WT-1-specific T cell immunotherapy

Abstract

The present disclosure provides binding proteins specific for human nephroblastoma protein 1 (WT-1) epitopes and host cells expressing the binding proteins. Polynucleotides encoding the binding proteins and vectors comprising the polynucleotides are also provided. Related methods and uses of the currently disclosed compositions are provided for treating diseases or conditions associated with WT-1 expression, such as various cancers.

Description

[0001] Statement regarding sequence listing

[0002] The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is 360056_473WO_SEQUENCE_LISTING.txt. The text file is 109 KB, was created on August 17, 2020, and was submitted electronically through EFS-Web. Background Art

[0003] T cell receptor (TCR) gene therapy is an emerging therapeutic approach that aims to overcome the obstacles associated with traditional T cell adoptive immunotherapy, such as the extensive time and labor required to isolate, characterize, and expand tumor antigen-specific T cells (Schmitt et al., Hum. Gene Ther. 20:1240, 2009). Another obstacle is that most of the identified tumor antigens that can be targeted by T cell immunotherapy are overexpressed self-proteins, so high-affinity T cells specific for these antigens are usually eliminated during thymic selection and are rare or absent in the peripheral component.

[0004] Strategies to enhance the affinity of TCRs for use in TCR gene therapy have been considered (eg, Udyavar et al., J. Immunol. 182:4439, 2009; Zhao et al., J. Immunol. 179:5845, 2007; Richman and Kranz, Biomol. Eng. 24:361, 2007).

[0005] Targeted T cell therapy achieves maximum clinical effect and the prerequisite with minimum immunotoxicity includes identification in, for example, malignant cell compartments with high expression and presentation but no disease-associated antigens significantly expressed in normal tissues. For example, several acute myeloid leukemia (AML)-associated antigens have been described, and it has been shown that Wilms tumor protein 1 (WT-1) expression levels in the leukemia stem cell (LSC) compartments of most AML patients are significantly higher than physiological hematopoietic stem cells (HSC). WT-1 has been used as a target in clinical trials of adoptive T cell transfer and peptide vaccination (see, for example, U.S. Patent number 7,342,092; 7,608,685; 7,622,119). In addition, it is reported that WT-1 expression is a marker of minimal residual disease, because the transcriptional levels of AML patients elevated in morphological remission indicate significant clinical relapse (Inoue et al., Blood 84:3071,1994; Ogawa et al., Blood 101:1698,2003).

[0006] Since WT-1 is an intracellular (usually nuclear) protein, immunotherapy targeting WT-1 has been conducted using agents designed to generate WT-1-specific CD8 + A cellular process of cytotoxic T lymphocyte (CTL) response that recognizes peptides presented on the cell surface via MHC class I. To induce a CTL response, intracellular proteins are typically degraded by the proteasome or endo / lysosome, and the resulting peptide fragments are bound to MHC class I or class II molecules. These peptide-MHC complexes are displayed on the cell surface, where they bind to T cells via peptide-MHC-TCR interactions. Peptides derived from WT-1 protein could theoretically be used in human vaccines to induce human leukocyte antigen (HLA)-restricted cytotoxic CD8 T cells capable of killing tumor cells. + T cells. However, since WT-1 is a self-protein, immunization may only elicit a response from T cells with low-affinity TCRs. In addition, antibodies against WT-1 can be detected in patients with hematopoietic malignancies and solid tumors, suggesting that WT-1 may be a highly immunogenic antigen (Gaiger et al., Clin. Cancer Res. 7 (Suppl. 3): 761, 2001).

[0007] Clearly, there is a need for alternative gene therapies for use as highly specific WT-1 targeted immunotherapies for various cancers, such as leukemias and tumors. The embodiments of the present disclosure address this need and provide other related advantages. Summary of the Invention

[0008] According to certain embodiments, the present disclosure provides a binding protein capable of binding to the RMFPNAPYL (SEQ ID NO: 94): human leukocyte antigen (HLA) complex, wherein the binding protein comprises: (a) a T cell receptor (TCR) alpha chain variable region (Vα) domain comprising a CDR3 amino acid sequence (CDR3α) according to any one of SEQ ID NOs.: 19, 22, 25, or 28, or a variant thereof; and a TCR beta chain variable region (Vβ) domain, wherein Vβ optionally comprises or consists of an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in any one of SEQ ID NOs.: 5-8 or 13-16; (b) a TCR Vβ domain comprising a CDR3 amino acid sequence (CDR3α) according to SEQ ID NOs.: 19, 22, 25, or 28, or a variant thereof; and ID NO.: 31, 34, 37 or 40 or a variant thereof; and a TCR Vα domain, wherein Vα optionally comprises or consists of an amino acid sequence that is at least 90% identical to the amino acid sequence shown in any one of SEQ ID NO.: 1-4 or 9-12; or (c) the TCR Vα domain of (a) and the TCR Vβ domain of (b).

[0009] In certain embodiments, the binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO.: 94): human leukocyte antigen (HLA) complex, generating pEC 50 IFNγ of 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 or more (e.g., an immune cell (e.g., a T cell, NK-T cell, or NK cell) comprising the binding protein has a pEC 50 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 or more SEQ ID NO.: 94: HLA IFNγ production).

[0010] In certain embodiments, the HLA comprises HLA-A*0201.

[0011] In certain embodiments, the binding protein is human, humanized, or chimeric.

[0012] In any embodiments disclosed herein, the binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO.: 94): human leukocyte antigen (HLA) complex, wherein the binding protein comprises a TCR Vβ domain and a TCR Vα domain, wherein: (i) the Vβ domain comprises, or consists of, an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence of any one of SEQ ID NOs.: 5-8 or 13-16; and / or (ii) the Vα domain comprises, or consists of, an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs.: 1-4 or 9-12.

[0013] In certain embodiments, the encoded binding protein is a TCR, CAR, or scTCR.

[0014] Also provided herein are modified immune cells comprising a heterologous polynucleotide encoding a binding protein disclosed herein. In certain embodiments, the immune cell comprises a T cell, an NK cell, an NK-T cell, or any combination thereof. In certain embodiments, the immune cell encoding the binding protein disclosed herein is activated in the presence of a peptide according to SEQ ID NO.: 94 (e.g., contained in a peptide:HLA complex). In certain embodiments, the immune cell encoding the binding protein disclosed herein is capable of killing a target cell expressing the SEQ ID NO: 94:HLA complex.

[0015] Also provided are isolated polynucleotides encoding binding proteins according to the present disclosure. In certain embodiments, the encoded binding protein comprises: (i) an amino acid sequence as set forth in any one of SEQ ID NOs.: 19, 22, 25, 28, 31, 34, 37, or 40; (ii) an amino acid sequence as set forth in any one of SEQ ID NOs.: 17, 20, 23, 26, 29, 32, 35, or 38; (iii) an amino acid sequence as set forth in any one of SEQ ID NOs.: 18, 21, 24, 27, 30, 33, 36, or 39; (iv) an amino acid sequence comprising, consisting of, or having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to an amino acid sequence as set forth in any one of SEQ ID NOs: 1-4 or 9-12; (v) an amino acid sequence comprising, consisting of, or having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to an amino acid sequence as set forth in any one of SEQ ID NOs: NO.: 5-8 or 13-16, or has at least 90% identity with the amino acid sequence; (vi) an amino acid sequence comprising, or consisting of, or having at least 90% identity with the amino acid sequence as shown in any one of SEQ ID NO.: 46-53; (vii) an amino acid sequence comprising, or consisting of, or having at least 90% identity with the amino acid sequence as shown in any one of SEQ ID NO.: 54-61; or any combination of (viii) (i)-(vii).

[0016] In certain embodiments, the polynucleotide comprises or consists of a polynucleotide having at least 75% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the nucleotide sequence set forth in any one of SEQ ID NOs.: 62-81. In certain embodiments, the polynucleotide is codon-optimized for expression in a host cell, such as an immune cell (e.g., a human T cell).

[0017] Also provided are vectors comprising a polynucleotide as described herein, as well as compositions comprising the modified cells, binding proteins, polynucleotides and / or vectors of the disclosure.

[0018] Also provided are methods for treating a subject having a disease or condition associated with WT-1 expression or activity, wherein the method comprises administering to the subject an effective amount of a modified immune cell, binding protein, polynucleotide, or vector as disclosed herein.

[0019] In certain embodiments, the disease or condition is a hematological malignancy or solid cancer. For example, the hematological malignancy to be treated can be acute lymphocytic leukemia (ALL), acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia (AML, including refractory and relapsed AML, and including acute myeloid leukemia, acute promyelocytic leukemia, acute granulocytic leukemia, acute myelomonocytic leukemia (for example, with or without eosinophilia), acute monocytic leukemia, acute erythroid leukemia and acute megakaryocytic leukemia), chronic myeloid leukemia (CML), chronic myeloid leukemia, chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL) or multiple myeloma (MM, including refractory and relapsed MM). Exemplary solid cancer to be treated can be bile duct cancer, bladder cancer, bone and soft tissue cancer, brain tumor, breast cancer (breast

[0013] The present invention relates to a type of cancer that is not intended to be treated with a tumour or other similar disease, and includes, but is not limited to, ovarian cancer (e.g., breast carcinoma, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonal carcinoma, endometrial cancer, esophageal cancer, stomach cancer, gastric adenocarcinoma, glioblastoma multiforme, glioblastoma, melanoma, diffuse peritoneal mesothelioma, malignant pleural mesothelioma, glioma, astrocytoma, gynecological cancer, head and neck squamous cell carcinoma, liver cancer, hepatocellular carcinoma, lung cancer, non-small cell carcinoma, malignant melanoma, osteosarcoma, ovarian cancer (e.g., epithelial ovarian cancer, ovarian carcinoma), fallopian tube cancer, endometrial cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytoma, primary thyroid cancer, prostate cancer, kidney cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, osteogenic sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, uterine carcinosarcoma, or uterine cancer.

[0020] These and other aspects and embodiments of the present disclosure will be further understood by reference to the following detailed description and accompanying drawings. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the application data sheet are incorporated herein by reference in their entirety, as if each were individually incorporated. If necessary, the aspects and embodiments of the present disclosure can be modified to employ concepts of the various patents, applications, and publications to provide further embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1Identification of WT-1 specific T cell lines is shown. Briefly, WT-1 specific polyclonal T cell lines (CD8+) obtained from 4 healthy donors were cultured with peptide-pulsed autologous dendritic cells, stained with CD8-independent (CD8i) WT-1 peptide / HLA-A tetramers, and high tetramer stained cells were sorted. Twenty-four (24) WT-1 specific clonotypes were identified. TCR repertoire analysis was performed by sequencing. The fold enrichment (y-axis) and CD3 surface expression (x-axis) of each clonotype in the classified and unclassified populations are shown.

[0022] Figures 2A-2C Shown is the expression of an exemplary WT-1 specific binding protein in a transduced host cell. Figure 1 Ten (10) WT-1-specific TCRs of the 24 clonotypes shown were used to transduce TCRα / TCRβ-deficient J76 Jurkat T cells. WT-1:HLA-A tetramer binding and CD3 expression of the transduced cells (shown in light grey) were compared to cells transduced with a reference WT-1-specific TCR (shown in dark grey) having an α chain according to SEQ ID NO.: 82 and a β chain according to SEQ ID NO.: 83 and described by Schmitt et al. (Nat. Biotechnol. 35: 1188, 2017).

[0023] Figure 3A and 3B The binding proteins of the present disclosure are shown to be functional in transduced host cells. (A) Jurkat cells expressing the Nur77-dt Tomato reporter gene (reporting antigen-specific signaling in human T cells; see Ahsouri and Weiss, J Immunol 198(2):657-668 (2017)) were transduced with a WT-1 specific TCR and incubated with APCs loaded with the indicated concentrations of WT-1 peptide for 24 hours. (B) Nur77 activating peptide ECs of the indicated TCRs 50 value.

[0024] Figures 4A-4C Further functional characterization of exemplary binding proteins of the present disclosure is shown. (A) Peptide / MHC tetramers bind to CD8+ T cells that were transduced to express WT-1-specific TCRs as shown. (B) IFNγ production (as measured by flow cytometry) was assessed by co-culturing expanded antigen-specific CD8+ T cells for 4 hours with T2 target cells pulsed with titrated concentrations of peptide. The percentage of IFN-γ+ T cells is shown. (C) The IFNγ peptide EC for each TCR was determined by fitting the percentage of IFNγ-producing cells to the dose-response curve by linear regression.50 .

[0025] Figure 5A and 5B Lysis of tumor cells by T cells expressing exemplary binding proteins of the present disclosure (TCR DL6 or DL10) is shown compared to lysis by T cells expressing a reference TCR (α chain according to SEQ ID NO.: 82; β chain according to SEQ ID NO.: 83). (A) Lysis of HLA-A2 transduced tumor cell line MDA-MB-468 cells. (B) Lysis of natural HLA-A2+ tumor line Panc-1 cells. (A) and (B) show the results of a 4 hour assay in which target cells were loaded with 51 Cr, TCR-transduced T cell-mediated killing was calculated by measuring Cr release in response to reduction of effector T cell dose relative to tumor (E:T; x-axis).

[0026] Figure 6 Shown is IFN-γ production by an exemplary TCR DL10 of the present disclosure in the presence of T2 target cells pulsed with peptides, compared to a reference TCR (α chain according to SEQ ID NO.: 82; β chain according to SEQ ID NO.: 83).

[0027] Figure 7 Figure 2 shows reduced growth of Panc-1 tumor cells in the presence of T cells expressing the exemplary TCR DL10 of the present disclosure or in the presence of T cells expressing a reference TCR (α chain according to SEQ ID NO.: 82; β chain according to SEQ ID NO.: 83) compared to tumor cells alone. Cell proliferation / survival was monitored over 7 days in the IncuCyte assay. Error bars represent standard error of the mean. DETAILED DESCRIPTION

[0028] In certain aspects, the present disclosure provides binding proteins (e.g., TCRs, CARs, scTCRs) specific for WT-1 peptide antigens associated with major histocompatibility complexes (MHCs) (e.g., human leukocyte antigens (HLAs)), polynucleotides encoding binding proteins, vectors, modified immune cells encoding and / or expressing binding proteins, and related compositions. The WT-1 specific compositions of the present disclosure can be used, for example, in adoptive immunotherapy to treat cancer. As background, most tumor targets for T cell-based immunotherapy are self-antigens because tumors originate from previously normal tissue. For example, such tumor-associated antigens (TAAs) may be expressed at high levels in cancer cells, but may not be expressed or may be minimally expressed in other cells. During T cell development in the thymus, T cells that weakly bind to self-antigens are allowed to survive in the thymus, where they can further develop to increase specificity to foreign invaders, while T cells that strongly bind to self-antigens are eliminated from the immune system because these cells produce an undesirable autoimmune response. Therefore, T cells are sorted by their relative ability to bind to antigens, preparing the immune system to respond to foreign invaders (i.e., recognizing non-self antigens) while preventing autoimmune reactions (i.e., recognizing self antigens). This tolerance mechanism limits naturally occurring T cells that can recognize tumor (self) antigens with high affinity, thereby eliminating T cells that can effectively eliminate tumor cells. Therefore, isolating T cells with high-affinity TCRs specific for tumor antigens is difficult because these cells are essentially eliminated by the immune system.

[0029] One advantage of the present disclosure is the provision of binding proteins specific for WT-1 peptides, wherein the binding protein is capable of binding to a WT-1 (SEQ ID NO.: 94):HLA (e.g., HLA-A*0201) complex. In certain embodiments, the binding protein is capable of binding to WT-1 independently of or in the absence of CD8, is capable of binding to CD3 protein more efficiently than endogenous TCR, is capable of signaling host cells expressing the binding protein to produce cytokines, or any combination thereof. In certain embodiments, the binding protein is capable of binding to a RMFPNAPYL (SEQ ID NO.: 94):HLA complex, producing pEC 50 and / or wherein the binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO.: 94):HLA complex and producing an IFNγ with a higher pEC compared to a TCR comprising the alpha chain amino acid sequence of SEQ ID NO: 82 and the beta chain amino acid sequence of SEQ ID NO: 83. 50 of IFNγ.

[0030] In certain embodiments, the compositions and methods described herein will have therapeutic utility for treating diseases and conditions associated with WT-1 expression (e.g., detectable WT-1 expression that is greater in magnitude than that detectable in normal or disease-free cells in a statistically significant manner). Such diseases include various diseases and conditions, such as hematological malignancies and solid cancers. Non-limiting examples of these and related uses are described herein, including in vitro, ex vivo, and in vivo stimulation of WT-1 antigen-specific T cell responses, for example, by using recombinant immune cells (e.g., T cells) encoding and / or expressing a heterologous binding protein specific for a WT-1 peptide (e.g., RMPFNAPYL, SEQ ID NO.: 94).

[0031] Before setting forth the present disclosure in more detail, it may be helpful to provide definitions of certain terms that will be used herein to facilitate understanding thereof. Additional definitions are set forth throughout this disclosure.

[0032] In this specification, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer in the range, and when appropriate, include its fraction (such as one tenth and one hundredth of an integer), unless otherwise stated. In addition, any numerical range related to any physical feature (such as polymer subunit, size or thickness) described herein should be understood to include any integer in the range, unless otherwise stated. As used herein, unless otherwise stated, the term "about" refers to ± 20% of the range, value or structure shown. It should be understood that the terms "one (a)" and "an" used herein refer to "one or more" listed components. The use of alternatives (for example, "or") should be understood to represent one, both or any combination thereof in the alternatives. As used herein, the terms "including", "having" and "comprising" are used synonymously, and these terms and their variants are intended to be interpreted as non-restrictive.

[0033] In addition, it should be understood that individual compounds or groups of compounds derived from various combinations of structures and substituents described herein are disclosed herein to the same extent as each compound or group of compounds is set forth. Therefore, the selection of a particular structure or a particular substituent is within the scope of this disclosure.

[0034] The term "consisting essentially of" is not equivalent to "comprising," but rather refers to specified materials or steps, or to materials or steps that do not materially affect the basic characteristics of the claimed invention. For example, a protein domain, region or module (e.g., a binding domain, hinge region, linker module) or a protein (which may have one or more domains, regions or modules) "consists essentially of" particular amino acids when the amino acid sequence of a domain, region, module or protein includes extensions, deletions, mutations or combinations thereof (e.g., amino acids at the amino or carboxyl termini or between domains), and these extensions, deletions, mutations or combinations thereof, taken together, contribute at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of the domain, region, module or protein and do not significantly affect (i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5% or 1%) the activity of the domain, region, module or protein (e.g., the target binding affinity of the binding protein).

[0035] As used herein, the term "host" refers to a cell (e.g., T cell) or microorganism that has been genetically modified with heterologous or exogenous nucleic acid molecules to produce a polypeptide of interest (e.g., enhanced affinity anti-WT-1 TCR). In certain embodiments, the host cell may optionally already have or be modified to include other genetic modifications (e.g., comprising a detectable marker; an endogenous TCR that is deleted, altered, or truncated; increased co-stimulatory factor expression) that confer desired properties related or unrelated to the biosynthesis of heterologous or exogenous proteins. In certain embodiments, the host cell is a human hematopoietic progenitor cell transduced with a heterologous or exogenous nucleic acid molecule encoding a TCR α chain specific for a WT-1 antigen peptide.

[0036] As used herein, "immune system cell" refers to any cell of the immune system that originates from hematopoietic stem cells in the bone marrow, which give rise to two major lineages, myeloid progenitor cells (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitor cells (which give rise to lymphoid cells such as T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4 + T cells, CD8 + T cells, CD4 - CD8 - Double negative T cells, gamma delta T cells, natural killer cells and dendritic cells. Macrophages and dendritic cells can be referred to as "antigen presenting cells" or "APCs," which are specialized cells that can activate T cells when the major histocompatibility complex (MHC) receptors on the surface of the APC interact with the TCR on the surface. In certain embodiments, the immune system cells are human immune system cells.

[0037] As used herein, "hematopoietic progenitor cells" are cells that can be derived from hematopoietic stem cells or fetal tissue and are capable of further differentiation into mature cell types (e.g., immune system cells). Exemplary hematopoietic progenitor cells include those with CD24 Lo Lin – CD117 + phenotype or those found in the thymus (called progenitor thymocytes).

[0038] The term "major histocompatibility complex" (MHC) refers to glycoproteins that deliver peptide antigens to the cell surface. MHC class I molecules are heterodimers with a transmembrane α chain (with three α domains) and non-covalently associated β2 microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, α and β, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides originating from the cytosol to the cell surface, where the peptide:MHC complex is bound by CD8 + T cell recognition. MHC class II molecules deliver peptides derived from the vesicle system to the cell surface, where they are recognized by CD4 + T cell recognition. Human MHC is called human leukocyte antigen (HLA).

[0039] A "T cell" is an immune system cell that matures in the thymus and produces the T cell receptor (TCR). T cells can be naive (not exposed to antigen; unlike T cells, which are CM Compared with T cells, the expression of CD62L, CCR7, CD28, CD3, CD127 and CD45RA increased, and the expression of CD45RO decreased), memory T cells (T M T cells (antigen experienced and long-lived) and effector cells (antigen experienced and cytotoxic). M It can be further divided into central memory T cell subsets (T CM , increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA compared to naive T cells), stem cell memory T cells, and effector memory T cells (T EM , with naive T cells or T CM Compared with T cells, the expression of CD62L, CCR7, CD28, and CD45RA was decreased, and the expression of CD127 was increased. E ) refers to antigen-experienced CD8 + Cytotoxic T lymphocytes, T CM In contrast, the expression of CD62L, CCR7, and CD28 was reduced, and granzyme and perforin were positive.

[0040] "T cell receptor" (TCR) refers to a member of the immunoglobulin superfamily (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3 rd Ed., Current Biology Publications, p. 4: 33, 1997), which is able to specifically bind to antigenic peptides bound to MHC receptors. TCRs can be found on the cell surface or in soluble form, and are typically composed of heterodimers having α and β chains (also referred to as TCRα and TCRβ, respectively), or γ and δ chains (also referred to as TCRγ and TCRδ, respectively). Like other immunoglobulins (e.g., antibodies), the extracellular portion of the TCR chain (e.g., α-chain, β-chain) contains two immunoglobulin domains: a variable domain at the N-terminus (e.g., α-chain variable domain or Vα, β-chain variable domain or Vβ; typically numbered based on Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," US Pat. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5 th ed.) amino acids 1 to 116; and one constant domain adjacent to the cell membrane (e.g., the α-chain constant domain or the C α , usually amino acids 117 to 259 based on Kabat; the β chain constant domain or C β , typically amino acids 117 to 295 based on Kabat). Like other immunoglobulins, the variable domain comprises complementarity determining regions (CDRs) separated by framework regions (FRs) (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. USA 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In certain embodiments, TCRs are present on the surface of T cells (or T lymphocytes) and bind to the CD3 complex. As used in the present disclosure, the source of TCRs can be from various animal species, such as humans, mice, rats, rabbits, or other mammals.

[0041] The term "variable region" or "variable domain" refers to the domain of an immunoglobulin superfamily binding protein (e.g., a TCR α-chain or β-chain (or the γ chain and δ chain for γδ TCRs)) that is involved in binding an immunoglobulin superfamily binding protein (e.g., TCR) to an antigen. In the case of TCRs, the variable domains of the α and β chains (Vα and Vβ, respectively) generally have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs. The Vα domain is encoded by two separate DNA segments, the variable gene segment and the joining gene segment (VJ); the Vβ domain is encoded by three separate DNA segments, the variable gene segment, the diversity gene segment, and the joining gene segment (VDJ). A single Vα or Vβ domain, or a functional fragment or portion thereof, may be sufficient to confer antigen binding specificity. In addition, the Vα or Vβ domain can be used to separate TCRs that bind to a specific antigen from those that bind to the antigen to screen a library of complementary Vα or Vβ domains, respectively.

[0042] The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to amino acid sequences within the variable region of an immunoglobulin (e.g., TCR) that confer antigen specificity and / or binding affinity and are separated from each other by framework regions. In general, each TCR α-chain variable region has three CDRs (αCDR1, αCDR2, αCDR3) and each TCR β-chain variable region has three CDRs (βCDR1, βCDR2, βCDR3). In TCRs, CDR3 is considered the primary CDR responsible for recognizing processed antigens. Typically, CDR1 and CDR2 primarily interact with MHC.

[0043] CDR1 and CDR2 are encoded within the variable gene segment of the TCR variable region encoding sequence, while CDR3 is encoded by a region spanning the variable and joining segments of Vα, or by a region spanning the variable, diversity, and joining segments of Vβ. Therefore, if the identity of the variable gene segment of Vα or Vβ is known, the sequences of their corresponding CDR1 and CDR2 can be inferred. Compared to CDR1 and CDR2, CDR3 is usually significantly more diverse due to the addition and loss of nucleotides during recombination.

[0044] TCR variable domain sequences can be aligned with numbering schemes (e.g., IMGT, Kabat, Chothia, Enhanced Chothia, Contact, and Aho), allowing annotation of equivalent residue positions and comparison of different molecules using, for example, ANARCI software (2016, Bioinformatics 15: 298-300). The numbering scheme provides a standardized description of the framework regions and CDRs in the TCR variable domain. Thus, it should be understood that a CDR from a TCR Vα or Vβ region or domain can have a specific sequence according to a specific numbering scheme, and can have a slightly shorter, longer, or shifted (e.g., partially overlapping) sequence according to a specific numbering scheme to a different numbering scheme. In certain disclosed embodiments, CDRs are determined using the IMGT numbering scheme; for example, using IMGT V-Quest (imgt.org / IMGTindex / V-QUEST.php).

[0045] "CD3" is known in the art as a six-chain multiprotein complex (see, Abbas and Lichtman, 2003; Janeway et al., p172 and 178, 1999). In mammals, the complex comprises a homodimer of one CD3γ chain, one CD3δ chain, two CD3ε chains, and one CD3ζ chain. CD3γ, CD3δ, and CD3ε chains are highly related immunoglobulin superfamily containing a single immunoglobulin domain. The transmembrane regions of CD3γ, CD3δ, and CD3ε chains are negatively charged, a property that is believed to allow these chains to bind to the positively charged regions of the T cell receptor chains. The intracellular tails of CD3γ, CD3δ, and CD3ε chains each contain a conserved motif, called an immunoreceptor tyrosine-based activation motif or ITAM, and each CD3ζ chain has three. Without wishing to be bound by theory, it is believed that ITAM is important for the signal transduction ability of the TCR complex. The CD3 used in the present disclosure can be from various animal species, including humans, mice, rats, or other mammals.

[0046] As used herein, "TCR complex" refers to a complex formed by the binding of CD3 to TCR. For example, a TCR complex can be composed of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRα chain, and a TCRβ chain. Alternatively, a TCR complex can be composed of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRγ chain, and a TCRδ chain.

[0047] As used herein, "component of a TCR complex" refers to a TCR chain (i.e., TCRα, TCRβ, TCRγ, or TCRδ), a CD3 chain (i.e., CD3γ, CD3δ, CD3ε, or CD3ζ), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRα and TCRβ, a complex of TCRγ and TCRδ, a complex of CD3ε and CD3δ, a complex of CD3γ and CD3ε, or a sub-TCR complex of TCRα, TCRβ, CD3γ, CD3δ, and two CD3ε chains).

[0048] As used herein, the term "CD8 co-receptor" or "CD8" refers to the cell surface glycoprotein CD8, which can be expressed by T cells as a homodimer containing two CD8 α chains, or as a heterodimer containing an α chain and a β chain. The CD8 co-receptor is believed to contribute to the activation of cytotoxic T cells (CD8 + ) function and exerts its effect through signal transduction of its cytoplasmic tyrosine phosphorylation pathway (Gao and Jakobsen, Immunol. Today 21: 630-636, 2000; Cole and Gao, Cell. Mol. Immunol. 1: 81-88, 2004). In particular, and without wishing to be bound by theory, it is believed that the CD8 co-receptor binds to the MHC-I protein complex expressed on the surface of antigen-expressing cells, and this binding in the context of TCR: antigen-MHC binding initiates or assists in the generation of T cell signaling pathways that generate immune responses (e.g., cytokine transcription and expression, calcium secretion, cytolytic activity, etc.) against antigen-expressing cells. In humans, eight (8) different CD8 β chain isoforms are known ("M1"-"M8"; see UniProtKB identifiers P10966-1, 2, 3, 4, 6, 7, 8 and 9); of these, isoforms 1, 2, 4 and 5 are thought to be associated with the cell membrane in nature, while isoforms 3, 6, 7 and 8 are thought to be associated with the extracellular region or to be secreted. Likewise, in humans, three CD8 α chain isoforms are known (see UniProtKB identifiers P01732-1, 2 and 3).

[0049] "CD4" refers to an immunoglobulin co-receptor glycoprotein that assists TCR in communicating with antigen-presenting cells (see Campbell & Reece, Biology 909 (Benjamin Cummings, Sixth Ed., 2002); UniProtKBP01730). CD4 is present on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells, and typically includes four immunoglobulin domains expressed on the cell surface (D1 (containing an Ig-like V-type domain), D2, D3, and D4 (containing Ig-like C2-type domains 1, 2, and 3, respectively). During antigen presentation, CD4 is recruited along with the TCR complex to bind to different regions of the MHCII molecule (CD4 binds to MHCII β2, while the TCR complex binds to MHCII α1 / β1). Without wishing to be bound by theory, it is believed that close proximity to the TCR complex allows the CD4-associated kinase molecule to phosphorylate the immunoreceptor tyrosine-based activation motif (ITAM) present on the cytoplasmic domain of CD3. This activity is thought to amplify the signal generated by activated TCRs to produce various types of T helper cells.

[0050] As used herein, a "binding domain" (also referred to as a "binding region" or "binding portion") refers to a peptide, oligopeptide, polypeptide, or protein that has the ability to specifically and non-covalently associate, unite, or combine with a target (e.g., WT-1 or WT-1 peptide:MHC complex). Binding domains include any naturally occurring, synthetic, semisynthetic, or recombinantly produced binding partner for a biomolecule, molecular complex (i.e., a complex comprising two or more biomolecules), or other target of interest. Exemplary binding domains include single-chain immunoglobulin variable regions (e.g., scTCR, scFv), receptor extracellular domains, ligands (e.g., cytokines, chemokines), or synthetic polypeptides selected for their specific ability to bind to a biomolecule, molecular complex, or other target of interest.

[0051] As used herein, "specifically binds" or "specifically for" refers to binding to a protein (e.g., a TCR receptor) or binding domain (or a fusion protein thereof) with a specific binding affinity equal to or greater than 10 5 M -1 (For this association reaction, the opening rate [k on ] and closing rate [k off ]) affinity or K a(i.e., an equilibrium association constant of a specific binding reaction with 1 / M units) to the target molecule without significant association or association with any other molecule or component in the sample. Binding proteins or binding domains (or fusion proteins thereof) can be classified as "high affinity" binding proteins or binding domains (or fusion proteins thereof) or "low affinity" binding proteins or binding domains (or fusion proteins thereof). A "high affinity" binding protein or binding domain is one that has at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , or at least 10 13 M -1 "Low affinity" binding proteins or binding domains are those with a low affinity for the a Up to 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 Alternatively, affinity can be defined as the affinity of the binding protein or binding domain to the M unit (e.g., 10 -5 M to 10 -13 The equilibrium dissociation constant (K) of the specific binding interaction d ).

[0052] In certain embodiments, a receptor or binding domain may have "enhanced affinity," which means that the selected or engineered receptor or binding domain binds to the target antigen more strongly than the wild-type (or parent) binding domain. For example, the enhanced affinity may be due to a K of the target antigen. a (equilibrium association constant) is higher than that of the wild-type binding domain due to the K d (dissociation constant) is less than the wild-type binding domain, the dissociation rate (k off ) is smaller than the wild-type binding domain, or a combination thereof. In certain embodiments, the enhanced affinity TCR can be codon-optimized to enhance expression in specific host cells such as T cells (Scholten et al., Clin. Immunol. 119: 135, 2006).

[0053] The term "functional avidity" refers to a biological measurement or activation threshold of the response of an in vitro immune cell (e.g., T cell, NK cell, NK-T cell) to a given concentration of a ligand, wherein the biological measurement can include cytokine production (e.g., IFNγ production, IL-2 production, etc.), cytotoxic activity, activation, and proliferation. For example, T cells that respond biologically (immunologically) to low antigen doses in vitro by producing cytokines, having cytotoxicity, expressing activation markers, or proliferating are considered to have high functional avidity, while T cells with lower functional avidity require a higher amount of antigen before inducing an immune response similar to that of high-affinity T cells. It is understood that functional avidity is different from affinity and avidity. Avidity refers to the strength of any given bond between a binding protein and its antigen / ligand. Some binding proteins are multivalent and bind to multiple antigens-in this case, the strength of the overall connection is avidity.

