative WT1 expression) were recognized by the RMF/A2 T-cell clones, apparently the RMF/A2 peptide is efficiently processed and presented if WT1 is artificially overexpressed. These data suggest that limited antitumor reactivity can be expected of RMF/A2-specific T cells and this corresponds to other observations.

Previously it was suggested that the 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.[30] Moreover, 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.[31] Finally, it also corresponds to the limited antitumor effects found in (pre)clinical studies targeting the RMF/A2 peptide [11, 32] Overall, the data suggests better clinical results may be achieved with the WT1 peptides that were identified in the HLA ligandome combined with potent T-cell clones.

The variation in antitumor reactivity against primary AML patient samples by the 12. 5HQVLP/At

TCR-T cells suggest variation in processing and presentation of HLA class | peptides between different samples. The 12.5H9V:P/At TCR-T cells efficiently recognize primary patient sample AML- 4443, from which initially the VLD/A1 peptide was eluted (Table 2), suggesting optimal processing and presentation of this peptide in sample AML-4443. The lack of recognition of the other 2 AML samples, with higher WT1 expression, could be an indication of limited processing and presentation of this 12 amino acids long VLD/A1 peptide, possibly hampered by the long length.

Furthermore, also for the HLA-A*02:01 and HLA-B*35:01 positive primary AML patient samples a variety in antitumor reactivity was observed which could not be explained by WT1 expression.

Both the ALL/A2 and TPY/B35 TCR-T cells and T-cell clones were reactive against all WT1 positive primary AML patient samples, whereas the VLD/A2 and HAA/B35 TCR-T cells and T-cell clones were not reactive against 2 of them (Figure 4). The data therefore suggest the ALL/A2 and

TPY/B35 peptides are processed and presented in more AML samples and therefore might be preferred targets to be able to treat more AML patients.

In this example it is demonstrated that safe and potent WT 1-specific T cells can be identified from the allo- HLA repertoire. Especially since these T cells were not subjected to the negative selection, the safety of the final TCRs was carefully evaluted. No on- and off-target toxicity was observed. By co-transducing the WT1-TCR engineered T cells with a suicide switch, such as the inducible caspase-9 (iC9) gene, prompt elimination of the engineered T cells can be induced. This approach was demonstrated for high-affinity PRAME-TCR transduced T cells in vivo.[33]

The identified WT1-TCRs demonstrated potent antitumor reactivity against AML and OVCA tumors. By 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. Besides the broad expression in various different tumors, several characteristics make WT 1 an interesting target. WT1 promotes cancer progression through the induction of tumor angiogenesis and metastasis formation.[7] In addition, WT1 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]

In summary, the 20.3D10VPA2 22 1H1ALMAZ 12.5H9V-P/AM and 17.2G4PYB35 TCRs were identified in a large- scale search for potent and safe WT 1-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 WT1 peptides identified from the HLA class associated ligandome of primary leukemia and OVCA patients, are expected to improve future vaccine and TCR gene therapy studies for WT 1-positive tumors.

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

Sequences

TCR 22.1H1; ALL peptide (ALLPAVPSL; SEQ ID NO 71); WT1, HLA-A*02:01

TCR AA

POLYP | or SEQUENCE

EPTIDE | NT a CDR1 TSDQSYG a CDR2 QGSYDEQN a CDR3 CAMRESTGGGNKLTF

B CDR2 FQNEAQ 6 |pCDR3 CASSSLSGAVHEKLFF

MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSD

7 a VJ AA | QSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANL

VISASQLGDSAMYFCAMRESTGGGNKLTFGTGTQLKVEL

ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCT

AGGACCTGGCATTGCCCAGAAGATAACTCAAACCCAACCAGGAATG

TTCGTGCAGGAAAAGGAGGCTGTGACTCTGGACTGCACATATGACA

CCAGTGATCAAAGTTATGGTCTATTCTGGTACAAGCAGCCCAGCAGT a VJ NT | GGGGAAATGATTTTTCTTATTTATCAGGGGTCTTATGACGAGCAAAAT

GCAACAGAAGGTCGCTACTCATTGAATTTCCAGAAGGCAAGAAAATC

CGCCAACCTTGTCATCTCCGCTTCACAACTGGGGGACTCAGCAATG

TATTTCTGTGCAATGAGAGAGTCTACGGGAGGAGGAAACAAACTCAC

CTTTGGGACAGGCACTCAGCTAAAAGTGGAACTC

MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISG

B VDJ AA | HVSLFWYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVST

LKIQRTQQEDSAVYLCASSSLSGAVHEKLFFGSGTQLSVL

ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGA

CAGATCACACAGGTGCTGGAGTCTCCCAGTCCCCTAGGTACAAAGT

CGCAAAGAGAGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCG

GGTCATGTATCCCTTTTTTGGTACCAACAGGCCCTGGGGCAGGGGC

B VDJ NT | CAGAGTTTCTGACTTATTTCCAGAATGAAGCTCAACTAGACAAATCG

GGGCTGCCCAGTGATCGCTTCTTTGCAGAAAGGCCTGAGGGATCCG

TCTCCACTCTGAAGATCCAGCGCACACAGCAGGAGGACTCCGCCGT

GTATCTCTGTGCCAGCAGCTCCTTATCGGGGGCGGTTCATGAAAAA

CTGTTTTTTGGCAGTGGAACCCAGCTCTCTGTCTTG

MSLSSLLKVVTASLWLGPGIAQKITQTQPGMFVQEKEAVTLDCTYDTSD a VJ QSYGLFWYKQPSSGEMIFLIYQGSYDEQNATEGRYSLNFQKARKSANL 11 and AA VISASQLGDSAMYFCAMRESTGGGNKLTFGTGTQLKVELNIQNPDPAV constant YQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDF

KSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFET

DTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

ATGTCACTTTCTAGCCTGCTGAAGGTGGTCACAGCTTCACTGTGGCT

AGGACCTGGCATTGCCCAGAAGATAACTCAAACCCAACCAGGAATG

TTCGTGCAGGAAAAGGAGGCTGTGACTCTGGACTGCACATATGACA

CCAGTGATCAAAGTTATGGTCTATTCTGGTACAAGCAGCCCAGCAGT

GGGGAAATGATTTTTCTTATTTATCAGGGSTCTTATGACGAGCAAAAT

GCAACAGAAGGTCGCTACTCATTGAATTTCCAGAAGGCAAGAAAATC

CGCCAACCTTGTCATCTCCGCTTCACAACTGGGGGACTCAGCAATG a VJ TATTTCTGTGCAATGAGAGAGTCTACGGGAGGAGGAAACAAACTCAC 12 and NT CTTTGGGACAGGCACTCAGCTAAAAGTGGAACTCAATATCCAGAACC constant CTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAA

GTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACA

AAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACA

TGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAA

CAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTC

CAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAG

CTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAA

CCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGG

TTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC

MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISG

HVSLFWYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVST

B VDJ LKIQRTQQEDSAVYLCASSSLSGAVHEKLFFGSGTQLSVLEDLNKVFPP

13 and AA | EVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWW/NGKEVHSGVS constant TDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLS

ENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILL

GKATLYAVLVSALVLMAMVKRKDF

ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGA

CAGATCACACAGGTGCTGGAGTCTCCCAGTCCCCTAGGTACAAAGT

CGCAAAGAGAGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCG

GGSTCATGTATCCCTTTTTTGGTACCAACAGGCCCTGGGGCAGGGGC

B VDJ CAGAGTTTCTGACTTATTTCCAGAATGAAGCTCAACTAGACAAATCG

14 and NT | GGGCTGCCCAGTGATCGCTTCTTTGCAGAAAGGCCTGAGGGATCCG constant TCTCCACTCTGAAGATCCAGCGCACACAGCAGGAGGACTCCGCCGT

GTATCTCTGTGCCAGCAGCTCCTTATCGGGGGCGGTTCATGAAAAA

CTGTTTTTTGGCAGTGGAACCCAGCTCTCTGTCTTGGAGGACCTGAA

CAAGGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCA

GAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAG

GCTTCTTCCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAA

GGAGGTGCACAGTGGGGTCAGCACGGACCCGCAGCCCCTCAAGGA

GCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTG

AGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCT

GTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCA

GGATAGGGCCAAACCCGTCACCCAGATCGTCAGCGCCGAGGCCTG

GGGTAGAGCAGACTGTGGCTTTACCTCGGTGTCCTACCAGCAAGGG

GTCCTGTCTGCCACCATCCTCTATGAGATCCTGCTAGGGAAGGCCA

CCCTGTATGCTGTGCTGGTCAGCGCCCTTGTGTTGATGGCCATGGT

CAAGAGAAAGGATTTC

Table 5: Sequences for TCR 22.1H1; HLA-A*02:01 (ALL/A2)

TCR 20.3D10; VLD peptide (VLDFAPPGA; SEQ ID NO: 72); WT1, HLA-A*02:01

TCR AA | SEQUENCE

POLYPE | or

PTIDE NT a CDR1 SSYSPS a CDR2 YTSAATLV a CDR3 CVVTHPNDYKLSF

B CDR2 FQNEAQ

B CDR3 CASSPEAGAGYNEQFF 21 a VJ AA | MLLLLVPVLEVIFTLGGTRAQSVTQLDSHVSVSEGTPVLLRCNYSSSYSPS

LFWYVQHPNKGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHM

SDAAEYFCVVTHPNDYKLSFGAGTTVTVRA

22 a VJ NT | ATGCTCCTGCTGCTCGTCCCAGTGCTCGAGGTGATTTTTACTCTGGGA

GGAACCAGAGCCCAGTCGGTGACCCAGCTTGACAGCCACGTCTCTGT

CTCTGAAGGAACCCCGGTGCTGCTGAGGTGCAACTACTCATCTTCTTAT

TCACCATCTCTCTTCTGGTATGTGCAACACCCCAACAAAGGACTCCAGC

TTCTCCTGAAGTACACATCAGCGGCCACCCTGGTTAAAGGCATCAACG

GTTTTGAGGCTGAATTTAAGAAGAGTGAAACCTCCTTCCACCTGACGAA

ACCCTCAGCCCATATGAGCGACGCGGCTGAGTACTTCTGTGTTGTGAC

TCATCCTAACGACTACAAGCTCAGCTTTGGAGCCGGAACCACAGTAAC

TGTAAGAGCA

23 B VDJ AA | MGTRLLCVWVLGFLGTDHTGAGVSQSPRYKVAKRGQDVALRCDPISGHV

SLFWYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQ

RTQQEDSAVYLCASSPEAGAGYNEQFFGPGTRLTVL

24 B VDJ NT | ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGAC

AGATCACACAGGTGCTGGAGTCTCCCAGTCCCCTAGGTACAAAGTCGC

AAAGAGAGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCA

TGTATCCCTTTTTTGGTACCAACAGGCCCTCGGGCAGGGGCCAGAGTT

TCTGACTTATTTCCAGAATGAAGCTCAACTAGACAAATCGGGGCTGCCC

AGTGATCGCTTCTTTGCAGAAAGGCCTGAGGGATCCGTCTCCACTCTG

AAGATCCAGCGCACACAGCAGGAGGACTCCGCCGTGTATCTCTGTGCC

AGCAGCCCCGAAGCGGGAGCAGGCTACAATGAGCAGTTCTTCGGGCC

AGGGACACGGCTCACCGTGCTA a VJand | AA | MLLLLVPVLEVIFTLGGTRAQSVTQLDSHVSVSEGTPVLLRCNYSSSYSPS constant LFWYVQHPNKGLQLLLKYTSAATLVKGINGFEAEFKKSETSFHLTKPSAHM