[0054] There are many correlations between functional avidity and the effectiveness of the immune response. Some in vitro studies have shown that different T cell functions (e.g., proliferation, cytokine production, etc.) can be triggered at different thresholds (see, for example, Betts et al., J. Immunol. 172: 6407, 2004; Langenkamp et al., Eur. J. Immunol. 32: 2046, 2002). Factors affecting functional avidity include (a) the affinity of the TCR for the pMHC complex, i.e., the strength of the interaction between the TCR and pMHC (Cawthon et al., J. Immunol. 167: 2577, 2001), (b) the expression level of the TCR and CD4 or CD8 co-receptors, and (c) the distribution and composition of signaling molecules (Viola and Lanzavecchia, Science 273: 104, 1996), as well as the expression level of molecules that weaken T cell function and TCR signaling.

[0055] The concentration of antigen required to induce a half-maximal response between baseline and maximal response after a specific exposure time is called the "half-maximal effective concentration" or "EC 50 ”.EC 50 The value is usually expressed as a molar (mol / L) amount, but it is usually converted to a logarithmic value as follows -log 10 (EC 50 )(See, for example, FIG. 4(C)). For example, if EC 50 Equal to 1μM (10 -6 M), then log 10 (EC 50 ) value is –6. Another value used is pEC 50 , which is defined as EC 50The negative logarithm (-log 10 (EC 50 )). In the above example, the EC is equal to 1 μM 50 With a pEC50 value of 6. In certain embodiments, the functional affinity of the binding protein of the present disclosure will be its ability to promote the production of IFNγ by immune cells (e.g., T cells, NK-T cells, NK cells), which can be measured using assays known in the art and / or described herein. A "high functional affinity" TCR or its binding domain refers to a protein having at least 10 -4 M, at least about 10 -5 M or at least about 10 -6 M's EC 50 Those TCRs or their binding domains.

[0056] In some embodiments, pEC 50 Used to describe the amount of peptide antigen required for 50% of immune cells to express the activation marker Nur77.

[0057] A variety of assays are known for identifying binding domains of the present disclosure that specifically bind to a particular target, and for determining the affinity of a binding domain or fusion protein, such as Western blot, ELISA, analytical ultracentrifugation, spectroscopy, and surface plasmon resonance. analysis (see, e.g., Scatchard et al., Ann. NY Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and US Patent Nos. 5,283,173, 5,468,614, or equivalents) and peptide:MHC tetramer staining.

[0058] The term "WT-1-specific immunoglobulin superfamily binding protein" or "WT-1-specific binding protein" refers, in certain embodiments, to a protein or polypeptide of the present disclosure that specifically binds to WT-1 or a peptide thereof, or a peptide optionally in complex with an HLA molecule. The term "WT-1 binding domain" or "WT-1 binding fragment" refers to the domain or portion of a WT-1-specific binding protein that is responsible for specific WT-1 binding. The WT-1-specific binding domain alone (i.e., without any other portion of the WT-1-specific binding protein) can be soluble. Exemplary WT-1 specific binding domains include those from WT-1 specific TCRs, such as those that can be found in a TCR or scTCR (e.g., single-chain αTCR proteins such as Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vα, or Vα-L-Vβ-Cβ, wherein Vα and Vβ are TCR α and β variable domains, respectively, Cα and Cβ are TCR α and β constant domains, respectively, and L is a linker) and scFv fragments as described herein, which can be derived from an anti-WT-1 TCR or antibody.

[0059] "WT-1 antigen" or "WT-1 peptide antigen" refers to a naturally occurring or synthetically produced portion of a WT-1 protein ranging in length from about 7 amino acids to about 15 amino acids that can form a complex with an MHC (e.g., HLA) molecule, and such a complex can bind to a TCR specific for the WT-1 peptide:MHC (e.g., HLA) complex. This has been well established (see, e.g., Murphy, Janeway's Immunobiology (8)). th Ed.) 2011 Garland Science, NY; Chapters 6, 9, and 16) The principles of antigen processing by antigen-presenting cells (APCs) (such as dendritic cells, macrophages, lymphocytes, or other cell types) and the principles of antigen presentation by APCs to T cells, including immunocompatibility (e.g., sharing at least one allelic form of an MHC gene associated with antigen presentation) between APCs and T cells and major histocompatibility complex (MHC)-restricted presentation. For example, processed antigenic peptides derived from the cytosol (e.g., tumor antigens, intracellular pathogens) are typically about 7 to about 11 amino acids in length and associate with class I MHC molecules, while peptides processed in vesicular systems (e.g., bacteria, viruses) vary in length from about 10 to about 25 amino acids and associate with class II MHC molecules. Since WT-1 is an internal host protein, WT-1 antigenic peptides will be presented in the context of class I MHC. In a specific embodiment, the WT-1 peptide is RMFPNAPYL (SEQ ID NO.: 94), which is known to be associated with human HLA class I (and more specifically, associated with allele HLA-A*201).

[0060] "Linker" refers to an amino acid sequence connecting two proteins, polypeptides, peptides, domains, regions or motifs, and can provide a spacer function compatible with the interaction of two sub-binding domains, so that the resulting polypeptide maintains specific binding affinity for the target molecule (e.g., scTCR) or retains signal activity (e.g., TCR complex). In certain embodiments, the linker is composed of, for example, about 2 to about 35 amino acids or about 4 to about 20 amino acids or about 8 to about 15 amino acids or about 15 to about 25 amino acids.

[0061] As used herein, "fusion protein" refers to a protein having at least two different domains or motifs in a single chain, wherein these domains or motifs are not naturally present together in the protein. Polynucleotides encoding fusion proteins can be constructed using PCR, recombinant engineering, etc., or such fusion proteins can be synthesized. Fusion proteins can also include other components, such as tags, linkers, or transduction markers. In certain embodiments, the fusion protein expressed or produced by a host cell (e.g., an immune cell, such as a T cell) is located on the cell surface, wherein the fusion protein is anchored to the cell membrane (e.g., through a transmembrane component or domain) and comprises an extracellular component (e.g., capable of binding to an MHC molecule) and an intracellular component (e.g., comprising a signaling domain, an effector domain, a costimulatory domain, or a portion or combination thereof).

[0062] "Linking amino acids" or "linking amino acid residues" refer to one or more (e.g., about 2-10) amino acid residues between two adjacent motifs, regions, or domains of a polypeptide (e.g., between a binding domain and an adjacent constant domain or between a TCR chain and an adjacent self-cleaving peptide). The connecting amino acids can be generated by the construct design of the fusion protein (e.g., amino acid residues generated using restriction enzyme sites during the construction of a nucleic acid molecule encoding the fusion protein).

[0063] By "altered domain" or "altered protein" is meant a motif, region, domain, peptide, polypeptide or protein that has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5%) sequence identity to a wild-type motif, region, domain, peptide, polypeptide or protein (e.g., a wild-type TCR alpha chain, TCR beta chain, TCR alpha constant domain, TCR beta constant domain).

[0064] As used herein, "nucleic acid" or "nucleic acid molecule" or "polynucleotide" refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments produced, for example, by polymerase chain reaction (PCR) or by in vitro translation, and fragments produced by any of ligation, cleavage, endonuclease action, or exonuclease action. In certain embodiments, the polynucleotides of the present disclosure are produced by PCR. Polynucleotides can be composed of naturally occurring nucleotide monomers (e.g., deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., α-enantiomeric forms of naturally occurring nucleotides), or a combination of the two. Modified nucleotides can be modified or replaced in the sugar moiety or in the pyrimidine or purine base moiety. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include phosphorothioates, phosphorodithioates, selenate phosphates, diselenate phosphates, aniline phosphorothioates, aniline phosphates, phosphoramidates, and the like. Polynucleotides can be single-stranded or double-stranded.

[0065] The term "isolated" refers to material removed from its original environment (e.g., the natural environment if it occurs in nature). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide separated from some or all coexisting materials in the natural system is isolated. Such a nucleic acid could be part of a vector and / or such a nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate) and still be isolated because such a vector or composition is not part of the natural environment for the nucleic acid or polypeptide. The term "gene" refers to a segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the "leader and trailer" coding regions, as well as intervening sequences (introns) between individual coding segments (exons).

[0066] Any host cell, binding protein, polynucleotide, or vector of the present disclosure may be "isolated."

[0067] As used herein, the terms "recombinant," "engineered," and "modified" refer to cells, microorganisms, nucleic acid molecules, or vectors that have been modified by the introduction of exogenous nucleic acid molecules, or to cells or microorganisms that have been altered so that expression of endogenous nucleic acid molecules or genes is controlled, dysregulated, or constitutive, wherein such alterations or modifications can be introduced by genetic engineering. Genetic alterations can include, for example, modifications that introduce nucleic acid molecules encoding one or more proteins or enzymes (which can include expression control elements, such as promoters), or additions, deletions, substitutions, or other functional disruptions or additions to the genetic material of the cell. Exemplary modifications include modifications in the coding region of a heterologous or homologous polypeptide from a reference or parent molecule, or a functional fragment thereof.

[0068] As used herein, "mutation" refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. Mutations can result in several different types of sequence changes, including substitutions, insertions, or deletions of nucleotides or amino acids. In certain embodiments, the mutation is a substitution of one or three codons or amino acids, a deletion of one to about five codons or amino acids, or a combination thereof.

[0069] "Conservative substitutions" are considered in the art to substitute one amino acid for another amino acid with similar properties. Exemplary conservative substitutions are well known in the art (see, for example, WO 97 / 09433, page 10; Lehninger, Biochemistry, 2 nd

[0015] The term "conservative" refers to a substitution of an amino acid residue at a particular position in a peptide or polypeptide. ... Similar amino acids can be included in the following categories: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine); amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine) and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline is considered more difficult to classify, as it has the same properties as amino acids with aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine). In some cases, replacing glutamic acid with glutamine or replacing aspartic acid with asparagine can be considered similar substitutions, as glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. As understood in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence of the polypeptide and its conserved amino acid substitutes to the sequence of a second polypeptide (e.g., using GENEWORKS, Align, BLAST algorithms, or other algorithms described herein and practiced in the art).

[0070] In certain embodiments, proteins (e.g., binding proteins) according to the present disclosure comprise variant sequences compared to a reference sequence (e.g., variant TCR CDRs compared to reference TCR CDRs disclosed herein). In certain embodiments, variant proteins, peptides, polypeptides, and amino acid sequences of the present disclosure may comprise one or more conservative substitutions relative to a reference amino acid sequence. Variants of the polynucleotides and polypeptides of the present disclosure are also contemplated. Variant nucleic acid molecules or polypeptides have at least 70%, 75%, 80%, 85%, 90%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.9% identity to a defined or reference polynucleotide or polypeptide (respectively) as described herein, or hybridize to a reference polynucleotide under stringent hybridization conditions of about 65-68°C, 0.015M sodium chloride, 0.0015M sodium citrate or at about 42°C, 0.015M sodium chloride, 0.0015M sodium citrate and 50% formamide. Nucleic acid molecule variants retain the ability to encode a binding protein or its binding domain that has the function described herein, e.g., specifically binds to a target molecule. For more details and explanation of hybridization reaction stringency, see Ausubel, FM (1995), Current Protocols in Molecular Biology. John Wiley & Sons, Inc. Furthermore, regarding how to identify DNA sequences by hybridization, those skilled in the art can follow the manuals Boehringer Mannheim GmbH (1993) The DIG System Users Guide for Filter Hybridization, Boehringer Mannheim GmbH, Mannheim, Germany and Liebl, W., Ehrmann, M., Ludwig, W. and Schleifer, KH (1991) International Journal of Systematic Bacteriology 41: 255-260.

[0071] Variants may also refer to fragments of a defined or reference sequence (e.g., portions resulting from truncation, cutting, etc.), and fragments may have any length shorter than the length of a defined or reference sequence. As used herein, "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide comprising only a domain, portion, or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50%, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% of the parent polypeptide activity level associated with the domain, portion, or fragment of the parent or reference compound, or provides a biological benefit (e.g., effector function). A "functional portion" or "functional fragment" of a polypeptide or encoded polypeptide of the present disclosure has "similar binding" or "similar activity" when the functional portion or fragment exhibits no more than 50% reduction in performance in a selected assay, such as an assay for measuring binding affinity or measuring effector function (e.g., cytokine release), as compared to a parent or reference polypeptide (preferably no more than 20% or 10%, or no more than a logarithmic difference in affinity compared to a parent or reference). In certain embodiments, the functional portion refers to a "signaling portion" of an effector molecule, effector domain, co-stimulatory molecule, or co-stimulatory domain.

[0072] In certain embodiments, a variant binding protein or a portion thereof (e.g., a binding domain) or fragment may comprise one or more amino acid substitutions relative to a parent or reference binding protein or domain, wherein the one or more amino acid substitutions, if present in the parent or reference binding domain or protein, remove, alter, or attenuate a potentially undesirable feature or property; for example, an amino acid sequence that is potentially immunogenic, or that may provide an undesirable glycosylation site, an undesirable deamidation site, an undesirable oxidation site, an undesirable isomerization site, or reduced thermodynamic stability, or that may result in mispairing or misfolding in the binding protein (e.g., unpaired cysteine ​​residues in close proximity). Amino acid sequences, patterns, and motifs that may provide undesirable features or properties are known (see, e.g., Seeliger et al., mAbs 7(3):505–515 (2015)).

[0073] In certain embodiments, amino acid substitutions include substitutions that remove somatic mutations (e.g., revert to germline-encoded amino acids). For example, in certain embodiments, variants of a reference CDR amino acid sequence or TCR variable domain sequence or TCR constant region sequence comprise substitutions to remove or attenuate potentially undesirable features or characteristics. It will be understood that such variants are selected so as not to impair or substantially impair the desired function (e.g., binding specificity and / or affinity for the WT-1 antigen: HLA complex).

[0074] The term "construct" refers to any polynucleotide comprising a recombinant nucleic acid. The construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome. A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. A vector may be, for example, a plasmid, a cosmid, a virus, an RNA vector, or a linear or circular DNA or RNA molecule, which may include chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acids. Exemplary vectors are those capable of autonomous replication (episomal vectors) or expression of the nucleic acid to which they are attached (expression vectors).

[0075] Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses (e.g., orthomyxoviruses (e.g., influenza viruses)), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses (e.g., picornaviruses and alphaviruses), and double-stranded DNA viruses, including adenoviruses, herpes viruses (e.g., herpes simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and pox viruses (e.g., cowpox, fowlpox, and canarypox). For example, other viruses include Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukemic sarcoma, mammalian C-type, B-type, D-type viruses, HTLV-BLV groups, lentiviruses, and foamy viruses (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0076] As used herein, "lentiviral vector" refers to an HIV-based lentiviral vector for gene delivery, which can be integrated or non-integrated, has a relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are typically produced after three (packaging, envelope, and transfer) or more plasmids are transiently transfected into production cells. Similar to HIV, lentiviral vectors enter target cells through the interaction of viral surface glycoproteins with cell surface receptors. Upon entry, viral RNA is reverse transcribed, which is mediated by the viral reverse transcriptase complex. The reverse transcription product is double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.

[0077] The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment such that the function of one nucleic acid molecule is affected by the other. For example, a promoter is operably linked to a coding sequence when the promoter is capable of affecting the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Unassociated" refers to related genetic elements that are not closely associated with each other and the function of one does not affect the other.

[0078] As used herein, "expression vector" refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence that can influence the expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter that influences transcription, an optional operator sequence that controls such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence that controls transcription and translation termination. The vector can be a plasmid, a phage particle, a virus, or a simple potential genome insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, can be integrated into the genome itself. In this manual, "plasmid," "expression plasmid," "virus," and "vector" are often used interchangeably.

[0079] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene, including transcription and translation.

[0080] The term "introduced" in the context of inserting a nucleic acid sequence into a cell refers to "transfection" or "transformation" or "transduction" and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell, where the nucleic acid molecule can be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

[0081] As used herein, "heterologous" or "exogenous" nucleic acid molecules, constructs or sequences refer to nucleic acid molecules or parts of nucleic acid molecules that are not native to the host cell but can be homologous to nucleic acid molecules or parts of nucleic acid molecules from the host. The source of heterologous or exogenous nucleic acid molecules, constructs or sequences can be from different genera or species. In certain embodiments, heterologous or exogenous nucleic acid molecules (i.e., non-endogenous or natural) are added to a host cell or host genome by, for example, conjugation, transformation, transfection, electroporation, etc., wherein the added molecules can be integrated into the host genome or exist as extrachromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and can exist in multiple copies. In addition, "heterologous" refers to non-natural enzymes, proteins or other activities encoded by exogenous nucleic acid molecules introduced into a host cell, even if the host cell encodes homologous proteins or activities. In addition, cells comprising "modified" or "heterologous" polynucleotides or binding proteins include the progeny of the cell, regardless of whether the progeny itself is transduced, transfected or otherwise manipulated or changed.

[0082] As described herein, more than one heterologous or exogenous nucleic acid molecules can be introduced into host cell proteins as separate nucleic acid molecules, as multiple individually controlled genes, as polycistronic nucleic acid molecules, as single nucleic acid molecules encoding fusions, or any combination thereof. For example, as disclosed herein, host cells can be modified to express two or more heterologous or exogenous nucleic acid molecules that encode desired TCRs (e.g., TCR α and TCR β) specific for WT-1 antigenic peptides. When two or more exogenous nucleic acid molecules are introduced into host cells, it should be understood that these two or more exogenous nucleic acid molecules can be integrated into host chromosomes as single nucleic acid molecules (e.g., on a single vector), on a separate vector, at a single site or multiple sites. The number of heterologous nucleic acid molecules or protein activities mentioned refers to the number of encoding nucleic acid molecules or the number of protein activities, rather than the number of separate nucleic acid molecules introduced into the host cell. Exogenous nucleic acid molecules (e.g., encoding the binding proteins of the present disclosure or CD8 co-receptors) can also be introduced into the genome of the host cell by gene editing techniques (e.g., using CRISPR-Cas systems, large-range nucleases, etc.).

[0083] As used herein, the term "endogenous" or "native" refers to a gene, protein, or activity that is normally present in a host cell. Furthermore, a gene, protein, or activity that is mutated, overexpressed, reorganized, duplicated, or otherwise altered compared to the parent gene, protein, or activity is still considered endogenous or native to that particular host cell. For example, an endogenous control sequence (e.g., a promoter, a translation attenuation sequence) from a first gene can be used to alter or regulate the expression of a second native gene or nucleic acid molecule, where the expression of the second native gene or nucleic acid molecule or the regulatory nucleic acid molecule is different from the normal expression or regulation in the parent cell.

[0084] The term "homologous" or "homolog" refers to a molecule or activity found or derived in a host cell, species or strain. For example, a heterologous or exogenous nucleic acid molecule can be homologous to a native host cell gene and can optionally have altered expression levels, a different sequence, an altered activity, or any combination thereof.

[0085] As used herein, "sequence identity" refers to the percentage identity of the amino acid residues in one sequence to the amino acid residues in another reference polypeptide sequence after aligning the sequences and, if necessary, introducing gaps to achieve maximum percentage sequence, and not considering any conservative substitutions as part of the sequence identity. Percent sequence identity values ​​can be generated using the NCBI BLAST2.0 software defined by Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402 (wherein the parameters are set to default values).

[0086] As understood in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence of a polypeptide and its conservative amino acid substitutes to the sequence of a second polypeptide (e.g., using GENEWORKS TM , Align, Clustal™, BLAST algorithm, etc.).

[0087] As used herein, "hyperproliferative disease" refers to excessive growth or proliferation compared to normal or non-diseased cells. Exemplary hyperproliferative diseases include tumors, cancers, tumor tissues, carcinomas, malignant precancerous cells, and non-neoplastic or non-malignant hyperproliferative diseases (e.g., adenomas, fibromas, lipomas, leiomyomas, hemangiomas, restenosis, and autoimmune diseases (e.g., rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, etc.). Certain diseases involving abnormal or excessive growth, which occur more slowly than in the case of hyperproliferative diseases, may be referred to as "proliferative diseases," including certain tumors, cancers, tumor tissues, carcinomas, sarcomas, malignant cells, malignant precancerous cells, and non-neoplastic or non-malignant diseases.

[0088] WT-1 antigen peptide-specific binding protein

[0089] In certain embodiments, the present disclosure provides a binding protein comprising (a) a T cell receptor (TCR) alpha chain variable region (Vα) domain comprising a CDR3 amino acid sequence (CDR3α) according to any one of SEQ ID NOs: 19, 22, 25 or 28, or a variant thereof, and a TCR Vβ domain; (b) a TCR Vβ domain comprising a CDR3 amino acid sequence (CDR3β) according to any one of SEQ ID NOs: 31, 34, 37 or 40, or a variant thereof, and a TCR Vα domain; or (c) the TCR Vα domain of (a) and the TCR Vβ domain of (b), wherein the binding protein is capable of binding to a RMFPNAPYL (SEQ ID NO.: 94):human leukocyte antigen (HLA) complex, wherein optionally, the HLA comprises HLA-A*0201.

[0090] In certain embodiments, the binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO.: 94):HLA complex, which produces pEC 50 IFNγ of 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 or more.

[0091] In certain embodiments, the Vβ domain of (a), (b) or (c) comprises or consists of an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identical to the amino acid sequence set forth in any one of SEQ ID NOs: 5-8 or 13-16, and / or the Vα domain of (a), (b) or (c) comprises or consists of any amino acid sequence that is at least 90% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4 or 9-12.

[0092] In certain embodiments, CDR3β comprises or consists of the amino acid sequence set forth in SEQ ID NO:31, and CDR3α comprises or consists of the amino acid sequence set forth in SEQ ID NO:19. In certain embodiments, CDR3β comprises or consists of the amino acid sequence set forth in SEQ ID NO:34, and CDR3α comprises or consists of the amino acid sequence set forth in SEQ ID NO:22. In certain embodiments, CDR3β comprises or consists of the amino acid sequence set forth in SEQ ID NO:37, and CDR3α comprises or consists of the amino acid sequence set forth in SEQ ID NO:25. In certain embodiments, CDR3β comprises or consists of the amino acid sequence set forth in SEQ ID NO:40, and CDR3a comprises or consists of the amino acid sequence set forth in SEQ ID NO:28.

[0093] In certain embodiments, the binding protein comprises a Vα domain comprising CDR1α, CDR2α, and CDR3α, and a Vβ domain comprising CDR1β, CDR2β, and CDR3β.

[0094] In certain embodiments, the Vβ domain comprises a CDR1β amino acid sequence as set forth in any one of SEQ ID NOs: 29, 32, 35, or 38, or a variant thereof, and / or a CDR2β amino acid sequence as set forth in any one of SEQ ID NOs: 30, 33, 36, or 39, or a variant thereof. In certain embodiments, the Vα domain comprises a CDR1α amino acid sequence as set forth in any one of SEQ ID NOs: 17, 20, 23, or 26, or a variant thereof, and / or a CDR2α amino acid sequence as set forth in any one of SEQ ID NOs: 18, 21, 24, or 27, or a variant thereof.

[0095] In certain embodiments, the TCR Vα and Vβ domains comprise CDR1α, CDR2α, CDR3α and CDR1β, CDR2β, CDR3β and the following amino acid sequences: (i) SEQ ID NOs.: 26-28 and 38-40, respectively; (ii) SEQ ID NOs.: 23, 27, 28 and 38-40, respectively; (iii) SEQ ID NOs.: 17-19 and 29-31, respectively; (iv) SEQ ID NOs.: 20-22 and 32-34, respectively; and (v) SEQ ID NOs.: 23-25 ​​and 35-37, respectively.

[0096] In any of the disclosed embodiments, the binding protein binds to the WT-1 peptide:HLA-A*201 complex. In any of the disclosed embodiments, the binding protein is capable of binding to the WT-1 peptide:HLA complex on the cell surface independent of or in the absence of CD8.

[0097] In certain embodiments, the binding proteins described herein include variant polypeptide species having one or more amino acid substitutions, insertions, or deletions in the amino acid sequence relative to the sequences of SEQ ID NOs: 1-61 as presented herein. In certain embodiments, the substitutions are or include conservative substitutions. Conservative amino acid substitutions are well known and can occur naturally or can be introduced during recombinant production of the binding protein. Amino acid substitutions, deletions, and additions can be introduced into proteins using mutagenesis methods known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY, 2001). Oligonucleotide-directed site-specific (or fragment-specific) mutagenesis procedures can be used to provide altered polynucleotides having specific codons altered according to the desired substitutions, deletions, or insertions. Alternatively, random or saturation mutagenesis techniques (e.g., alanine scanning mutagenesis, error-prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis) can be used to prepare immunogenic polypeptide variants (see, e.g., Sambrook et al., supra).

[0098] In certain embodiments, species (or variants) of particular binding proteins specific for WT-1 may include amino acid sequences that are at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the exemplary amino acid sequences disclosed herein (e.g., SEQ ID NOs.: 1-61).

[0099] In certain embodiments, a Vβ domain having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of any one of SEQ ID NOs.: 1-4 or 9-12, and / or a Vα domain having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs.: 5-8 or 13-16, is provided, wherein the binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO: 94):HLA complex. In other embodiments, any one or more of the β or α CDR amino acid sequences provided herein can be present in the Vβ domain and / or the Vα domain, respectively.

[0100] In certain embodiments, the binding protein comprises a Vβ domain comprising or consisting of an amino acid sequence at least 90% identical to any one of SEQ ID NOs.: 5-8 or 13-16, and / or a Vα domain comprising or consisting of an amino acid sequence at least 90% identical to any one of SEQ ID NOs.: 1-4 or 9-12, provided that (a) at least three or four CDRs are not mutated, (b) the CDRs that do have mutations have only a maximum of two amino acid substitutions, a maximum of five consecutive amino acid deletions, or a combination thereof, and (c) the binding protein retains its ability to bind to the RMFPNAPYL (SEQ ID NO: 94):HLA complex, optionally producing a pEC 50 IFNγ of 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 or more.

[0101] In certain embodiments, the binding protein includes a TCR variable domain comprising an amino acid sequence encoded by human TCR V, D and / or J alleles. As background, during lymphocyte development, Vα exons are assembled from different variable and connection gene segments (VJ), and Vβ exons are assembled from different variable, diversity and connection gene segments (VDJ). The TCRα chromosome site has 70-80 variable gene segments and 61 connection gene segments. The TCRβ chromosome site has 52 variable gene segments and two independent clusters, each cluster comprising a diversity gene segment and six or seven connection gene segments. Functional Vα and Vβ gene exons are produced by the recombination of variable gene segments with Vα connection gene segments and the recombination of variable gene segments with diversity gene segments and Vβ connection gene segments. The nucleotide and amino acid sequences of TCR gene segments according to various alleles are known in the art and can be found on the ImMunoGeneTics website; for example, at imgt.org / IMGTrepertoire / LocusGenes / listIG_TR / TR / human / Hu_TRgroup.html.

[0102] It should be understood that although the polynucleotide encoding the binding protein may comprise the same nucleotide sequence as that disclosed herein in accordance with the TCR gene fragment, any nucleotide sequence encoding the amino acid sequence of the gene fragment may be used in the context of the present disclosure.

[0103] In certain embodiments, the binding protein comprises a Vβ domain comprising (i) an amino acid sequence according to the TRBJ02-03 gene segment; and / or (ii) an amino acid sequence according to the TRBV06-05 gene segment, an amino acid sequence according to the TRBV07-09 gene segment, or an amino acid sequence according to the TRBV20-01 gene segment. In certain embodiments, the binding protein comprises a Vα domain comprising (i) an amino acid sequence according to the TRAJ43 gene segment; and / or (ii) an amino acid sequence according to the TRAV20-02 gene segment; an amino acid sequence encoded by the TRAV38DV08 gene segment; or an amino acid sequence according to the TRAV38-01 gene segment. In a related embodiment, the Vβ domain comprises an amino acid sequence according to the TRBJ02-03 gene segment, and the Vα domain comprises an amino acid sequence according to the TRAJ43 gene segment; the Vβ domain comprises an amino acid sequence according to the TRBV06-05 gene fragment, and the Vα domain comprises an amino acid sequence according to the TRAV20-02 gene fragment; the Vβ domain comprises an amino acid sequence according to the TRBV07-09 gene fragment, and the Vα domain comprises an amino acid sequence according to the TRAV38DV08 gene fragment; or the Vβ domain comprises an amino acid sequence according to the TRBV20-01 gene fragment, and the Vα domain comprises an amino acid sequence according to the TRAV38-01 gene fragment.

[0104] In the aforementioned embodiments, the amino acid sequence length of the TRA or TRB gene segment is 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 amino acids, including any length of amino acids in between these exemplary lengths (e.g., 11, 12, 13, 14 amino acids, etc.).

[0105] In certain embodiments, a binding protein capable of binding to RMFPNAPYL (SEQ ID NO.: 94): HLA complex is provided, wherein the binding protein comprises a TCR Vα domain and a TCR Vβ domain. In certain embodiments, the Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 1 or 9, and the Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 5 or 13. In certain embodiments, the Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 2 or 10, and the Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 6 or 14. In certain embodiments, the Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 3 or 11, and the Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 7 or 15. In certain embodiments, the Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 4 or 12, and the Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO.: 8 or 16.

[0106] In any of the disclosed embodiments, the binding protein may comprise an α chain constant domain (Cα) or a fragment thereof and / or a β chain constant domain (Cβ) or a fragment or portion thereof. In certain embodiments, Cα comprises or consists of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to any one of SEQ ID NOs.: 41-44. In certain embodiments, Cβ comprises or consists of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 45.

[0107] In other embodiments, the TCR Cβ comprises a cysteine ​​amino acid in place of the native serine at amino acid position 57 (e.g., GV(S→C)TD), and the TCR Ca comprises a cysteine ​​amino acid in place of the native threonine at amino acid position 48 (e.g., DK(T→C)VL; see, e.g., Cohen et al., Cancer Res. 67(8):3898-3903 (2007)).

[0108] In certain embodiments, the binding protein is a T cell receptor (TCR), a chimeric antigen receptor, or an antigen binding fragment of a TCR, any of which can be chimeric, humanized, or human. In other embodiments, the antigen binding fragment of a TCR comprises a single-chain TCR (scTCR) or a chimeric antigen receptor (CAR).

[0109] A "chimeric antigen receptor" (CAR) is a fusion protein designed to contain two or more naturally occurring amino acid sequences linked together in a manner that does not occur naturally or in the host cell, and that can function as a receptor when present on the cell surface. A CAR can include an extracellular portion comprising an antigen binding domain (e.g., derived from or obtained from an immunoglobulin or immunoglobulin-like molecule, such as a TCR binding domain derived from or obtained from a TCR specific for a cancer antigen, a scFv derived from or obtained from an antibody, or an antigen binding domain derived from or obtained from a killer immune receptor of a NK cell) connected to a transmembrane domain and one or more intracellular signaling domains (optionally comprising a costimulatory domain) (see, e.g., Sadelain et al., Cancer Discov., 3(4):388 (2013); see also Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2016), Stone et al., Cancer Immunol. Immunother., 63(11):1163 (2014) and Walseng et al., Scientific Reports 7:10713 (2017), wherein CAR constructs and methods of making the same are incorporated herein by reference). The CARs of the present disclosure that specifically bind to the WT-1 antigen (e.g., in the context of a peptide:HLA complex) comprise a TCR Va domain and a Vβ domain, or functional fragments or portions thereof.

[0110] In certain embodiments, the WT-1-specific binding protein is a TCR. In related embodiments, the binding protein (a) comprises a TCR alpha chain that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to, or comprises or consists of, the amino acid sequence set forth in SEQ ID NO: 50 or 46; and a TCR beta chain that is at least 90% identical to, comprises or consists of, the amino acid sequence set forth in SEQ ID NO: 58 or 54; (b) comprises a TCR alpha chain that is at least 90% identical to, comprises or consists of, the amino acid sequence set forth in SEQ ID NO: 51 or 47; and a TCR beta chain that is at least 90% identical to, comprises or consists of, the amino acid sequence set forth in SEQ ID NO: 59 or 55; (c) comprises a TCR alpha chain that is at least 90% identical to, comprises or consists of, the amino acid sequence set forth in SEQ ID NO: NO: 52 or 48, which has at least 90% identity, comprises or consists of the amino acid sequence shown in NO: 52 or 48; and a TCRβ chain, which TCRβ chain has at least 90% identity, comprises or consists of the amino acid sequence shown in SEQ ID NO: 60 or 56; or (d) comprises a TCRα chain, which TCRα chain has at least 90% identity, comprises or consists of the amino acid sequence shown in SEQ ID NO: 53 or 49; and a TCRβ chain, which TCRβ chain has at least 90% identity, comprises or consists of the amino acid sequence shown in SEQ ID NO: 61 or 57.

[0111] In certain embodiments, the binding protein comprises a fusion protein comprising (i) an extracellular component comprising a binding domain comprising a T cell receptor (TCR) α chain variable (Vα) domain and a T cell receptor (TCR) β chain variable (Vβ) domain, wherein the Vα domain comprises a CDR3 amino acid sequence (CDR3α) according to any one of SEQ ID NOs: 19, 22, 25, or 28, or a variant thereof, and the TCR Vβ domain comprises a CDR3 amino acid sequence (CDR3β) according to any one of SEQ ID NOs: 31, 34, 37, or 40, or a variant thereof; (ii) an intracellular component; and (iii) a transmembrane component located between the extracellular component and the intracellular component. In certain embodiments, the fusion protein is a CAR.

[0112] In certain embodiments, the binding protein comprises a fusion protein comprising (i) an extracellular component comprising a binding domain that specifically binds to the RMFPNAPYL (SEQ ID NO: 94) peptide:HLA complex, wherein the binding domain comprises a T-cell receptor (TCR) alpha chain variable (Vα) domain and a T-cell receptor (TCR) beta chain variable (Vβ) domain, wherein the Vα domain comprises or consists of an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 1, 2, 3, 4, 9, 10, 11, or 12, or a variant thereof, and the Vβ domain comprises or consists of an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 5, 6, 7, 8, 13, 14, 15, or 16, or a variant thereof, optionally with the proviso that: (a) at least three or four CDRs are not mutated, (b) the CDRs that do have mutations have no more than two amino acid substitutions, no more than five consecutive amino acid deletions, or a combination thereof; and (c) the fusion protein retains its ability to bind to the RMFPNAPYL (SEQ ID NO: 94) peptide:HLA complex. NO: 94): capacity of the HLA complex; (ii) an intracellular component; and (iii) a transmembrane component located between the extracellular and intracellular components.

[0113] It will be understood that any of the presently disclosed binding domains, including the exemplary CDR and variable domain sequences and variants thereof, can be included in a fusion protein according to the present disclosure.

[0114] In any of the embodiments described herein, the encoded polypeptides of the present disclosure may comprise a "signal peptide" (also referred to as a leader sequence, leader peptide, or transit peptide). The signal peptide targets the newly synthesized polypeptide to the appropriate location inside or outside the cell. The amino acid sequences of exemplary proteins or polypeptides of the present disclosure comprising a signal peptide are provided, for example, in SEQ ID NOs.: 1-8, 46-49, and 54-57. The signal peptide can be removed from the polypeptide during or once localization or secretion is completed. Polypeptides having a signal peptide are referred to herein as "proproteins," and polypeptides from which the signal peptide has been removed are referred to herein as "mature" proteins or polypeptides. In any of the embodiments disclosed herein, the binding protein or fusion protein comprises or is a mature protein, or is or comprises a proprotein. The amino acid sequences of exemplary mature proteins or polypeptides of the present disclosure are provided in SEQ ID NOs.: 9-16, 50-53, and 58-61.

[0115] In certain embodiments, the binding proteins of the present disclosure comprise one or more linking amino acids.

[0116] In certain embodiments, the binding protein comprises a linker. In certain embodiments, the linker consists of, for example, about 2 to about 35 amino acids, or about 4 to about 20 amino acids, or about 8 to about 15 amino acids, or about 15 to about 25 amino acids. Exemplary linkers include glycine-serine linkers (e.g., SEQ ID NOs.: 95 and 96).

[0117] In certain embodiments, a composition comprising a WT-specific binding protein according to any one of the preceding embodiments and a pharmaceutically acceptable carrier, diluent, or excipient is provided.

[0118] In certain embodiments, the WT-1 specific binding protein is provided in a soluble form (see, e.g., Walseng et al., PLoS One doi: 10.1371 / journal.pone.0119559 (2015)) and can optionally be conjugated to a cytotoxic agent and / or a detectable agent. For example, a method for isolating and purifying a recombinantly produced soluble TCR can include obtaining a supernatant from a suitable host cell / vector system that secretes the recombinant soluble TCR into a culture medium, and then concentrating the culture medium using a commercially available filter. After concentration, the concentrate can be applied to a single suitable purification matrix or a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse phase HPLC steps can be used to further purify the recombinant polypeptide. These purification methods can also be used when isolating the immunogen from its natural environment. Methods for large-scale production of one or more isolated / recombinant soluble TCRs described herein include batch cell cultures that are monitored and controlled to maintain appropriate culture conditions. The purification of soluble TCRs can be carried out according to the methods described herein and known in the art and in accordance with the laws and guidelines of domestic and foreign regulatory agencies.

[0119] In certain embodiments, nucleic acid molecules encoding protein-binding proteins specific to WT-1 are used to transfect / transduce host cells (e.g., T cells) for adoptive transfer therapy. Advances in TCR sequencing have been described (e.g., Robins et al., 2009 Blood 114:4099; Robins et al., 2010 Sci. Translat. Med. 2:47ra64, PMID:20811043; Robins et al., 2011 (Sept. 10) J. Imm. Meth. Epub ahead of print, PMID:21945395; Warren et al., 2011 Genome Res. 21:790), and can be used in implementing the process according to the disclosed embodiments. Similarly, methods for transfecting / transducing T cells with a desired nucleic acid have been described (e.g., US 2004 / 0087025) as well as adoptive transfer procedures using T cells of the desired antigen specificity (e.g., Schmitt et al., Hum. Gen. 20:1240, 2009; Dossett et al., Mol. Ther. 17:742, 2009; Till et al., Blood 112:2261, 2008; Wang et al., Hum. Gene Ther. 18:712, 2007; Kuball et al., Blood 109:2331, 2007; US 2004 / 0087025). 2011 / 0243972; US 2011 / 0189141; Leen et al., Ann. Rev. Immunol. 25:243, 2007), and thus based on the teachings herein, including the teachings of enhanced affinity TCRs specific for the WT-1 peptide antigen RMFPNAPYL (SEQ ID NO.: 94) complexed with HLA, it is expected that these methodologies are applicable to the embodiments of the present disclosure.

[0120] In certain embodiments, the WT-1 specific binding protein or domain as described herein can be expressed by host T cells and can be functionally characterized according to any of a large number of methodologies recognized in the art for measuring T cell activity (including determining the binding, activation or induction of T cells, and also including determining antigen-specific T cell responses). In certain embodiments, the binding protein is capable of promoting antigen-specific T cell responses against human WT-1 in a class I HLA restricted manner. In other embodiments, the class I HLA restricted response is associated with a transporter unrelated to antigen processing (TAP). In certain embodiments, the antigen-specific T cell response includes CD4 + Helper T lymphocyte (Th) responses and CD8 +At least one of a cytotoxic T lymphocyte (CTL) response. In a related embodiment, the CTL response is directed against WT-1 overexpressing cells. Other examples of methods for measuring T cell activity include determining T cell proliferation, T cell cytokine release, antigen-specific T cell stimulation, MHC-restricted T cell stimulation, CTL activity (e.g., by detecting T cells released from preloaded targets), T cell proliferation, T cell cytokine release, antigen-specific T cell stimulation, MHC-restricted T cell stimulation, T cell proliferation, T cell proliferation, T cell cytokine release ... 51 Cr cells), changes in expression of T cell phenotypic markers, and other measurements of T cell function. Procedures for performing these and similar assays can be found, for example, in Lefkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998). Reference can also be made to Current Protocols in Immunology; Weir, Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA (1986); Mishell and Shigii (eds.) Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA (1979); Green and Reed, Science 281: 1309 (1998) and references cited therein.

[0121] "MHC-peptide tetramer staining" refers to an assay for detecting antigen-specific T cells, characterized by a tetramer of MHC molecules, each molecule comprising an identical peptide having an amino acid sequence homologous (e.g., identical or related) to at least one antigen (e.g., WT-1), wherein the complex is capable of binding to a T cell receptor specific for the cognate antigen. Each MHC molecule can be labeled with a biotin molecule. The biotinylated MHC / peptide is tetramerized by adding streptavidin and can be fluorescently labeled. The tetramers can be detected by flow cytometry using fluorescent labels. In certain embodiments, the MHC-peptide tetramer assay is used to detect or select the enhanced affinity TCRs of the present disclosure.

[0122] Cytokine levels can be determined according to methods described herein and practiced in the art, including, for example, ELISA, ELISPOT, intracellular cytokine staining and flow cytometry and combinations thereof (e.g., intracellular cytokine staining and flow cytometry). Immune cell proliferation and clonal expansion caused by antigen-specific triggering or stimulation of an immune response can be determined by isolating lymphocytes, such as circulating lymphocytes in a peripheral blood cell sample or cells from lymph nodes, stimulating cells with antigens and measuring cytokine production, cell proliferation and / or cell viability, such as by incorporating tritiated thymidine or non-radioactive assays, such as MTT assays, etc. The effects of the immunogens described herein on the balance between Th1 and Th2 immune responses can be examined, for example, by measuring the levels of Th1 cytokines (e.g., IFN-γ, IL-12, IL-2, and TNF-β) and type 2 cytokines (e.g., IL-4, IL-5, IL-9, IL-10, and IL-13).

[0123] Polynucleotides and vectors

[0124] Also provided are polynucleotides encoding binding proteins according to the present disclosure or fragments or portions thereof (e.g., isolated polynucleotides). Separated or recombinant polynucleotides encoding binding proteins specific for WT-1 as described herein can be produced and prepared according to various methods and techniques in the field of molecular biology or polypeptide purification.

[0125] In certain embodiments, the polynucleotide is codon optimized for efficient expression in the target host cell. Codon optimization can be performed using known techniques and tools, such as using OptimumGene TM tool; GeneOptimizer TM (Sigma) etc. Codon optimized sequences include partially codon optimized (i.e., at least one codon optimized for expression in a host cell) and fully codon optimized sequences. Codon optimization for expression in certain immune host cells is disclosed in, for example, Scholten et al., Clin. Immunol. 119: 135, 2006.

[0126] As will be appreciated by those skilled in the art, polynucleotides may refer to any form of single-stranded or double-stranded DNA, cDNA, or RNA, and may include positive and negative strands of mutually complementary nucleic acids, including antisense DNA, cDNA, and RNA. Also included are siRNA, microRNA, RNA-DNA hybrids, ribozymes, and various other natural or synthetic forms of DNA or RNA.

[0127] In some embodiments, the polynucleotide encoding a binding protein of the present disclosure comprises a polynucleotide having at least 75% identity (e.g., 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the polynucleotide sequence set forth in any one of SEQ ID NOs: 62-81.

[0128] In certain embodiments, the polynucleotide encoding the binding protein comprises (i) a TCR β chain encoding polynucleotide that is at least 75% identical to the polynucleotide sequence set forth in SEQ ID NO:74, and a TCR α chain encoding polynucleotide that is at least 75% identical to the polynucleotide sequence set forth in SEQ ID NO:66; (ii) a TCR β chain encoding polynucleotide that is at least 75% identical to the polynucleotide sequence set forth in SEQ ID NO:75, and a TCR α chain encoding polynucleotide that is at least 75% identical to the polynucleotide sequence set forth in SEQ ID NO:67; (iii) a TCR β chain encoding polynucleotide that is at least 75% identical to the polynucleotide sequence set forth in SEQ ID NO:76, and a TCR α chain encoding polynucleotide that is at least 75% identical to the polynucleotide sequence set forth in SEQ ID NO:68; or (iv) a TCR β chain encoding polynucleotide that is at least 75% identical to the polynucleotide sequence set forth in SEQ ID NO:77, and a TCR α chain encoding polynucleotide that is at least 75% identical to the polynucleotide sequence set forth in SEQ ID NO:69.

[0129] In some embodiments, a single polynucleotide encodes a binding protein as described herein, or alternatively, a binding protein can be encoded by more than one polynucleotide. In other words, a component or portion of a binding protein can be encoded by two or more polynucleotides, which can be contained on a single nucleic acid molecule or can be contained on two or more nucleic acid molecules.

[0130] In certain embodiments, the polynucleotides encoding two or more components or parts of the associated protein of the present disclosure are included in two or more coding sequences operably connected in a single open reading frame. This arrangement can advantageously allow the coordinated expression of desired gene products, such as expressing the α chain and β chain of TCR simultaneously so that they are produced in a ratio of about 1: 1. In certain embodiments, two or more substituent gene products of the associated protein of the present disclosure, such as TCR (for example, α chain and β chain) or CAR, are expressed as separate molecules and associate after translation. In other embodiments, two or more substituent gene products of the associated protein of the present disclosure are expressed as single polypeptides, wherein part is separated by a cuttable or removable segment. For example, self-cleaving peptides that can be used to express separable polypeptides encoded by a single polynucleotide or vector are known in the art and include, for example, Porcine teschovirus-1 2A (P2A-1) peptide, Porcine teschovirus-2 2A (P2A-2) peptide, Thoesa asigna virus 2A (T2A) peptide, Equine rhinitis A virus (ERAV) 2A (E2A) peptide, and foot-and-mouth disease virus 2A (F2A) peptide. Exemplary nucleotide and amino acid sequences of self-cleaving peptides are provided in SEQ ID NOs: 84-93. In certain embodiments, the polynucleotide encoding the self-cleaving peptide comprises or consists of a polynucleotide sequence having at least 75% identity to the polynucleotide sequence shown in any one of SEQ ID NOs: 89-93. In certain embodiments, the encoded self-cleaving peptide comprises or consists of an amino acid sequence that is at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of any one of SEQ ID NOs: 84-88.

[0131] It will be understood that various arrangements of polynucleotides encoding two polypeptides of interest separated by a self-cleaving peptide (e.g., polynucleotide encoding a β-chain-self-cleaving peptide-polynucleotide encoding an α-chain; polynucleotide encoding an α-chain-self-cleaving peptide-polynucleotide encoding a β-chain) are contemplated.

[0132] Accordingly, in some embodiments, the polynucleotide encoding the binding protein has a structure comprising or having (polynucleotide encoding the TCRβ domain (e.g., chain))-(polynucleotide encoding the self-cleaving peptide)-(polynucleotide encoding the TCRβ domain (e.g., chain)) from the 5'-end to the 3'-end.

[0133] In certain embodiments, the polynucleotide encoding the binding protein comprises or consists of a polynucleotide sequence that is at least 75% (e.g., 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the polynucleotide sequence set forth in any one of SEQ ID NOs: 78-81. In certain embodiments, the polynucleotide comprises a polynucleotide sequence comprising or consisting of the polynucleotide sequence set forth in any one of SEQ ID NOs: 78-81.

[0134] In other aspects, an isolated polynucleotide encoding a binding protein is provided, wherein the encoded binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO.: 94):HLA complex and comprises a TCR Vα domain and a TCR Vβ domain, comprising: (i) an amino acid sequence as set forth in any one of SEQ ID NOs: 19, 22, 25, 28, 31, 34, 37, or 40; (ii) an amino acid sequence as set forth in any one of SEQ ID NOs: 17, 20, 23, 26, 29, 32, 35, or 38; (iii) an amino acid sequence as set forth in any one of SEQ ID NOs: 18, 21, 24, 27, 30, 33, 36, or 39; (iv) an amino acid sequence that is identical to an amino acid sequence as set forth in SEQ ID NOs: or (viii) any combination of (i) to (vii).

[0135] In other embodiments, the encoded binding protein is expressed as part of a transgenic construct, and the immune cells encoded and / or modified can encode: one or more additional auxiliary proteins, such as a safety switch protein; a label, a selection marker; a CD8 co-receptor β chain; a CD8 co-receptor α chain or both; or any combination thereof. Polynucleotides and transgenic constructs that can be used to encode and express binding proteins and auxiliary components (e.g., one or more of a safety switch protein, a selection marker, a CD8 co-receptor β-chain or a CD8 co-receptor α-chain) are described in PCT Publication WO 2018 / 058002, and the polynucleotides, transgenic constructs, and auxiliary components (including their nucleotide and amino acid sequences) are incorporated herein by reference. It should be understood that any one or all of the binding proteins, safety switch proteins, labels, selection markers, CD8 co-receptor β chains or CD8 co-receptor α chains of the present disclosure can be encoded by a polynucleotide sequence or that the polynucleotide sequence is a separate nucleic acid molecule or is present on a separate nucleic acid molecule.

[0136] Exemplary safety switch proteins include, for example, a truncated EGF receptor polypeptide (huEGFRt), which lacks the extracellular N-terminal ligand-binding domain and intracellular receptor tyrosine kinase activity but retains its native amino acid sequence, has a type I transmembrane cell surface localization, and has a conformationally intact binding epitope for the pharmaceutical-grade anti-EGFR monoclonal antibody, cetuximab (Erbitux); tEGF receptor (tEGFr; Wang et al., Blood 118:1255-1263, 2011); caspase polypeptides (e.g., iCasp9; Straathof et al., Blood 105:4247-4254, 2005; Di Stasi et al., N. Engl. J. Med. 365: 1673-1683, 2011; Zhou and Brenner, Exp. Hematol. pii: S0301-472X(16)30513-6. doi: 10.1016 / j.exphem.2016.07.011), RQR8 (Philip et al., Blood 124: 1277-1287, 2014); a 10-amino acid tag derived from human c-myc protein (Myc) (Kieback et al., Proc. Natl. Acad. Sci. USA 105: 623-628, 2008); and marker / safety switch polypeptides such as RQR (CD20+CD34; Philip et al., 2014).

[0137] Other auxiliary components that can be used in the modified immune cells of the present disclosure include tags or selectable markers that allow cells to be identified, sorted, isolated, enriched, or tracked. For example, labeled immune cells with desired characteristics (e.g., antigen-specific TCRs and safety switch proteins) can be sorted from unlabeled cells in a sample and more efficiently activated and expanded to be included in a product of desired purity.

[0138] As used herein, the term "selectable marker" includes nucleic acid constructs (and encoded gene products) that confer recognizable changes to cells, thereby allowing detection and positive selection of immune cells transduced with a polynucleotide comprising a selectable marker. RQR is a selectable marker comprising a major CD20 extracellular loop and two minimal CD34 binding sites. In some embodiments, the polynucleotide encoding RQR comprises a polynucleotide encoding a 16-amino acid CD34 minimal epitope. In some embodiments, the CD34 minimal epitope is incorporated into the amino-terminal position of the CD8 co-receptor stem domain (Q8). In other embodiments, the CD34 minimal binding site sequence can be combined with a target epitope of CD20 to form a compact marker / suicide gene (RQR8) for T cells (Philip et al., 2014, incorporated herein by reference). This construct allows selection of immune cells expressing the construct, for example, using a CD34-specific antibody bound to magnetic beads (Miltenyi), and utilizing the clinically accepted drug antibody rituximab, which allows selective deletion of engineered T cells expressing the transgene (Philip et al., 2014).

[0139] Other exemplary selection markers include several truncated type I transmembrane proteins that are not normally expressed on T cells: truncated low-affinity nerve growth factor, truncated CD19, and truncated CD34 (see, e.g., Di Stasi et al., N. Engl. J. Med. 365:1673-1683, 2011; Mavilio et al., Blood 83:1988-1997, 1994; Fehse et al., Mol. Ther. 1:448-456, 2000; each of which is incorporated herein in its entirety). A useful property of CD19 and CD34 is that they are readily available in MiltenyiCliniMACs. TMThe availability of a selection system that can be used to classify clinical levels of these markers. However, CD19 and CD34 are relatively large surface proteins that may affect the vector packaging capacity and transcription efficiency of the integration vector. Surface markers comprising extracellular, non-signaling domains or various proteins (e.g., CD19, CD34, LNGFR) can also be used. Any selection marker can be used, and should be acceptable to good manufacturing practices. In certain embodiments, a polynucleotide encoding a gene product of interest (e.g., a binding protein of the present disclosure, such as TCR or CAR) is expressed. Other examples of selection markers include, for example, reporter genes, such as GFP, EGFP, β-gal, or chloramphenicol acetyltransferase (CAT). In certain embodiments, the selection markers of, for example, CD34 are expressed by cells, and CD34 can be used to select enrichment or separation (e.g., by immunomagnetic selection) for the transduction of interest used in the methods described herein. As used herein, the CD34 marker is distinguished from anti-CD34 antibodies or, for example, scFv, TCR, or other antigen recognition moieties in conjunction with CD34.

[0140] In certain embodiments, the selectable marker comprises an RQR polypeptide, truncated low affinity nerve growth factor (tNGFR), truncated CD19 (tCD19), truncated CD34 (tCD34), or any combination thereof.

[0141] As background, inclusion of CD4 in immunotherapy cell products + T cells can provide antigen-induced IL-2 secretion and enhance the introduction of cytotoxic CD8 + T cell persistence and function (see, e.g., Kennedy et al., Immunol. Rev. 222: 129 (2008); Nakanishi et al., Nature 462(7272): 510 (2009)). In certain instances, CD4 + Class I restricted TCRs in T cells may require the transfer of CD8 co-receptors to enhance TCR sensitivity to class I HLA peptide complexes. The CD4 co-receptor is structurally different from CD8 and has been shown to be an ineffective substitute for the CD8 co-receptor (see, e.g., Stone & Kranz, Front. Immunol. 4: 244 (2013); see also Cole et al., Immunology 137(2): 139 (2012). Therefore, another accessory protein for the compositions and methods of the present disclosure comprises a CD8 co-receptor or a component thereof.

[0142] In certain embodiments, the engineered immune cell comprising a heterologous polynucleotide encoding a binding protein of the present disclosure may further comprise a heterologous polynucleotide encoding a CD8 co-receptor protein or a beta chain or alpha chain component thereof.

[0143] In some embodiments, the encoded CD8 co-receptor comprises an alpha chain or a fragment or variant thereof. The amino acid sequence of the human CD8 co-receptor alpha chain precursor is known and provided, for example, at UniProtKB-P30433 (see also UniProtKB-P31783; -P10732; and -P10731).

[0144] In some embodiments, the encoded CD8 co-receptor comprises a beta chain or a fragment or variant thereof. The amino acid sequence of the human CD8 co-receptor beta chain precursor is known and provided, for example, at UniProtKB-P10966 (see also UniProtKB-Q9UQ56; -E9PD41; Q8TD28; and -P30434; and -P05541).

[0145] Without wishing to be bound by theory, it is believed that the distance from the host cell surface is important for the function of the RQR polypeptide as a selectable marker / safety switch (Philip et al., 2010 (supra)). In some embodiments, the encoded RQR polypeptide is contained in the β chain, α chain, or both of the encoded CD8 co-receptor, or in a fragment or variant of one or both. In specific embodiments, the modified immune cell comprises a heterologous polynucleotide encoding iCasp9 and a heterologous polynucleotide encoding a recombinant CD8 co-receptor protein comprising a β chain containing an RQR polypeptide and further comprising a CD8 α chain.

[0146] The isolated polynucleotide of the present disclosure may further comprise a polynucleotide encoding a safety switch protein, a selectable marker, a CD8 co-receptor β chain, or a CD8 co-receptor α chain as disclosed herein, or may comprise a polynucleotide encoding any combination thereof.

[0147] In certain embodiments, the modified immune cell of the present disclosure further comprises: (i) a heterologous polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor α chain, wherein optionally, the encoded polypeptide is or comprises a CD8 co-receptor α chain; (ii) a heterologous polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor β chain, wherein optionally, the encoded polypeptide is or comprises a CD8 co-receptor β chain; or (iii) the polynucleotide of (i) and the polynucleotide of (ii), wherein optionally, the host cell comprises a CD4+ T cell, a CD8+ T cell, or both.

[0148] In other embodiments, the modified immune cell comprises: (a) a heterologous polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor α chain, wherein optionally, the encoded polypeptide is or comprises a CD8 co-receptor α chain; (b) a heterologous polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor β chain, wherein optionally, the encoded polypeptide is or comprises a CD8 co-receptor β chain; (c) a polynucleotide encoding a self-cleaving peptide located between the polynucleotide of (a) and the polynucleotide of (b).

[0149] In some embodiments, the polynucleotide encoding the binding protein further comprises a polynucleotide encoding a selectable marker.

[0150] Standard techniques can be used for recombinant DNA, peptide and oligonucleotide synthesis, immunoassays and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as generally accomplished in the art or as described herein. These and related techniques and procedures can generally be performed according to conventional methods well known in the art, and as described in various general and more specific references in the microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunological techniques cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: APractical Approach, vol.I&II (IRL Press, Oxford Univ. Press USA, 1985); CurrentProtocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M.Shevach,Warren Strober 2001 John Wiley&Sons,NY,NY);Real-Time PCR:Current Technology and Applications,Edited by Julie Logan,KirstinEdwards and Nick Saunders,2009,Caister Academic Press,Norfolk,UK;Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992);Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology(AcademicPress,New York,1991);Oligonucleotide Synthesis(N.Gait,Ed.,1984);Nucleic AcidHybridization(B.Hames&S.Higgins,Eds.,1985);Transcription and Translation(B.Hames&S.Higgins,Eds.,1984);Animal Cell Culture(R.Freshney,Ed.,1986);Perbal,A Practical Guide to Molecular Cloning(1984);Next-Generation GenomeSequencing(Janitz,2008 Wiley-VCH);PCR Protocols(Methods in Molecular Biology)(Park,Ed.,3. rdEdition,2010 Humana Press);Immobilized Cells And Enzymes(IRLPress,1986);the treatise,Methods In Enzymology(Academic Press,Inc.,N.Y.);GeneTransfer Vectors For Mammalian Cells(J.H.Miller and M.P.Calos eds.,1987,ColdSpring Harbor Laboratory);Harlow and Lane,Antibodies,(Cold Spring HarborLaboratory Press,Cold Spring Harbor,N.Y.,1998);Immunochemical Methods In CellAnd Molecular Biology(Mayer and Walker,eds.,Academic Press,London,1987);Handbook Of Experimental Immunology,Volumes I-IV(D.M.Weir andCC Blackwell,eds.,1986);Roitt,Essential Immunology,6th Edition,(Blackwell ScientificPublications,Oxford,1988);Embryonic Stem Cells:Methods and Protocols(Methodsin Molecular Biology)(Kurstad Turksen,Ed.,2002);Embryonic Stem CellProtocols:Volume I:Isolation and Characterization(Methods in MolecularBiology)(Kurstad Turksen,Ed.,2006);Embryonic Stem Cell Protocols:Volume II:Differentiation Models(Methods in Molecular Biology)(Kurstad Turksen,Ed., 2006); Human Embryonic Stem Cell Protocols (Methods in Molecular Biology) (Kursad Turksen Ed., 2006); Mesenchymal Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Darwin J. Prockop, Donald G. Phinney, and Bruce A. Bunnell Eds., 2008); Hematopoietic Stem Cell Protocols (Methods in Molecular Medicine) (Christopher A. Klug, and Craig T. Jordan Eds., 2001); Hematopoietic StemCell Protocols (Methods in Molecular Biology) (Kevin D. Bunting Ed., 2008) NeuralStem Cells: Methods and Protocols (Methods in Molecular Biology) (Leslie P. Weiner Ed., 2008). .

[0151] Certain embodiments include polynucleotides contained in vectors. Those skilled in the art can easily determine suitable vectors for use with certain embodiments disclosed herein. Typical vectors can include another nucleic acid capable of transporting or polynucleotides capable of replicating in a host organism. Some examples of vectors include plasmids, viral vectors, cosmids, etc. Some vectors may be capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins), while other vectors can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, some vectors can guide the expression of genes effectively connected thereto. According to relevant embodiments, it should be further understood that if one or more agents (e.g., polynucleotides encoding proteins specific to WT-1 or variants thereof, as described herein) are co-administered to a subject, each agent may be present in a separate or identical vector, and multiple vectors (each vector comprising different agents or identical agents) may be introduced into a cell or cell group or administered to a subject.

[0152] In certain embodiments, the polynucleotides of the present disclosure can be operably linked to certain elements of the vector. For example, polynucleotide sequences can be operably linked that are required to achieve expression and processing of the coding sequence to which they are linked. Expression control sequences can include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that may enhance protein secretion. Expression control sequences can be operably linked if they are adjacent to a gene of interest and an expression control sequence that acts in trans or remotely to control the gene of interest.

[0153] In certain embodiments, the vector comprises a plasmid vector or a viral vector (e.g., a vector selected from a lentiviral vector or a γ-retroviral vector). Viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, negative-strand RNA viruses, such as orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses, such as picornaviruses and alphaviruses, and double-stranded DNA viruses, including adenoviruses, herpes viruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus), and pox viruses (e.g., cowpox, fowlpox, and canarypox). For example, other viruses include Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis sarcoma, mammalian C-, B-, and D-type viruses, the HTLV-BLV group, lentiviruses, and foamy viruses (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0154] "Retrovirus" is a virus with an RNA genome that is reverse transcribed into DNA using a reverse transcriptase, and the reverse transcribed DNA is then incorporated into the host cell genome. "Gammaretrovirus" refers to a genus of the Retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus. As used herein, "lentiviral vector" refers to a HIV-based lentiviral vector for gene delivery that can be integrated or non-integrated, has a relatively large packaging capacity, and can transduce a range of different cell types. Slow viral vectors are typically produced after three (packaging, envelope, and transfer) or more plasmids are transiently transfected into production cells. Similar to HIV, slow viral vectors enter target cells through the interaction of viral surface glycoproteins with cell surface receptors. Upon entry, viral RNA is reverse transcribed, which is mediated by the viral reverse transcriptase complex. The reverse transcription product is a double-stranded linear viral DNA, which is the substrate for viral integration into the infected cell DNA.

[0155] In certain embodiments, the viral vector may be a gammaretrovirus, such as a Moloney murine leukemia virus (MLV)-derived vector. In other embodiments, the viral vector may be a more complex retrovirus-derived vector, such as a lentivirus-derived vector. HIV-1-derived vectors fall into this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (sheep lentivirus). Methods for using retroviral and lentiviral vectors and packaging cells to transduce mammalian host cells with viral particles containing TCR or CAR transgenes are known in the art and have been previously described in, for example, U.S. Patent 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors can also be used for polynucleotide delivery, including DNA viral vectors, including, for example, adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex virus (HSV), including amplicon vectors, replication-defective HSV, and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998).

[0156] Other vectors developed for gene therapy applications can also be used with the compositions and methods of the present disclosure. Such vectors include those derived from baculoviruses and alpha viruses (Jolly, DJ. 1999. Emerging Viral Vectors. pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as Sleeping Beauty or other transposon vectors).

[0157] When the viral vector genome comprises multiple polynucleotides to be expressed as separate transcripts in a host cell, the viral vector may also comprise additional sequences between the two (or more) transcripts that allow bicistronic or polycistronic expression. Examples of such sequences for viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptides, or any combination thereof.

[0158] In certain embodiments, carrier can deliver polynucleotide or transgenic construct to host cell (for example, hematopoietic progenitor cell or human immune system cell).In a specific embodiment, carrier can deliver polynucleotide or transgenic construct to human immune system cell, such as CD4+T cell, CD8+T cell, CD4-CD8-double negative T cell, stem cell memory T cell, γδT cell, natural killer cell, dendritic cell or its any combination.In other embodiments, carrier can deliver transgenic construct to immature T cell, central memory T cell, effector memory T cell or its any combination.In certain embodiments, the carrier of polynucleotide of encoding the present disclosure or transgenic construct can further include the polynucleotide of encoding nuclease, and the nuclease can be used for performing chromosome knockout (for example, CRISPR-Cas nuclease or another nuclease) in host cell, or can be used for delivering therapeutic polynucleotide or transgenic or its part to host cell in gene therapy replacement or gene repair therapy.Or, the nuclease for chromosome knockout or gene replacement or gene repair therapy can be delivered to host cell independently of the carrier of polynucleotide of encoding content of the present disclosure or transgenic construct.

[0159] host cells

[0160] Also provided herein are host cells comprising heterologous polynucleotides of the present disclosure (e.g., encoding any binding protein or fusion protein of the present disclosure and optionally encoding one or more additional proteins, such as a selectable marker, a self-cleaving peptide, a CD8 co-receptor polypeptide, or any combination thereof) and / or expressing any binding protein disclosed herein. Such host cells can be generated, for example, by transfection or transduction with a vector of the present disclosure, and / or by gene editing.

[0161] In some embodiments, the host cell comprises an immune system cell, which can be any human immune system cell, such as those exemplary immune system cells described herein. In certain embodiments, the host cell comprises a T cell, a NK cell, a NK-T cell, or any combination thereof. In other embodiments, the T cell comprises a CD8 + T cells, CD4 + T cells or both.

[0162] Thus, in certain embodiments, the modified immune cell comprises a heterologous polynucleotide encoding a binding protein according to the present disclosure, wherein the encoded binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO.: 94): human leukocyte antigen (HLA) complex.