SDAAEYFCVVTHPNDYKLSFGAGTTVTVRANIQNPDPAVYQLRDSKSSDK

SVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSD

FACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL

LLKVAGFNLLMTLRLWSS

26 aVJand | NT | ATGCTCCTGCTGCTCGTCCCAGTGCTCGAGGTGATTTTTACTCTGGGA constant GGAACCAGAGCCCAGTCGGTGACCCAGCTTGACAGCCACGTCTCTGT

CTCTGAAGGAACCCCGGTGCTGCTGAGGTGCAACTACTCATCTTCTTAT

TCACCATCTCTCTTCTGGTATGTGCAACACCCCAACAAAGGACTCCAGC

TTCTCCTGAAGTACACATCAGCGGCCACCCTGGTTAAAGGCATCAACG

GTTTTGAGGCTGAATTTAAGAAGAGTGAAACCTCCTTCCACCTGACGAA

ACCCTCAGCCCATATGAGCGACGCGGCTGAGTACTTCTGTGTTGTGAC

TCATCCTAACGACTACAAGCTCAGCTTTGGAGCCGGAACCACAGTAAC

TGTAAGAGCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAG

AGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGAT

TCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAG

ACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTG

CTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCA

ACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTC

CTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTA

AACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAG

TGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC

27 B VDJ | AA | MGTRLLCWVVLGFLGTDHTGAGVSQSPRYKVAKRGODVALRCDPISGHV and SLFWYQQALGQGPEFLTYFQNEAQLDKSGLPSDRFFAERPEGSVSTLKIQ constant RTQQEDSAVYLCASSPEAGAGYNEQFFGPGTRLTVLEDLKNVFPPEVAVF

EPSEAEISHTQKATLVCLATGFYPDHVELSWMWNGKEVHSGVSTDPQPLK

EQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQD

RAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVS

ALVLMAMVKRKDSRG

28 B VDJ| NT | ATGGGCACCAGGCTCCTCTGCTGGGTGGTCCTGGGTTTCCTAGGGAC and AGATCACACAGGTGCTGGAGTCTCCCAGTCCCCTAGGTACAAAGTCGC constant AAAGAGAGGACAGGATGTAGCTCTCAGGTGTGATCCAATTTCGGGTCA

TGTATCCCTTTTTTGGTACCAACAGGCCCTCGGGCAGGGGCCAGAGTT

TCTGACTTATTTCCAGAATGAAGCTCAACTAGACAAATCGGGGCTGCCC

AGTGATCGCTTCTTTGCAGAAAGGCCTGAGGGATCCGTCTCCACTCTG

AAGATCCAGCGCACACAGCAGGAGGACTCCGCCGTGTATCTCTGTGCC

AGCAGCCCCGAAGCGGGAGCAGGCTACAATGAGCAGTTCTTCGGGCC

AGGGACACGGCTCACCGTGCTAGAGGACCTGAAAAACGTGTTCCCACC

CGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCA

AAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGT

GGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCA

GCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCC

AGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCA

GAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTC

GGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGA

TCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCC

GAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATC

TTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTG

CTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC

Table 6: Sequences for TCR 20.3D10; HLA-A*02:01 (VLD/A2)

TCR 23.2G9; VLD peptide (VLDFAPPGA; SEQ ID NO: 72); WT1, HLA-A*02:01

TCR AA | SEQUENCE

POLYPE | or

PTIDE NT a CDR1 DRGSQS a CDR2 IYSNGD a CDR3 CASREAGSYQLTF

B CDR2 YSLEER

B CDR3 CASSSHLGGANEQYF a VJ AA | MKSLRVLLVILWLQLSVWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRG

SQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ

PSDSATYLCASREAGSYQLTFGKGTKLSVIP

36 a VJ NT | ATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCT

GGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTC

AGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGAC

CGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCC

CTGAGTTGATAATGTTCATATACTCCAATGGTGACAAAGAAGATGGAAG

GTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATC

AGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCTCTAGG

GAGGCTGGGAGTTACCAACTCACTTTCGGGAAGGGGACCAAACTCTCG

GTCATACCA

37 B VDJ AA | MGCRLLCCAVLCLLGAGELVPMETGVTQTPRHLVMGMTNKKSLKCEQHL

GHNAMYWYKGQSAKKPLELMFVYSLEERVENNSVPSRFSPECPNSSHLFL

HLHTLQPEDSALYLCASSSHLGGANEQYFGPGTRLTVT

38 B VDJ NT | ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGC

GGGTGAGTTGGTCCCCATGGAAACGGGAGTTACGCAGACACCAAGAC

ACCTGGTCATGGGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACA

TCTGGGTCATAACGCTATGTATTGGTACAAGCAAAGTGCTAAGAAGCCA

CTGGAGCTCATGTTTGTCTACAGTCTTGAAGAACGGGTTGAAAACAACA

GTGTGCCAAGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCACTTATT

CCTTCACCTACACACCCTGCAGCCAGAAGACTCGGCCCTGTATCTCTG

CGCCAGCAGCTCTCATCTCGGGGGCGCTAACGAGCAGTACTTCGGGC

CGGGCACCAGGCTCACGGTCACA

39 aVJand | AA | MKSLRYLLVILWLQLSWVWSQQKEVEGQNSGPLSVPEGAIASLNCTYSDRG constant SQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQ

PSDSATYLCASREAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDK

SVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSD

FACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRIL

LLKVAGFNLLMTLRLWSS

40 aVJand | NT | ATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCT constant GGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTC

AGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGAC

CGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCC

CTGAGTTGATAATGTTCATATACTCCAATGGTGACAAAGAAGATGGAAG

GTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATC

AGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCTCTAGG

GAGGCTGGGAGTTACCAACTCACTTTCGGGAAGGGGACCAAACTCTCG

GTCATACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGA

GACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATT

CTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGA

CAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGC

TGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAA

CAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCC

TGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAA

ACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGT

GGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC

41 B VDJ AA | MGCRLLCCAVLCLLGAGELVPMETGVTQTPRHLVMGMTNKKSLKCEQHL and GHNAMYWYKQSAKKPLELMFVYSLEERVENNSVPSRFSPECPNSSHLFL

HLHTLQPEDSALYLCASSSHLGGANEQYFGPGTRLTVTEDLKNVFPPEVA constant VFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQP

LKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQ

DRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLV

SALVLMAMVKRKDSRG

42 B VDJ NT | ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGC and GGGTGAGTTGGTCCCCATGGAAACGGGAGTTACGCAGACACCAAGAC

ACCTGGTCATGGGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACA constant TCTGGGTCATAACGCTATGTATTGGTACAAGCAAAGTGCTAAGAAGCCA

CTGGAGCTCATGTTTGTCTACAGTCTTGAAGAACGGGTTGAAAACAACA

GTGTGCCAAGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCACTTATT

CCTTCACCTACACACCCTGCAGCCAGAAGACTCGGCCCTGTATCTCTG

CGCCAGCAGCTCTCATCTCGGGGGCGCTAACGAGCAGTACTTCGGGC

CGGGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCA

CCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACC

CAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCAC

GTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGT

CAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACT

CCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGG

CAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTC

TCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCA

GATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCT

CCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGA

TCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCG

TGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC

Table 7: Sequences for TOR 23.2G9; HLA-A*02:01 (VLD/A2)

TCR AA | SEQUENCE

POLYP | or

EPTIDE | NT 49 a VJ AA | MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDS

SSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRI

ADTQTGDSAIYFCAVSSGGYQKVTFGIGTKLQVIP a VJ NT | ATGAAGACATTTGCTGGATTTTCGTTCCTGTTTTTGTGGCTGCAGCT

GGACTGTATGAGTAGAGGAGAGGATGTGGAGCAGAGTCTTTTCCTG

AGTGTCCGAGAGGGAGACAGCTCCGTTATAAACTGCACTTACACAG

ACAGCTCCTCCACCTACTTATACTGGTATAAGCAAGAACCTGGAGCA

GGTCTCCAGTTGCTGACGTATATTTTTTCAAATATGGACATGAAACAA

GACCAAAGACTCACTGTTCTATTGAATAAAAAGGATAAACATCTGTCT

CTGCGCATTGCAGACACCCAGACTGGGGACTCAGCTATCTACTTCT

GTGCAGTTTCCTCTGGGGGTTACCAGAAAGTTACCTTTGGAATTGGA

ACAAAGCTCCAAGTCATCCCA

51 B VDJ AA | MGTRLFFYVALCLLWTGHMDAGITQSPRHKVTETGTPVTLRCHQTENH

RYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDGYSVSRSKTEDFLLT

LESATSSQTSVYFCAISDPYRGEQYFGPGTRLTVT

52 B VDJ NT | ATGGGCACAAGGTTGTTCTTCTATGTGGCCCTTTGTCTCCTGTGGAC

AGGACACATGGATGCTGGAATCACCCAGAGCCCAAGACACAAGGTC

ACAGAGACAGGAACACCAGTGACTCTGAGATGTCACCAGACTGAGA

ACCACCGCTATATGTACTGGTATCGACAAGACCCGGGGCATGGGCT

GAGGCTGATCCATTACTCATATGGTGTTAAAGATACTGACAAAGGAG

AAGTCTCAGATGGCTATAGTGTCTCTAGATCAAAGACAGAGGATTTC

CTCCTCACTCTGGAGTCCGCTACCAGCTCCCAGACATCTGTGTACTT

CTGTGCCATCAGTGATCCGTACAGGGGCGAGCAGTACTTCGGGCCG

GGCACCAGGCTCACGGTCACA

53 a VJ AA | MKTFAGFSFLFLWLQLDCMSRGEDVEQSLFLSVREGDSSVINCTYTDS and SSTYLYWYKQEPGAGLQLLTYIFSNMDMKQDQRLTVLLNKKDKHLSLRI constant ADTQTGDSAIYFCAVSSGGYQKVTFGIGTKLQVIPNIQNPDPAVYQLRD

SKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSA

VAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLN

FQNLSVIGFRILLLKVAGFNLLMTLRLWSS

54 a VJ NT | ATGAAGACATTTGCTGGATTTTCGTTCCTGTTTTTGTGGCTGCAGCT and GGACTGTATGAGTAGAGGAGAGGATGTGGAGCAGAGTCTTTTCCTG constant AGTGTCCGAGAGGGAGACAGCTCCGTTATAAACTGCACTTACACAG

ACAGCTCCTCCACCTACTTATACTGGTATAAGCAAGAACCTGGAGCA

GGTCTCCAGTTGCTGACGTATATTTTTTCAAATATGGACATGAAACAA

GACCAAAGACTCACTGTTCTATTGAATAAAAAGGATAAACATCTGTCT

CTGCGCATTGCAGACACCCAGACTGGGGACTCAGCTATCTACTTCT

GTGCAGTTTCCTCTGGGGGTTACCAGAAAGTTACCTTTGGAATTGGA

ACAAAGCTCCAAGTCATCCCAAATATCCAGAACCCTGACCCTGCCGT

GTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTAT

TCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTG

ATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGAC

TTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGC

ATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCT

TCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAG

CTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTG

GGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATG

ACGCTGCGGCTGTGGTCCAGC

B VDJ AA | MGTRLFFYVALCLLWTGHMDAGITQSPRHKVTETGTPVTLRCHQTENH and RYMYWYRQDPGHGLRLIHYSYGVKDTDKGEVSDGYSVSRSKTEDFLLT constant LESATSSQTSVYFCAISDPYRGEQYFGPGTRLTVTEDLKNVFPPEVAVF

EPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQP

LKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEW

TQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATL

YAVLVSALVLMAMVKRKDSRG

56 B VDJ NT | ATGGGCACAAGGTTGTTCTTCTATGTGGCCCTTTGTCTCCTGTGGAC and AGGACACATGGATGCTGGAATCACCCAGAGCCCAAGACACAAGGTC constant ACAGAGACAGGAACACCAGTGACTCTGAGATGTCACCAGACTGAGA

ACCACCGCTATATGTACTGGTATCGACAAGACCCGGGGCATGGGCT

GAGGCTGATCCATTACTCATATGGTGTTAAAGATACTGACAAAGGAG

AAGTCTCAGATGGCTATAGTGTCTCTAGATCAAAGACAGAGGATTTC

CTCCTCACTCTGGAGTCCGCTACCAGCTCCCAGACATCTGTGTACTT

CTGTGCCATCAGTGATCCGTACAGGGGCGAGCAGTACTTCGGGCCG

GGCACCAGGCTCACGGTCACAGAGGACCTGAAAAACGTGTTCCCAC

CCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACAC

CCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGAC

CACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGT

GGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTC

AATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCA

CCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTT

CTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAA

ACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGA

CTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCC

ACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGT

GCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGAT

TCCAGAGGC

Table 8: sequence for TCR 12.5H9; HLA-A*01:01 (VLD/A1)

TCR 12.5H9; VLD peptide (VLDFAPPGASAY; SEQ ID NO: 74); WT1, HLA-A*01:01

TCR AA | SEQUENCE

POLYP | or

EPTIDE | NT a CDR1 TTSDR a CDR2 LLSNGAV a CDR3 CAVGRNNDMRF

B CDR2 FYNNEI

B CDR3 CASTEAFF 63 a VJ AA | MKKLLAMILWLQLDRLSGELKVEQNPLFLSMQEGKNYTIYCNYSTTSDR

LYWYRQDPGKSLESLFVLLSNGAVKQEGRLMASLDTKARLSTLHITAAV

HDLSATYFCAVGRNNDMRFGAGTRLTVKP

64 a VJ NT | ATGAAGAAGCTACTAGCAATGATTCTGTGGCTTCAACTAGACCGGTT

AAGTGGAGAGCTGAAAGTGGAACAAAACCCTCTGTTCCTGAGCATG

CAGGAGGGAAAAAACTATACCATCTACTGCAATTATTCAACCACTTC

AGACAGACTGTATTGGTACAGGCAGGATCCTGGGAAAAGTCTGGAA

TCTCTGTTTGTGTTGCTATCAAATGGAGCAGTGAAGCAGGAGGGAC

GATTAATGGCCTCACTTGATACCAAAGCCCGTCTCAGCACCCTCCAC

ATCACAGCTGCCGTGCATGACCTCTCTGCCACCTACTTCTGTGCCGT

GGGCCGAAACAATGACATGCGCTTTGGAGCAGGGACCAGACTGACA

GTAAAACCA

65 B VDJ AA | MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHL

YFYWYRQILGOKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIR

STKLEDSAMYFCASTEAFFGOGTRLTVV

B VDJ NT | ATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTTGAAAGC

AGGACTCACAGAACCTGAAGTCACCCAGACTCCCAGCCATCAGGTC

ACACAGATGGGACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTA

ATCACTTATACTTCTATTGGTACAGACAAATCTTGGGGCAGAAAGTC

GAGTTTCTGGTTTCCTTTTATAATAATGAAATCTCAGAGAAGTCTGAA

ATATTCGATGATCAATTCTCAGTTGAAAGGCCTGATGGATCAAATTTC

ACTCTGAAGATCCGGTCCACAAAGCTGGAGGACTCAGCCATGTACT

TCTGTGCCAGCACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACA

GTTGTA

67 a VJ AA | MKKLLAMILWLQLDRLSGELKVEQNPLFLSMQEGKNYTIYCNYSTTSDR and LYWYRQDPGKSLESLFVLLSNGAVKQEGRLMASLDTKARLSTLHITAAV constant HDLSATYFCAVGRNNDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSD

KSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSN

KSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSV

IGFRILLLKVAGFNLLMTLRLWSS a VJ NT | ATGAAGAAGCTACTAGCAATGATTCTGTGGCTTCAACTAGACCGGTT and AAGTGGAGAGCTGAAAGTGGAACAAAACCCTCTGTTCCTGAGCATG constant CAGGAGGGAAAAAACTATACCATCTACTGCAATTATTCAACCACTTC

AGACAGACTGTATTGGTACAGGCAGGATCCTGGGAAAAGTCTGGAA

TCTCTGTTTGTGTTGCTATCAAATGGAGCAGTGAAGCAGGAGGGAC

GATTAATGGCCTCACTTGATACCAAAGCCCGTCTCAGCACCCTCCAC

ATCACAGCTGCCGTGCATGACCTCTCTGCCACCTACTTCTGTGCCGT

GGGCCGAAACAATGACATGCGCTTTGGAGCAGGGACCAGACTGACA

GTAAAACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGA

GAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTT

GATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATC

ACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAA

CAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAAC

GCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCC

AGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAG

ATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATC

CTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGC

TGTGGTCCAGC

B VDJ AA | MDTWLVCWAIFSLLKAGLTEPEVTQTPSHQVTQMGQEVILRCVPISNHL and YFYWYRQILGQKVEFLVSFYNNEISEKSEIFDDQFSVERPDGSNFTLKIR constant STKLEDSAMYFCASTEAFFGQGTRLTVVEDLNKVFPPEVAVFEPSEAEI

SHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVSTDPQPLKEQPA

LNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAK

PVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSA

LVLMAMVKRKDF

70 B VDJ NT | ATGGATACCTGGCTCGTATGCTGGGCAATTTTTAGTCTCTTGAAAGC and AGGACTCACAGAACCTGAAGTCACCCAGACTCCCAGCCATCAGGTC constant ACACAGATGGGACAGGAAGTGATCTTGCGCTGTGTCCCCATCTCTA

ATCACTTATACTTCTATTGGTACAGACAAATCTTGGGGCAGAAAGTC

GAGTTTCTGGTTTCCTTTTATAATAATGAAATCTCAGAGAAGTCTGAA

ATATTCGATGATCAATTCTCAGTTGAAAGGCCTGATGGATCAAATTTC

ACTCTGAAGATCCGGTCCACAAAGCTGGAGGACTCAGCCATGTACT

TCTGTGCCAGCACTGAAGCTTTCTTTGGACAAGGCACCAGACTCACA

GTTGTAGAGGACCTGAACAAGGTGTTCCCACCCGAGGTCGCTGTGT

TTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACT

GGTGTGCCTGGCCACAGGCTTCTTCCCCGACCACGTGGAGCTGAG

CTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACGGA

CCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATAC

TGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAAC

CCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGG

AGAATGACGAGTGGACCCAGGATAGGGCCAAACCCGTCACCCAGAT

CGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTTACCTC

GGTGTCCTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAG

ATCCTGCTAGGGAAGGCCACCCTGTATGCTGTGCTGGTCAGCGCCC

TTGTGTTGATGGCCATGGTCAAGAGAAAGGATTTC

Table 9: Sequences for TCR 17.2G4; HLA- B*35:01 (TPY/B35)

SEQ ID NO: 71 — ALLPAVPSL

SEQ ID NO: 72 — VLDFAPPGA

SEQ ID NO: 73 — TPYSSDNLY

SEQ ID NO: 74 — VLDFAPPGASAY

SEQ ID NO: 75 — AGACCCACACCAGGACTCAT — WT1: forward;

SEQ ID NO: 76 — GATGCATGTTGTGATGGCGG — WT1 reverse;

SEQ ID NO: 77 — ACTGAACAGTCACCGACGAG - GUSB forward;

SEQ ID NO: 78 — GGAACGCTGCACTTTTTGGT — GUSB reverse;

SEQ ID NO: 79 -GTTTCCGCAACATCTCTCGC — PSMB4 forward;

SEQ ID NO: 80 — CATCAATCACCATCTGGCCG — PSMB reverse;

SEQ ID NO: 81 — TGAGAGGAGACTTCGATGAGAATC — VPS29 forward;

SEQ ID NO: 82 — TCTGCAACAGGGCTAAGCTG — VPS29 reverse;

SEQ ID NO: 83 — NLVPMVATV — murinized CMV-specific TCR peptide presented in HLA-

A*02:01;

SEQ ID NO: 84 —- WTEGQSNHSTGY;

SEQ ID NO: 85 — FGPPPPSQA ;

SEQ ID NO: 86 — AQFPNHSFK;

SEQ ID NO: 87 — HAAQFPNHSF;

SEQ ID NO: 88 — RMFPNAPYL;

SEQ ID NO: 89 - CMTWNQMNL; and

SEQ ID NO: 90 — RWPSCQKKEF.

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SEQLTXT

SEQUENCE LISTING

<110> Academisch Ziekenhuis Leiden (h.o.d.n. LUMC) <120> T CELL RECEPTORS DIRECTED AGAINST TRANSCRIPTION FACTOR WT1 AND

USES THEREOF

<130> P337828NL <160> 90 <170> PatentIn version 3.5 <210> 1 <211> 7 <212> PRT <213> Homo sapiens <400> 1

Thr Ser Asp Gln Ser Tyr Gly 1 5 <210> 2 <211> 8 <212> PRT <213> Homo sapiens <400> 2

Gln Gly Ser Tyr Asp Glu Gln Asn 1 5 <210> 3 <211> 15 <212> PRT <213> Homo sapiens <400> 3

Cys Ala Met Arg Glu Ser Thr Gly Gly Gly Asn Lys Leu Thr Phe 1 5 10 15 <210> 4 <211> 5 <212> PRT <213> Homo sapiens <400> 4

Pagina 1

SEQLTXT

Ser Gly His Val Ser 1 5 <210> 5 <211> 6 <212> PRT <213> Homo sapiens <400> 5

Phe Gln Asn Glu Ala Gln 1 5 <210> 6 <211> 16 <212> PRT <213> Homo sapiens <400> 6

Cys Ala Ser Ser Ser Leu Ser Gly Ala Val His Glu Lys Leu Phe Phe 1 5 10 15 <210> 7 <211> 136 <212> PRT <213> Homo sapiens <400> 7

Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 1 5 10 15

Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe

Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser

Asp Gln Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 60

Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Glu Gln Asn Ala Thr 65 70 75 80