[0163] In certain embodiments, the encoded binding protein is capable of binding to SEQ ID NO.: 94: HLA complex, producing pEC 50 IFNγ of 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 or more.

[0164] In certain embodiments, the peptide:HLA complex comprises a peptide:HLA-A*201 complex.

[0165] In any of the disclosed embodiments, when the modified immune cell population is co-cultured with antigen presenting cells (e.g., T2 cells, Jurkat cells, dendritic cells expressing HLA (e.g., HLA-A*0201)), the peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO.: 94) is administered in the co-culture at a dose of 10 -4 μM or 10 -5 μM or 10 -6In certain embodiments, when the population of modified immune cells is co-cultured with antigen presenting cells (also referred to as "target cells" in some cases), a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO.: 94) is administered in the co-culture at a concentration of about 10 μM. -2 Under conditions where the peptide concentration is pulsed at μM, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of the modified immune cells in the population are Nur77 + (ie, expressing Nur77), wherein the antigen presenting cells optionally express include T2 cells, Jurkat cells, or both, and are optionally HLA-A*0201+.

[0166] In some embodiments, when the modified immune cell population is co-cultured with antigen presenting cells, a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO.: 94) is administered to the co-culture at a dose of 10 -3 Under conditions where the peptide concentration was pulsed at μM, 10%, 15%, 20% or more of the modified immune cells in the population were Nur77 + .

[0167] In some embodiments, the binding protein has a pEC50 of 1.0, 1.1, 1.2, 1.3, 1.4, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6 or more for Nur77 expression (SEQ ID NO.: 94: HLA, e.g., HLA-A*0201).

[0168] In any of the disclosed embodiments, when the modified immune cells and MDA-MB-468 cells are present in a sample at a ratio of 30:1, the modified immune cells are capable of killing at least 11%, 12%, 13%, 14%, 15%, 20%, 25% or more of the HLA-A*0201 + In certain embodiments, when the modified immune cells and MDA-MB-468 cells are present in a sample at a ratio of 10:1, the modified immune cells are capable of killing at least 10% or more of the HLA-A*0201 + In certain embodiments, when the modified immune cells and MDA-MB-468 cells are present in a sample at a 1:1 ratio, the modified immune cells are capable of killing at least 5% or more of the HLA-A*0201 + MDA-MB-468 cells.

[0169] In any of the disclosed embodiments, lethality can be determined using a 4-hour co-culture assay, as described herein, optionally using labeled chromium (e.g., 51 Cr).

[0170] In certain embodiments, when the modified immune cells are present at a ratio of 30:1 to Panc1 cells, the modified immune cells are capable of killing 21%, 22%, 23%, 24%, 25% or more of the Panc1 cells in the sample. In other embodiments, when the modified immune cells are present at a ratio of 10:1 to Panc1 cells, the modified immune cells are capable of killing 10%, 15% or more of the Panc1 cells in the sample. In other embodiments, when the modified immune cells are present at a ratio of 9:1, 8:1, 7:1, 6:1 or 5:1 to Panc1 cells, the modified immune cells are capable of killing 10%, 15% or more of the Panc1 cells in the sample.

[0171] In certain embodiments, the modified immune cells: have a gene specific for HLA-A*0201 in a 4-hour co-culture + Increased killing activity of MDA-MB-468 cells and / or against Panc1 cells, and / or improved IFN-γ production after 4 hours of co-culture with peptide-pulsed target cells, and / or further reducing the proliferation of Panc-1 cancer cells (e.g., for more than 60, 75, 100, 125, 150 or more hours, as measured using an IncuCyte assay) compared to reference immune cells comprising a polynucleotide encoding a T cell receptor comprising a TCR alpha chain having the amino acid sequence set forth in SEQ ID NO: 82 and a TCR beta chain having the amino acid sequence set forth in SEQ ID NO: 83.

[0172] In some embodiments, the modified immune cells of the present disclosure prevent or substantially prevent the growth and / or proliferation of Panc-1 cancer cells for greater than 50, 100, 150 or more hours, wherein optionally, the modified immune cells and Panc-1 cancer cells are present at an effector:target cell ratio of 8:1.

[0173] In any of the disclosed embodiments, lethality can be determined using a 4-hour co-culture assay, as described herein, optionally using labeled chromium (e.g. 51 Cr).

[0174] It should be understood that, except for the encoded binding protein, the "reference" immune cell is the same immune cell type and is phenotypically identical or substantially identical to the subject's modified immune cell. For example, for a modified CD8+ human T cell, the reference immune cell is a CD8+ human T cell and can, for example, be from the same source or donor as the modified immune cell.

[0175] In any of the above embodiments, the encoded binding protein can be a TCR, a CAR, or a scTCR. In any of the above embodiments, the encoded binding protein is capable of binding to the WT-1 peptide (SEQ ID NO.: 94): HLA complex on the cell surface independent of CD8 or in the absence of CD8.

[0176] In any of the disclosed embodiments, the binding protein expressed by the modified immune cells can comprise a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or any combination thereof.

[0177] Any suitable immune cell can be modified to include a heterologous polynucleotide encoding a binding protein of the present disclosure, including, for example, a T cell, a NK cell, a NK-T cell, a macrophage, a monocyte, or a dendritic cell. In some embodiments, the modified immune cell includes a CD4 + T cells, CD8 + T cells or both. In some embodiments, the modified immune cells comprise CD4-CD8- double negative T cells or γδ T cells. In some embodiments, the T cells are naive T cells, central memory T cells, effector memory T cells, or any combination thereof. Methods for transfecting / transducing T cells with desired nucleic acids have been described (e.g., U.S. Patent Application Publication No. US 2004 / 0087025) as have adoptive transfer procedures using T cells with desired target specificity (e.g., Schmitt et al., Hum. Gen. 20:1240, 2009; Dossett et al., Mol. Ther. 17:742, 2009; Till et al., Blood 112:2261, 2008; Wang et al., Hum. Gene Ther. 18:712, 2007; Kuball et al., Blood 109:2331, 2007; US 2011 / 0243972; US 2011 / 0189141; Leen et al., Ann. Rev. Immunol. 25:243, 2007), and it is contemplated that these methods will be adapted for use in the presently disclosed embodiments based on the teachings herein.

[0178] Any suitable method can be used to transfect or transduce cells, such as T cells, or to administer the polynucleotides or compositions of the present method. Known methods for polynucleotide delivery to host cells include, for example, using cationic polymers, lipid molecules and some commercial products, such as IN-VIVO-JET PEI. Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, ultrasound loading, liposome-mediated transfection, receptor-mediated transduction, microparticle bombardment, transposon-mediated transfer, etc. A further method of transfecting or transducing host cells uses the vector described in further detail herein. In certain embodiments, host cells are gene-edited to include in their genome a polynucleotide encoding a binding protein (or other protein or polypeptide) as provided herein.

[0179] In any of the foregoing embodiments, the modified immune cells can be modified to reduce or eliminate the expression of one or more endogenous genes encoding polypeptides involved in immune signaling or other related activities (e.g., by chromosomal gene knockout as described herein), and / or to include heterologous polynucleotides as provided herein. Exemplary gene knockouts include gene knockouts encoding PD-1, LAG-3, CTLA4, TIM3, HLA molecules, TCR molecules, etc. Without wishing to be bound by theory, some endogenously expressed immune cell proteins may be recognized as foreign by the allogeneic host receiving the modified immune cells, which may result in the elimination of the modified immune cells (e.g., HLA alleles), or the immune activity of the modified immune cells (e.g., PD-1, LAG-3, CTLA4) may be lowered, or the binding proteins of the heterologous expression of the present disclosure may be interfered with (e.g., the endogenous TCR of the modified T cells, which bind to non-WT-1 antigens and may interfere with the modified immune cells binding to cells expressing WT-1 antigens), or may compete with heterologous binding proteins for expression.

[0180] Thus, reducing or eliminating the expression or activity of such endogenous genes or proteins can increase the activity of the binding protein, tolerance, expression, or persistence of the modified immune cells in an autologous or allogeneic host environment, and can allow for universal administration of the cells (e.g., to any recipient, regardless of HLA type). In certain embodiments, the modified immune cells are donor cells (e.g., allogeneic) or autologous cells. In certain embodiments, the modified immune cells of the present disclosure comprise a chromosomal knockout of one or more genes encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, an HLA component (e.g., a gene encoding α1 macroglobulin, α2 macroglobulin, α3 macroglobulin, β1 microglobulin, or β2 microglobulin), or a TCR component (e.g., a gene encoding a TCR variable region or a TCR constant region) (see, e.g., Torikai et al., Nature Sci. Rep. 6:21757 (2016); Torikai et al., Blood 119(24):5697 (2012); and Torikai et al., Blood 122(8):1341 (2013), the entire contents of which are incorporated herein by reference for their gene editing techniques, compositions, and adoptive cell therapies).

[0181] As used herein, the term "chromosomal gene knockout" refers to a genetic alteration or introduction of an inhibitor in a host cell that prevents (e.g., reduces, delays, inhibits, or eliminates) the production of a functionally active endogenous polypeptide product by the host cell. Alterations that result in chromosomal gene knockout can include, for example, introduction of nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletions, and strand breaks, as well as heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in the host cell.

[0182] In certain embodiments, chromosomal gene knockout or gene knock-in is achieved by chromosome editing of the host cell. Chromosome editing can be performed using, for example, an endonuclease. As used herein, "endonuclease" refers to an enzyme that can catalyze the cleavage of phosphodiester bonds within a polynucleotide chain. In certain embodiments, an endonuclease can cut the target gene, thereby inactivating or "knocking out" the target gene. The endonuclease can be a naturally occurring, recombinant, genetically modified, or fused endonuclease. The nucleic acid chain breaks caused by endonucleases are typically repaired by different mechanisms of homologous recombination or non-homologous end joining (NHEJ). During homologous recombination, donor nucleic acid molecules can be used for donor gene "knocking in", target gene "knocking out", and optionally inactivating the target gene by donor gene knocking in or target gene knockout events. NHEJ is an error-prone repair process that typically results in changes in the DNA sequence of the cleavage site, such as replacement, deletion, or addition of at least one nucleotide. NHEJ can be used for "knocking out" the target gene. Examples of endonucleases include zinc finger nucleases, TALE-nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.

[0183] As used herein, "zinc finger nuclease" (ZFN) refers to a fusion protein comprising a zinc finger DNA binding domain fused to a non-specific DNA cleavage domain (such as Fok1 endonuclease). Each zinc finger motif of approximately 30 amino acids binds to approximately 3 DNA base pairs, and the amino acids on certain residues can be changed to change the triplet sequence specificity (see, for example, Desjarlais et al., Proc. Natl. Acad. Sci. 90: 2256-2260, 1993; Wolfe et al., J. Mol. Biol. 285: 1917-1934, 1999). Multiple zinc finger motifs can be connected in series to produce binding specificity to a desired DNA sequence (e.g., a region ranging in length from about 9 to about 18 base pairs). As background, ZFN mediates genome editing by catalyzing the formation of site-specific DNA double-strand breaks (DSBs) in the genome, and promotes targeted integration of transgenics containing flanking sequences homologous to the genome at the DSB site for homology-directed repair. Alternatively, the DSB generated by the ZFN can be repaired by non-homologous end joining (NHEJ), an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the cleavage site to knock out the target gene. In certain embodiments, gene knockout comprises insertion, deletion, mutation, or a combination thereof performed using a ZFN molecule.

[0184] As used herein, "transcription activator-like effector nuclease" (TALEN) refers to a fusion protein comprising a TALE DNA binding domain and a DNA cleavage domain (e.g., FokI endonuclease). A "TALE DNA binding domain" or "TALE" consists of one or more TALE repeat domains / units, each typically having a highly conserved 33-35 amino acid sequence with different 12th and 13th amino acids. The TALE repeat domain is involved in the binding of TALE to a target DNA sequence. Different amino acid residues, referred to as repeat variable residues (RVDs), are associated with specific nucleotide recognition. The natural (canonical) codes for the DNA recognition of these TALEs have been determined so that the HD (histidine-aspartic acid) sequences at positions 12 and 13 of TALE result in TALE binding to cytosine (C), NG (asparagine-glycine) binding to T nucleotides, NI (asparagine-isoleucine) binding to A nucleotides, NN (asparagine-asparagine) binding to G or A nucleotides, and NG (asparagine-glycine) binding to T nucleotides. Non-classical (atypical) RVDs are also known (see, for example, U.S. Patent Publication US 2011 / 0301073, which is incorporated herein by reference in its entirety). TALEN can be used to direct site-specific double-strand breaks (DSBs) in the T cell genome. Non-homologous end joining (NHEJ) connects the DNA from both sides of the double-strand break, with little or no sequence overlap for annealing, thereby introducing errors in knocking out gene expression. Alternatively, homology-directed repair can introduce a transgene at the DSB site, provided that homologous flanking sequences are present in the transgene. In certain embodiments, gene knockout comprises insertion, deletion, mutation, or a combination thereof, and is performed using TALEN molecules.

[0185] As used herein, "Clustered Regularly Interspaced Short Palindromic Repeats / Cas" (CRISPR / Cas) nuclease system refers to a system that uses CRISPR RNA (crRNA)-guided Cas nucleases to recognize target sites within the genome (called protospacers) through base pairing complementarity, and then cuts the DNA if a short, conserved protospacer-associated motif (PAM) follows the 3' of the complementary target sequence. Based on the sequence and structure of the Cas nuclease, CRISPR / Cas systems are divided into three types (i.e., type I, type II, and type III). The crRNA-guided surveillance complexes in types I and III require multiple Cas subunits. The most studied type II system contains at least three components: an RNA-guided Cas9 nuclease, crRNA, and a trans-acting crRNA (tracrRNA). TracrRNA contains a duplex-forming region. CrRNA and TracrRNA form a duplex that can interact with the Cas9 nuclease and guide the Cas9 / crRNA:tracrRNA complex to a specific site on the target DNA by Watson-Crick base pairing between the protospacer on the crRNA and the target DNA upstream of the PAM. The Cas9 nuclease cuts double-strand breaks within the region defined by the crRNA spacer. NHEJ repair results in insertions and / or deletions that disrupt the expression of the target locus. Alternatively, a transgene with a homologous flanking sequence can be introduced into the DSB site by homology-directed repair. CrRNA and TracrRNA can be engineered into a single guide RNA (sgRNA or gRNA) (see, for example, Jinek et al., Science 337:816-21, 2012). In addition, the region of the guide RNA complementary to the target site can be altered or programmed to target the desired sequence (Xie et al., PLOS One 9:e100448, 2014; U.S. Patent Application No. US 2014 / 0068797, U.S. Patent Application No. US 2014 / 0186843; U.S. Patent No. 8,697,359, and PCT Application No. WO 2015 / 071474; each of which is incorporated by reference). In certain embodiments, gene knockout comprises insertion, deletion, mutation, or a combination thereof, and is performed using the CRISPR / Cas nuclease system.

[0186] Exemplary gRNA sequences and methods of using them to knock out endogenous genes encoding immune cell proteins include those described in Ren et al., Clin. Cancer Res. 23(9): 2255-2266 (2017), the gRNAs, Cas9 DNAs, vectors, and gene knockout technologies of which are incorporated herein by reference in their entirety.

[0187] Alternative Cas nucleases can be used, including but not limited to Cas 12, Cas 13, and Cas 14 nucleases and variants thereof. For example, the Cas nucleases disclosed in WO 2019 / 178427, which is incorporated herein by reference in its entirety (including the Cas nucleases, CRISPR-Cas systems, and related methods disclosed therein), can be used.

[0188] As used herein, "meganucleases," also known as "homing endonucleases," refer to endodeoxyribonucleases characterized by a large recognition site (a double-stranded DNA sequence of about 12 to about 40 base pairs). Based on sequence and structural motifs, meganucleases can be divided into five families: LAGLIDADG (SEQ ID NO: 97), GIY-YIG (SEQ ID NO: 98), HNH, His-Cys box, and PD-(D / E)XK (SEQ ID NO: 99). Exemplary meganucleases include I-Scel, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-ScelI, I-PpoI, I-ScelIII, I-Crel, I-TevI, I-TevII, and I-TevIII, whose recognition sequences are known (see, e.g., U.S. Pat. Nos. 5,420,032 and 6,833,252; Belfort et al., Nucleic Acids Res. 25:3379-3388, 1997; Dujon et al., Gene 82:115-118, 1989; Perler et al., Nucleic Acids Res. 22:1125-1127, 1994; Jasin, Trends Genet. 12:224-228, 1996; Gimble et al., J. Mol. Biol. 263:163-180, 1996; Argast et al., J. Mol. Biol. 280:345-353, 1998).

[0189] In certain embodiments, naturally occurring meganucleases can be used to facilitate site-specific genomic modification of a target selected from PD-1, LAG3, TIM3, CTLA4, TIGIT, an HLA encoding gene, or a TCR component encoding gene. In other embodiments, engineered meganucleases with novel binding specificities for target genes are used for site-specific genome modification (see, e.g., Porteus et al., Nat. Biotechnol. 23:967-73, 2005; Sussman et al., J. Mol. Biol. 342:31-41, 2004; Epinat et al., Nucleic Acids Res. 31:2952-62, 2003; Chevalier et al., Molec. Cell 10:895-905, 2002; Ashworth et al., Nature 441:656-659, 2006; Paques et al., Curr. Gene Ther. 7:49-66, 2007; U.S. Patent Publication Nos. US 2007 / 0117128; US 2006 / 0206949; US 2006 / 0153826; US 2006 / 0078552; and US 2004 / 0002092). In other embodiments, chromosomal gene knockouts are generated using homing endonucleases modified with the modular DNA binding domains of TALENs to create fusion proteins termed megaTALs. MegaTALs can be used not only to knock out one or more target genes, but also to introduce (knock-in) heterologous or exogenous polynucleotides when used in combination with an exogenous donor template encoding a polypeptide of interest.

[0190] In certain embodiments, the chromosomal gene knockout comprises an inhibitory nucleic acid molecule introduced into a host cell (e.g., an immune cell) that comprises a heterologous polynucleotide encoding an antigen-specific receptor that specifically binds a tumor-associated antigen, wherein the inhibitory nucleic acid molecule encodes a target-specific inhibitor, and wherein the encoded target-specific inhibitor inhibits endogenous gene expression in the host immune cell (i.e., PD-1, TIM3, LAG3, CTLA4, TIGIT, an HLA component, or a TCR component, or any combination thereof).

[0191] After using a knockout procedure or reagent, chromosomal gene knockout can be directly confirmed by DNA sequencing of host immune cells. Chromosomal gene knockout can also be inferred from the absence of gene expression (e.g., absence of the gene-encoded mRNA or polypeptide product) after knockout.

[0192] Any of the aforementioned gene editing techniques can be used to introduce the polynucleotides of the present disclosure (e.g., encoding binding proteins and / or CD8 co-receptor polypeptides) into the host cell genome. In certain embodiments, heterologous polynucleotides are introduced into the loci encoding endogenous TCR components, HLA components, PD-1, LAG-3, CTLA4, TIM3 or TIGIT, or safe harbor loci (e.g., Rosa26, AAVS1, CCR5, etc.). In certain embodiments, heterologous polynucleotides encoding binding proteins and / or encoding CD8 co-receptor polypeptides are introduced into the host cell TRAC locus. In other embodiments, the chromosome knockout of the host cell TRBC locus is introduced.

[0193] Thus, in certain embodiments, a host cell (e.g., a modified immune cell) is provided that comprises a heterologous polynucleotide encoding a binding protein of the present disclosure, a CD8 co-receptor of the present disclosure, or both in the endogenous TRAC locus. In other embodiments, the host cell comprises a chromosomal knockout of the endogenous TRBC locus.

[0194] In another aspect, provided herein are compositions comprising the modified immune cells (and / or polynucleotides, vectors, or binding proteins) of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient.

[0195] Also provided herein are unit doses comprising an effective amount of modified immune cells or compositions comprising modified immune cells. In certain embodiments, a unit dose comprises (i) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4+ T cells and (ii) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD8+ T cells in a ratio of about 1:1, wherein the unit dose contains a reduced amount of or is substantially free of naive T cells (i.e., the percentage of the naive T cell population present in the unit dose is less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% compared to a patient sample having a comparable number of PBMCs).

[0196] In some embodiments, a unit dose comprises (i) a composition comprising at least about 50% modified CD4+ T cells, and (ii) a composition comprising at least about 50% modified CD8+ T cells, at a ratio of about 1:1, wherein the unit dose contains a reduced amount of or substantially no naive T cells. In other embodiments, a unit dose comprises (i) a composition comprising at least about 60% modified CD4+ T cells, and (ii) a composition comprising at least about 60% modified CD8+ T cells, at a ratio of about 1:1, wherein the unit dose contains a reduced amount of or substantially no naive T cells. In other embodiments, a unit dose comprises (i) a composition comprising at least about 70% modified CD4+ T cells, and (ii) a composition comprising at least about 70% modified CD8+ T cells, at a ratio of about 1:1, wherein the unit dose contains a reduced amount of or substantially no naive T cells. In some embodiments, a unit dose comprises (i) a composition comprising at least about 80% modified CD4+ T cells and (ii) a composition comprising at least about 80% modified CD8+ T cells in a ratio of about 1:1, wherein the unit dose contains a reduced amount of, or substantially no, naive T cells. In some embodiments, a unit dose comprises (i) a composition comprising at least about 85% modified CD4+ T cells and (ii) a composition comprising at least about 85% modified CD8+ T cells in a ratio of about 1:1, wherein the unit dose contains a reduced amount of, or substantially no, naive T cells. In some embodiments, a unit dose comprises (i) a composition comprising at least about 90% modified CD4+ T cells and (ii) a composition comprising at least about 90% modified CD8+ T cells in a ratio of about 1:1, wherein the unit dose contains a reduced amount of, or substantially no, naive T cells.

[0197] It is understood that the unit doses of the present disclosure can comprise modified immune cells as described herein (i.e., expressing a binding protein specific for the WT-1 antigen according to SEQ ID NO: 94) and modified immune cells expressing a binding protein specific for a different antigen (e.g., a different WT-1 antigen (e.g., comprising SEQ ID NO.: 100)) or for an antigen from a different protein or target, such as BCMA, CD3, CEACAM6, c-Met, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis A, Lewis Y, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A (e.g., including MAGE-A1, MAGE-A3, and MAGE-A4), mesothelin, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, HLA, tumor- or pathogen-associated peptides bound to HLA, hTERT peptides bound to HLA, tyrosinase peptides bound to HLA, KRAS peptides bound to HLA, LTβR, LIFRβ, LRP5, MU C1, OSMRβ, TCRα, TCRβ, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD79a, CD79b, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, TLR7, TLR9, PTCH1, HA1-H, Robo1, alpha-fetoprotein (AFP), Frizzled, OX40, PRAME, BRAF, core binding factor, MR-1 (Crowther et al., Nature Immunol. 21: 175-185 (2020)) and SSX-2, etc. For example, a unit dose can contain a modified CD8 expressing a binding protein that specifically binds to the WT-1:HLA complex. + T cells and modified CD4 T cells expressing a binding protein (e.g., CAR) that specifically binds to the CD19 antigen + T cells (and / or modified CD8+ T cells).

[0198] In any embodiment described herein, the unit doses comprise equal or approximately equal amounts of engineered CD45RA - CD3 +CD8 + and modified CD45RA - CD3 + CD4 + TM cells.

[0199] In some embodiments, a composition is provided comprising (i) a modified immune cell as disclosed herein, and further comprising (ii) an immune cell comprising a polynucleotide encoding a binding protein comprising TCR Vα and TCR Vβ and capable of specifically binding to the VLDFAPPGA (SEQ ID NO.: 100):HLA complex, wherein optionally, HLA comprises HLA-A*0201, and wherein optionally, the modified immune cell of (i) and the immune cell of (ii) are each independently selected from T cells, NK cells and NK-T cells.

[0200] In certain other embodiments, the immune cell binding protein in (ii) comprises: (a) the CDR1a, CDR2a, CDR3a, CDR1β, CDR2β and CDR3β amino acid sequences of SEQ ID NOs.: 101-103 and 105-107, respectively, and wherein optionally, (1) Vα has at least 90% identity with, comprises or consists of the amino acid sequence shown in SEQ ID NO.: 104, and / or (2) Vβ has at least 90% identity with, comprises or consists of the amino acid sequence shown in SEQ ID NO.: 108; (b) the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β amino acid sequences of SEQ ID NOs.: 109-110 and 113-115, and wherein optionally, (1) Vα has at least 90% identity with, comprises or consists of the amino acid sequence shown in SEQ ID NO.: 109; NO.: 112 has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity, comprises or consists of the amino acid sequence shown in SEQ ID NO.: 116, and / or (2) Vβ has at least 90% identity, comprises or consists of the amino acid sequence shown in SEQ ID NO.: 116; (c) the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β amino acid sequences of SEQ ID NOs.: 117-119 and 121-123, and wherein optionally, (1) Vα has at least 90% identity, comprises or consists of the amino acid sequence shown in SEQ ID NO.: 120 and / or (2) Vβ has at least 90% identity, comprises or consists of the amino acid sequence shown in SEQ ID NO.: 124.

[0201] use

[0202] In certain aspects, the present disclosure relates to a method for treating a disease or condition associated with WT-1 expression by administering an effective amount of a binding protein, polynucleotide, vector, cell or composition according to any embodiment of the present disclosure to a human subject in need thereof.

[0203] "Treat" or "treatment" or "amelioration" refers to the medical management of a disease, disorder or condition in a subject (e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, rat). Therapeutic or prophylactic / preventative benefits include improving clinical outcome; reducing or alleviating symptoms associated with the disease; reducing the occurrence of symptoms; improving quality of life; prolonging the disease-free state; reducing the extent of the disease, stabilizing the disease state; delaying disease progression; remission; survival; prolonging survival; or any combination thereof.

[0204] As used herein, "therapeutically effective amount" or "effective amount" refers to the amount of modified immune cells sufficient to produce a therapeutic effect, including improved clinical outcome; alleviation or reduction of symptoms associated with the disease; reduction in the occurrence of symptoms; improved quality of life; longer disease-free state; reduction in disease severity, stable disease state; delayed disease progression; remission; survival; or prolonging survival in a statistically significant manner.

[0205] As used herein, "statistically significant" means a p-value of 0.050 or less when calculated using Student's T-test, and indicates that it is unlikely that the particular event or result measured occurred by chance.

[0206] The subjects that can be treated according to the methods of the present disclosure are typically humans and other primate subjects, such as monkeys and apes for veterinary and / or research purposes. Mice or rats can also be used for research purposes. In any of the above embodiments, the subject can be a human subject. The subject can be male or female and can be of any suitable age, including infants, teenagers, adolescents, adults and elderly subjects. The cells according to the present disclosure can be administered in a manner suitable for the disease, condition or illness to be treated as determined by a person skilled in the art of medicine. In any of the above embodiments, the modified immune cells or unit doses as described herein are administered intravenously, intraperitoneally, intratumorally, into the bone marrow, into the lymph nodes or into the cerebrospinal fluid in order to encounter target cells (e.g., leukemia cells). The appropriate dose, appropriate duration and frequency of administration of the composition will be determined by multiple factors, such as the patient's condition; the size, type and severity of the disease, condition or illness; the specific form of the active ingredient; and the method of administration, etc.

[0207] When referring to a single active ingredient, administered alone, or a cell expressing a single active ingredient, a therapeutically effective amount refers to the effect of that ingredient or cell expressing that ingredient alone. When referring to a combination, a therapeutically effective amount refers to the combined amount of the active ingredient or a co-active ingredient of the combination and the cell expressing the active ingredient that results in the therapeutic effect, whether administered sequentially or simultaneously. A combination can also include cells expressing more than one active ingredient.

[0208] As used herein, the term "adoptive immune therapy" or "adoptive immunotherapy" refers to the administration of naturally occurring or genetically engineered disease or antigen-specific immune cells (e.g., T cells). Adoptive cellular immunotherapy can be autologous (immune cells come from the recipient), allogeneic (immune cells come from a donor of the same species), or syngeneic (immune cells come from a donor that is genetically identical to the recipient).

[0209] Conditions associated with WT-1 expression include any disorder or condition in which there is low activity, excessive activity, or inappropriate activity of WT-1 cells or molecular events, and are typically caused by abnormally high (statistically significant) levels of WT-1 expression in diseased cells (e.g., leukemia cells) relative to normal cells. Subjects suffering from such conditions or conditions will benefit from treatment with the compositions or methods of the presently described embodiments. Thus, some conditions associated with WT-1 overexpression may include acute and chronic conditions and diseases, such as those pathological conditions that predispose a subject to a particular condition.

[0210] Diseases and disorders associated with WT-1 expression include the presence of dysplastic, cancerous, and / or transformed cells in a subject, including, for example, neoplastic cells, tumors, non-contact-inhibited or oncogenically transformed cells, and the like (e.g., solid cancers; hematologic cancers, including lymphomas and leukemias, such as acute myeloid leukemia, chronic myeloid leukemia, and the like), which are known in the art and have established diagnostic and classification criteria (e.g., Hanahan and Weinberg, 2011 Cell 144:646; Hanahan and Weinberg 2000 Cell 100:57; Cavallo et al., 2011 Canc. Immunol. Immunother. 60:319; Kyrigideis et al., 2010 J. Carcinog. 9:3). In certain embodiments, such cancer cells can be cells of acute myeloid leukemia, B-cell lymphoblastic leukemia, T-cell lymphoblastic leukemia, or myeloma, including cancer stem cells capable of initiating and continuously transplanting any of these types of cancer (see, for example, Park et al. 2009 Molec. Therap. 17: 219). According to certain embodiments, almost any type of cancer characterized by overexpression of WT-1 can be treated using the compositions and methods disclosed herein, including hematological cancers (e.g., leukemias, including acute myeloid leukemia (AML), T or B cell lymphomas, myeloma, etc.). In addition, "cancer" can refer to any cell that accelerates proliferation, including solid tumors, ascites tumors, blood or lymphoid or other malignant tumors; connective tissue malignancies; metastatic diseases; minimal residual disease after organ or stem cell transplantation; multidrug-resistant cancers, primary or secondary malignant tumors, angiogenesis associated with malignant tumors, or other forms of cancer. Specific embodiments are also contemplated within the present disclosure in which only one of the above-mentioned types of diseases is included, or specific disorders may be excluded regardless of whether they are characterized by WT-1 overexpression.

[0211] Some examples of conditions associated with WT-1 overexpression include hyperproliferative and proliferative diseases in subjects, including tumors, neoplasms, cancers, malignancies, and the like. In addition to activated or proliferating cells, hyperproliferative or proliferative diseases can also include abnormalities or disorders in cell death processes, whether through necrosis or apoptosis. Such abnormalities in cell death processes may be associated with a variety of conditions, including cancer (including primary, secondary malignant tumors, and metastatic tumors) or other conditions.

[0212] In certain embodiments, current method can be used for treating hematological malignancies or solid cancers.Exemplary hematological malignancies include acute lymphocytic leukemia (ALL), acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia (AML, including refractory and recurrent AML, and including acute myeloid leukemia, acute promyelocytic leukemia, acute granulocytic leukemia, acute myelomonocytic leukemia (for example, with or without eosinophilia), acute monocytic leukemia, acute erythroid leukemia and acute megakaryocytic leukemia), chronic myeloid leukemia (CML), chronic myeloid leukemia, chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL) or multiple myeloma (MM, including refractory and recurrent MM).

[0213] In certain embodiments, the methods of the present disclosure can be used to treat solid cancers, such as bile duct cancer, bladder cancer, bone and soft tissue cancer, brain tumors, breast cancer, breast carcinoma), cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonal carcinoma, endometrial cancer, esophageal cancer, stomach cancer, gastric adenocarcinoma, glioblastoma multiforme, glioblastoma, melanoma, diffuse peritoneal mesothelioma, malignant pleural mesothelioma, glioma, astrocytoma, gynecological cancer, head and neck squamous cell carcinoma, liver cancer, hepatocellular carcinoma, lung cancer, non-small cell lung cancer, malignant melanoma, osteosarcoma, ovarian cancer (e.g., epithelial ovarian cancer, ovarian carcinoma), fallopian tube cancer, endometrial cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytoma, primary thyroid cancer, prostate cancer, kidney cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, osteogenic sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, uterine carcinosarcoma, or uterine cancer.

[0214] Also provided are pharmaceutical compositions comprising binding proteins, polynucleotides, vectors, host cells and / or modified immune cells for use according to any of the methods described herein (optionally in combination with immune cells encoding binding proteins capable of binding to SEQ ID NO.: 100: HLA complex). As determined by one skilled in the medical field, the pharmaceutical composition can be administered in a manner appropriate to the disease or condition to be treated (or prevented). The appropriate dose of the composition and the appropriate duration and frequency of administration will be determined by the following factors: the patient's health, the patient's size (i.e., weight, mass, or body area), the type and severity of the patient's symptoms, the specific form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provides the composition in an amount sufficient to provide a therapeutic and / or preventive benefit (e.g., as described herein, including improved clinical outcomes, such as more frequent complete or partial remissions, or longer disease-free and / or overall survival, or a reduction in the severity of symptoms).