Pagina 2

SEQLTXT

Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 85 90 95

Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 100 105 110

Ala Met Arg Glu Ser Thr Gly Gly Gly Asn Lys Leu Thr Phe Gly Thr 115 120 125

Gly Thr Gln Leu Lys Val Glu Leu 130 135 <210> 8 <211> 408 <212> DNA <213> Homo sapiens <400> 8 atgtcacttt ctagcctgct gaaggtggtc acagcttcac tgtggctagg acctggcatt 60 gcccagaaga taactcaaac ccaaccagga atgttcgtgc aggaaaagga ggctgtgact 120 ctggactgca catatgacac cagtgatcaa agttatggtc tattctggta caagcagccc 180 agcagtgggg aaatgatttt tcttatttat caggggtctt atgacgagca aaatgcaaca 240 gaaggtcgct actcattgaa tttccagaag gcaagaaaat ccgccaacct tgtcatctcc 300 gcttcacaac tgggggactc agcaatgtat ttctgtgcaa tgagagagtc tacgggagga 360 ggaaacaaac tcacctttgg gacaggcact cagctaaaag tggaactc 408 <210> 9 <211> 135 <212> PRT <213> Homo sapiens <400> 9

Met Gly Thr Arg Leu Leu Cys Trp Val Val Leu Gly Phe Leu Gly Thr 1 5 10 15

Asp His Thr Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala

Pagina 3

SEQLTXT

Lys Arg Gly Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His

Val Ser Leu Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro 65 70 75 80

Ser Asp Arg Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu 85 90 95

Lys Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala 100 105 110

Ser Ser Ser Leu Ser Gly Ala Val His Glu Lys Leu Phe Phe Gly Ser 115 120 125

Gly Thr Gln Leu Ser Val Leu 130 135 <210> 10 <211> 405 <212> DNA <213> Homo sapiens <400> 10 atgggcacca ggctcctctg ctgggtggtc ctgggtttcc tagggacaga tcacacaggt 60 gctggagtct cccagtcccc taggtacaaa gtcgcaaaga gaggacagga tgtagctctc 120 aggtgtgatc caatttcggg tcatgtatcc cttttttggt accaacaggc cctggggcag 180 gggccagagt ttctgactta tttccagaat gaagctcaac tagacaaatc ggggctgccc 240 agtgatcgct tctttgcaga aaggcctgag ggatccgtct ccactctgaa gatccagcgc 300 acacagcagg aggactccgc cgtgtatctc tgtgccagca gctccttatc gggggeggtt 360 catgaaaaac tgttttttgg cagtggaacc cagctctctg tcttg 405 <210> 11

Pagina 4

SEQLTXT

<211> 277 <212> PRT <213> Artificial Sequence <220> <223> TCR V alpha domain with a constant domain <400> 11

Met Ser Leu Ser Ser Leu Leu Lys Val Val Thr Ala Ser Leu Trp Leu 1 5 10 15

Gly Pro Gly Ile Ala Gln Lys Ile Thr Gln Thr Gln Pro Gly Met Phe

Val Gln Glu Lys Glu Ala Val Thr Leu Asp Cys Thr Tyr Asp Thr Ser

Asp Gln Ser Tyr Gly Leu Phe Trp Tyr Lys Gln Pro Ser Ser Gly Glu 60

Met Ile Phe Leu Ile Tyr Gln Gly Ser Tyr Asp Glu Gln Asn Ala Thr 65 70 75 80

Glu Gly Arg Tyr Ser Leu Asn Phe Gln Lys Ala Arg Lys Ser Ala Asn 85 90 95

Leu Val Ile Ser Ala Ser Gln Leu Gly Asp Ser Ala Met Tyr Phe Cys 100 105 110

Ala Met Arg Glu Ser Thr Gly Gly Gly Asn Lys Leu Thr Phe Gly Thr 115 120 125

Gly Thr Gln Leu Lys Val Glu Leu Asn Ile Gln Asn Pro Asp Pro Ala 130 135 140

Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu 145 150 155 160

Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser 165 170 175

Pagina 5

SEQLTXT

Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp 180 185 190

Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala 195 200 205

Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe 210 215 220

Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe 225 230 235 240

Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe 245 250 255

Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu 260 265 270

Arg Leu Trp Ser Ser 275 <210> 12 <211> 831 <212> DNA <213> Artificial Sequence <220> <223> TCR alpha chain of clone 22.1H1 <400> 12 atgtcacttt ctagcctgct gaaggtggtc acagcttcac tgtggctagg acctggcatt 60 gcccagaaga taactcaaac ccaaccagga atgttcgtgc aggaaaagga ggctgtgact 120 ctggactgca catatgacac cagtgatcaa agttatggtc tattctggta caagcagccc 180 agcagtgggg aaatgatttt tcttatttat caggggtctt atgacgagca aaatgcaaca 240 gaaggtcgct actcattgaa tttccagaag gcaagaaaat ccgccaacct tgtcatctcc 300 gcttcacaac tgggggactc agcaatgtat ttctgtgcaa tgagagagtc tacgggagga 360 ggaaacaaac tcacctttgg gacaggcact cagctaaaag tggaactcaa tatccagaac 420

Pagina 6

SEQLTXT cctgaccctg ccgtgtacca gctgagagac tctaaatcca gtgacaagtc tgtctgccta 480 ttcaccgatt ttgattctca aacaaatgtg tcacaaagta aggattctga tgtgtatatc 540 acagacaaaa ctgtgctaga catgaggtct atggacttca agagcaacag tgctgtggcc 600 tggagcaaca aatctgactt tgcatgtgca aacgccttca acaacagcat tattccagaa 660 gacaccttct tccccagccc agaaagttcc tgtgatgtca agctggtcga gaaaagcttt 720 gaaacagata cgaacctaaa ctttcaaaac ctgtcagtga ttgggttccg aatcctcctc 780 ctgaaagtgg ccgggtttaa tctgctcatg acgctgcggc tgtggtccag c 831 <210> 13 <211> 312 <212> PRT <213> Artificial Sequence <220> <223> TCR V beta domain and a constant domain <400> 13

Met Gly Thr Arg Leu Leu Cys Trp Val Val Leu Gly Phe Leu Gly Thr 1 5 10 15

Asp His Thr Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala

Lys Arg Gly Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His

Val Ser Leu Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro 65 70 75 80

Ser Asp Arg Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu 85 90 95

Lys Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala 100 105 110

Pagina 7

SEQLTXT

Ser Ser Ser Leu Ser Gly Ala Val His Glu Lys Leu Phe Phe Gly Ser 115 120 125

Gly Thr Gln Leu Ser Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro 130 135 140

Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln 145 150 155 160

Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val 165 170 175

Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser 180 185 190

Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg 195 200 205

Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn 210 215 220

Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu 225 230 235 240

Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val 245 250 255

Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser 260 265 270

Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu 275 280 285

Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met 290 295 300

Ala Met Val Lys Arg Lys Asp Phe 305 310

Pagina 8

SEQLTXT

<210> 14 <211> 936 <212> DNA <213> Artificial Sequence <220> <223> TCR beta chain of clone 22.1H1 <400> 14 atgggcacca ggctcctctg ctgggtggtc ctgggtttcc tagggacaga tcacacaggt 60 gctggagtct cccagtcccc taggtacaaa gtcgcaaaga gaggacagga tgtagctctc 120 aggtgtgatc caatttcggg tcatgtatcc cttttttggt accaacaggc cctggggcag 180 gggccagagt ttctgactta tttccagaat gaagctcaac tagacaaatc ggggctgccc 240 agtgatcgct tctttgcaga aaggcctgag ggatccgtct ccactctgaa gatccagcgc 300 acacagcagg aggactccgc cgtgtatctc tgtgccagca gctccttatc gggggeggtt 360 catgaaaaac tgttttttgg cagtggaacc cagctctctg tcttggagga cctgaacaag 420 gtgttcccac ccgaggtcgc tgtgtttgag ccatcagaag cagagatctc ccacacccaa 480 aaggccacac tggtgtgcct ggccacaggc ttcttccccg accacgtgga gctgagctgg 540 tgggtgaatg ggaaggaggt gcacagtggg gtcagcacgg acccgcagcc cctcaaggag 600 cagcccgccc tcaatgactc cagatactgc ctgagcagcc gcctgagggt ctcggccacc 660 ttctggcaga acccccgcaa ccacttccgc tgtcaagtcc agttctacgg gctctcggag 720 aatgacgagt ggacccagga tagggccaaa cccgtcaccc agatcgtcag cgccgaggec 780 tggggtagag cagactgtgg ctttacctcg gtgtcctacc agcaaggggt cctgtctgcc 840 accatcctct atgagatcct gctagggaag gccaccctgt atgctgtgct ggtcagcgcc 900 cttgtgttga tggccatggt caagagaaag gatttc 936 <210> 15 <211> 6 <212> PRT <213> Homo sapiens <400> 15

Ser Ser Tyr Ser Pro Ser 1 5

Pagina 9

SEQLTXT

<210> 16 <211> 8 <212> PRT <213> Homo sapiens <400> 16

Tyr Thr Ser Ala Ala Thr Leu Val 1 5 <210> 17 <211> 13 <212> PRT <213> Homo sapiens <400> 17

Cys Val Val Thr His Pro Asn Asp Tyr Lys Leu Ser Phe 1 5 10 <210> 18 <211> 5 <212> PRT <213> Homo sapiens <400> 18

Ser Gly His Val Ser 1 5 <210> 19 <211> 6 <212> PRT <213> Homo sapiens <400> 19

Phe Gln Asn Glu Ala Gln 1 5 <210> 20 <211> 16 <212> PRT <213> Homo sapiens <400> 20

Pagina 10

SEQLTXT

Cys Ala Ser Ser Pro Glu Ala Gly Ala Gly Tyr Asn Glu Gln Phe Phe 1 5 10 15 <210> 21 <211> 132 <212> PRT <213> Homo sapiens <400> 21

Met Leu Leu Leu Leu Val Pro Val Leu Glu Val Ile Phe Thr Leu Gly 1 5 10 15

Gly Thr Arg Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val

Ser Glu Gly Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr

Ser Pro Ser Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln 60

Leu Leu Leu Lys Tyr Thr Ser Ala Ala Thr Leu Val Lys Gly Ile Asn 65 70 75 80

Gly Phe Glu Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr 85 90 95

Lys Pro Ser Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Val Val 100 105 110

Thr His Pro Asn Asp Tyr Lys Leu Ser Phe Gly Ala Gly Thr Thr Val 115 120 125

Thr Val Arg Ala 130 <210> 22 <211> 396 <212> DNA <213> Homo sapiens

Pagina 11

SEQLTXT

<400> 22 atgctcctgc tgctcgtccc agtgctcgag gtgattttta ctctgggagg aaccagagcc 60 cagtcggtga cccagcttga cagccacgtc tctgtctctg aaggaacccc ggtgctgctg 120 aggtgcaact actcatcttc ttattcacca tctctcttct ggtatgtgca acaccccaac 180 aaaggactcc agcttctcct gaagtacaca tcagcggcca ccctggttaa aggcatcaac 240 ggttttgagg ctgaatttaa gaagagtgaa acctccttcc acctgacgaa accctcagcc 300 catatgagcg acgcggctga gtacttctgt gttgtgactc atcctaacga ctacaagctc 360 agctttggag ccggaaccac agtaactgta agagca 396 <210> 23 <211> 135 <212> PRT <213> Homo sapiens <400> 23