[0215] As described herein, an effective amount of a pharmaceutical composition refers to an amount sufficient to achieve the desired clinical outcome or beneficial treatment at the dosage and within the desired time period. An effective amount can be delivered in one or more administrations. If administered to a subject known or confirmed to have a disease or disease state, the term "therapeutic amount" can be used to refer to treatment, while a "prophylactically effective amount" can be used to describe the administration of an effective amount to a subject as a preventative procedure who is susceptible to or at risk of developing a disease or disease state (e.g., recurrence).

[0216] In certain embodiments, the method comprises administering a modified immune cell as provided herein to an immune cell that has received, is receiving, or will receive a polynucleotide encoding a binding protein comprising TCR Vα and TCR Vβ and capable of specifically binding to a VLDFAPPGA (SEQ ID NO.: 100):HLA complex, wherein optionally, HLA comprises HLA-A*0201, and wherein optionally, the modified immune cell and the immune cell are each independently selected from a T cell, a NK cell, and a NK-T cell. In certain embodiments, the binding protein capable of specifically binding to the VLDFAPPGA (SEQ ID NO.: 100):HLA complex comprises: (a) the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β amino acid sequences of SEQ ID NOs.: 101-103 and 105-107, respectively, and wherein optionally, (1) Vα is at least 90% identical to, comprises, or consists of, the amino acid sequence set forth in SEQ ID NO.: 104 and / or (2) Vβ is at least 90% identical to, comprises, or consists of, the amino acid sequence set forth in SEQ ID NO.: 108;

[0217] (b) the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β amino acid sequences of SEQ ID NOs: 109-110 and 113-115, and wherein, optionally, (1) Vα is at least 90% identical to, comprises or consists of the amino acid sequence shown in SEQ ID NO.: 112, and / or (2) Vβ is at least 90% identical to, comprises or consists of the amino acid sequence shown in SEQ ID NO.: 116; or (c) the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β amino acid sequences of SEQ ID NOs.: 117-119 and 121-123, and wherein, optionally, (1) Vα is at least 90% identical to, comprises or consists of the amino acid sequence shown in SEQ ID NO.: 120, and / or (2) Vβ is at least 90% identical to, comprises or consists of the amino acid sequence shown in SEQ ID NO.: The amino acid sequence shown in NO.: 124 has at least 90% identity, comprises or consists of the amino acid sequence.

[0218] In general, appropriate dosage and treatment regimen provide active molecules or cells in an amount sufficient to provide benefit. Compared to untreated subjects, this reaction can be monitored by establishing improved clinical outcomes (e.g., more frequent remissions, complete or partial or longer disease-free survival) in treated subjects. An increase in pre-existing immune responses to tumor proteins is generally associated with improved clinical outcomes. This immune response can generally be assessed using standard proliferation, cytotoxicity or cytokine assays, which are routine in the art and can be performed using samples obtained from subjects before and after treatment.

[0219] For prophylactic use, the dosage should be sufficient to prevent, delay the onset of, or lessen the severity of the disease or disorder associated with the disease. The prophylactic benefit of the compositions administered according to the methods described herein can be determined by conducting preclinical (including in vitro and in vivo animal studies) and clinical studies and analyzing the data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can be readily practiced by those skilled in the art.

[0220] Certain treatments or prevention methods contemplated herein include administering a host cell (which may be autologous, allogeneic, or homologous) comprising a desired nucleic acid molecule acid molecule stably integrated into a cell chromosome or present as an extrachromosomal nucleic acid as described herein. For example, autologous, allogeneic, or homologous immune system cells (e.g., T cells, antigen presenting cells, natural killer cells) may be used to produce such a cell composition in vitro, to administer the desired targeting WT-1 T cell composition as adoptive immunotherapy. In certain embodiments, the immune cell is a CD4+ T cells, CD8 + T cells, CD4 - CD8 - Double negative T cells, γδ T cells, natural killer cells, dendritic cells or any combination thereof. In other embodiments, the immune cells are naive T cells, central memory T cells, effector memory T cells or any combination thereof.

[0221] The amount of cells in a composition or unit dose is at least one cell (e.g., a modified CD8 + T cell subsets (e.g., optionally comprising memory and / or naive CD8 + T cells); a modified CD4 + T cell subsets (e.g., optionally comprising memory and / or naive CD4 + T cells) or more typically greater than 10 2 cells, e.g., up to 10 4 Up to 10 5 Up to 10 6 Up to 10 7 Up to 10 8 Up to 10 9 Up to 10 10 or up to 10 11 In certain embodiments, the cells are expressed in an amount of about 10 4 to about 10 11 cells / m 2 range of application, preferably about 10 5 to about 10 9 cells / m 2 In some embodiments, the administered dose comprises up to about 3.3 x 10 5 In some embodiments, the administered dose comprises up to about 1×10 6 In some embodiments, the administered dose comprises up to about 3.3×10 6 In some embodiments, the administered dose comprises up to about 1×10 7 In certain embodiments, the modified immune cells are prepared to contain up to about 5 x 10 4 cells / kg, 5x10 5 cells / kg, 5x10 6 cells / kg or up to approximately 5 x 10 7 cells / kg. In certain embodiments, the 4 cells / kg, 5×10 5 cells / kg, 5×10 6cells / kg or up to approximately 5 × 10 7 The modified immune cells are administered to a subject at a dose of 10 cells / kg. The number of cells will depend on the ultimate use of the composition and the cell types contained therein. For example, cells modified to contain binding proteins will contain at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more such cells. For the purposes provided herein, the volume of cells is typically one liter or less, 500 milliliters or less, 250 milliliters or less, or 100 milliliters or less. In embodiments, the density of the desired cells is typically greater than 10 4 cells / ml and usually greater than 10 7 cells / ml, usually 10 8 cells / ml or greater. The cells can be administered as a single infusion or as multiple infusions over a period of time. Clinically relevant numbers of immune cells can be distributed across multiple infusions that cumulatively equal or exceed 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or 10 11 In certain embodiments, a unit dose of modified immune cells can be co-administered with hematopoietic stem cells from an allogeneic donor (e.g., simultaneously or contemporaneously). In some embodiments, one or more modified immune cells contained in the unit dose are autologous to the subject.

[0222] The pharmaceutical compositions described herein may be presented in unit dose or multidose containers, such as sealed ampoules or vials. Such containers may be frozen to maintain the stability of the formulation prior to use. In certain embodiments, a unit dose comprises a recombinant host cell as described herein in a dose of about 10 4 cells / m 2 to about 10 11 cells / m 2 Suitable dosing and treatment regimens are developed for use of the particular compositions described herein in a variety of treatment regimens, including, for example, parenteral or intravenous administration or formulations.

[0223] In certain embodiments, the pharmaceutical composition comprising the modified immune cells disclosed herein further comprises a pharmaceutically acceptable carrier, diluent, or excipient. The term "pharmaceutically acceptable excipient or carrier" or "physiologically acceptable excipient or carrier" refers to a biocompatible excipient, such as normal saline, which is described in more detail herein, which is suitable for administration to humans or other non-human mammalian subjects and is generally considered safe or does not cause serious adverse events.

[0224] Suitable excipients include water, saline, glucose, glycerol, and combinations thereof. In embodiments, the composition comprising the fusion protein or host cells disclosed herein further comprises a suitable infusion medium. Suitable infusion media can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter), 5% glucose in water, and lactated Ringer's solution can be used. The infusion medium can be supplemented with human serum albumin or other human serum components.

[0225] If the subject composition is administered parenterally, the composition may also include a sterile aqueous or oily solution or suspension. Suitable non-toxic parenteral acceptable diluents or solvents include water, Ringer's solution, isotonic saline solution, 1,3-butanediol, ethanol, propylene glycol, or polyethylene glycol mixed with water. The aqueous solution or suspension may further comprise one or more buffers, such as sodium acetate, sodium citrate, sodium borate, or sodium tartrate. Of course, any material used to prepare any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amount used. In addition, the active compound can be incorporated into sustained-release formulations and preparations. As used herein, dosage unit form refers to a physically discrete unit suitable as a unit dose for a subject to be treated; each unit may contain a predetermined amount of recombinant cells or active compound, calculated to produce the desired therapeutic effect in combination with an appropriate pharmaceutical carrier.

[0226] As used herein, administration of a composition or therapy refers to its delivery to a subject, regardless of the route or mode of delivery. Administration can be performed continuously or intermittently and parenterally. Administration can be used to treat a subject who has been confirmed to have a recognized condition, disease or disease state, or to treat a subject who is susceptible to or at risk of developing such a condition, disease or disease state. Co-administration with adjuvant therapy can include the simultaneous and / or continuous delivery of multiple agents in any order and any dosing regimen (e.g., immune cells modified with one or more cytokines; immunosuppressive therapies, such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low doses of mycophenolic acid prodrugs, or any combination thereof).

[0227] In certain embodiments, multiple doses of the modified immune cells or binding proteins as described herein are administered to the subject, which can be administered at intervals of about 2 to about 4 weeks. In other embodiments, the cytokine is administered continuously, provided that the modified immune cells are administered to the subject at least three or four times prior to the administration of the cytokine. In certain embodiments, the cytokine (e.g., IL-2, IL-15, IL-21) is administered subcutaneously. In other embodiments, the subject being treated is further receiving immunosuppressive therapy, such as a calcineurin inhibitor, a corticosteroid, a microtubule inhibitor, a low dose of mycophenolic acid prodrug, or any combination thereof.

[0228] Treatment or prevention methods disclosed herein can be applied to subjects as part of a course of treatment or regimen, which may include additional treatment before or after administering the immediately disclosed unit dose, cell or composition. For example, in certain embodiments, the subject receiving the modified immune cell of a unit dose is receiving or has previously received hematopoietic cell transplantation (HCT; including myeloablative and non-myeloablative HCT). In certain embodiments, the modified immune cell can be administered for at least two to at least three months after non-myeloablative hematopoietic cell transplantation. The techniques and regimens for performing HCT are known in the art, and may include transplanting any suitable donor cell, such as cells derived from cord blood, bone marrow or peripheral blood, hematopoietic stem cells, mobilized stem cells, or cells from amniotic fluid. Therefore, in certain embodiments, the modified immune cell of the present disclosure can be administered together with hematopoietic stem cells in modified HCT therapy or shortly after administering hematopoietic stem cells. In certain embodiments, HCT includes donor hematopoietic cells, which include chromosome knockouts of genes encoding HLA components, chromosome knockouts of genes encoding TCR components, or both.

[0229] In some embodiments, the subject receiving the modified immune cell or binding protein has previously received lymphodepleting chemotherapy. In other embodiments, the lymphodepleting chemotherapy comprises cyclophosphamide, fludarabine, antithymocyte globulin, or a combination thereof.

[0230] In certain embodiments, the subject has previously received treatment for AML, or is at risk of developing or progressing AML.

[0231] The method according to the present disclosure may further include administering one or more additional agents to treat a disease or condition in a combination therapy. In some embodiments, the method further includes administering the modified immune cells or binding proteins with immune checkpoint inhibitors (in parallel, simultaneously or sequentially). In some embodiments, the method includes administering modified immune cells with agonists of stimulating immune checkpoint agents. In other embodiments, the combination therapy includes administering the modified immune cells together with secondary therapies, such as chemotherapeutic agents, radiotherapy, surgery, antibodies or any combination thereof.

[0232] As used herein, the term "immunosuppressant" or "immunosuppressive agent" refers to one or more cells, proteins, molecules, compounds, or complexes that provide an inhibitory signal to help control or suppress an immune response. For example, immunosuppressants include molecules that: partially or completely block immune stimulation; reduce, prevent, or delay immune activation; or increase, activate, or upregulate immune suppression. Exemplary immunosuppressive agents that are targeted (e.g., using immune checkpoint inhibitors) include PD-1, PD-L1, PD-L2, LAG3, CTLA4, B7-H3, B7-H4, CD244 / 2B4, HVEM, BTLA, CD160, TIM3, GAL9, KIR, PVR1G (CD112R), PVRL2, adenosine, A2aR, immunosuppressive cytokines (e.g., IL-10, IL-4, IL-1RA, IL-35), IDO, arginase, VISTA, TIGIT, LAIR1, CEACAM-1, CEACAM-3, CEACAM-5, Treg cells, or any combination thereof.

[0233] Immunosuppressants The inhibitor (also known as an immune checkpoint inhibitor) can be a compound, an antibody, an antibody fragment or a fusion polypeptide (e.g., an Fc fusion, such as CTLA4-Fc or LAG3-Fc), an antisense molecule, a ribozyme or RNAi molecule, or a low molecular weight organic molecule. In any embodiments disclosed herein, the method can include immune cells or binding proteins modified with one or more inhibitors of any of the following immunosuppressive components (alone or in any combination).

[0234] In certain embodiments, the modified immune cells or binding proteins are used in combination with a PD-1 inhibitor, such as a PD-1 specific antibody or binding fragment thereof, such as pidilizumab, nivolumab, pembrolizumab, MEDI0680 (formerly known as AMP-514), AMP-224, BMS-936558, or any combination thereof. In other embodiments, the modified immune cells or binding proteins of the present disclosure are used in combination with a PD-L1 specific antibody or binding fragment thereof, such as BMS-936559, durvalumab (MEDI4736), atezolizumab (RG7446), avelumab (MSB0010718C), MPDL3280A, or any combination thereof.

[0235] In certain embodiments, the modified immune cells or binding proteins of the present disclosure are used in combination with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof.

[0236] In certain embodiments, the modified immune cells or binding proteins are used in combination with CTLA4 inhibitors. In specific embodiments, the modified immune cells or binding proteins are used in combination with CTLA4-specific antibodies or binding fragments thereof (e.g., ipilimumab, tremelimumab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), or any combination thereof).

[0237] In certain embodiments, the modified immune cells or binding proteins are used in combination with a B7-H3 specific antibody or binding fragment thereof (e.g., enoxizumab (MGA271), 376.96, or both). The B7-H4 antibody binding fragment can be a scFv or a fusion protein thereof, such as those described in, for example, Dangaj et al., Cancer Res. 73:4820, 2013, U.S. Patent No. 9,574,000, and PCT Patent Publication Nos. WO / 201640724A1 and WO2013 / 025779A1.

[0238] In certain embodiments, modified immune cells or binding proteins are used in combination with CD244 inhibitors.

[0239] In certain embodiments, the modified immune cells or binding proteins are used in combination with inhibitors of BLTA, HVEM, CD160, or any combination thereof. Anti-CD-160 antibodies are described, for example, in PCT Publication No. WO 2010 / 084158.

[0240] In certain embodiments, the modified immune cells or binding proteins are used in combination with a TIM3 inhibitor.

[0241] In certain embodiments, the modified immune cells or binding proteins are used in combination with a Gal9 inhibitor.

[0242] In certain embodiments, the modified immune cells or binding proteins are used in combination with an adenosine signaling inhibitor, such as a decoy adenosine receptor.

[0243] In certain embodiments, modified immune cells or binding proteins are used in combination with A2aR inhibitors.

[0244] In certain embodiments, the modified immune cells or binding proteins are used in combination with a KIR inhibitor, such as lirilumab (BMS-986015).

[0245] In certain embodiments, modified immune cells are used in combination with suppressive cytokines (typically cytokines other than TGFβ) or inhibitors of Treg development or activity.

[0246] In certain embodiments, the modified immune cells or binding proteins are used in combination with an IDO inhibitor, such as L-1-methyltryptophan, epacadostat (INCB024360; Liu et al., Blood 115:3520-30, 2010), ebselen (Terentis et al., Biochem. 49:591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research 104th Annual Meeting 2013; Apr 6-10, 2013), 1-methyl-tryptophan (1-MT)-tira-pazamine, or any combination thereof.

[0247] In certain embodiments, the modified immune cells or binding proteins are used in combination with an arginase inhibitor, such as N(Ω)-nitro-L-arginine methyl ester (L-NAME), N-ω-hydroxy-nor-l-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine ​​(BEC), or any combination thereof.

[0248] In certain embodiments, the modified immune cells or binding proteins are used in combination with a VISTA inhibitor, such as CA-170 (Curis, Lexington, Mass).

[0249] In certain embodiments, the modified immune cells are coadministered with a TIGIT inhibitor such as COM902 (Compugen, Toronto, Ontario Canada), a CD155 inhibitor such as COM701 (Compugen), or both.

[0250] In certain embodiments, the modified immune cells or binding proteins are used in combination with inhibitors of PVRIG, PVRL2, or both. Anti-PVRIG antibodies are described, for example, in PCT Publication No. WO 2016 / 134333. Anti-PVRL2 antibodies are described, for example, in PCT Publication No. WO 2017 / 021526.

[0251] In certain embodiments, the modified immune cells or binding proteins are used in combination with a LAIR1 inhibitor.

[0252] In certain embodiments, the modified immune cells or binding proteins are used in combination with inhibitors of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.

[0253] In certain embodiments, the modified immune cells or binding proteins are used in combination with agents that increase the activity of stimulatory immune checkpoint molecules (ie, agonists). For example, the modified immune cells or binding proteins can be combined with CD137 (4-1BB) agonists (e.g., urerulumab), CD134 (OX-40) agonists (e.g., MEDI6469, MEDI6383 or MEDI0562), lenalidomide, pomalidomide, CD27 agonists (e.g., CDX-1127), CD28 agonists (e.g., TGN1412, CD80 or CD86), CD40 agonists (e.g., CP-870,893, rhuCD40L or SGN-40), CD122 agonists (e.g., IL-2), agonists of GITR (e.g., PCT Patent Publication No. WO 2016 / 054638), agonists of ICOS (CD278) (e.g., GSK3359609, mAb 88.2, JTX-2011, Icos 145-1, Icos 314-8 or any combination thereof). In any embodiment disclosed herein, the method may include administering the modified immune cell or binding protein together with one or more agonists (alone or in any combination) of a stimulatory immune checkpoint molecule including any of the above.

[0254] In certain embodiments, the combination therapy comprises a modified immune cell or binding protein and an adjuvant therapy comprising one or more of: an antibody or antigen-binding fragment thereof specific for a cancer antigen expressed by a non-inflamed solid tumor, radiation therapy, surgery, a chemotherapeutic agent, a cytokine, RNAi, or any combination thereof.

[0255] In certain embodiments, the combined treatment method comprises administering the modified immune cells or binding proteins and further administering radiation therapy or surgery. Radiation therapy is well known in the art and includes X-ray therapy, such as gamma irradiation, and radiopharmaceutical therapy. Surgery and surgical techniques suitable for treating a given cancer in a subject are well known to those of ordinary skill in the art.

[0256] In certain embodiments, the combined treatment method comprises administering the modified immune cell or binding protein and further administering a chemotherapeutic agent. Chemotherapeutic agents include, but are not limited to, chromatin function inhibitors, topoisomerase inhibitors, microtubule inhibitory drugs, DNA damaging agents, antimetabolites (e.g., folic acid antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), DNA synthesis inhibitors, DNA interactors (e.g., intercalators), and DNA repair inhibitors. Exemplary chemotherapeutic agents include, but are not limited to, the following groups: antimetabolites / anticancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents, including natural products, such as vinca alkaloids (vinblastine, vincristine, and vinorelbine); microtubule disrupting agents, such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, and nab-termitazole. , epothilones and naviridine, epipodophyllotoxins (etoposide, teniposide), DNA damaging drugs (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cyclophosphamide, actinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylpyridamole oxaliplatin, ifosfamide, melphalan, methylclomethamine, mitomycin, mitoxantrone, promycin, nitrourea-paclitaxel, taxotere, temozolomide, teniposide, triethylenethiophosphamide, and etoposide (VP) 16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; enzymes (L-asparaginase, which metabolizes L-asparagine systemically and deprives cells that do not have the ability to synthesize their own asparagine); antiplatelet drugs; antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimine and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates - busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozotocin), triazine-dacarbazine (DTIC); antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutamine; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide), and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts, and other thrombin inhibitors); fibrinolytics (such as tissue plasminogen activator, streptokinase, and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin);Immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (TNP470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab, rituximab); chimeric antigen receptors; cell cycle inhibitors and differentiation inducers (retinoic acid); mTOR inhibitors, topoisomerase inhibitors (polyclonal antibodies); Doxorubicin (doxorubicin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); growth factor signaling kinase inhibitors; mitochondrial dysfunction inducers, toxins such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheria toxin, and caspase activators; and chromatin disruptors.

[0257] Cytokines can be used to manipulate the host's immune response to anti-cancer activity. See, for example, Floros & Tarhini, Semin. Oncol. 42(4): 539-548, 2015. Cytokines that can be used to promote immune anti-cancer or anti-tumor responses include, for example, IFN-α, IL-2, IL-3, IL-4, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-24, and GM-CSF, which can be used alone or in any combination with the modified immune cells or binding proteins disclosed herein.

[0258] In other aspects, methods for making compositions of the present disclosure are provided. In certain embodiments, the methods include combining (i) an aliquot of host cells transduced with a vector of the present disclosure with (ii) a pharmaceutically acceptable carrier. In certain embodiments, the vector of the present disclosure is used to transfect / transduce host cells (e.g., T cells) for adoptive transfer therapy (e.g., targeting cancer antigens).

[0259] In certain embodiments, the method further comprises, before aliquoting, cultivating the transduced host cells and selecting the transduced cells into which the vector has been incorporated (i.e., expressed). In other embodiments, the method comprises, after cultivation and selection and before aliquoting, amplifying the transduced host cells. In any embodiment of the present method, the composition or unit dose of manufacture can be frozen for later use. Any suitable host cell can be used to prepare a composition or unit dose according to the present method, including, for example, hematopoietic stem cells, T cells, primary T cells, T cell lines, NK cells or NK-T cells. In a specific embodiment, the method comprises a host cell, which is a CD8+T cell, a CD4+T cell or both.

[0260] The present disclosure also includes the following exemplary embodiments.

[0261] Example 1. A modified immune cell comprising a heterologous polynucleotide encoding a binding protein, wherein the encoded binding protein comprises: (a) a T cell receptor (TCR) α chain variable region (Vα) domain comprising a CDR3 amino acid sequence (CDR3α) according to any one of SEQ ID NOs: 28, 19, 22 or 25, or a variant thereof, and a TCR β chain variable (Vβ) domain; or (b) a TCR Vβ domain comprising a CDR3 amino acid sequence (CDR3β) according to any one of SEQ ID NOs.: 40, 31, 34 or 37, or a variant thereof, and a TCR Vα domain; or (c) the TCR Vα domain of (a) and the TCR Vβ domain of (b), wherein the encoded binding protein is capable of binding to a RMFPNAPYL (SEQ ID NO.: 94):human leukocyte antigen (HLA) complex, wherein the HLA optionally comprises HLA-A*0201.

[0262] Example 2. The modified immune cell according to Example 1, wherein the encoded binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO.: 94): HLA complex to produce pEC 50 IFNγ of 4.0 or higher, or 4.5 or higher.

[0263] Example 3. The modified immune cell according to Example 1 or 2, wherein the encoded binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO.: 94): HLA complex to produce pEC 50 IFNγ of 5.0 or higher.

[0264] Example 4. The modified immune cell according to any one of Examples 1-3, wherein the encoded binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO.: 94): HLA complex to produce pEC50 IFNγ of 5.5 or higher.

[0265] Example 5. The modified immune cell according to any one of Examples 1-4, wherein the encoded binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO.: 94): HLA complex to produce pEC 50 IFNγ of 6.0 or higher.

[0266] Example 6. The modified immune cell according to any one of Examples 1-5, wherein the encoded binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO.: 94): HLA complex to produce pEC 50 IFNγ of 6.5 or higher.

[0267] Example 7. The modified immune cell according to any one of Examples 1-6, wherein the HLA comprises HLA-A*201.

[0268] Example 8. The modified immune cell according to any one of Examples 1-7, wherein when a population of the modified immune cells is co-cultured with antigen presenting cells (such as HLA-A*0201+) for 4 hours, 50% or more of the modified immune cells in the population produce IFN-γ, and the modified immune cells are cultured in the co-culture with a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO.: 94) at a dose of 10 -4 μM or 10 -5 μM or 10 -6 The method comprises the step of: administering the antigen-presenting cells to a concentration of about 1 μM peptide pulse, wherein the antigen-presenting cells optionally comprise T2 cells.

[0269] Embodiment 9. The modified immune cell according to any one of embodiments 1-8, wherein when the population of the modified immune cells is co-cultured with antigen presenting cells (e.g., for about 24 hours), a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO.: 94) is administered in the co-culture at a rate of 10 -2 Under conditions of a peptide concentration pulse of μM, 10% or more of the modified immune cells in the population were Nur77 + , wherein the antigen-presenting cells optionally comprise T2 cells, Jurkat cells, or both.

[0270] Example 10. The modified immune cell according to Example 9, wherein when the population of modified immune cells is co-cultured with antigen presenting cells (e.g., for about 24 hours), a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO.: 94) is administered in the co-culture at a rate of 10 -2Under conditions of a peptide concentration pulse of μM, 15% or more of the modified immune cells in the population were Nur77 + .

[0271] Example 11. The modified immune cell according to Example 9 or 10, wherein when the population of the modified immune cells is co-cultured with antigen presenting cells (e.g., for about 24 hours), a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO.: 94) is administered in the co-culture at a rate of 10 -2 Under conditions of a peptide concentration pulse of μM, 20% or more of the modified immune cells in the population were Nur77 + .

[0272] Example 12. The modified immune cell according to any one of Examples 9-11, wherein when the population of modified immune cells is co-cultured with antigen presenting cells (e.g., for about 24 hours), a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO.: 94) is administered in the co-culture at a rate of 10 -2 Under conditions of a peptide concentration pulse of μM, 40% or more of the modified immune cells in the population were Nur77 + .

[0273] Example 13. The modified immune cell according to any one of Examples 9-12, wherein when the population of modified immune cells is co-cultured with antigen presenting cells (e.g., for about 24 hours), a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO.: 94) is administered in the co-culture at a rate of 10 -2 Under conditions of a peptide concentration pulse of μM, 50% or more of the modified immune cells in the population were Nur77 + .

[0274] Embodiment 14. The modified immune cell according to any one of embodiments 1-13, wherein when the population of modified immune cells is co-cultured with antigen presenting cells (e.g., for about 24 hours), a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO.: 94) is administered in the co-culture at a rate of 10 -3 Under conditions of a peptide concentration pulse of μM, 10% or more of the modified immune cells in the population were Nur77 + .

[0275] Example 15. The modified immune cell according to Example 14, wherein when the population of the modified immune cells is co-cultured with antigen presenting cells (e.g., for about 24 hours), a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO.: 94) is administered in the co-culture at a rate of 10-3 Under conditions of a peptide concentration pulse of μM, 15% or more of the modified immune cells in the population were Nur77 + .

[0276] Example 16. The modified immune cell according to Example 14 or 15, wherein when the population of modified immune cells is co-cultured with antigen presenting cells (e.g., for about 24 hours), a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO.: 94) is administered in the co-culture at a rate of 10 -3 Under conditions of a peptide concentration pulse of μM, 20% or more of the modified immune cells in the population were Nur77 + .

[0277] Example 17. The modified immune cell according to any one of Examples 1-16, wherein when the modified immune cell and MDA-MB-468 cells are present in a sample at a ratio of 30:1, the modified immune cell is capable of killing 11% or more of the HLA-A*0201 in the sample. + MDA-MB-468 cells.

[0278] Example 18. The modified immune cell according to Example 17, wherein when the modified immune cell and MDA-MB-468 cells are present in a sample at a ratio of 30:1, the modified immune cell is capable of killing 12% or more of the HLA-A*0201 in the sample. + MDA-MB-468 cells.

[0279] Example 19. The modified immune cell according to Example 17 or 18, wherein when the modified immune cell and MDA-MB-468 cells are present in a sample at a ratio of 30:1, the modified immune cell is capable of killing 14% or more of the HLA-A*0201 in the sample. + MDA-MB-468 cells.

[0280] Example 20. The modified immune cell according to any one of Examples 17-19, wherein when the modified immune cell and MDA-MB-468 cells are present in a sample at a ratio of 30:1, the modified immune cell is capable of killing 15% or more of the HLA-A*0201 in the sample. + MDA-MB-468 cells.

[0281] Example 21. The modified immune cell according to any one of Examples 17-20, wherein when the modified immune cell and MDA-MB-468 cells are present in a sample at a ratio of 30:1, the modified immune cell is capable of killing 20% ​​or more of the HLA-A*0201 in the sample. + MDA-MB-468 cells.

[0282] Example 22. The modified immune cell according to any one of Examples 17-21, wherein when the modified immune cell and MDA-MB-468 cells are present in a sample at a ratio of 30:1, the modified immune cell is capable of killing 25% or more of the HLA-A*0201 in the sample. + MDA-MB-468 cells.

[0283] Example 23. The modified immune cell according to any one of Examples 1-22, wherein when the modified immune cell and MDA-MB-468 cells are present in a sample at a ratio of 10:1, the modified immune cell is capable of killing 10% or more of the HLA-A*0201 in the sample. + MDA-MB-468 cells.

[0284] Example 24. The modified immune cell according to any one of Examples 1-23, wherein when the modified immune cell and MDA-MB-468 cells are present in a sample at a ratio of 1:1, the modified immune cell is capable of killing 5% or more of the HLA-A*0201 in the sample. + MDA-MB-468 cells.

[0285] Embodiment 25. The modified immune cell according to any one of embodiments 1-24, wherein when the modified immune cell and the Panc1 cell are present in a ratio of 30:1, the modified immune cell is capable of killing 21% or more of the Panc1 cells in a sample.

[0286] Embodiment 26. The modified immune cell according to embodiment 25, wherein when the modified immune cell and the Panc1 cell are present at a ratio of 30:1, the modified immune cell is capable of killing 22% or more of the Panc1 cells in a sample.

[0287] Embodiment 27. The modified immune cell according to embodiment 25 or 26, wherein when the modified immune cell and the Panc1 cell are present at a ratio of 30:1, the modified immune cell is capable of killing 23% or more of the Panc1 cells in a sample.

[0288] Embodiment 28. The modified immune cell according to any one of embodiments 25-27, wherein when the modified immune cell and the Pancl cell are present in a ratio of 30:1, the modified immune cell is capable of killing 24% or more of the Pancl cells in a sample.

[0289] Embodiment 29. The modified immune cell according to any one of embodiments 25-28, wherein when the modified immune cell and the Panc1 cell are present in a ratio of 30:1, the modified immune cell is capable of killing 25% or more of the Panc1 cells in a sample.

[0290] Embodiment 30. The modified immune cell of any one of embodiments 1-29, wherein the modified immune cell has a higher affinity for HLA-A*0201 than a reference immune cell comprising a polynucleotide encoding a T cell receptor in a 4-hour co-culture. + The MDA-MB-468 cells and / or Panc1 cells have increased cytotoxic activity, wherein the T cell receptor comprises a TCR alpha chain having the amino acid sequence shown in SEQ ID NO.: 82 and a TCR beta chain having the amino acid sequence shown in SEQ ID NO.: 83.

[0291] Embodiment 31. The modified immune cell of any one of embodiments 1-30, wherein the encoded binding protein is capable of binding to the WT-1 peptide:HLA complex on the cell surface independent of CD8 or in the absence of CD8.

[0292] Embodiment 32. A modified immune cell according to any one of embodiments 1-31, wherein: (i) the encoded Vβ domain comprises or consists of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO.: 16, 5, 6, 7, 8, 13, 14 or 15; and / or (ii) the encoded Vα domain comprises or consists of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO.: 12, 1, 2, 3, 4, 9, 10, 11.

[0293] Embodiment 33. The modified immune cell of any one of embodiments 1-32, wherein the encoded Vβ domain comprises or consists of an amino acid sequence that is at least 90% identical to any one of SEQ ID NO.: 16, 5, 6, 7, 8, 13, 14, or 15, and / or the encoded Vα domain comprises or consists of an amino acid sequence that is at least 90% identical to any one of SEQ ID NO.: 12, 1, 2, 3, 4, 9, 10, or 11, with the proviso that: (i) at least three or four CDRs are not mutated; (ii) the CDRs that do have mutations have only a maximum of two amino acid substitutions, a maximum of five consecutive amino acid deletions, or a combination thereof; and (iii) the encoded binding protein retains its ability to bind to the RMFPNAPYL (SEQ ID NO.: 94):HLA complex, optionally producing interferon-γ (IFNγ) with a pEC50 of 4.0 or greater.