Met Gly Thr Arg Leu Leu Cys Trp Val Val Leu Gly Phe Leu Gly Thr 1 5 10 15

Asp His Thr Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala

Lys Arg Gly Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His

Val Ser Leu Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro 65 70 75 80

Ser Asp Arg Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu 85 90 95

Lys Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala 100 105 110

Pagina 12

SEQLTXT

Ser Ser Pro Glu Ala Gly Ala Gly Tyr Asn Glu Gln Phe Phe Gly Pro 115 120 125

Gly Thr Arg Leu Thr Val Leu 130 135 <210> 24 <211> 405 <212> DNA <213> Homo sapiens <400> 24 atgggcacca ggctcctctg ctgggtggtc ctgggtttcc tagggacaga tcacacaggt 60 gctggagtct cccagtcccc taggtacaaa gtcgcaaaga gaggacagga tgtagctctc 120 aggtgtgatc caatttcggg tcatgtatcc cttttttggt accaacaggc cctcgggcag 180 gggccagagt ttctgactta tttccagaat gaagctcaac tagacaaatc ggggctgccc 240 agtgatcgct tctttgcaga aaggcctgag ggatccgtct ccactctgaa gatccagcgc 300 acacagcagg aggactccgc cgtgtatctc tgtgccagca gccccgaagc gggagcaggc 360 tacaatgagc agttcttcgg gccagggaca cggctcaccg tgcta 405 <210> 25 <211> 273 <212> PRT <213> Artificial Sequence <220> <223> TCR V alpha domain with a constant domain <400> 25

Met Leu Leu Leu Leu Val Pro Val Leu Glu Val Ile Phe Thr Leu Gly 1 5 10 15

Gly Thr Arg Ala Gln Ser Val Thr Gln Leu Asp Ser His Val Ser Val

Ser Glu Gly Thr Pro Val Leu Leu Arg Cys Asn Tyr Ser Ser Ser Tyr

Ser Pro Ser Leu Phe Trp Tyr Val Gln His Pro Asn Lys Gly Leu Gln

Pagina 13

SEQLTXT

60

Leu Leu Leu Lys Tyr Thr Ser Ala Ala Thr Leu Val Lys Gly Ile Asn 65 70 75 80

Gly Phe Glu Ala Glu Phe Lys Lys Ser Glu Thr Ser Phe His Leu Thr 85 90 95

Lys Pro Ser Ala His Met Ser Asp Ala Ala Glu Tyr Phe Cys Val Val 100 105 110

Thr His Pro Asn Asp Tyr Lys Leu Ser Phe Gly Ala Gly Thr Thr Val 115 120 125

Thr Val Arg Ala Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu 130 135 140

Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe 145 150 155 160

Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile 165 170 175

Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn 180 185 190

Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala 195 200 205

Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu 210 215 220

Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr 225 230 235 240

Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu 245 250 255

Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser

Pagina 14

SEQLTXT

260 265 270

Ser <210> 26 <211> 819 <212> DNA <213> Artificial Sequence <220> <223> TCR alpha chain of clone 290.3D190 <400> 26 atgctcctgc tgctcgtccc agtgctcgag gtgattttta ctctgggagg aaccagagcc 60 cagtcggtga cccagcttga cagccacgtc tctgtctctg aaggaacccc ggtgctgctg 120 aggtgcaact actcatcttc ttattcacca tctctcttct ggtatgtgca acaccccaac 180 aaaggactcc agcttctcct gaagtacaca tcagcggcca ccctggttaa aggcatcaac 240 ggttttgagg ctgaatttaa gaagagtgaa acctccttcc acctgacgaa accctcagcc 300 catatgagcg acgcggctga gtacttctgt gttgtgactc atcctaacga ctacaagctc 360 agctttggag ccggaaccac agtaactgta agagcaaata tccagaaccc tgaccctgcc 420 gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 480 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 540 gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 600 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 660 cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 720 aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 780 gggtttaatc tgctcatgac gctgcggctg tggtccagc 819 <210> 27 <211> 314 <212> PRT <213> Artificial Sequence <220> <223> TCR V beta domain and a constant domain

Pagina 15

SEQLTXT

<400> 27

Met Gly Thr Arg Leu Leu Cys Trp Val Val Leu Gly Phe Leu Gly Thr 1 5 10 15

Asp His Thr Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala

Lys Arg Gly Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His

Val Ser Leu Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe 60

Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro 65 70 75 80

Ser Asp Arg Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu 85 90 95

Lys Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala 100 105 110

Ser Ser Pro Glu Ala Gly Ala Gly Tyr Asn Glu Gln Phe Phe Gly Pro 115 120 125

Gly Thr Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro 130 135 140

Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln 145 150 155 160

Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val 165 170 175

Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser 180 185 190

Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg

Pagina 16

SEQLTXT

195 200 205

Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn 210 215 220

Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu 225 230 235 240

Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val 245 250 255

Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser 260 265 270

Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu 275 280 285

Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met 290 295 300

Ala Met Val Lys Arg Lys Asp Ser Arg Gly 305 310 <210> 28 <211> 942 <212> DNA <213> Artificial Sequence <220> <223> TCR beta chain of clone 20.3D10 <400> 28 atgggcacca ggctcctctg ctgggtggtc ctgggtttcc tagggacaga tcacacaggt 60 gctggagtct cccagtcccc taggtacaaa gtcgcaaaga gaggacagga tgtagctctc 120 aggtgtgatc caatttcggg tcatgtatcc cttttttggt accaacaggc cctcgggcag 180 gggccagagt ttctgactta tttccagaat gaagctcaac tagacaaatc ggggctgccc 240 agtgatcgct tctttgcaga aaggcctgag ggatccgtct ccactctgaa gatccagcgc 300 acacagcagg aggactccgc cgtgtatctc tgtgccagca gccccgaagc gggagcaggc 360

Pagina 17

SEQLTXT tacaatgagc agttcttcgg gccagggaca cggctcaccg tgctagagga cctgaaaaac 420 gtgttcccac ccgaggtcgc tgtgtttgag ccatcagaag cagagatctc ccacacccaa 480 aaggccacac tggtgtgcct ggccacaggc ttctaccccg accacgtgga gctgagctgg 540 tgggtgaatg ggaaggaggt gcacagtggg gtcagcacag acccgcagcc cctcaaggag 600 cagcccgccc tcaatgactc cagatactgc ctgagcagcc gcctgagggt ctcggccacc 660 ttctggcaga acccccgcaa ccacttccgc tgtcaagtcc agttctacgg gctctcggag 720 aatgacgagt ggacccagga tagggccaaa cctgtcaccc agatcgtcag cgccgaggcc 780 tggggtagag cagactgtgg cttcacctcc gagtcttacc agcaaggggt cctgtctgcc 840 accatcctct atgagatctt gctagggaag gccaccttgt atgccgtgct ggtcagtgcc 900 ctcgtgctga tggccatggt caagagaaag gattccagag gc 942 <210> 29 <211> 6 <212> PRT <213> Homo sapiens <400> 29

Asp Arg Gly Ser Gln Ser 1 5 <210> 30 <211> 6 <212> PRT <213> Homo sapiens <400> 30

Ile Tyr Ser Asn Gly Asp 1 5 <210> 31 <211> 13 <212> PRT <213> Homo sapiens <400> 31

Cys Ala Ser Arg Glu Ala Gly Ser Tyr Gln Leu Thr Phe 1 5 10

Pagina 18

SEQLTXT

<210> 32 <211> 5 <212> PRT <213> Homo sapiens <400> 32

Leu Gly His Asn Ala 1 5 <210> 33 <211> 6 <212> PRT <213> Homo sapiens <400> 33

Tyr Ser Leu Glu Glu Arg 1 5 <210> 34 <211> 15 <212> PRT <213> Homo sapiens <400> 34

Cys Ala Ser Ser Ser His Leu Gly Gly Ala Asn Glu Gln Tyr Phe 1 5 10 15 <210> 35 <211> 132 <212> PRT <213> Homo sapiens <400> 35

Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15

Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu

Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp

Pagina 19

SEQLTXT

Arg Gly Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser 60

Pro Glu Leu Ile Met Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly 65 70 75 80

Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu 85 90 95

Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Ser 100 105 110

Arg Glu Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys Leu 115 120 125

Ser Val Ile Pro 130 <210> 36 <211> 396 <212> DNA <213> Homo sapiens <400> 36 atgaaatcct tgagagtttt actagtgatc ctgtggcttc agttgagctg ggtttggagc 60 caacagaagg aggtggagca gaattctgga cccctcagtg ttccagaggg agccattgcc 120 tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 180 tctgggaaaa gccctgagtt gataatgttc atatactcca atggtgacaa agaagatgga 240 aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 300 cagcccagtg attcagccac ctacctctgt gcctctaggg aggctgggag ttaccaactc 360 actttcggga aggggaccaa actctcggtc atacca 396 <210> 37 <211> 136 <212> PRT <213> Homo sapiens

Pagina 20

SEQLTXT

<400> 37

Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala 1 5 10 15

Gly Glu Leu Val Pro Met Glu Thr Gly Val Thr Gln Thr Pro Arg His

Leu Val Met Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His

Leu Gly His Asn Ala Met Tyr Trp Tyr Lys Gln Ser Ala Lys Lys Pro 60

Leu Glu Leu Met Phe Val Tyr Ser Leu Glu Glu Arg Val Glu Asn Asn 65 70 75 80

Ser Val Pro Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser His Leu 85 90 95

Phe Leu His Leu His Thr Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu 100 105 119

Cys Ala Ser Ser Ser His Leu Gly Gly Ala Asn Glu Gln Tyr Phe Gly 115 120 125

Pro Gly Thr Arg Leu Thr Val Thr 130 135 <210> 38 <211> 408 <212> DNA <213> Homo sapiens <400> 38 atgggctgca ggctgctctg ctgtgcggtt ctctgtctcc tgggagcggg tgagttggtc 60 cccatggaaa cgggagttac gcagacacca agacacctgg tcatgggaat gacaaataag 120 aagtctttga aatgtgaaca acatctgggt cataacgcta tgtattggta caagcaaagt 180 gctaagaagc cactggagct catgtttgtc tacagtcttg aagaacgggt tgaaaacaac 240

Pagina 21

SEQLTXT agtgtgccaa gtcgcttctc acctgaatgc cccaacagct ctcacttatt ccttcaccta 300 cacaccctgc agccagaaga ctcggccctg tatctctgcg ccagcagctc tcatctcggg 360 ggcgctaacg agcagtactt cgggccgggc accaggctca cggtcaca 408 <210> 39 <211> 273 <212> PRT <213> Artificial Sequence <220> <223> TCR V alpha domain with a constant domain <400> 39

Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15

Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu

Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp

Arg Gly Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser 60

Pro Glu Leu Ile Met Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly 65 70 75 80

Arg Phe Thr Ala Gln Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu 85 90 95

Ile Arg Asp Ser Gln Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Ser 100 105 119

Arg Glu Ala Gly Ser Tyr Gln Leu Thr Phe Gly Lys Gly Thr Lys Leu 115 120 125

Ser Val Ile Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu 130 135 140

Pagina 22

SEQLTXT

Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe 145 150 155 160

Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile 165 170 175

Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn 180 185 190

Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala 195 200 205

Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu 210 215 220

Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr 225 230 235 240

Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu 245 250 255

Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 260 265 270

Ser <210> 40 <211> 819 <212> DNA <213> Artificial Sequence <220> <223> TCR alpha chain of clone 23.2G9 <400> 40 atgaaatcct tgagagtttt actagtgatc ctgtggcttc agttgagctg ggtttggagc 60 caacagaagg aggtggagca gaattctgga cccctcagtg ttccagaggg agccattgcc 120 tctctcaact gcacttacag tgaccgaggt tcccagtcct tcttctggta cagacaatat 180