[0294] Embodiment 34. A modified immune cell according to any one of embodiments 1-33, wherein: (i) the encoded CDR3β comprises or consists of the amino acid sequence shown in SEQ ID NO.: 40, and the encoded CDR3α comprises or consists of the amino acid sequence shown in SEQ ID NO.: 28; (ii) the encoded CDR3β comprises or consists of the amino acid sequence shown in SEQ ID NO: 31, and the encoded CDR3α comprises or consists of the amino acid sequence shown in SEQ ID NO: 19; (iii) the encoded CDR3β comprises or consists of the amino acid sequence shown in SEQ ID NO: 34, and the encoded CDR3α comprises or consists of the amino acid sequence shown in SEQ ID NO: 22; or (iv) the encoded CDR3β comprises or consists of the amino acid sequence shown in SEQ ID NO: 37, and the encoded CDR3α comprises or consists of the amino acid sequence shown in SEQ ID NO: 25.

[0295] Embodiment 35. A modified immune cell according to any one of embodiments 1-34, wherein: (i) the encoded Vβ domain comprises a CDR1β amino acid sequence of any one of SEQ ID NO.: 38, 29, 32 or 35 and / or a CDR2β amino acid sequence of any one of SEQ ID NO.: 39, 30, 33 or 36; and / or (ii) the encoded Vα domain comprises a CDR1α amino acid sequence of any one of SEQ ID NO.: 26, 17, 20 or 23, and / or a CDR2α amino acid sequence of any one of SEQ ID NO.: 27, 18, 21 or 24.

[0296] Example 36. A modified immune cell according to any one of Examples 1-35, wherein the encoded TCR Vα and Vβ domains comprise the following CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β amino acid sequences, respectively: (i) SEQ ID NOs.: 26-28 and 38-40, respectively; (ii) SEQ ID NOs.: 23, 27, 28 and 38-40, respectively; (iii) SEQ ID NOs.: 17-19 and 29-31, respectively; (iv) SEQ ID NOs.: 20-22 and 32-34, respectively; or (v) SEQ ID NOs.: 23-25 ​​and 35-37, respectively.

[0297] Example 37. A modified immune cell according to any one of Examples 1-36, wherein the encoded TCR Vβ domain comprises: (i) an amino acid sequence according to the TRBJ02-03 gene segment; and / or (ii) an amino acid sequence according to the TRBV06-05 gene fragment, an amino acid sequence according to the TRBV07-09 gene fragment, or an amino acid sequence according to the TRBV20-01 gene fragment.

[0298] Example 38. A modified immune cell according to any one of Examples 1-37, wherein the encoded TCR Vα domain comprises: (i) an amino acid sequence according to the TRAJ43 gene segment; and / or (ii) an amino acid sequence according to the TRAV20-02 gene segment; an amino acid sequence according to the TRAV38DV08 gene fragment; or an amino acid sequence according to the TRAV38-01 gene fragment.

[0299] Example 39. A modified immune cell according to Example 38, wherein: (i) the encoded TCR Vβ domain comprises an amino acid sequence according to the TRBJ02-03 gene segment; and (ii) the encoded TCR Vα domain comprises an amino acid sequence according to the TRAJ43 gene fragment.

[0300] Embodiment 40. A modified immune cell according to embodiment 38, wherein: (i) the encoded TCR Vβ domain comprises an amino acid sequence according to the TRBV06-05 gene segment; and (ii) the encoded TCR Vα domain comprises an amino acid sequence according to the TRAV20 gene segment.

[0301] Example 41. A modified immune cell according to Example 38, wherein: (i) the encoded TCR Vβ domain comprises an amino acid sequence according to the TRBV07-09 gene segment; and (ii) the encoded TCR Vα domain comprises an amino acid sequence according to the TRAV38DV08 gene fragment.

[0302] Example 42. A modified immune cell according to Example 38, wherein: (i) the encoded TCR Vβ domain comprises an amino acid sequence according to the TRBV20-01 gene segment; and (ii) the encoded TCR Vα domain comprises an amino acid sequence according to the TRAV38-01 gene fragment.

[0303] Embodiment 43. A modified immune cell according to any one of embodiments 1-42, wherein: (i) the encoded Vβ domain comprises or consists of the amino acid sequence shown in SEQ ID NO.: 16 or 8, and the encoded Vα domain comprises or consists of the amino acid sequence shown in SEQ ID NO.: 12 or 4; (ii) the encoded Vβ domain comprises or consists of the amino acid sequence shown in SEQ ID NO.: 13 or 5, and the encoded Vα domain comprises or consists of the amino acid sequence shown in SEQ ID NO.: 9 or 1; (iii) the encoded Vβ domain comprises or consists of the amino acid sequence shown in SEQ ID NO.: 14 or 6, and the encoded Vα domain comprises or consists of the amino acid sequence shown in SEQ ID NO.: 10 or 2; or (iv) the encoded Vβ domain comprises or consists of the amino acid sequence shown in SEQ ID NO.: 15 or 7, and the encoded Vα domain comprises or consists of the amino acid sequence shown in SEQ ID NO.: 11 or 3.

[0304] Example 44. A modified immune cell according to any one of Examples 1-43, wherein the encoded binding protein further comprises: (i) a TCRα chain constant domain (Cα) or a fragment thereof; and / or (ii) a TCRβ chain constant domain (Cβ) or a fragment thereof.

[0305] Embodiment 45. The modified immune cell of embodiment 44, wherein the encoded Ca comprises or consists of an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs.: 41-44.

[0306] Embodiment 46. The modified immune cell of embodiment 44 or 45, wherein the encoded Cβ comprises or consists of an amino acid sequence that is at least 90% identical to SEQ ID NO.: 45.

[0307] Embodiment 47. The modified immune cell of any one of embodiments 44-46, wherein the encoded binding protein comprises: (i) a TCR β chain having at least 90% identity with, or comprising or consisting of, the amino acid sequence of SEQ ID NO.: 61 or 57; and a TCR α chain having at least 90% identity with, or comprising or consisting of, the amino acid sequence of SEQ ID NO.: 53 or 49; (ii) a TCR β chain having at least 90% identity with, or comprising or consisting of, the amino acid sequence of SEQ ID NO.: 58 or 54; and a TCR α chain having at least 90% identity with, or comprising or consisting of, the amino acid sequence of SEQ ID NO.: 50 or 46; (iii) a TCR β chain having at least 90% identity with, or comprising or consisting of, the amino acid sequence of SEQ ID NO.: 51 or 57; NO.: 59 or 55, which has at least 90% identity, or includes or consists of the amino acid sequence shown in SEQ ID NO.: 59 or 55; and a TCR alpha chain, which TCR alpha chain has at least 90% identity, or includes or consists of the amino acid sequence shown in SEQ ID NO.: 51 or 52; or (iv) a TCR beta chain, which TCR beta chain has at least 90% identity, or includes or consists of the amino acid sequence shown in SEQ ID NO.: 60 or 56; and a TCR alpha chain, which TCR alpha chain has at least 90% identity, or includes or consists of the amino acid sequence shown in SEQ ID NO.: 52 or 48.

[0308] Embodiment 48. The modified immune cell of any one of embodiments 1-47, wherein the encoded binding protein is a TCR, a chimeric antigen receptor (CAR), or a single-chain TCR (scTCR).

[0309] Example 49. The modified immune cell of any one of Examples 1-48, wherein the immune cell is a human immune system cell.

[0310] Example 50. The modified immune cell according to any one of Examples 1-49, wherein the immune cell is a T cell, optionally a CD4 + T cells, CD8 + T cells, CD4 - CD8 - Double negative T cells, or γδ T cells, natural killer cells, NK-T cells, dendritic cells or any combination thereof.

[0311] Example 51. The modified immune cell according to Example 50, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, a stem cell memory T cell, or any combination thereof.

[0312] Embodiment 52. The modified immune cell of any one of embodiments 1-51, wherein the polynucleotide encoding the binding protein is codon-optimized for expression in the immune cell.

[0313] Embodiment 53. The modified immune cell of any one of embodiments 1-52, wherein the polynucleotide encoding the binding protein comprises a polynucleotide having at least 75% identity to the polynucleotide sequence shown in any one of SEQ ID NOs.: 62-77.

[0314] Embodiment 54. The modified immune cell of embodiment 53, wherein the polynucleotide encoding the binding protein comprises: (i) a polynucleotide having at least 75% identity to the polynucleotide sequence of SEQ ID NO.: 77, and a polynucleotide having at least 75% identity to the polynucleotide sequence of SEQ ID NO.: 69; (ii) a polynucleotide having at least 75% identity to the polynucleotide sequence of SEQ ID NO.: 74, and a polynucleotide having at least 75% identity to the polynucleotide sequence of SEQ ID NO.: 66; (iii) a polynucleotide having at least 75% identity to the polynucleotide sequence of SEQ ID NO.: 75, and a polynucleotide having at least 75% identity to the polynucleotide sequence of SEQ ID NO.: 67; or (iv) a polynucleotide having at least 75% identity to the polynucleotide sequence of SEQ ID NO.: 76, and a polynucleotide having at least 75% identity to the polynucleotide sequence of SEQ ID NO.: 68.

[0315] Embodiment 55. The modified immune cell of any one of embodiments 1-54, wherein the polynucleotide encoding the binding protein further comprises a polynucleotide encoding a self-cleaving peptide located between the polynucleotide encoding Vβ and the polynucleotide encoding Vα.

[0316] Embodiment 56. The modified immune cell according to embodiment 55, wherein the encoded self-cleaving peptide comprises or consists of an amino acid sequence that is at least 90% identical to the amino acid sequence shown in any one of SEQ ID NOs.: 84-88.

[0317] Embodiment 57. The modified immune cell according to embodiment 55 or 56, wherein the polynucleotide encoding the self-cleaving peptide comprises or consists of a polynucleotide sequence that is at least 75% identical to the polynucleotide sequence shown in any one of SEQ ID NOs.: 89-93.

[0318] Example 58. A modified immune cell according to any one of Examples 55-57, wherein the polynucleotide encoding the binding protein has a structure from the 5'-end to the 3'-end of (polynucleotide encoding the TCRβ chain)-(polynucleotide encoding the self-cleaving peptide)-(polynucleotide encoding the TCRα chain).

[0319] Embodiment 59. The modified immune cell of any one of embodiments 55-58, wherein the polynucleotide encoding the binding protein comprises a polynucleotide having at least 75% identity to the polynucleotide sequence shown in any one of SEQ ID NOs.: 78-81.

[0320] Example 60. The modified immune cell according to Example 53, wherein the polynucleotide encoding the binding protein comprises a polynucleotide comprising or consisting of a polynucleotide sequence as shown in any one of SEQ ID NOs.: 78-81.

[0321] Example 61. A modified immune cell according to any one of Examples 1-60, further comprising: (i) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 co-receptor α chain, wherein optionally, the encoded polypeptide is or comprises a CD8 co-receptor α chain; (ii) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 co-receptor β chain, wherein optionally, the encoded polypeptide is or comprises a CD8 co-receptor β chain; or (iii) the polynucleotide of (i) and the polynucleotide of (ii), wherein optionally, the host cell comprises a CD4+ T cell.

[0322] Example 62. A host cell according to Example 61, comprising: (a) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 co-receptor α chain, wherein optionally, the encoded polypeptide is or comprises a CD8 co-receptor α chain; (b) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 co-receptor β chain, wherein, optionally, the encoded polypeptide is or comprises a CD8 co-receptor β chain; and (c) a polynucleotide encoding a self-cleaving peptide located between the polynucleotide of (a) and the polynucleotide of (b).

[0323] Example 63. A modified immune cell according to any one of Examples 1-62, wherein the immune cell comprises a chromosomal gene knockout of: PD-1 gene; LAG3 gene; TIM3 gene; CTLA4 gene; HLA component genes; TCR component genes; or any combination thereof.

[0324] Example 64. A modified immune cell according to Example 63, wherein the chromosomal gene knockout includes the knockout of an HLA component gene, and the HLA component gene is selected from: α1 macroglobulin gene; α2 macroglobulin gene; α3 macroglobulin gene; β1 microglobulin gene; or β2 microglobulin gene; or any combination thereof.

[0325] Example 65. A modified immune cell according to Example 63 or 64, wherein the chromosomal gene knockout includes the knockout of a TCR component gene, and the TCR component gene is selected from: TCRα variable region gene; TCRβ variable region gene; TCR constant region gene; or any combination thereof.

[0326] Example 66. An isolated polynucleotide encoding a binding protein, wherein the encoded binding protein comprises a TCR Vα domain and a TCR Vβ domain and is capable of binding to a RMFPNAPYL (SEQ ID NO.: 94):HLA complex, wherein: (i) the encoded Vα domain comprises a CDR3 amino acid sequence as set forth in any one of SEQ ID NOs.: 28, 19, 22, or 25; (ii) the encoded Vβ domain comprises a CDR3 amino acid sequence as set forth in any one of SEQ ID NOs.: 40, 31, 34, or 37; (iii) the encoded Vα domain comprises a CDR1 amino acid sequence as set forth in any one of SEQ ID NOs.: 26, 17, 20, or 23; (iv) the encoded Vβ domain comprises a CDR1 amino acid sequence as set forth in any one of SEQ ID NOs.: 38, 32, or 35; (v) the encoded Vα domain comprises a CDR2 amino acid sequence as set forth in any one of SEQ ID NOs.: 41, 43, 44, or 46. NO.: 27, 18, 21 or 24; (vi) the encoded Vβ domain comprises the CDR2 amino acid sequence of any one of SEQ ID NO.: 39, 30, 33 or 36; (vii) the encoded Vα domain comprises or consists of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO.: 4, 1-3, 12 or 9-11; (viii) the encoded Vβ domain comprises or consists of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO.: 8, 5-7, 16 or 13-15; (ix) the encoded TCR Vα is contained in a TCRα chain, wherein the TCRα chain comprises or consists of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NO.: 53 and 46-52; (x) the encoded TCR Vβ is contained in a TCRβ chain, wherein the TCRβ chain comprises or consists of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO.: The amino acid sequence shown in any one of NO.: 61 and 54-60 has at least 90% identical amino acid sequence composition; or any combination of (xi)(i)-(x).

[0327] Embodiment 67. The isolated polynucleotide of embodiment 66, wherein the polynucleotide is codon-optimized for expression in a host cell, wherein optionally, the host cell is a human T cell, NK cell, or NK-T cell.

[0328] Embodiment 68. The isolated polynucleotide of embodiment 66 or 67, wherein the polynucleotide comprises a polynucleotide having at least 75% identity to the polynucleotide sequence shown in any one of SEQ ID NOs: 62-81.

[0329] Embodiment 69. A vector comprising the polynucleotide of any one of embodiments 66-68.

[0330] Embodiment 70. The vector of embodiment 69, wherein the vector is a lentiviral vector or a retroviral vector.

[0331] Embodiment 71. A host cell comprising the polynucleotide of any one of embodiments 66-68 and capable of expressing the encoded binding protein.

[0332] Example 72. A composition comprising: (i) the modified immune cell of any one of Examples 1-65; (ii) the polynucleotide of any one of Examples 66-68; (iii) the vector of Example 69 or 70; and / or (iv) the host cell of Example 71; and a pharmaceutically acceptable carrier, excipient, or diluent.

[0333] Example 73. A composition according to Example 72, comprising: (i) a modified immune cell of any one of Examples 1-65; and (ii) an immune cell comprising a polynucleotide encoding a binding protein comprising TCR Vα and TCRVβ and capable of specifically binding to a VLDFAPPGA (SEQ ID NO.: 100):HLA complex, wherein optionally, HLA comprises HLA-A*0201, and wherein optionally, the modified immune cell of (i) and the immune cell of (ii) are each independently selected from T cells, NK cells and NK-T cells.

[0334] Example 74. A composition according to Example 73, wherein the immune cell binding protein in (ii) comprises: (a) the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β amino acid sequences of SEQ ID NOs.: 101-103 and 105-107, and wherein optionally, (1) Vα has at least 90% identity with, comprises or consists of the amino acid sequence of SEQ ID NO.: 104, and / or (2) Vβ has at least 90% identity with, comprises or consists of the amino acid sequence of SEQ ID NO.: 108; (b) the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β amino acid sequences of SEQ ID NOs.: 109-110 and 113-115, and wherein optionally, (1) Vα has at least 90% identity with, comprises or consists of the amino acid sequence of SEQ ID NO.: 109 NO.: 112 has at least 90% identity, comprises or consists of the amino acid sequence shown in NO.: 112, and / or (2) Vβ has at least 90% identity, comprises or consists of the amino acid sequence shown in SEQ ID NO.: 116; or (c) the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β amino acid sequences of SEQ ID NO.: 117-119 and 121-123, and optionally, (1) Vα has at least 90% identity, comprises or consists of the amino acid sequence shown in SEQ ID NO.: 120, and / or (2) Vβ has at least 90% identity, comprises or consists of the amino acid sequence shown in SEQ ID NO.: 124.

[0335] Example 75. A method of treating a subject having a disease or condition associated with WT-1 expression, comprising administering to the subject an effective amount of: (i) the modified immune cell of any one of Examples 1-65; (ii) the polynucleotide of any one of Examples 66-68; (iii) the vector of Example 69 or 70; (iv) the host cell of Example 71; and / or (v) the composition of any one of Examples 72-74.

[0336] Example 76. The method of Example 75, comprising administering the modified immune cell of any one of Examples 1-65 to a subject who has received, is receiving, or will receive an immune cell comprising a polynucleotide encoding a binding protein comprising TCR Vα and TCR Vβ and capable of specifically binding to a VLDFAPPGA (SEQ ID NO.: 100):HLA complex, wherein optionally, HLA comprises HLA-A*0201, and wherein optionally, the modified immune cell and the immune cell are each independently selected from T cells, NK cells, and NK-T cells.

[0337] Example 77. A method according to Example 76, wherein the binding protein capable of specifically binding to the VLDFAPPGA (SEQ ID NO.: 100): HLA complex comprises: (a) the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β amino acid sequences of SEQ ID NOs.: 101-103 and 105-107, and wherein optionally, (1) Vα has at least 90% identity with, or comprises or consists of, the amino acid sequence shown in SEQ ID NO.: 104, and / or (2) Vβ has at least 90% identity with, or comprises or consists of, the amino acid sequence shown in SEQ ID NO.: 108; (b) the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β amino acid sequences of SEQ ID NOs.: 109-110 and 113-115, and wherein optionally, (1) Vα has at least 90% identity with, or comprises or consists of, the amino acid sequence shown in SEQ ID NO.: 109 NO.: 112 has at least 90% identity, or comprises or consists of the amino acid sequence shown in SEQ ID NO.: 116, and / or (2) Vβ has at least 90% identity, or comprises or consists of the amino acid sequence shown in SEQ ID NO.: 116; or (c) the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β amino acid sequences of SEQ ID NO.: 117-119 and 121-123, and optionally, (1) Vα has at least 90% identity, or comprises or consists of the amino acid sequence shown in SEQ ID NO.: 120, and / or (2) Vβ has at least 90% identity, or comprises or consists of the amino acid sequence shown in SEQ ID NO.: 124.

[0338] Embodiment 78. The method of any one of embodiments 75-77, wherein the disease or condition is a hematological malignancy or a solid cancer.

[0339] Example 79. A method according to Example 78, wherein the hematological malignancy is selected from acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia (AML, including refractory and relapsed AML, and including acute myeloid leukemia, acute promyelocytic leukemia, acute granulocytic leukemia, acute myelomonocytic leukemia (e.g., with or without eosinophilia), acute monocytic leukemia, acute erythroid leukemia and acute megakaryocytic leukemia), chronic myeloid leukemia (CML), chronic myeloid leukemia, chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL) or multiple myeloma (MM, including refractory and relapsed MM).

[0340] Example 80. The method of Example 78, wherein the solid cancer is selected from bile duct cancer, bladder cancer, bone and soft tissue cancer, brain tumor, breast cancer, breast carcinoma), cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonal carcinoma, endometrial cancer, esophageal cancer, stomach cancer, gastric adenocarcinoma, glioblastoma multiforme, glioblastoma, melanoma, diffuse peritoneal mesothelioma, malignant pleural mesothelioma, glioma, astrocytoma, gynecological cancer, head and neck squamous cell carcinoma, liver cancer, hepatocellular carcinoma, lung cancer, non-small cell lung cancer, malignant melanoma, osteosarcoma, ovarian cancer (e.g., epithelial ovarian cancer, ovarian carcinoma), fallopian tube cancer, endometrial cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytoma, primary thyroid cancer, prostate cancer, kidney cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, osteogenic sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, uterine carcinosarcoma, or uterine cancer.

[0341] Embodiment 81. The method of any one of embodiments 75-80, wherein the modified immune cell is modified ex vivo to contain the heterologous polynucleotide.

[0342] Embodiment 82. The method of any one of embodiments 75-81, wherein the modified immune cells are allogeneic, syngeneic, or autologous to the subject.

[0343] Example 83. The method of any one of Examples 75-82, wherein the modified immune cell is a hematopoietic progenitor cell or a human immune system cell.

[0344] Example 84. The method of Example 83, wherein the immune system cell is a CD4 + T cells, CD8 + T cells, CD4- CD8 - Double negative T cells, gamma delta T cells, natural killer cells, dendritic cells, or any combination thereof.

[0345] Example 85. The method of Example 84, wherein the T cells are naive T cells, central memory T cells, effector memory T cells, or any combination thereof.

[0346] Example 86. The method of any one of Examples 75-85, wherein said method comprises administering multiple doses of said modified immune cells to said subject.

[0347] Embodiment 87. The method of embodiment 86, wherein the multiple doses are administered at an administration interval of about 2 to about 4 weeks.

[0348] Example 88. The method of any one of Examples 75-87, wherein the modified immune cells are grown at about 10 4 cells / kg to about 10 11 A dose of cells / kg is administered to the subject.

[0349] Example 89. The method of any one of Examples 75-88, further comprising administering a cytokine to the subject.

[0350] Example 90. The method of Example 89, wherein the cytokine is IL-2, IL-15, IL-21 or any combination thereof.

[0351] Example 91. The method of Example 90, wherein the cytokine is IL-2 and is administered simultaneously or sequentially with the modified immune cells.

[0352] Example 92. The method of Example 91, wherein the cytokines are administered sequentially, provided that the subject is administered the modified immune cells at least three or four times prior to administration of the cytokines.

[0353] Example 93. The method of any one of Examples 89-92, wherein the cytokine is IL-2 and is administered subcutaneously.

[0354] Embodiment 94. The method of any one of embodiments 75-93, wherein the subject is further receiving immunosuppressive therapy.

[0355] Example 95. The method of Example 94, wherein the immunosuppressive therapy is selected from a calcineurin inhibitor, a corticosteroid, a microtubule inhibitor, a low dose of mycophenolic acid prodrug, or any combination thereof.

[0356] Example 96. The method of any one of Examples 75-95, wherein the subject has received a non-myeloablative or myeloablative hematopoietic cell transplant.

[0357] Example 97. The method of Example 96, wherein the modified immune cells are administered to the subject at least three months after the non-myeloablative hematopoietic cell transplant.

[0358] Example 98. The method of Example 97, wherein the modified immune cells are administered to the subject at least two months after the myeloablative hematopoietic cell transplant.

[0359] Embodiment 99. The method of any one of embodiments 75-98, wherein the subject has previously received treatment for AML.

[0360] Example 100. A composition comprising: (i) a modified immune cell of any one of Examples 1-65; (ii) a polynucleotide of any one of Examples 66-68; (iii) a vector of Example 69 or 70; and / or (iv) a host cell of Example 71, for treating a disease or condition associated with WT-1 expression.

[0361] Example 101. A composition comprising: (i) a modified immune cell of any one of Examples 1-65; (ii) a polynucleotide of any one of Examples 66-68; (iii) a vector of Example 69 or 70; and / or (iv) a host cell of Example 71, for use in the manufacture of a medicament for treating a disease or condition associated with WT-1 expression.

[0362] Embodiment 102. The composition for use according to embodiment 100 or 101, wherein the disease or condition is a hematological malignancy or a solid cancer.

[0363] Example 103. A composition for use according to Example 102, wherein the hematological malignancy is selected from acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia (AML, including refractory and relapsed AML, and including acute myeloid leukemia, acute promyelocytic leukemia, acute granulocytic leukemia, acute myelomonocytic leukemia (e.g., with or without eosinophilia), acute monocytic leukemia, acute erythroid leukemia and acute megakaryocytic leukemia), chronic myeloid leukemia (CML), chronic myeloid leukemia, chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL) or multiple myeloma (MM, including refractory and relapsed MM).

[0364] Example 104. The composition for use according to Example 102, wherein the solid cancer is selected from bile duct cancer, bladder cancer, bone and soft tissue cancer, brain tumor, breast cancer, breast cancer, carcinoma), cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonal carcinoma, endometrial cancer, esophageal cancer, stomach cancer, gastric adenocarcinoma, glioblastoma multiforme, glioblastoma, melanoma, diffuse peritoneal mesothelioma, malignant pleural mesothelioma, glioma, astrocytoma, gynecological cancer, head and neck squamous cell carcinoma, liver cancer, hepatocellular carcinoma, lung cancer, non-small cell lung cancer, malignant melanoma, osteosarcoma, ovarian cancer (e.g., epithelial ovarian cancer, ovarian carcinoma), fallopian tube cancer, endometrial cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytoma, primary thyroid cancer, prostate cancer, kidney cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, osteogenic sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, uterine carcinosarcoma, or uterine cancer.

[0365] Example 105. A binding protein comprising a T cell receptor (TCR) alpha chain variable (Vα) domain and a TCR beta chain variable (Vβ) domain, wherein: the Vα domain comprises: (i) a CDR3 amino acid sequence (CDR3α) of any one of SEQ ID NOs.: 28, 19, 22, 25, or 28, or a variant thereof; (ii) a CDR1 amino acid sequence (CDR1α) of any one of SEQ ID NOs.: 26, 17, 20, or 23; (iii) a CDR2 amino acid sequence (CDR2a) of any one of SEQ ID NOs.: 27, 18, 21, or 24; (iv) an amino acid sequence that is at least 90% identical to an amino acid sequence of any one of SEQ ID NOs: 1-4 or 9-12; or any combination of (v) (i)-(iv); and the Vβ domain comprises: (vi) a CDR3 amino acid sequence of SEQ ID NOs.: 28, 19, 22, 25, or 28, or a variant thereof. NO: 40, 31, 34 or 37 or a variant thereof; (vii) the CDR1 amino acid sequence (CDR1β) of any one of SEQ ID NO.: 38, 29, 32 or 35; (viii) the CDR2 amino acid sequence (CDR2β) of any one of SEQ ID NO.: 39, 30, 33 or 36; (ix) an amino acid sequence that is at least 90% identical to the amino acid sequence shown in any one of SEQ ID NO: 5-8 or 13-16; or (x) any combination of (vi)-(x), wherein the binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO: 94):HLA complex, wherein optionally, the HLA comprises HLA-A*0201.

[0366] Example 106. The binding protein according to Example 105 further comprises: (1) a TCRα chain constant domain (Cα) or a fragment thereof; and / or (2) a TCRβ chain constant domain (Cβ) or a fragment thereof.

[0367] Embodiment 107. The binding protein of embodiment 106, wherein the Ca comprises or consists of the amino acid sequence of any one of SEQ ID NOs.: 41-44.

[0368] Embodiment 108. The binding protein according to embodiment 106 or 107, wherein the Cβ comprises or consists of the amino acid sequence of SEQ ID NO.: 45.

[0369] Embodiment 109. An isolated polynucleotide encoding the binding protein of any one of embodiments 105-108.

[0370] Embodiment 110. The polynucleotide of embodiment 109, which is codon-optimized for expression in a host cell.

[0371] Embodiment 111. A modified immune cell comprising the polynucleotide of any one of embodiments 109-110, wherein the polynucleotide is heterologous to the immune cell.

[0372] sequence

[0373] SEQ ID NO: 1 (#4 α chain variable domain with leader sequence)

[0374] MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNIGNHDMRFGAGTRLTVKPN

[0375] SEQ ID NO: 2 (#6 α chain variable domain with leader sequence)

[0376] MEKMLECAFIVLQLGWLSGEDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVQTMDGNQFYFGTGTSLTVIPN

[0377] SEQ ID NO: 3 (#7 α chain variable domain with leader sequence)

[0378] MACPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCASPGTYKYIFGTGTRLKVLAN

[0379] SEQ ID NO: 4 (#10 α chain variable domain with leader sequence)

[0380] MTRVSLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSESNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFNPWENYGQNFVFGPGTRLSVLPY

[0381] SEQ ID NO: 5 (#4 β chain variable domain with leader sequence)

[0382] MGCRLLCCAVLCLLGAVPIDTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSQGTSGADTQYFPGGTRLTVLE

[0383] SEQ ID NO: 6 (#6 β chain variable domain with leader sequence)

[0384] MSIGLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYSLWDLQETQYFPGTRLLVLE

[0385] SEQ ID NO: 7 (#7 β chain variable domain with leader sequence)

[0386] MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSFSDGGATDTQYFPGGTRLTVLE

[0387] SEQ ID NO: 8 (#10 β chain variable domain with leader sequence)

[0388] MLLLLLLLGPAGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARPHSLTDTQYFPGGTRLTVLE

[0389] SEQ ID NO: 9 (α chain variable domain without leader sequence #4)

[0390] QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNIGNHDMRFGAGTRLTVKPN

[0391] SEQ ID NO: 10 (α chain variable domain of #6 with leader sequence removed)

[0392] EDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKERLKATLTKKESFLHITAPKPEDSATYLCAVQTMDGNQFYFGTGTSLTVIPN

[0393] SEQ ID NO: 11 (α chain variable domain of #7 with leader sequence removed)

[0394] AQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFASSPGTYKYIFGTGTRLKVLAN

[0395] SEQ ID NO: 12 (α chain variable domain without leader sequence of #10)

[0396] AQTVTQSQPEMSVQEAETVTLSCTYDTSESNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFNPWENYGQNFVFGPGTRLSVLPY

[0397] SEQ ID NO: 13 (beta chain variable domain of #4 with leader sequence removed)

[0398] DTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSQGTSGADTQYFPGGTRLTVLE

[0399] SEQ ID NO: 14 (beta chain variable domain of #6 with leader sequence removed)

[0400] NAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYSLWDLQETQYFPGTRLLVLE

[0401] SEQ ID NO: 15 (beta chain variable domain of #7 with leader sequence removed)

[0402] DTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSFSDGGATDTQYFPGGTRLTVLE

[0403] SEQ ID NO: 16 (beta chain variable domain without leader sequence of #10)

[0404] GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARPHSLTDTQYFPGGTRLTVLE

[0405] SEQ ID NO: 17 (#4 CDR1α)

[0406] DRGSQS

[0407] SEQ ID NO:18(#4 CDR2α)

[0408] IYSNGD

[0409] SEQ ID NO:19(#4 CDR3α)

[0410] CAVNIGNHDMRF

[0411] SEQ ID NO:20(#6 CDR1α)

[0412] VSGLRG

[0413] SEQ ID NO:21(#6 CDR2α)

[0414] LYSAGEE

[0415] SEQ ID NO:22(#6 CDR3α)

[0416] CAVQTMDGNQFYF

[0417] SEQ ID NO:23(#7 CDR1α)

[0418] TSESDYY

[0419] SEQ ID NO:24(#7 CDR2α)

[0420] QEAYKQQN

[0421] SEQ ID NO:25(#7 CDR3α)

[0422] CASSPGTYKYIF

[0423] SEQ ID NO:26(#10 CDR1α)

[0424] TSESNYY

[0425] SEQ ID NO:27(#10 CDR2α)

[0426] QEAYKQQN

[0427] SEQ ID NO:28(#10 CDR3α)

[0428] CAFNPWENYGQNFVF

[0429] SEQ ID NO:29(#4 CDR1β)

[0430] MGHRA

[0431] SEQ ID NO: 30 (#4 CDR2β)

[0432] YSYEKL

[0433] SEQ ID NO: 31 (#4 CDR3β)

[0434] CASQGTSGADTQYF

[0435] SEQ ID NO: 32 (#6 CDR1β)

[0436] MNHEY

[0437] SEQ ID NO: 33 (#6 CDR2β)

[0438] VGAGI

[0439] SEQ ID NO: 34 (#6 CDR3β)<000!120>

[0440] CASSYSLWDLQETQYF

[0441] SEQ ID NO: 35 (#7 CDR1β)

[0442] SEHNR

[0443] SEQ ID NO: 36 (#7 CDR2β)

[0444] FQNEAQ

[0445] SEQ ID NO: 37 (#7 CDR3β)

[0446] CASSFSDGGATDTQYF

[0447] SEQ ID NO: 38 (#10 CDR1β)

[0448] DFQATT

[0449] SEQ ID NO: 39 (#10 CDR2β)