Pagina 23

SEQLTXT tctgggaaaa gccctgagtt gataatgttc atatactcca atggtgacaa agaagatgga 240 aggtttacag cacagctcaa taaagccagc cagtatgttt ctctgctcat cagagactcc 300 cagcccagtg attcagccac ctacctctgt gcctctaggg aggctgggag ttaccaactc 360 actttcggga aggggaccaa actctcggtc ataccaaata tccagaaccc tgaccctgcc 420 gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 480 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 540 gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 600 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 660 cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 720 aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 780 gggtttaatc tgctcatgac gctgcggctg tggtccagc 819 <210> 41 <211> 315 <212> PRT <213> Artificial Sequence <220> <223> TCR V beta domain and a constant domain <400> 41

Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala 1 5 10 15

Gly Glu Leu Val Pro Met Glu Thr Gly Val Thr Gln Thr Pro Arg His

Leu Val Met Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His

Leu Gly His Asn Ala Met Tyr Trp Tyr Lys Gln Ser Ala Lys Lys Pro 60

Leu Glu Leu Met Phe Val Tyr Ser Leu Glu Glu Arg Val Glu Asn Asn 65 70 75 80

Pagina 24

SEQLTXT

Ser Val Pro Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser His Leu 85 90 95

Phe Leu His Leu His Thr Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu 100 105 110

Cys Ala Ser Ser Ser His Leu Gly Gly Ala Asn Glu Gln Tyr Phe Gly 115 120 125

Pro Gly Thr Arg Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro 130 135 140

Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr 145 150 155 160

Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His 165 170 175

Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val 180 185 190

Ser Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser 195 200 205

Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln 210 215 220

Asn Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser 225 230 235 240

Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile 245 250 255

Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu 260 265 270

Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu 275 280 285

Pagina 25

SEQLTXT

Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu 290 295 300

Met Ala Met Val Lys Arg Lys Asp Ser Arg Gly 305 310 315 <210> 42 <211> 945 <212> DNA <213> Artificial Sequence <220> <223> TCR beta chain of clone 23.2G9 <400> 42 atgggctgca ggctgctctg ctgtgcggtt ctctgtctcc tgggagcggg tgagttggtc 60 cccatggaaa cgggagttac gcagacacca agacacctgg tcatgggaat gacaaataag 120 aagtctttga aatgtgaaca acatctgggt cataacgcta tgtattggta caagcaaagt 180 gctaagaagc cactggagct catgtttgtc tacagtcttg aagaacgggt tgaaaacaac 240 agtgtgccaa gtcgcttctc acctgaatgc cccaacagct ctcacttatt ccttcaccta 300 cacaccctgc agccagaaga ctcggccctg tatctctgcg ccagcagctc tcatctcggg 360 ggcgctaacg agcagtactt cgggccgggc accaggctca cggtcacaga ggacctgaaa 420 aacgtgttcc cacccgaggt cgctgtgttt gagccatcag aagcagagat ctcccacacc 480 caaaaggcca cactggtgtg cctggccaca ggcttctacc ccgaccacgt ggagctgagc 540 tggtgggtga atgggaagga ggtgcacagt ggggtcagca cagacccgca gcccctcaag 600 gagcagcccg ccctcaatga ctccagatac tgcctgagca gccgcctgag ggtctcggcc 660 accttctggc agaacccccg caaccacttc cgctgtcaag tccagttcta cgggctctcg 720 gagaatgacg agtggaccca ggatagggcc aaacctgtca cccagatcgt cagcgccgag 780 gcctggggta gagcagactg tggcttcacc tccgagtctt accagcaagg ggtcctgtct 840 gccaccatcc tctatgagat cttgctaggg aaggccacct tgtatgccgt gctggtcagt 900 gccctcgtgc tgatggccat ggtcaagaga aaggattcca gaggc 945 <210> 43

Pagina 26

SEQLTXT

<211> 6 <212> PRT <213> Homo sapiens <400> 43

Asp Ser Ser Ser Thr Tyr 1 5 <210> 44 <211> 7 <212> PRT <213> Homo sapiens <400> 44

Ile Phe Ser Asn Met Asp Met 1 5 <210> 45 <211> 13 <212> PRT <213> Homo sapiens <400> 45

Cys Ala Val Ser Ser Gly Gly Tyr Gln Lys Val Thr Phe 1 5 10 <210> 46 <211> 5 <212> PRT <213> Homo sapiens <400> 46

Glu Asn His Arg Tyr 1 5 <210> 47 <211> 6 <212> PRT <213> Homo sapiens <400> 47

Ser Tyr Gly Val Lys Asp 1 5

Pagina 27

SEQLTXT

<210> 48 <211> 13 <212> PRT <213> Homo sapiens <400> 48

Cys Ala Ile Ser Asp Pro Tyr Arg Gly Glu Gln Tyr Phe 1 5 10 <210> 49 <211> 132 <212> PRT <213> Homo sapiens <400> 49

Met Lys Thr Phe Ala Gly Phe Ser Phe Leu Phe Leu Trp Leu Gln Leu 1 5 10 15

Asp Cys Met Ser Arg Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser

Val Arg Glu Gly Asp Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser

Ser Ser Thr Tyr Leu Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu 60

Gln Leu Leu Thr Tyr Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln 65 70 75 80

Arg Leu Thr Val Leu Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg 85 90 95

Ile Ala Asp Thr Gln Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Val 100 105 110

Ser Ser Gly Gly Tyr Gln Lys Val Thr Phe Gly Ile Gly Thr Lys Leu 115 120 125

Pagina 28

SEQLTXT

Gln Val Ile Pro 130 <210> 50 <211> 396 <212> DNA <213> Homo sapiens <400> 50 atgaagacat ttgctggatt ttcgttcctg tttttgtggc tgcagctgga ctgtatgagt 60 agaggagagg atgtggagca gagtcttttc ctgagtgtcc gagagggaga cagctccgtt 120 ataaactgca cttacacaga cagctcctcc acctacttat actggtataa gcaagaacct 180 ggagcaggtc tccagttgct gacgtatatt ttttcaaata tggacatgaa acaagaccaa 240 agactcactg ttctattgaa taaaaaggat aaacatctgt ctctgcgcat tgcagacacc 300 cagactgggg actcagctat ctacttctgt gcagtttcct ctgggggtta ccagaaagtt 360 acctttggaa ttggaacaaa gctccaagtc atccca 396 <210> 51 <211> 131 <212> PRT <213> Homo sapiens <400> 51

Met Gly Thr Arg Leu Phe Phe Tyr Val Ala Leu Cys Leu Leu Trp Thr 1 5 10 15

Gly His Met Asp Ala Gly Ile Thr Gln Ser Pro Arg His Lys Val Thr

Glu Thr Gly Thr Pro Val Thr Leu Arg Cys His Gln Thr Glu Asn His

Arg Tyr Met Tyr Trp Tyr Arg Gln Asp Pro Gly His Gly Leu Arg Leu 60

Ile His Tyr Ser Tyr Gly Val Lys Asp Thr Asp Lys Gly Glu Val Ser 65 70 75 80

Pagina 29

SEQLTXT

Asp Gly Tyr Ser Val Ser Arg Ser Lys Thr Glu Asp Phe Leu Leu Thr 85 90 95

Leu Glu Ser Ala Thr Ser Ser Gln Thr Ser Val Tyr Phe Cys Ala Ile 100 105 110

Ser Asp Pro Tyr Arg Gly Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu 115 120 125

Thr Val Thr 130 <210> 52 <211> 393 <212> DNA <213> Homo sapiens <400> 52 atgggcacaa ggttgttctt ctatgtggcc ctttgtctcc tgtggacagg acacatggat 60 gctggaatca cccagagccc aagacacaag gtcacagaga caggaacacc agtgactctg 120 agatgtcacc agactgagaa ccaccgctat atgtactggt atcgacaaga cccggggcat 180 gggctgagge tgatccatta ctcatatggt gttaaagata ctgacaaagg agaagtctca 240 gatggctata gtgtctctag atcaaagaca gaggatttcc tcctcactct ggagtccgct 300 accagctccc agacatctgt gtacttctgt gccatcagtg atccgtacag gggcgagcag 360 tacttcgggc cgggcaccag gctcacggtc aca 393 <210> 53 <211> 273 <212> PRT <213> Artificial Sequence <220> <223> TCR V alpha domain with a constant domain <400> 53

Met Lys Thr Phe Ala Gly Phe Ser Phe Leu Phe Leu Trp Leu Gln Leu 1 5 10 15

Asp Cys Met Ser Arg Gly Glu Asp Val Glu Gln Ser Leu Phe Leu Ser

Pagina 30

SEQLTXT

Val Arg Glu Gly Asp Ser Ser Val Ile Asn Cys Thr Tyr Thr Asp Ser

Ser Ser Thr Tyr Leu Tyr Trp Tyr Lys Gln Glu Pro Gly Ala Gly Leu 60

Gln Leu Leu Thr Tyr Ile Phe Ser Asn Met Asp Met Lys Gln Asp Gln 65 70 75 80

Arg Leu Thr Val Leu Leu Asn Lys Lys Asp Lys His Leu Ser Leu Arg 85 90 95

Ile Ala Asp Thr Gln Thr Gly Asp Ser Ala Ile Tyr Phe Cys Ala Val 100 105 110

Ser Ser Gly Gly Tyr Gln Lys Val Thr Phe Gly Ile Gly Thr Lys Leu 115 120 125

Gln Val Ile Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu 130 135 140

Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe 145 150 155 160

Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile 165 170 175

Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn 180 185 190

Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala 195 200 205

Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu 210 215 220

Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr

Pagina 31

SEQLTXT

225 230 235 240

Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu 245 250 255

Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 260 265 270

Ser <210> 54 <211> 819 <212> DNA <213> Artificial Sequence <220> <223> TCR alpha chain of clone 17.2G4 <400> 54 atgaagacat ttgctggatt ttcgttcctg tttttgtggc tgcagctgga ctgtatgagt 60 agaggagagg atgtggagca gagtcttttc ctgagtgtcc gagagggaga cagctccgtt 120 ataaactgca cttacacaga cagctcctcc acctacttat actggtataa gcaagaacct 180 ggagcaggtc tccagttgct gacgtatatt ttttcaaata tggacatgaa acaagaccaa 240 agactcactg ttctattgaa taaaaaggat aaacatctgt ctctgcgcat tgcagacacc 300 cagactgggg actcagctat ctacttctgt gcagtttcct ctgggggtta ccagaaagtt 360 acctttggaa ttggaacaaa gctccaagtc atcccaaata tccagaaccc tgaccctgcc 420 gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 480 gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 540 gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 600 tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 660 cccagcccag aaagttcctg tgatgtcaag ctggtcgaga aaagctttga aacagatacg 720 aacctaaact ttcaaaacct gtcagtgatt gggttccgaa tcctcctcct gaaagtggcc 780 gggtttaatc tgctcatgac gctgcggctg tggtccagc 819

Pagina 32

SEQLTXT

<210> 55 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> TCR V beta domain and a constant domain <400> 55

Met Gly Thr Arg Leu Phe Phe Tyr Val Ala Leu Cys Leu Leu Trp Thr 1 5 10 15

Gly His Met Asp Ala Gly Ile Thr Gln Ser Pro Arg His Lys Val Thr

Glu Thr Gly Thr Pro Val Thr Leu Arg Cys His Gln Thr Glu Asn His

Arg Tyr Met Tyr Trp Tyr Arg Gln Asp Pro Gly His Gly Leu Arg Leu 60

Ile His Tyr Ser Tyr Gly Val Lys Asp Thr Asp Lys Gly Glu Val Ser 65 70 75 80

Asp Gly Tyr Ser Val Ser Arg Ser Lys Thr Glu Asp Phe Leu Leu Thr 85 90 95

Leu Glu Ser Ala Thr Ser Ser Gln Thr Ser Val Tyr Phe Cys Ala Ile 100 105 110

Ser Asp Pro Tyr Arg Gly Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu 115 120 125

Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val 130 135 140

Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu 145 150 155 160

Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp

Pagina 33

SEQLTXT

165 170 175

Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180 185 190

Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser 195 200 205

Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His 210 215 220

Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp 225 230 235 240

Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala 245 250 255

Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly 260 265 270

Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285

Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300

Arg Lys Asp Ser Arg Gly 305 310 <210> 56 <211> 930 <212> DNA <213> Artificial Sequence <220> <223> TCR beta chain of clone 17.2G4 <400> 56 atgggcacaa ggttgttctt ctatgtggcc ctttgtctcc tgtggacagg acacatggat 60 gctggaatca cccagagccc aagacacaag gtcacagaga caggaacacc agtgactctg 120

Pagina 34

SEQLTXT agatgtcacc agactgagaa ccaccgctat atgtactggt atcgacaaga cccggggcat 180 gggctgagge tgatccatta ctcatatggt gttaaagata ctgacaaagg agaagtctca 240 gatggctata gtgtctctag atcaaagaca gaggatttcc tcctcactct ggagtccgct 300 accagctccc agacatctgt gtacttctgt gccatcagtg atccgtacag gggcgagcag 360 tacttcgggc cgggcaccag gctcacggtc acagaggacc tgaaaaacgt gttcccaccc 420 gaggtcgctg tgtttgagcc atcagaagca gagatctccc acacccaaaa ggccacactg 480 gtgtgcctgg ccacaggctt ctaccccgac cacgtggagc tgagctggtg ggtgaatggg 540 aaggaggtgc acagtggggt cagcacagac ccgcagcccc tcaaggagca gcccgccctc 600 aatgactcca gatactgcct gagcagccgc ctgagggtct cggccacctt ctggcagaac 660 ccccgcaacc acttccgctg tcaagtccag ttctacgggc tctcggagaa tgacgagtgg 720 acccaggata gggccaaacc tgtcacccag atcgtcagcg ccgaggcctg gggtagagca 780 gactgtggct tcacctccga gtcttaccag caaggggtcc tgtctgccac catcctctat 840 gagatcttgc tagggaaggc caccttgtat gccgtgctgg tcagtgccct cgtgctgatg 900 gccatggtca agagaaagga ttccagaggc 930 <210> 57 <211> 5 <212> PRT <213> Homo sapiens <400> 57

Thr Thr Ser Asp Arg 1 5 <210> 58 <211> 7 <212> PRT <213> Homo sapiens <400> 58

Leu Leu Ser Asn Gly Ala Val 1 5 <210> 59

Pagina 35

SEQLTXT

<211> 11 <212> PRT <213> Homo sapiens <400> 59

Cys Ala Val Gly Arg Asn Asn Asp Met Arg Phe 1 5 10 <210> 60 <211> 5 <212> PRT <213> Homo sapiens <400> 60

Ser Asn His Leu Tyr 1 5 <210> 61 <211> 6 <212> PRT <213> Homo sapiens <400> 61

Phe Tyr Asn Asn Glu Ile 1 5 <210> 62 <211> 8 <212> PRT <213> Homo sapiens <400> 62

Cys Ala Ser Thr Glu Ala Phe Phe 1 5 <210> 63 <211> 127 <212> PRT <213> Homo sapiens <400> 63

Met Lys Lys Leu Leu Ala Met Ile Leu Trp Leu Gln Leu Asp Arg Leu 1 5 10 15

Pagina 36

SEQLTXT

Ser Gly Glu Leu Lys Val Glu Gln Asn Pro Leu Phe Leu Ser Met Gln

Glu Gly Lys Asn Tyr Thr Ile Tyr Cys Asn Tyr Ser Thr Thr Ser Asp

Arg Leu Tyr Trp Tyr Arg Gln Asp Pro Gly Lys Ser Leu Glu Ser Leu 60

Phe Val Leu Leu Ser Asn Gly Ala Val Lys Gln Glu Gly Arg Leu Met 65 70 75 80

Ala Ser Leu Asp Thr Lys Ala Arg Leu Ser Thr Leu His Ile Thr Ala 85 90 95

Ala Val His Asp Leu Ser Ala Thr Tyr Phe Cys Ala Val Gly Arg Asn 100 105 110

Asn Asp Met Arg Phe Gly Ala Gly Thr Arg Leu Thr Val Lys Pro 115 120 125 <210> 64 <211> 381 <212> DNA <213> Homo sapiens <400> 64 atgaagaagc tactagcaat gattctgtgg cttcaactag accggttaag tggagagctg 60 aaagtggaac aaaaccctct gttcctgagc atgcaggagg gaaaaaacta taccatctac 120 tgcaattatt caaccacttc agacagactg tattggtaca ggcaggatcc tgggaaaagt 180 ctggaatctc tgtttgtgtt gctatcaaat ggagcagtga agcaggaggg acgattaatg 240 gcctcacttg ataccaaagc ccgtctcagc accctccaca tcacagctgc cgtgcatgac 300 ctctctgcca cctacttctg tgccgtgggc cgaaacaatg acatgcgctt tggagcaggg 360 accagactga cagtaaaacc a 381 <210> 65

Pagina 37

SEQLTXT

<211> 127 <212> PRT <213> Homo sapiens <400> 65

Met Asp Thr Trp Leu Val Cys Trp Ala Ile Phe Ser Leu Leu Lys Ala 1 5 10 15

Gly Leu Thr Glu Pro Glu Val Thr Gln Thr Pro Ser His Gln Val Thr

Gln Met Gly Gln Glu Val Ile Leu Arg Cys Val Pro Ile Ser Asn His

Leu Tyr Phe Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Lys Val Glu Phe 60

Leu Val Ser Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Phe 65 70 75 80

Asp Asp Gln Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu 85 90 95

Lys Ile Arg Ser Thr Lys Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala 100 105 119

Ser Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val 115 120 125 <210> 66 <211> 381 <212> DNA <213> Homo sapiens <400> 66 atggatacct ggctcgtatg ctgggcaatt tttagtctct tgaaagcagg actcacagaa 60 cctgaagtca cccagactcc cagccatcag gtcacacaga tgggacagga agtgatcttg 120 cgctgtgtcc ccatctctaa tcacttatac ttctattggt acagacaaat cttggggcag 180 aaagtcgagt ttctggtttc cttttataat aatgaaatct cagagaagtc tgaaatattc 240

Pagina 38

SEQLTXT gatgatcaat tctcagttga aaggcctgat ggatcaaatt tcactctgaa gatccggtcc 300 acaaagctgg aggactcagc catgtacttc tgtgccagca ctgaagcttt ctttggacaa 360 ggcaccagac tcacagttgt a 381 <210> 67 <211> 268 <212> PRT <213> Artificial Sequence <220> <223> TCR V alpha domain with a constant domain <400> 67

Met Lys Lys Leu Leu Ala Met Ile Leu Trp Leu Gln Leu Asp Arg Leu 1 5 10 15

Ser Gly Glu Leu Lys Val Glu Gln Asn Pro Leu Phe Leu Ser Met Gln

Glu Gly Lys Asn Tyr Thr Ile Tyr Cys Asn Tyr Ser Thr Thr Ser Asp

Arg Leu Tyr Trp Tyr Arg Gln Asp Pro Gly Lys Ser Leu Glu Ser Leu 60

Phe Val Leu Leu Ser Asn Gly Ala Val Lys Gln Glu Gly Arg Leu Met 65 70 75 80

Ala Ser Leu Asp Thr Lys Ala Arg Leu Ser Thr Leu His Ile Thr Ala 85 90 95

Ala Val His Asp Leu Ser Ala Thr Tyr Phe Cys Ala Val Gly Arg Asn 100 105 119

Asn Asp Met Arg Phe Gly Ala Gly Thr Arg Leu Thr Val Lys Pro Asn 115 120 125

Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser 130 135 140

Pagina 39

SEQLTXT

Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn 145 150 155 160

Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Thr Val 165 170 175

Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp 180 185 190

Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile 195 200 205

Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val 210 215 220

Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln 225 230 235 240

Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly 245 250 255

Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 <210> 68 <211> 804 <212> DNA <213> Artificial Sequence <220> <223> TCR alpha chain of clone 12.5H9 <400> 68 atgaagaagc tactagcaat gattctgtgg cttcaactag accggttaag tggagagctg 60 aaagtggaac aaaaccctct gttcctgagc atgcaggagg gaaaaaacta taccatctac 120 tgcaattatt caaccacttc agacagactg tattggtaca ggcaggatcc tgggaaaagt 180 ctggaatctc tgtttgtgtt gctatcaaat ggagcagtga agcaggaggg acgattaatg 240 gcctcacttg ataccaaagc ccgtctcagc accctccaca tcacagctgc cgtgcatgac 300

Pagina 40

SEQLTXT ctctctgcca cctacttctg tgccgtgggc cgaaacaatg acatgcgctt tggagcaggg 360 accagactga cagtaaaacc aaatatccag aaccctgacc ctgccgtgta ccagctgaga 420 gactctaaat ccagtgacaa gtctgtctgc ctattcaccg attttgattc tcaaacaaat 480 gtgtcacaaa gtaaggattc tgatgtgtat atcacagaca aaactgtgct agacatgagg 540 tctatggact tcaagagcaa cagtgctgtg gcctggagca acaaatctga ctttgcatgt 600 gcaaacgcct tcaacaacag cattattcca gaagacacct tcttccccag cccagaaagt 660 tcctgtgatg tcaagctggt cgagaaaagc tttgaaacag atacgaacct aaactttcaa 720 aacctgtcag tgattgggtt ccgaatcctc ctcctgaaag tggccgggtt taatctgctc 780 atgacgctgc ggctgtggtc cagc 804 <210> 69 <211> 304 <212> PRT <213> Artificial Sequence <220> <223> TCR V beta domain and a constant domain <400> 69

Met Asp Thr Trp Leu Val Cys Trp Ala Ile Phe Ser Leu Leu Lys Ala 1 5 10 15

Gly Leu Thr Glu Pro Glu Val Thr Gln Thr Pro Ser His Gln Val Thr

Gln Met Gly Gln Glu Val Ile Leu Arg Cys Val Pro Ile Ser Asn His

Leu Tyr Phe Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Lys Val Glu Phe 60

Leu Val Ser Phe Tyr Asn Asn Glu Ile Ser Glu Lys Ser Glu Ile Phe 65 70 75 80

Asp Asp Gln Phe Ser Val Glu Arg Pro Asp Gly Ser Asn Phe Thr Leu 85 90 95

Pagina 41

SEQLTXT

Lys Ile Arg Ser Thr Lys Leu Glu Asp Ser Ala Met Tyr Phe Cys Ala 100 105 119

Ser Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val Glu 115 120 125

Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser 130 135 140

Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala 145 150 155 160

Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly 165 170 175

Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln Pro Leu Lys Glu 180 185 190

Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg 195 200 205

Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln 210 215 220

Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg 225 230 235 240

Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala 245 250 255

Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly Val Leu Ser Ala 260 265 270

Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val 275 280 285

Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Phe 290 295 300

Pagina 42

SEQLTXT

<210> 70 <211> 912 <212> DNA <213> Artificial Sequence <220> <223> TCR beta chain of clone 12.5H9 <400> 70 atggatacct ggctcgtatg ctgggcaatt tttagtctct tgaaagcagg actcacagaa 60 cctgaagtca cccagactcc cagccatcag gtcacacaga tgggacagga agtgatcttg 120 cgctgtgtcc ccatctctaa tcacttatac ttctattggt acagacaaat cttggggcag 180 aaagtcgagt ttctggtttc cttttataat aatgaaatct cagagaagtc tgaaatattc 240 gatgatcaat tctcagttga aaggcctgat ggatcaaatt tcactctgaa gatccggtcc 300 acaaagctgg aggactcagc catgtacttc tgtgccagca ctgaagcttt ctttggacaa 360 ggcaccagac tcacagttgt agaggacctg aacaaggtgt tcccacccga ggtcgctgtg 420 tttgagccat cagaagcaga gatctcccac acccaaaagg ccacactggt gtgcctggcc 480 acaggcttct tccccgacca cgtggagctg agctggtggg tgaatgggaa ggaggtgcac 540 agtggggtca gcacggaccc gcagcccctc aaggagcagc ccgccctcaa tgactccaga 600 tactgcctga gcagccgcct gagggtctcg gccaccttct ggcagaaccc ccgcaaccac 660 ttccgctgtc aagtccagtt ctacgggctc tcggagaatg acgagtggac ccaggatagg 720 gccaaacccg tcacccagat cgtcagcgcc gaggcctggg gtagagcaga ctgtggcttt 780 acctcggtgt cctaccagca aggggtcctg tctgccacca tcctctatga gatcctgcta 840 gggaaggcca ccctgtatgc tgtgctggtc agcgcccttg tgttgatggc catggtcaag 900 agaaaggatt tc 912 <210> 71 <211> 9 <212> PRT <213> Homo sapiens <400> 71

Ala Leu Leu Pro Ala Val Pro Ser Leu 1 5

Pagina 43

SEQLTXT

<2105 72 <211> 9 <212> PRT <213> Homo sapiens <400> 72

Val Leu Asp Phe Ala Pro Pro Gly Ala 1 5 <2105 73 <2115 9 <212> PRT <213> Homo sapiens <400> 73

Thr Pro Tyr Ser Ser Asp Asn Leu Tyr 1 5 <210> 74 <211> 12 <212> PRT <213> Homo sapiens <400> 74

Val Leu Asp Phe Ala Pro Pro Gly Ala Ser Ala Tyr 1 5 10 <21e> 75 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> WT1 forward primer <400> 75 agacccacac caggactcat 20 <210> 76 <211> 20 <212> DNA <213> Artificial Sequence

Pagina 44

SEQLTXT

<220> <223> WT1 reverse primer <400> 76 gatgcatgtt gtgatggcgg 20 <21e> 77 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GUSB forward primer <400> 77 actgaacagt caccgacgag 20 <21e> 78 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GUSB reverse primer <400> 78 ggaacgctgc actttttggt 20 <210> 79 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PSMB4 forward primer <400> 79 gtttccgcaa catctctcgc 20 <210> 80 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PSMB4 reverse primer <400> 80

Pagina 45

SEQLTXT catcaatcac catctggccg 20 <21e> 81 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> VPS29 forward primer <400> 81 tgagaggaga cttcgatgag aatc 24 <21e> 82 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> VPS29 reverse primer <400> 82 tctgcaacag ggctaagctg 20 <21e> 83 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> murinized CMV-specific TCR peptide <400> 83

Asn Leu Val Pro Met Val Ala Thr Val 1 5 <210> 84 <211> 12 <212> PRT <213> Homo sapiens <400> 84

Trp Thr Glu Gly Gln Ser Asn His Ser Thr Gly Tyr 1 5 10

Pagina 46

SEQLTXT

<210> 85 <211> 9 <212> PRT <213> Homo sapiens <400> 85

Phe Gly Pro Pro Pro Pro Ser Gln Ala 1 5 <210> 86 <211> 9 <212> PRT <213> Homo sapiens <400> 86

Ala Gln Phe Pro Asn His Ser Phe Lys 1 5 <210> 87 <211> 10 <212> PRT <213> Homo sapiens <400> 87

His Ala Ala Gln Phe Pro Asn His Ser Phe 1 5 10 <210> 88 <211> 9 <212> PRT <213> Homo sapiens <400> 88

Arg Met Phe Pro Asn Ala Pro Tyr Leu 1 5 <210> 89 <211> 9 <212> PRT <213> Homo sapiens <400> 89

Cys Met Thr Trp Asn Gln Met Asn Leu

Pagina 47

SEQLTXT

1 5 <210> 90 <211> 9 <212> PRT <213> Homo sapiens <400> 90

Arg Trp Pro Ser Cys Gln Lys Lys Phe 1 5

Pagina 48

Claims

CONCLUSIONS

Isolated nucleic acid compound encoding a transcription factor Wilms' tumor 1 (WT1) antigen-specific binding protein having a TCR a chain variable (Va) domain and a TCR B chain variable (VB) domain, the compound comprising:

i. a nucleic acid sequence encoding 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; and a nucleic acid sequence encoding a TCR VB domain comprising a CDR3 amino acid sequence having at least 80% sequence identity to SEQ ID NO: 6, or a functional fragment thereof, wherein the CDR3 sequences together specifically bind to WT1; or ii. a nucleic acid sequence encoding 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 encoding a TCR VB 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; or iii. a nucleic acid sequence encoding 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 encoding a TCR VB 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 WT1; or iv. a nucleic acid sequence encoding 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 encoding a TCR VB 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 WT1;

v. a nucleic acid sequence encoding 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 encoding a TCR VB 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.

2. Nucleic acid composition according to any one of the preceding claims, wherein:

i. the CDR3 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 3, and the CDR3 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 6; or i. the CDR3 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 17, and the CDR3 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 20; or il. the CDR3 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 31, and the CDR3 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 34; or iv. the CDR3 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: and the CDR3 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 48; or

V. the CDR3 of the Va domain comprises or consists of the amino acid sequence of SEQ ID NO: 59, and the CDR3 of the VB domain comprises or consists of the amino acid sequence of SEQ ID NO: 62.

The nucleic acid composition of any one of the preceding claims, wherein: the Va domain comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of SEQ ID NO: 7; and the VB domain comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of SEQ ID NO: 9;

ii. the Va domain comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of SEQ ID NO: 21; and the VB domain comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of SEQ ID NO: 21; or iii. the Va domain comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of SEQ ID NO: 35; and the VB domain comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of SEQ ID NO: 37; or iv. the Va domain comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of SEQ ID NO: 49; and the VB domain comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of SEQ ID NO: 51; or v. the Va domain comprises an amino acid sequence having at least 80% sequence identity to, comprising, or consisting of SEQ ID NO: 63; and the VB domain comprises an amino acid sequence having a sequence

identity of at least 80% from, including, or consisting of SEQ ID NO: 65.

A nucleic acid composition according to any one of the preceding claims, wherein the WT1 antigen comprises 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 nucleic acid composition of claim 4, wherein the encoded binding protein is capable of specifically binding a peptide:HLA complex selected from the group consisting of: an 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 nucleic acid composition according to any one of the preceding claims, wherein the nucleic acid sequence is codon-optimized for expression in a host cell, and optionally wherein the host cell is a human cell

7. Nucleic acid composition according to any one of the preceding claims, further comprising a TCR α chain constant domain and/or a TCR B chain constant domain.

The nucleic acid composition of any one of the preceding claims, wherein the encoded binding protein comprises a TCR, an antigen-binding fragment of a TCR, a chimeric antigen receptor (CAR), or an ImmTAC.

The nucleic acid composition of claim 8, wherein the antigen-binding fragment of a TCR is a single chain TCR (scTCR) or a chimeric TCR dimer, wherein the antigen-binding fragment of the TCR is linked to an alternative transmembrane and intracellular binding domain.

A vector system comprising a nucleic acid composition according to any one of claims 1 to 9.

The vector system of claim 10, wherein 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, canarypox virus, minicircle virus, and synthetic DNA or RNA.

12. Modified cell, comprising a nucleic acid composition according to any one of claims 1 to 9, or a vector system according to claim 10 or 11.

The modified cell of claim 12, wherein the modified cell is selected from the group consisting of a CD8 T cell, a CD4 T cell, an NK cell, a gamma-delta T cell, a hematopoietic stem cell, a inducible pluripotent stem cell, a progenitor cell, a T cell line, and an NK-92 cell line.

The modified cell of claim 12 or 13, wherein the modified cell is a human cell.

A pharmaceutical composition comprising a nucleic acid composition according to any one of claims 1 to 9, a vector system according to claim 10 or 11, or a modified cell according to any one of claims 12 to 14, and a pharmaceutically acceptable excipient, a pharmaceutically acceptable adjuvant , a pharmaceutically acceptable diluent, and/or a pharmaceutically acceptable carrier.

A pharmaceutical composition according to claim 15, for use in inducing or enhancing an immune response in a human subject diagnosed with a WT 1-associated disease or condition.

A pharmaceutical composition according to claim 15, for use in stimulating a cell-mediated immune response in a target cell population or tissue in a human subject.

A pharmaceutical composition according to claim 15 for use in conferring anti-humor immunity on a human subject.

A pharmaceutical composition according to claim 15, for use in treating a human subject suffering from a disease or condition associated with an elevated level of HLA-restricted WT1 antigen.

A pharmaceutical composition for use according to any one of claims 16 to 19, wherein the human subject has at least one tumor.

A pharmaceutical composition for use according to any one of claims 17 to 20, wherein the subject has been diagnosed with a WT 1-associated disease or condition.

A pharmaceutical composition for use according to any one of claims 16 or 21, wherein the WT 1-associated disease or condition is a hematological malignancy or a solid tumor.

A pharmaceutical composition for use according to claim 22, wherein the hematological malignancy is selected from the group consisting of: acute myeloid leukemia (AML), multiple myeloma, plasma cell leukemia, acute lymphocytic leukemia (ALL), and B-cell lymphoma, optionally wherein B-cell lymphoma was selected from the group consisting of: diffuse large B-cell lymphoma (DLBCL), high grade B-cell lymphoma, Mantle cell lymphoma (MCL), follicular lymphoma (FL), hairy cell leukemia, and Burkitt lymphoma.

A pharmaceutical composition for use according to claim 22, wherein the hematological malignancy is AML.

A pharmaceutical composition for use according to claim 22, wherein the solid tumor is selected from the group consisting of: ovarian cancer, mesothelioma, uterine cancer, testicular cancer, pancreatic cancer, lung cancer, kidney cancer, thymoma, sarcoma, prostate cancer, colorectal cancer, breast cancer, cervical cancer, stomach cancer, melanoma, bladder cancer, and kidney cancer.

26. Method for generating a binding protein capable of specifically binding to a peptide comprising a WT1 antigen, and not binding to a peptide not comprising the WT1 antigen, comprising contacting a cell and a nucleic acid composition according to any one of claims 1 to 9, in conditions in which the nucleic acid composition is taken up and expressed by the cell.

The method of claim 26, wherein the method is ex-vivo.

28. Isolated nucleic acid sequence comprising or consisting of the nucleotide sequence of any 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.

29. Isolated nucleic acid sequence comprising or consisting of the nucleotide sequence of any 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.