[0450] SNEGSKA

[0451] SEQ ID NO: 40 (#10 CDR3β)

[0452] [[ID=6!]]CSARPHLTDTQYF ,

[0453] SEQ ID NO: 41 (#4 TCRα constant domain)

[0454] It should be noted that there seems to be a small error in the original text where "CASSFSDGGATDTQYF" has an exclamation mark in the translated text at position 29. Also, "CSARPHLTDTQYF" has a "!" instead of a "1" in the original text. These should be corrected in the original text for a more accurate translation.IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0455] SEQ ID NO: 42 (#6 TCRα constant domain)

[0456] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0457] SEQ ID NO: 43 (#7 TCRα constant domain)

[0458] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0459] SEQ ID NO: 44 (#10 TCRα constant domain)

[0460] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0461] SEQ ID NO: 45 (TCRβ constant domain)

[0462] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0463] SEQ ID NO: 46 (α-chain with leader sequence of #4)

[0464] MKSLRVLLVILWLQLSWVWSQQKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNIGNHDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0465] SEQ ID NO: 47 (α-chain with leader sequence of #6)

[0466] MEKMLECAFIVLWLQLGWLSGEDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKEKERLKATLTKKESFLHITAPKPEDSATYLCAVQTMDGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0467] SEQ ID NO: 48 (α-chain with leader sequence of #7)

[0468] MACSPGFLWALVISTCLEFSMAQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCASSPGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFQWSPESSCDVLLKVAKSDFNLLMFRFQ

[0469] SEQ ID NO: 49 (α-chain with leader sequence of #10)

[0470] MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSESNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFNPWENYGQNFVFGPGTRLSVLPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0471] SEQ ID NO: 50 (α-chain without leader sequence of #4)

[0472] QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNIGNHDMRFGAGTRLTVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFTLRLWSPESSCDVLLVEKSFETDTNLNFQNLSVLGFRIL

[0473] SEQ ID NO: 51 (α-chain without leader sequence of #6)

[0474] EDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKERLKATLTKKESFLHITAPKPEDSATYLCAVQTMDGNQFYFGTGTSLTVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVLLVEKSFETDTNLNFQNLNSSFR

[0475] SEQ ID NO: 52 (α-chain without leader sequence of #7)

[0476] AQTVTQSQPEMSVQEAETVTLSCTYDTSESDYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDAAMYFCASSPGTYKYIFGTGTRLKVLANIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSRLWSSVIGFRILL

[0477] SEQ ID NO: 53 (α-chain without leader sequence of #10)

[0478] AQTVTQSQPEMSVQEAETVTLSCTYDTSESNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFNPWENYGQNFVFGPGTRLSVLPYIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTLNFKSPESSCDVKLVEKSGFDTNLNFQWSVSLVA

[0479] SEQ ID NO: 54 (β-chain with leader sequence of #4)

[0480] MGCRLLCCAVLCLLGAVPIDTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSQGTSGADTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0481] SEQ ID NO: 55 (β-chain with leader sequence #6)

[0482] MSIGLLCCAALSLLWAGPVNAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYSLWDLQETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0483] SEQ ID NO: 56 (β-chain with leader sequence #7)

[0484] MGTSLLCWMALCLLGADHADTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSFSDGGATDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0485] SEQ ID NO: 57 (β-chain with a leader sequence in #10)

[0486] MLLLLLLLGPAGSGLGAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARPHSLTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCL

[0487] ATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0488] SEQ ID NO: 58 (β-chain without a leader sequence in #4)

[0489] DTEVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSQGTSGADTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0490] SEQ ID NO: 59 (β-chain without leader sequence #6)

[0491] NAGVTQTPKFQVLKTGQSMTLQCAQDMNHEYMSWYRQDPGMGLRLIHYSVGAGITDQGEVPNGYNVSRSTTEDFPLRLLSAAPSQTSVYFCASSYSLWDLQETQYFGPGTRLLVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0492] SEQ ID NO: 60 (β-chain without leader sequence #7)

[0493] DTGVSQDPRHKITKRGQNVTFRCDPISEHNRLYWYRQTLGQGPEFLTYFQNEAQLEKSRLLSDRFSAERPKGSFSTLEIQRTEQGDSAMYLCASSFSDGGATDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0494] SEQ ID NO: 61 (β-chain without the leading sequence, #10)

[0495] GAVVSQHPSWVICKSGTSVKIECRSLDFQATTMFWYRQFPKQSLMLMATSNEGSKATYEQGVEKDKFLINHASLTLSTLTVTSAHPEDSSFYICSARPHSLTDTQYFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0496] SEQ ID NO: 62 (α-gene fragment, #4)

[0497] ATGAAATCCTTGAGAGTTTTACTAGTGATCCTGTGGCTTCAGTTGAGCTGGGTTTGGAGCCAACAGAAGGAGGTGGAGCAGAATTCTGGACCCCTCAGTGTTCCAGAGGGAGCCATTGCCTCTCTCAACTGCACTTACAGTGACCGAGGTTCCCAGTCCTTCTTCTGGTACAGACAATATTCTGGGAAAAGCCCTGAGTTGATAATGTTCATATACTCCAATGGTGACAAAGAAGATGGAAGGTTTACAGCACAGCTCAATAAAGCCAGCCAGTATGTTTCTCTGCTCATCAGAGACTCCCAGCCCAGTGATTCAGCCACCTACCTCTGTGCCGTGAACATAGGAAACCATGACATGCGCTTTGGAGCAGGGACCAGACTGACAGTAAAACCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA

[0498] SEQ ID NO: 63 (Gene fragment of #6α)

[0499] ATGGAGAAAATGTTGGAGTGTGCATTCATAGTCTTGTGGCTTCAGCTTGGCTGGTTGAGTGGAGAAGACCAGGTGACGCAGAGTCCCGAGGCCCTGAGACTCCAGGAGGGAGAGAGTAGCAGTCTCAACTGCAGTTACACAGTCAGCGGTTTAAGAGGGCTGTTCTGGTATAGGCAAGATCCTGGGAAAGGCCCTGAATTCCTCTTCACCCTGTATTCAGCTGGGGAAGAAAAGGAGAAAGAAAGGCTAAAAGCCACATTAACAAAGAAGGAAAGCTTTCTGCACATCACAGCCCCTAAACCTGAAGACTCAGCCACTTATCTCTGTGCTGTGCAGACCATGGACGGTAACCAGTTCTATTTTGGGACAGGGACAAGTTTGACGGTCATTCCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA

[0500] SEQ ID NO: 64 (Fragment of #7α gene)

[0501] ATGGCATGCCCTGGCTTCCTGTGGGCACTTGTGATCTCCACCTGTCTTGAATTTAGCATGGCTCAGACAGTCACTCAGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACCGTGACCCTGAGCTGCACATATGACACCAGTGAGAGTGATTATTATTTATTCTGGTACAAGCAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCAACAGAATGCAACAGAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCTCAAGATCTCAGACTCACAGCTGGGGGATGCCGCGATGTATTTCTGTGCTTCCAGTCCAGGAACCTACAAATACATCTTTGGAACAGGCACCAGGCTGAAGGTTTTAGCAAATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA

[0502] SEQ ID NO: 65 (Fragment of #10α gene)

[0503] ATGACACGAGTTAGCTTGCTGTGGGCAGTCGTGGTCTCCACCTGTCTTGAATCCGGCATGGCCCAGACAGTCACTCAGTCTCAACCAGAGATGTCTGTGCAGGAGGCAGAGACTGTGACCCTGAGTTGCACATATGACACCAGTGAGAGTAATTATTATTTGTTCTGGTACAAACAGCCTCCCAGCAGGCAGATGATTCTCGTTATTCGCCAAGAAGCTTATAAGCAACAGAATGCAACGGAGAATCGTTTCTCTGTGAACTTCCAGAAAGCAGCCAAATCCTTCAGTCTCAAGATCTCAGACTCACA

[0504] GCTGGGGGACACTGCGATGTATTTCTGTGCTTTCAACCCTTGGGAGAACTATGGTCAGAATTTTGTCTTTGGTCCCGGAACCAGATTGTCCGTGCTGCCCTATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA

[0505] SEQ ID NO: 66 (#4α gene fragment, codon optimized)

[0506] ATGAAGAGCCTGAGAGTCCTGCTGGTGATTTTGTGGCTGCAGCTGTCTTGGGTTTGGTCTCAGCAGAAAGAAGTGGAGCAGAATAGCGGCCCTCTGTCTGTTCCTGAAGGCGCTATTGCTAGCCTGAATTGCACATACAGCGATAGAGGATCTCAGAGCTTCTTCTGGTACCGGCAGTACAGCGGCAAGAGCCCAGAACTGATCATGTTCATCTACAGCAATGGCGACAAGGAGGATGGCAGGTTTACAGCCCAGCTGAACAAGGCCAGCCAGTATGTTTCTCTGCTGATCAGAGATAGCCAGCCTAGCGATTCTGCCACCTACCTGTGTGCCGTGAACATCGGAAATCACGACATGAGATTTGGAGCCGGCACAAGACTGACCGTGAAGCCCAATATCCAGAACCCTGATCCTGCTGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA

[0507] SEQ ID NO: 67 (#6α gene fragment, codon-optimized)

[0508] ATGGAGAAGATGCTGGAGTGTGCGTTCATCGTTCTGTGGCTGCAACTTGGATGGCTGTCTGGAGAGGATCAGGTTACACAGTCTCCTGAAGCCCTGAGACTGCAAGAAGGAGAAAGCTCTAGCCTGAACTGCAGCTACACAGTGTCTGGACTGAGAGGCCTGTTCTGGTACAGACAGGATCCTGGAAAAGGCCCAGAGTTCCTGTTTACCCTGTATTCTGCCGGCGAGGAGAAGGAGAAAGAGAGACTGAAAGCTACCCTGACCAAGAAGGAGAGCTTCCTGCACATTACCGCCCCCAAACCTGAGGATTCTGCCACATATCTGTGTGCTGTGCAGACCATGGATGGCAACCAGTTCTACTTCGGCACAGGCACATCTCTGACCGTTATCCCCAATATCCAGAACCCTGATCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA

[0509] [[ID=]3]SEQ ID NO: 68 (#7α gene fragment, codon-optimized)

[0510] ATGGCTTGTCCTGGATTCTTATGGGCTCTGGTGATCAGCACCTGTCTGGAGTTCTCTATGGCCCAGACAGTGACACAGTCTCAGCCTGAAATGTCTGTGCAGGAAGCCGAAACCGTGACACTGTCTTGCACCTACGATACAAGCGAGAGCGACTACTACCTGTTCTGGTACAAGCAGCCTCCCTCTAGGCAGATGATCCTGGTGATTAGACAGGAGGCCTACAAACAGCAGAATGCCACCGAGAACCGGTTTAGCGTGAACTTCCAGAAAGCCGCCAAGAGCTTCAGCCTGAAAATCTCTGACAGCCAGCTGGGAGATGCTGCCATGTACTTTTGTGCCAGCTCTCCAGGCACCTACAAGTACATTTTTGGCACCGGCACCAGACTGAAGGTGCTGGCCAATATCCAGAATCCCGATCCTGCCGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA

[0511] SEQ ID NO: 69 (#10α gene fragment, codon-optimized)

[0512] ATGACCAGAGTTAGCCTGTTATGGGCTGTGGTGGTGAGCACATGTCTGGAATCTGGAATGGCCCAGACAGTGACACAGTCTCAGCCTGAAATGTCTGTGCAGGAAGCCGAAACCGTTACACTGAGCTGCACCTACGATACAAGCGAGAGCAACTACTACCTGTTCTGGTACAAGCAGCCCCCTTCTAGGCAGATGATCCTGGTGATCAGACAGGAGGCCTATAAACAGCAGAATGCCACCGAGAACCGGTTTAGCGTGAACTTCCAGAAAGCCGCCAAGAGCTTCAGCCTGAAAATCTCTGACAGCCAGCTGGGCGATACAGCCATGTACTTTTGTGCCTTCAACCCCTGGGAGAACTATGGCCAGAATTTCGTGTTCGGCCCTGGCACCAGACTGTCTGTTCTGCCTTATATCCAGAACCCCGATCCTGCTGTGTACCAGCTGCGGGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAGACCAACGTGTCCCAGAGCAAGGACAGCGACGTGTACATCACCGATAAGTGCGTGCTGGACATGCGGAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGTCCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATTATCCCCGAGGACACATTCTTCCCAAGCCCCGAGAGCAGCTGCGACGTGAAGCTGGTGGAAAAGAGCTTCGAGACAGACACCAACCTGAACTTCCAGAACCTCAGCGTGATCGGCTTCCGGATCCTGCTGCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGCGGCTGTGGTCCAGCTGA

[0513] SEQ ID NO: 70 (#4β gene fragment)

[0514] ATGGGCTGCAGGCTGCTCTGCTGTGCGGTTCTCTGTCTCCTGGGAGCAGTTCCCATAGACACTGAAGTTACCCAGACACCAAAACACCTGGTCATGGGAATGACAAATAAGAAGTCTTTGAAATGTGAACAACATATGGGGCACAGGGCTATGTATTGGTACAAGCAGAAAGCTAAGAAGCCACCGGAGCTCATGTTTGTCTACAGCTATGAGAAACTCTCTATAAATGAAAGTGTGCCAAGTCGCTTCTCACCTGAATGCCCCAACAGCTCTCTCTTAAACCTTCACCTACACGCCCTGCAGCCAGAAGACTCAGCCCTGTATCTCTGCGCCAGCAGCCAAGGGACTAGCGGGGCAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCC

[0515] GACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG

[0516] SEQ ID NO: 71 (#6β gene fragment)

[0517] ATGAGCATCGGCCTCCTGTGCTGTGCAGCCTTGTCTCTCCTGTGGGCAGGTCCAGTGAATGCTGGTGTCACTCAGACCCCAAAATTCCAGGTCCTGAAGACAGGACAGAGCATGACACTGCAGTGTGCCCAGGATATGAACCATGAATACATGTCCTGGTATCGACAAGACCCAGGCATGGGGCTGAGGCTGATTCATTACTCAGTTGGTGCTGGTATCACTGACCAAGGAGAAGTCCCCAATGGCTACAATGTCTCCAGATCAACCACAGAGGATTTCCCGCTCAGGCTGCTGTCGGCTGCTCCCTCCCAGACATCTGTGTACTTCTGTGCCAGCAGTTACTCTCTTTGGGACCTTCAAGAGACCCAGTACTTCGGGCCAGGCACGCGGCTCCTGGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG

[0518] SEQ ID NO: 72 (#7β gene fragment)

[0519] ATGGGCACCAGCCTCCTCTGCTGGATGGCCCTGTGTCTCCTGGGGGCAGATCACGCAGATACTGGAGTCTCCCAGGACCCCAGACACAAGATCACAAAGAGGGGACAGAATGTAACTTTCAGGTGTGATCCAATTTCTGAACACAACCGCCTTTATTGGTACCGACAGACCCTGGGGCAGGGCCCAGAGTTTCTGACTTACTTCCAGAATGAAGCTCAACTAGAAAAATCAAGGCTGCTCAGTGATCGGTTCTCTGCAGAGAGGCCTAAGGGATCTTTCTCCACCTTGGAGATCCAGCGCACAGAGCAGGGGGACTCGGCCATGTATCTCTGTGCCAGCAGCTTTTCAGACGGGGGGGCTACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAG

[0520] GCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG

[0521] SEQ ID NO: 73 (Fragment of #10β gene)

[0522] ATGCTGCTGCTTCTGCTGCTTCTGGGGCCAGCAGGCTCCGGGCTTGGTGCTGTCGTCTCTCAACATCCGAGCTGGGTTATCTGTAAGAGTGGAACCTCTGTGAAGATCGAGTGCCGTTCCCTGGACTTTCAGGCCACAACTATGTTTTGGTATCGTCAGTTCCCGAAACAGAGTCTCATGCTGATGGCAACTTCCAATGAGGGCTCCAAGGCCACATACGAGCAAGGCGTCGAGAAGGACAAGTTTCTCATCAACCATGCAAGCCTGACCTTGTCCACTCTGACAGTGACCAGTGCCCATCCTGAAGACAGCAGCTTCTACATCTGCAGTGCTAGACCCCATTCTCTCACAGATACGCAGTATTTTGGCCCAGGCACCCGGCTGACAGTGCTCGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCAGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGCTAG

[0523] SEQ ID NO: 74 (#4β gene fragment, codon-optimized)

[0524] ATGGGCTGTAGACTGTTGTGTTGTGCTGTGCTGTGTCTGTTGGGAGCTGTGCCTATCGATACAGAGGTGACCCAGACCCCTAAACATCTGGTTATGGGCATGACCAACAAGAAGAGCCTGAAGTGCGAGCAGCACATGGGCCATAGGGCCATGTATTGGTATAAGCAGAAGGCCAAGAAACCTCCTGAGCTGATGTTCGTGTACAGCTACGAGAAGCTGAGCATCAACGAGAGCGTGCCCAGCAGATTTTCTCCTGAGTGCCCTAATTCTAGCCTGCTGAATCTGCACCTGCATGCTCTGCAGCCTGAGGATTCTGCTCTGTACCTGTGTGCTTCTTCTCAGGGCACATCTGGAGCTGATACACAGTACTTCGGACCTGGCACAAGACTGACAGTGCTGGAAGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC

[0525] SEQ ID NO: 75 (#6β gene fragment, codon-optimized)

[0526] ATGTCTATCGGTCTGCTGTGCTGTGCTGCTCTTTCTCTGCTTTGGGCTGGACCTGTGAATGCTGGAGTTACACAAACCCCCAAGTTCCAAGTGCTGAAGACAGGACAGAGCATGACCCTGCAGTGTGCTCAGGACATGAATCACGAGTACATGAGCTGGTACAGACAGGATCCTGGAATGGGCCTGAGGCTGATCCACTACTCTGTTGGAGCCGGAATTACAGATCAGGGAGAAGTGCCAAATGGCTACAACGTGAGCAGGAGCACAACCGAGGACTTCCCCTTAAGACTGTTGTCTGCTGCTCCATCTCAGACAAGCGTGTACTTTTGCGCCAGCTCCTACTCTCTGTGGGATCTGCAGGAAACCCAGTACTTTGGACCAGGCACAAGACTGTTAGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC

[0527] SEQ ID NO: 76 (#7β gene fragment, codon-optimized)

[0528] ATGGGCACATCTCTTCTCTGCTGGATGGCTCTTTGTCTGCTTGGAGCCGATCATGCCGATACAGGAGTTAGCCAGGATCCTAGACACAAGATCACCAAGAGAGGCCAGAATGTGACCTTCCGGTGCGATCCTATCTCTGAGCACAACAGGCTGTACTGGTACAGACAAACACTGGGACAAGGACCTGAGTTCCTGACCTACTTCCAGAACGAAGCCCAGCTGGAGAAGTCTAGACTTCTGAGCGACAGATTTAGCGCCGAGAGACCTAAAGGCAGCTTTAGCACCCTGGAGATCCAGAGAACAGAACAGGGCGATTCTGCCATGTACCTGTGTGCTAGCAGCTTTTCTGATGGAGGCGCCACCGATACACAGTATTTCGGACCTGGCACAAGACTGACAGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCACGTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC

[0529] SEQ ID NO: 77 (#10β gene fragment, codon-optimized)

[0530] ATGCTGCTTCTTCTCCTCCTTCTCGGACCTGCTGGATCTGGATTAGGAGCTGTTGTGTCTCAGCACCCTTCTTGGGTGATCTGTAAAAGCGGCACAAGCGTGAAGATCGAGTGCAGAAGCCTGGACTTTCAGGCCACAACCATGTTCTGGTATAGGCAGTTCCCCAAGCAGTCTCTGATGCTGATGGCCACCTCTAATGAGGGCTCTAAGGCCACATATGAACAGGGAGTGGAGAAGGACAAGTTCCTGATCAACCACGCCTCTCTGACCCTGTCTACCCTGACAGTTACATCTGCCCACCCTGAGGATAGCAGCTTTTACATCTGTAGCGCCAGACCTCACAGCCTGACCGATACACAGTACTTTGGCCCTGGCACAAGACTGACAGTGTTAGAAGACCTGAAGAACGTGTTCCCCCCAGAGGTGGCCGTGTTCGAGCCTAGCGAGGCCGAGATCAGCCACACCCAGAAAGCCACCCTCGTGTGCCTGGCCACCGGCTTTTACCCCGACCAC

[0531] GTGGAACTGTCTTGGTGGGTCAACGGCAAAGAGGTGCACAGCGGCGTCTGCACCGACCCCCAGCCCCTGAAAGAGCAGCCCGCCCTGAACGACAGCCGGTACTGTCTGAGCAGCAGACTGAGAGTGTCCGCCACCTTCTGGCAGAACCCCCGGAACCACTTCAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAGGACCGGGCCAAGCCCGTGACCCAGATCGTGTCTGCTGAGGCCTGGGGCAGAGCCGATTGCGGCTTCACCAGCGAGAGCTACCAGCAGGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGTCCGCCCTGGTGCTGATGGCCATGGTCAAGCGGAAGGACAGCCGGGGC

[0532] SEQ ID NO: 78 (#4β-P2A-#4α, codon-optimized)

[0533]

[0534] SEQ ID NO: 79 (#6β-P2A-#6α, codon optimized)

[0535]

[0536] SEQ ID NO: 80 (#7β-P2A-#7α, codon optimized)

[0537]

[0538] SEQ ID NO: 81 (#10β-P2A-#10α, codon optimized)

[0539]

[0540] SEQ ID NO: 82 (Reference TCR α chain)

[0541] MTSIRAVFIFLWLQLDLVNGENVEQHPSTLSVQEGDSAVIKCTYSDSASNYFPWYKQELGKRPQLIIDIRSNVGEKKDQRIAVTLNKTAKHFSLHITETQPEDSAVYFCAATEDYQLIWGAGTKLIIKPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0542] SEQ ID NO: 83 (Reference TCR β chain)

[0543] MSNQVLCCVVLCFLGANTVDGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIVNDFQKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSPGALYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0544] SEQ ID NO: 84 (Porcine teschovirus-1 2A (P2A-1) peptide)

[0545] GSGATNFSLLKQAGDVEENPGP

[0546] SEQ ID NO: 85 (Porcine teschovirus-2 2A (P2A-2) peptide)

[0547] SGATNFSLKQAGDVEENPGP

[0548] SEQ ID NO: 86 (Thosea asigna virus 2A (T2A) peptide)

[0549] GSGEGRGSLLTCGDVEENPGP

[0550] SEQ ID NO: 87 (Equine Rhinitis Virus Type A (ERAV) 2A (E2A) peptide)

[0551] GSGQCTNYALLKLAGDVESNPGP

[0552] SEQ ID NO: 88 (Foot-and-mouth disease virus 2A (F2A) peptide)

[0553] GSGVKQTLNFDLLKLAGDVESNPGP

[0554] SEQ ID NO: 89 (Porcine Teschovirus-1 2A (P2A-1) peptide-nt sequence)

[0555] GGAAGTGGAGCTACGAATTTTTTTTATTAAAACAAGCAGGAGATGTTGAGGAGAATCCCGGTCCA

[0556] SEQ ID NO: 90 (Porcine Teschovirus-2 2A (P2A-2) peptide-nt sequence)

[0557] AGCGGCGCCACCAACTTCAGCCTGCTGAAACAGGCCGGCGACGTGGAAGAGAACCCTGGCCCT

[0558] SEQ ID NO: 91 (Thosea asigna virus 2A (T2A) peptide-nt sequence)

[0559] GGAAGCGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGACCT

[0560] SEQ ID NO: 92 (Equine Rhinitis Virus Type A (ERAV) 2A (E2A) peptide-nt sequence)

[0561] GGAAGCGGGACAGTGTACTAATTATGCTCCTTTGAAATTGGCTGGAGATGTTGAGAGCAACCCTGGACCT

[0562] SEQ ID NO: 93 (Foot-and-mouth disease virus 2A (F2A) peptide-nt sequence)

[0563] GGAAGCGGGGTGAACAGACTTTGAATTTTGACCTTCTCAAGTTGGCGGGAGACGTGGAGTCCAACCCTGGACCT

[0564] SEQ ID NO: 94 (WT-1 peptide antigen)

[0565] RMFPNAPYL

[0566] SEQ ID NO: 95 (GS linker)

[0567] GGGS

[0568] SEQ ID NO: 96

[0569] GlyxSery, where X and Y are each independently a non-zero integer

[0570] SEQ ID NO: 97—Meganuclease recognition site

[0571] Laglidag

[0572] SEQ ID NO: 98—Meganuclease recognition site

[0573] GIY-YIG

[0574] SEQ ID NO: 99—Meganuclease recognition site

[0575] PD-(D / E)XK

[0576] SEQ ID NO.: 100 (WT-1 aa 37-45)

[0577] VLDFAPPGA

[0578] SEQ ID NO.: 101 (TCR 10.1CDR1α)

[0579] DSAIYN

[0580] SEQ ID NO.: 102 (TCR 10.1CDR2α)

[0581] IQSSQRE

[0582] SEQ ID NO.: 103 (TCR 10.1CDR3α)

[0583] CAVKETSGSRLTF

[0584] SEQ ID NO.: 104 (TCR 10.1 Vα)

[0585] KQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVKETSGSRLTFGEGTQLTVNP

[0586] SEQ ID NO.:105(TCR 10.1 CDR1β)

[0587] SGHVS

[0588] SEQ ID NO.:106(TCR 10.1 CDR2β)

[0589] FNYEAQ

[0590] SEQ ID NO.:107(TCR 10.1 CDR3β)

[0591] CASSLTGSYEQYF

[0592] SEQ ID NO.:108(TCR 10.1 Vβ)

[0593] GAGVSQSPRYKVTKRGQDVALRCDPISGHVSLYWYRQALGQGPEFLTYFNYEAQQDKSGLPNDRFSAERPEGSISTLTIQRTEQRDSAMYRCASSLTGSYEQYFGPGTRLTVTE

[0594] SEQ ID NO.:109(TCR 13.1 CDR1α)

[0595] NSMFDY

[0596] SEQ ID NO.:110(TCR 13.1 CDR2α)

[0597] ISSIKDK

[0598] SEQ ID NO.:111(TCR 13.1 CDR3α)

[0599] CAASGIGDYKLSF

[0600] SEQ ID NO.:112(TCR 13.1 Vα)

[0601] DQQVKQNSPSLSVQEGRISILNCDYTNSMFDYFLWYKKYPAEGPTFLISISSIKDKNEDGRFTVFLNKSAKHLSLHIVPSQPGDSAVYFCAASGIGDYKLSFGAGTTVTVRAN

[0602] SEQ ID NO.:113(TCR 13.1 CDR1β)

[0603] PRHDT

[0604] SEQ ID NO.:114(TCR 13.1 CDR2β)

[0605] FYEKMQ

[0606] SEQ ID NO.:115(TCR 13.1 CDR3β)

[0607] CASSLRLGRETQYF

[0608] SEQ ID NO.:116(TCR 13.1 Vβ)

[0609] AAGVIQSPRHLIKEKRETATLKCYPIPRHDTVYWYQQGPGQDPQFLISFYEKMQSDKGSIPDRFSAQQFSDYHSELNMSSLELGDSALYFCASLRLGRETQYFGPGTRLLVLE

[0610] SEQ ID NO.:117(TCR 16.1 CDR1α)

[0611] VSGLRG

[0612] SEQ ID NO.:118(TCR 16.1 CDR2α)

[0613] LYSAGEE

[0614] SEQ ID NO.:119(TCR 16.1 CDR3α)

[0615] CAVITGFQKLVF

[0616] SEQ ID NO.:120(TCR 16.1 Vα)

[0617] EDQVTQSPEALRLQEGESSSLNCSYTVSGLRGLFWYRQDPGKGPEFLFTLYSAGEEKERLKATLTKKESFLHITAPKPEDSATYLCAVITGFQKLVFGTGTRLLVSPN

[0618] SEQ ID NO.: 121 (TCR 16.1 CDR1β)

[0619] MNHEY

[0620] SEQ ID NO.: 122 (TCR 16.1 CDR2β)

[0621] SMNVEV

[0622] SEQ ID NO.: 123 (TCR 16.1 CDR3β)

[0623] CASSFSGGTYEQYF

[0624] SEQ ID NO.: 124 (TCR 16.1 Vβ)

[0625] EAQVTQNPRYLITVTGKKLTVTCSQNMNHEYMSWYRQDPGLGLRQIYYSMNVEVTDKGDVPEGYKVSRKEKRNFPLILESPSPNQTSLYFCASFSGGTYEQYFPGGTRLTVTE

[0626] Examples

[0627] Example 1

[0628] Identification and characterization of WT-1 peptide-specific TCRs

[0629] CD8 +T cells were cultured with autologous dendritic cells (DCs) pulsed with the WT-1 peptide RMFPNAPYL (SEQ ID NO: 94) (Ho, Greenberg et al., 2006). T cell lines were pooled, stained with CD8-independent (CD8i) WT-1 peptide:HLA tetramers and high tetramer staining cells were sorted. A total of 40 polyclonal T cell lines with high tetramer staining were identified. The sorted T cell samples and a portion of the total unsorted sample were analyzed by TCR repertoire analysis (Adaptive Biotechnologies) to quantify each TCR clonotype. A portion of the high tetramer staining cells was also analyzed by single cell RNA sequencing and TCR pairing (10X Genomics). By comparing the fold enrichment of each clonotype in the sorted and unsorted populations and the CD3 surface expression of each clonotype, 24 TCR clonotypes were identified as WT-1 specific ( Figure 1 ). Of these, 10 were selected for synthesis. TCRα / TCRβ-deficient J76 Jurkat T cells were transduced to express the candidate TCRs. The affinity of each candidate for WT-1 was assessed by measuring relative WT-1 peptide:HLA tetramer staining compared to CD3 expression as a marker of total transgenic TCR surface expression. The results were compared with those of cells expressing a WT-1 peptide:HLA-specific TCR having an α chain according to SEQ ID NO: 82 and a β chain according to SEQ ID NO: 83. Data are shown in Figure 5. Figures 2A-2C shown.

[0630] Activation of Jurkat cells transduced with a TCR expressing the Nur77-Tomato activation tag was assessed in the presence of peptide. Figure 3A shown.

[0631] CD8+ T cells isolated from donor PMBCs were transduced to express one of eight TCRs selected for further study. One week later, transduced cells were stained for WT-1 peptide:HLA tetramer and sorted for CD8 expression ( Figure 4A ). Amplification of tetramers + CD8 + The expanded antigen-specific cells were cultured with T2 target cells pulsed with titrated concentrations of WT-1 peptide for 4 h, and the production of IFNγ was determined by flow cytometry ( Figure 4B The percentage of IFNγ-producing cells was fitted to the dose-response curves by nonlinear regression to calculate the peptide EC for each TCR. 50 ( Figure 4C ).

[0632] Regarding the ability to induce tumor cell lysis, the expression of the protein with the lowest EC50 T cells expressing the two TCRs with the same values ​​were compared with T cells expressing the reference WT-1 specific TCR. + Tumor cell line Panc-1 loading 51 TCR-mediated killing by TCR-transduced T cells was calculated by measuring Cr release in response to dose reduction of effector T cells relative to tumor cell targets ( Figure 5A and 5B ).

[0633] IFN-γ production by T cells expressing the exemplary TCR DL10 was further investigated ( Figure 6 ) and the ability to reduce cell growth in tumor cell lines ( Figure 7 For IFN-γ production, primary CD8+ T cells were transduced with reference TCR or TCRDL10 and cultured with peptide-pulsed T2 target cells, as shown in Figure 6 As shown. After 4 hours, IFNγ production was assessed by intracellular flow cytometry, and the percentage IFNγ positivity was fitted to the dose-response curve by nonlinear regression in Graphpad Prism. To assess the ability to control tumor growth, primary CD8+ T cells were transduced with a reference TCR (including a TCRα chain having the amino acid sequence shown in SEQ ID NO: 82 and a TCRβ chain having the amino acid sequence shown in SEQ ID NO: 83) or with TCRDL10 and cultured in triplicate with the HLA-A2+WT-1+ pancreatic tumor cell line Panc-1 (transduced to express red fluorescent protein) at a ratio of 8:1 in the IncuCyte assay, and cell proliferation / survival was monitored over 7 days. Error bars represent the standard error of the mean.

[0634] The various embodiments described above can be combined to provide other embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the application data sheet (including U.S. patent application No. 62 / 889,519 filed on August 20, 2019) are incorporated herein by reference in their entirety. If necessary, aspects of the embodiments can be modified to adopt the concepts of the various patents, applications, and publications to provide other embodiments.

[0635] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to include all possible embodiments and the full scope of equivalents to the claims. Therefore, the claims are not limited by this disclosure. Sequence Listing <110> Fred Hutchinson Cancer Research Center T.M. Schmidt A.G. Shapiy P.D. Greenberg <120> 360056.473WO <130> WT-1-specific T cell immunotherapy <140> PCT <141> 2020-08-19 <150> US 62 / 889,519 <151> 2019-08-20 <160> 124 <170> FastSEQ for Windows Version 4.0 <210> 1 <211> 132 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 4 α chain variable domain with leader sequence <400> 1 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 20 25 30 Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp 35 40 45 Arg Gly Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser 50 55 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 Val 100 105 110 Asn Ile Gly Asn His Asp Met Arg Phe Gly Ala Gly Thr Arg Leu Thr 115 120 125 Val Lys Pro Asn 130 <210> 2 <211> 132 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 6 α chain variable domain with leader sequence <400> 2 Met Glu Lys Met Leu Glu Cys Ala Phe Ile Val Leu Trp Leu Gln Leu 1 5 10 15 Gly Trp Leu Ser Gly Glu Asp Gln Val Thr Gln Ser Pro Glu Ala Leu 20 25 30 Arg Leu Gln Glu Gly Glu Ser Ser Ser Leu Asn Cys Ser Tyr Thr Val 35 40 45 Ser Gly Leu Arg Gly Leu Phe Trp Tyr Arg Gln Asp Pro Gly Lys Gly 50 55 60 Pro Glu Phe Leu Phe Thr Leu Tyr Ser Ala Gly Glu Glu Lys Glu Lys 65 70 75 80 Glu Arg Leu Lys Ala Thr Leu Thr Lys Lys Glu Ser Phe Leu His Ile 85 90 95 Thr Ala Pro Lys Pro Glu Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln 100 105 110 Thr Met Asp Gly Asn Gln Phe Tyr Phe Gly Thr Gly Thr Ser Leu Thr 115 120 125 Val Ile Pro Asn 130 <210> 3 <211> 134 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 7 α chain variable domain with leader sequence <400> 3 Met Ala Cys Pro Gly Phe Leu Trp Ala Leu Val Ile Ser Thr Cys Leu 1 5 10 15 Glu Phe Ser Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser 20 25 30 Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser 35 40 45 Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln 50 55 60 Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr 65 70 75 80 Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser 85 90 95 Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys 100 105 110 Ala Ser Ser Pro Gly Thr Tyr Lys Tyr Ile Phe Gly Thr Gly Thr Arg 115 120 125 Leu Lys Val Leu Ala Asn 130 <210> 4 <211> 137 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 10 α chain variable domain with leader sequence <400> 4 Met Thr Arg Val Ser Leu Leu Trp Ala Val Val Val Ser Thr Cys Leu 1 5 10 15 Glu Ser Gly Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser 20 25 30 Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser 35 40 45 Glu Ser Asn Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln 50 55 60 Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr 65 70 75 80 Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser 85 90 95 Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Thr Ala Met Tyr Phe Cys 100 105 110 Ala Phe Asn Pro Trp Glu Asn Tyr Gly Gln Asn Phe Val Phe Gly Pro 115 120 125 Gly Thr Arg Leu Ser Val Leu Pro Tyr 130 135 <210> 5 <211> 134 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 4 α chain variable domain with leader sequence <400> 5 Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala 1 5 10 15 Val Pro Ile Asp Thr Glu Val Thr Gln Thr Pro Lys His Leu Val Met 20 25 30 Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His Met Gly His 35 40 45 Arg Ala Met Tyr Trp Tyr Lys Gln Lys Ala Lys Lys Pro Pro Glu Leu 50 55 60 Met Phe Val Tyr Ser Tyr Glu Lys Leu Ser Ile Asn Glu Ser Val Pro 65 70 75 80 Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser Leu Leu Asn Leu His 85 90 95 Leu His Ala Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser 100 105 110 Ser Gln Gly Thr Ser Gly Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr 115 120 125 Arg Leu Thr Val Leu Glu 130 <210> 6 <211> 135 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 6 β chain variable domain with leader sequence <400> 6 Met Ser Ile Gly Leu Leu Cys Cys Ala Ala Leu Ser Leu Leu Trp Ala 1 5 10 15 Gly Pro Val Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu 20 25 30 Lys Thr Gly Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu 50 55 60 Ile His Tyr Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro 65 70 75 80 Asn Gly Tyr Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg 85 90 95 Leu Leu Ser Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser 100 105 110 Ser Tyr Ser Leu Trp Asp Leu Gln Glu Thr Gln Tyr Phe Gly Pro Gly 115 120 125 Thr Arg Leu Leu Val Leu Glu 130 135 <210> 7 <211> 136 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No.7 β chain variable domain with leader sequence <400> 7 Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala 1 5 10 15 Asp His Ala Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr 20 25 30 Lys Arg Gly Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His 35 40 45 Asn Arg Leu Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe 50 55 60 Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu 65 70 75 80 Ser Asp Arg Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu 85 90 95 Glu Ile Gln Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala 100 105 110 Ser Ser Phe Ser Asp Gly Gly Ala Thr Asp Thr Gln Tyr Phe Gly Pro 115 120 125 Gly Thr Arg Leu Thr Val Leu Glu 130 135 <210> 8 <211> 132 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 10 β chain variable domain with leader sequence <400> 8 Met Leu Leu Leu Leu Leu Leu Leu Gly Pro Ala Gly Ser Gly Leu Gly 1 5 10 15 Ala Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly Thr 20 25 30 Ser Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr Met 35 40 45 Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala Thr 50 55 60 Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys Asp 65 70 75 80 Lys Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr Val 85 90 95 Thr Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg 100 105 110 Pro His Ser Leu Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Leu Glu 130 <210> 9 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 4 α chain variable domain without leader sequence <400> 9 Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 1 5 10 15 Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser 20 25 30 Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met 35 40 45 Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln 50 55 60 Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln 65 70 75 80 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Ile Gly Asn His 85 90 95 Asp Met Arg Phe Gly Ala Gly Thr Arg Leu Thr Val Lys Pro Asn 100 105 110 <210> 10 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 6 α chain variable domain without leader sequence <400> 10 Glu Asp Gln Val Thr Gln Ser Pro Glu Ala Leu Arg Leu Gln Glu Gly 1 5 10 15 Glu Ser Ser Ser Leu Asn Cys Ser Tyr Thr Val Ser Gly Leu Arg Gly 20 25 30 Leu Phe Trp Tyr Arg Gln Asp Pro Gly Lys Gly Pro Glu Phe Leu Phe 35 40 45 Thr Leu Tyr Ser Ala Gly Glu Glu Lys Glu Lys Glu Arg Leu Lys Ala 50 55 60 Thr Leu Thr Lys Lys Glu Ser Phe Leu His Ile Thr Ala Pro Lys Pro 65 70 75 80 Glu Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln Thr Met Asp Gly Asn 85 90 95 Gln Phe Tyr Phe Gly Thr Gly Thr Ser Leu Thr Val Ile Pro Asn 100 105 110 <210> 11 <211> 114 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 7 α chain variable domain without leader sequence <400> 11 Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser Val Gln Glu Ala 1 5 10 15 Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser Glu Ser Asp Tyr 20 25 30 Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Ile Leu Val 35 40 45 Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr Glu Asn Arg Phe 50 55 60 Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser Leu Lys Ile Ser 65 70 75 80 Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys Ala Ser Ser Pro 85 90 95 Gly Thr Tyr Lys Tyr Ile Phe Gly Thr Gly Thr Arg Leu Lys Val Leu 100 105 110 Ala Asn <210> 12 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 10 α chain variable domain without leader sequence <400> 12 Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser Val Gln Glu Ala 1 5 10 15 Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser Glu Ser Asn Tyr 20 25 30 Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Ile Leu Val 35 40 45 Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr Glu Asn Arg Phe 50 55 60 Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser Leu Lys Ile Ser 65 70 75 80 Asp Ser Gln Leu Gly Asp Thr Ala Met Tyr Phe Cys Ala Phe Asn Pro 85 90 95 Trp Glu Asn Tyr Gly Gln Asn Phe Val Phe Gly Pro Gly Thr Arg Leu 100 105 110 Ser Val Leu Pro Tyr 115 <210> 13 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 4 β chain variable domain without leader sequence <400> 13 Asp Thr Glu Val Thr Gln Thr Pro Lys His Leu Val Met Gly Met Thr 1 5 10 15 Asn Lys Lys Ser Leu Lys Cys Glu Gln His Met Gly His Arg Ala Met 20 25 30 Tyr Trp Tyr Lys Gln Lys Ala Lys Lys Pro Pro Glu Leu Met Phe Val 35 40 45 Tyr Ser Tyr Glu Lys Leu Ser Ile Asn Glu Ser Val Pro Ser Arg Phe 50 55 60 Ser Pro Glu Cys Pro Asn Ser Ser Leu Leu Asn Leu His Leu His Ala 65 70 75 80 Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser Ser Gln Gly 85 90 95 Thr Ser Gly Ala Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr 100 105 110 Val Leu Glu 115 <210> 14 <211> 116 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 6 β chain variable domain without leader sequence <400> 14 Asn Ala Gly Val Thr Gln Thr Pro Lys Phe Gln Val Leu Lys Thr Gly 1 5 10 15 Gln Ser Met Thr Leu Gln Cys Ala Gln Asp Met Asn His Glu Tyr Met 20 25 30 Ser Trp Tyr Arg Gln Asp Pro Gly Met Gly Leu Arg Leu Ile His Tyr 35 40 45 Ser Val Gly Ala Gly Ile Thr Asp Gln Gly Glu Val Pro Asn Gly Tyr 50 55 60 Asn Val Ser Arg Ser Thr Thr Glu Asp Phe Pro Leu Arg Leu Leu Ser 65 70 75 80 Ala Ala Pro Ser Gln Thr Ser Val Tyr Phe Cys Ala Ser Ser Tyr Ser 85 90 95 Leu Trp Asp Leu Gln Glu Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu 100 105 110 Leu Val Leu Glu 115 <210> 15 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 7 β chain variable domain without leader sequence <400> 15 Asp Thr Gly Val Ser Gln Asp Pro Arg His Lys Ile Thr Lys Arg Gly 1 5 10 15 Gln Asn Val Thr Phe Arg Cys Asp Pro Ile Ser Glu His Asn Arg Leu 20 25 30 Tyr Trp Tyr Arg Gln Thr Leu Gly Gln Gly Pro Glu Phe Leu Thr Tyr 35 40 45 Phe Gln Asn Glu Ala Gln Leu Glu Lys Ser Arg Leu Leu Ser Asp Arg 50 55 60 Phe Ser Ala Glu Arg Pro Lys Gly Ser Phe Ser Thr Leu Glu Ile Gln 65 70 75 80 Arg Thr Glu Gln Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Phe 85 90 95 Ser Asp Gly Gly Ala Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg 100 105 110 Leu Thr Val Leu Glu 115 <210> 16 <211> 117 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 10 β chain variable domain without leader sequence <400> 16 Gly Ala Val Val Ser Gln His Pro Ser Trp Val Ile Cys Lys Ser Gly 1 5 10 15 Thr Ser Val Lys Ile Glu Cys Arg Ser Leu Asp Phe Gln Ala Thr Thr 20 25 30 Met Phe Trp Tyr Arg Gln Phe Pro Lys Gln Ser Leu Met Leu Met Ala 35 40 45 Thr Ser Asn Glu Gly Ser Lys Ala Thr Tyr Glu Gln Gly Val Glu Lys 50 55 60 Asp Lys Phe Leu Ile Asn His Ala Ser Leu Thr Leu Ser Thr Leu Thr 65 70 75 80 Val Thr Ser Ala His Pro Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala 85 90 95 Arg Pro His Ser Leu Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg 100 105 110 Leu Thr Val Leu Glu 115 <210> 17 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 4 CDR1α <400> 17 Asp Arg Gly Ser Gln Ser 1 5 <210> 18 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 4 CDR2α <400> 18 Ile Tyr Ser Asn Gly Asp 1 5 <210> 19 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 4 CDR3α <400> 19 Cys Ala Val Asn Ile Gly Asn His Asp Met Arg Phe 1 5 10 <210> 20 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 6 CDR1α <400> 20 Val Ser Gly Leu Arg Gly 1 5 <210> twenty one <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 6 CDR2α <400> twenty one Leu Tyr Ser Ala Gly Glu Glu 1 5 <210> twenty two <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 6 CDR3α <400> twenty two Cys Ala Val Gln Thr Met Asp Gly Asn Gln Phe Tyr Phe 1 5 10 <210> twenty three <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 7 CDR1α <400> twenty three Thr Ser Glu Ser Asp Tyr Tyr 1 5 <210> twenty four <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 7 CDR2α <400> twenty four Gln Glu Ala Tyr Lys Gln Gln Asn 1 5 <210> 25 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 7 CDR3α <400> 25 Cys Ala Ser Ser Pro Gly Thr Tyr Lys Tyr Ile Phe 1 5 10 <210> 26 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 10 CDR1α <400> 26 Thr Ser Glu Ser Asn Tyr Tyr 1 5 <210> 27 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 10 CDR2α <400> 27 Gln Glu Ala Tyr Lys Gln Gln Asn 1 5 <210> 28 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 10 CDR3α <400> 28 Cys Ala Phe Asn Pro Trp Glu Asn Tyr Gly Gln Asn Phe Val Phe 1 5 10 15 <210> 29 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 4 CDR1β <400> 29 Met Gly His Arg Ala 1 5 <210> 30 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 4 CDR2β <400> 30 Tyr Ser Tyr Glu Lys Leu 1 5 <210> 31 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 4 CDR3β <400> 31 Cys Ala Ser Ser Gln Gly Thr Ser Gly Ala Asp Thr Gln Tyr Phe 1 5 10 15 <210> 32 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 6 CDR1β <400> 32 Met Asn His Glu Tyr 1 5 <210> 33 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 6 CDR2β <400> 33 Ser Val Gly Ala Gly Ile 1 5 <210> 34 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 6 CDR3β <400> 34 Cys Ala Ser Ser Tyr Ser Leu Trp Asp Leu Gln Glu Thr Gln Tyr Phe 1 5 10 15 <210> 35 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 7 CDR1β <400> 35 Ser Glu His Asn Arg 1 5 <210> 36 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 7 CDR2β <400> 36 Phe Gln Asn Glu Ala Gln 1 5 <210> 37 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 7 CDR3β <400> 37 Cys Ala Ser Ser Phe Ser Asp Gly Gly Ala Thr Asp Thr Gln Tyr Phe 1 5 10 15 <210> 38 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 10 CDR1β <400> 38 Asp Phe Gln Ala Thr Thr 1 5 <210> 39 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 10 CDR2β <400> 39 Ser Asn Glu Gly Ser Lys Ala 1 5 <210> 40 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 10 CDR3β <400> 40 Cys Ser Ala Arg Pro His Ser Leu Thr Asp Thr Gln Tyr Phe 1 5 10 <210> 41 <211> 140 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 4 TCR α constant domain <400> 41 Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser 1 5 10 15 Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn 20 25 30 Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val 35 40 45 Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp 50 55 60 Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile 65 70 75 80 Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val 85 90 95 Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln 100 105 110 Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly 115 120 125 Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 130 135 140 <210> 42 <211> 140 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 6 TCR α constant domain <400> 42 Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser 1 5 10 15 Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn 20 25 30 Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val 35 40 45 Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp 50 55 60 Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile 65 70 75 80 Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val 85 90 95 Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln 100 105 110 Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly 115 120 125 Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 130 135 140 <210> 43 <211> 140 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence No. 7 TCR α Constant Domain <400> 43 Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser 1 5 10 15 Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn 20 25 30 Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val 35 40 45 Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp 50 55 60 Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile 65 70 75 80 Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val 85 90 95 Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln 100 105 110 Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly 115 120 125 Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 130 135 140 <210> 44 <211> 140 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 10 TCR α constant domain <400> 44 Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser 1 5 10 15 Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn 20 25 30 Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val 35 40 45 Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp 50 55 60 Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile 65 70 75 80 Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val 85 90 95 Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln 100 105 110 Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly 115 120 125 Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 130 135 140 <210> 45 <211> 178 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic sequence TCR β constant domain <400> 45 Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser 1 5 10 15 Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala 20 25 30 Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly 35 40 45 Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys Glu 50 55 60 Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg 65 70 75 80 Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln 85 90 95 Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg 100 105 110 Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala 115 120 125 Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala 130 135 140 Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val 145 150 155 160 Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Ser 165 170 175 Arg Gly <210> 46 <211> 272 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 4 α chain with leader sequence <400> 46 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 20 25 30 Ser Val Pro Glu Gly Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp 35 40 45 Arg Gly Ser Gln Ser Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser 50 55 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 Val 100 105 110 Asn Ile Gly Asn His Asp Met Arg Phe Gly Ala Gly Thr Arg Leu Thr 115 120 125 Val Lys Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg 130 135 140 Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp 145 150 155 160 Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr 165 170 175 Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser 180 185 190 Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe 195 200 205 Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser 210 215 220 Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn 225 230 235 240 Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu 245 250 255 Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 270 <210> 47 <211> 272 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 6 α chain with leader sequence <400> 47 Met Glu Lys Met Leu Glu Cys Ala Phe Ile Val Leu Trp Leu Gln Leu 1 5 10 15 Gly Trp Leu Ser Gly Glu Asp Gln Val Thr Gln Ser Pro Glu Ala Leu 20 25 30 Arg Leu Gln Glu Gly Glu Ser Ser Ser Leu Asn Cys Ser Tyr Thr Val 35 40 45 Ser Gly Leu Arg Gly Leu Phe Trp Tyr Arg Gln Asp Pro Gly Lys Gly 50 55 60 Pro Glu Phe Leu Phe Thr Leu Tyr Ser Ala Gly Glu Glu Lys Glu Lys 65 70 75 80 Glu Arg Leu Lys Ala Thr Leu Thr Lys Lys Glu Ser Phe Leu His Ile 85 90 95 Thr Ala Pro Lys Pro Glu Asp Ser Ala Thr Tyr Leu Cys Ala Val Gln 100 105 110 Thr Met Asp Gly Asn Gln Phe Tyr Phe Gly Thr Gly Thr Ser Leu Thr 115 120 125 Val Ile Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg 130 135 140 Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp 145 150 155 160 Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr 165 170 175 Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser 180 185 190 Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe 195 200 205 Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser 210 215 220 Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn 225 230 235 240 Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu 245 250 255 Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 270 <210> 48 <211> 274 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 7 α chain with leader sequence <400> 48 Met Ala Cys Pro Gly Phe Leu Trp Ala Leu Val Ile Ser Thr Cys Leu 1 5 10 15 Glu Phe Ser Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser 20 25 30 Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser 35 40 45 Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln 50 55 60 Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr 65 70 75 80 Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser 85 90 95 Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys 100 105 110 Ala Ser Ser Pro Gly Thr Tyr Lys Tyr Ile Phe Gly Thr Gly Thr Arg 115 120 125 Leu Lys Val Leu Ala Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln 130 135 140 Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp 145 150 155 160 Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr 165 170 175 Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser 180 185 190 Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn 195 200 205 Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro 210 215 220 Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp 225 230 235 240 Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu 245 250 255 Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp 260 265 270 Ser Ser <210> 49 <211> 277 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence No. 10 α chain with leader sequence <400> 49 Met Thr Arg Val Ser Leu Leu Trp Ala Val Val Val Ser...

Claims

1. A modified immune cell comprising a heterologous polynucleotide encoding a binding protein, wherein the encoded binding protein comprises: A T cell receptor TCR alpha chain variable Vα domain comprising the complementary determining regions CDR1α, CDR2α and CDR3α amino acid sequences according to SEQ ID NOs.: 26, 27 and 28, respectively; and A TCR β chain variable Vβ domain comprising CDR1β, CDR2β and CDR3β amino acid sequences according to SEQ ID NOs.: 38, 39 and 40, respectively, in, The encoded binding protein is capable of binding to the RMFPNAPYL: human leukocyte antigen HLA-A*0201 complex shown in SEQ ID NO.:

94.

2. The modified immune cell according to claim 1, wherein The encoded binding protein can bind to the RMFPNAPYL:HLA-A*0201 complex shown in SEQ ID NO.: 94, producing pEC 50 IFNγ of 4.0 or higher.

3. The modified immune cell according to claim 1, wherein The encoded binding protein can bind to the RMFPNAPYL:HLA-A*0201 complex shown in SEQ ID NO.: 94, producing pEC 50 IFNγ of 5.0 or higher.

4. The modified immune cell according to claim 1, wherein The encoded binding protein can bind to the RMFPNAPYL:HLA-A*0201 complex shown in SEQ ID NO.: 94, producing pEC 50 IFNγ of 6.0 or higher.

5. The modified immune cell according to claim 1, wherein The encoded binding protein can bind to the RMFPNAPYL:HLA-A*0201 complex shown in SEQ ID NO.: 94, producing pEC 50 IFNγ of 6.5 or higher.

6. The modified immune cell according to claim 1, wherein When the population of modified immune cells was co-cultured with antigen presenting cells for 4 hours, a peptide comprising or consisting of the amino acid sequence RMFPNAPYL as shown in SEQ ID NO.: 94 was used in the co-culture at a rate of 10 -4 μM, 10 -5 μM or 10 -6 Under the conditions of the peptide concentration pulse of μM, 50% or more of the modified immune cells in the population produced IFN-γ.

7. The modified immune cell according to claim 6, wherein the antigen presenting cell comprises a T2 cell.

8. The modified immune cell according to claim 1, wherein When the population of modified immune cells is co-cultured with antigen presenting cells, a peptide comprising or consisting of the amino acid sequence RMFPNAPYL shown in SEQ ID NO.: 94 is administered to the co-culture at a rate of 10 -2 Under conditions of a peptide concentration pulse of μM, 10% or more of the modified immune cells in the population were Nur77 + .

9. The modified immune cell of claim 8, wherein the antigen presenting cells comprise T2 cells, Jurkat cells, or both.

10. The modified immune cell according to claim 8, wherein When the population of modified immune cells is co-cultured with antigen presenting cells, a peptide comprising or consisting of the amino acid sequence RMFPNAPYL shown in SEQ ID NO.: 94 is administered to the co-culture at a rate of 10 -2 Under conditions of a peptide concentration pulse of μM, 20% or more of the modified immune cells in the population were Nur77 + .

11. The modified immune cell according to claim 8, wherein When the population of modified immune cells is co-cultured with antigen presenting cells, a peptide comprising or consisting of the amino acid sequence RMFPNAPYL shown in SEQ ID NO.: 94 is administered to the co-culture at a rate of 10 -2 Under conditions of a peptide concentration pulse of μM, 40% or more of the modified immune cells in the population were Nur77 + .

12. The modified immune cell according to claim 1, wherein When the population of modified immune cells is co-cultured with antigen presenting cells, a peptide comprising or consisting of the amino acid sequence RMFPNAPYL shown in SEQ ID NO.: 94 is administered to the co-culture at a rate of 10 -3 Under conditions of a peptide concentration pulse of μM, 10% or more of the modified immune cells in the population were Nur77 + .

13. The modified immune cell according to claim 12, wherein When the population of modified immune cells is co-cultured with antigen presenting cells, a peptide comprising or consisting of the amino acid sequence RMFPNAPYL shown in SEQ ID NO.: 94 is administered to the co-culture at a rate of 10 -3 Under conditions of a peptide concentration pulse of μM, 20% or more of the modified immune cells in the population were Nur77 + .

14. The modified immune cell according to claim 1, wherein When the modified immune cells and MDA-MB-468 cells were present in a sample at a ratio of 30:1, the modified immune cells were able to kill 11% or more of the HLA-A*0201 + MDA-MB-468 cells.

15. The modified immune cell according to claim 14, wherein When the modified immune cells and MDA-MB-468 cells were present in a sample at a ratio of 30:1, the modified immune cells were able to kill 15% or more of the HLA-A*0201 + MDA-MB-468 cells.

16. The modified immune cell according to claim 14, wherein When the modified immune cells and MDA-MB-468 cells were present in a sample at a ratio of 30:1, the modified immune cells were able to kill 20% or more of the HLA-A*0201 in the sample. + MDA-MB-468 cells.

17. The modified immune cell according to claim 1, wherein When the modified immune cells and MDA-MB-468 cells are present in a sample at a ratio of 1:1, the modified immune cells are able to kill 5% or more of the HLA-A*0201 + MDA-MB-468 cells.

18. The modified immune cell according to claim 1, wherein When the modified immune cells and Panc1 cells were present at a ratio of 30:1, the modified immune cells were able to kill 21% or more of the Panc1 cells in the sample.

19. The modified immune cell according to claim 18, wherein When the modified immune cells and the Panc1 cells were present at a ratio of 30:1, the modified immune cells were able to kill 23% or more of the Panc1 cells in the sample.

20. The modified immune cell according to claim 18, wherein When the modified immune cells and the Panc1 cells are present at a ratio of 30:1, the modified immune cells are able to kill 25% or more of the Panc1 cells in a sample.

21. The modified immune cell according to claim 1, wherein Compared to reference immune cells comprising a polynucleotide encoding a T cell receptor, the modified immune cells have a higher affinity for HLA-A*0201 in a 4-hour co-culture. + The invention discloses a T cell receptor for MDA-MB-468 cells and / or Panc1 cells, wherein the T cell receptor comprises a TCR alpha chain having an amino acid sequence shown in SEQ ID NO.: 82 and a TCR beta chain having an amino acid sequence shown in SEQ ID NO.:

83.

22. The modified immune cell according to claim 1, wherein The encoded binding protein is capable of binding to the RMFPNAPYL:HLA-A*0201 complex shown in SEQ ID NO.: 94 on the cell surface independently of CD8 or in the absence of CD8.

23. The modified immune cell according to claim 1, wherein: (i) the encoded Vβ domain comprises or consists of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs.: 16 and 8; and / or (ii) the encoded Vα domain comprises or consists of an amino acid sequence that is at least 90% identical to the amino acid sequence of any one of SEQ ID NOs.: 12 and 4.

24. The modified immune cell according to claim 1, wherein The encoded TCR Vβ domain comprises an amino acid sequence according to the TRBJ02-03 gene segment.

25. The modified immune cell according to claim 1, wherein The encoded TCR Vα domain comprises an amino acid sequence according to the TRAJ43 gene segment.

26. The modified immune cell according to claim 25, wherein: (i) the encoded TCR Vβ domain comprises an amino acid sequence according to the TRBJ02-03 gene segment; and (ii) The encoded TCR Vα domain comprises an amino acid sequence according to the TRAJ43 gene fragment.

27. The modified immune cell according to claim 1, wherein: The encoded Vβ domain comprises or consists of the amino acid sequence shown in SEQ ID NO.: 16 or 8, and the encoded Vα domain comprises or consists of the amino acid sequence shown in SEQ ID NO.: 12 or 4.

28. The modified immune cell according to claim 1, wherein the encoded Vβ domain comprises the amino acid sequence shown in SEQ ID NO.: 16, and the encoded Vα domain comprises the amino acid sequence shown in SEQ ID NO.:

12.

29. The modified immune cell according to claim 1, wherein the encoded Vβ domain consists of the amino acid sequence shown in SEQ ID NO.: 16, and the encoded Vα domain consists of the amino acid sequence shown in SEQ ID NO.:

12.

30. The modified immune cell according to claim 1, wherein The encoded binding protein further comprises: (i) TCR alpha chain constant domain Cα or a fragment thereof; and / or (ii) TCR β chain constant domain Cβ or a fragment thereof.

31. The modified immune cell according to claim 30, wherein The encoded Ca comprises or consists of an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs.: 41-44.

32. The modified immune cell according to claim 30, wherein The encoded Cβ comprises or consists of an amino acid sequence having at least 90% identity to SEQ ID NO.:

45.

33. The modified immune cell according to claim 30, wherein The encoded binding protein comprises a TCRβ chain, which has at least 90% identity with the amino acid sequence shown in SEQ ID NO.: 61 or 57, or includes or consists of the amino acid sequence shown in SEQ ID NO.: 61 or 57; and a TCRα chain, which has at least 90% identity with the amino acid sequence shown in SEQ ID NO.: 53 or 49, or includes or consists of the amino acid sequence shown in SEQ ID NO.: 53 or 49.

34. The modified immune cell according to claim 30, wherein the encoded binding protein comprises a TCR β chain and a TCR α chain, wherein the TCR β chain comprises the amino acid sequence shown in SEQ ID NO.: 61, and the TCR α chain comprises the amino acid sequence shown in SEQ ID NO.:

53.

35. The modified immune cell according to claim 30, wherein the encoded binding protein comprises a TCR β chain and a TCR α chain, wherein the TCR β chain consists of the amino acid sequence shown in SEQ ID NO.: 61, and the TCR α chain consists of the amino acid sequence shown in SEQ ID NO.:

53.

36. The modified immune cell according to any one of claims 1-29 and 31-34, wherein The encoded binding protein is TCR, chimeric antigen receptor CAR or single-chain TCR.

37. The modified immune cell of claim 36, wherein the encoded binding protein is a TCR.

38. The modified immune cell of claim 36, wherein the immune cell is a human T cell.

39. The modified immune cell of claim 38, wherein the immune cell is a human CD8+ T cell.

40. The modified immune cell of claim 38, wherein the immune cell is a human CD4+ T cell.

41. The modified immune cell of claim 37, wherein the immune cell is a human T cell.

42. The modified immune cell of claim 41, wherein the immune cell is a human CD8+ T cell.

43. The modified immune cell of claim 41, wherein the immune cell is a human CD4+ T cell.

44. The modified immune cell according to claim 1, wherein The immune cells are human immune system cells.

45. The modified immune cell according to claim 1, wherein The immune cells are T cells.

46. ​​The modified immune cell according to claim 45, wherein the T cell is a CD4 + T cells, CD8 + T cells, CD4 - CD8 - Double negative T cells, or γδ T cells, natural killer cells, NK-T cells, dendritic cells or any combination thereof.

47. The modified immune cell of claim 46, wherein the T cell is a CD4 + T cells, CD8 + T cells or both.

48. The modified immune cell according to claim 45, wherein The T cells are naive T cells, central memory T cells, effector memory T cells, stem cell memory T cells or any combination thereof.

49. The modified immune cell according to claim 1, wherein The polynucleotide encoding the binding protein is codon-optimized for expression in the immune cell.

50. The modified immune cell according to claim 1, wherein The polynucleotide encoding the binding protein comprises a polynucleotide having at least 75% identity to the polynucleotide sequence shown in any one of SEQ ID NOs.: 65, 69, 73 and 77.

51. The modified immune cell according to claim 50, wherein The polynucleotide encoding the binding protein comprises: A polynucleotide having at least 75% identity to the polynucleotide sequence shown in SEQ ID NO.: 77, and a polynucleotide having at least 75% identity to the polynucleotide sequence shown in SEQ ID NO.:

69.

52. The modified immune cell according to claim 1, wherein The polynucleotide encoding the binding protein further comprises a polynucleotide encoding a self-cleaving peptide located between the polynucleotide encoding Vβ and the polynucleotide encoding Vα.

53. The modified immune cell according to claim 52, wherein The encoded self-cleaving peptide comprises or consists of an amino acid sequence that is at least 90% identical to the amino acid sequence shown in any one of SEQ ID NOs.: 84-88.

54. The modified immune cell according to claim 52, wherein The polynucleotide encoding the self-cleaving peptide comprises or consists of a polynucleotide sequence having at least 75% identity to the polynucleotide sequence shown in any one of SEQ ID NOs.: 89-93.

55. The modified immune cell according to claim 52, wherein The polynucleotide encoding the binding protein has the following structure from the 5'-end to the 3'-end: a polynucleotide encoding a TCR β chain - a polynucleotide encoding a self-cleaving peptide - a polynucleotide encoding a TCR α chain.

56. The modified immune cell according to claim 55, wherein The polynucleotide encoding the binding protein comprises a polynucleotide having at least 75% identity with the polynucleotide sequence shown in SEQ ID NO.:

81.

57. The modified immune cell according to claim 56, wherein The polynucleotide encoding the binding protein comprises a polynucleotide comprising or consisting of a polynucleotide sequence shown in SEQ ID NO.:

81.

58. The modified immune cell of claim 1, further comprising: (i) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha chain; (ii) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor β chain; or (iii) the polynucleotide of (i) and the polynucleotide of (ii).

59. The modified immune cell of claim 58, comprising: (a) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha chain; (b) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor β chain; and (c) A polynucleotide encoding a self-cleaving peptide located between the polynucleotide of (a) and the polynucleotide of (b).

60. The modified immune cell of claim 58, wherein: (i) the encoded polypeptide comprising the extracellular portion of the CD8 co-receptor alpha chain is or comprises the CD8 co-receptor alpha chain; and / or (ii) the encoded polypeptide comprising the extracellular part of the CD8 co-receptor β chain is or comprises the CD8 co-receptor β chain.

61. The modified immune cell of claim 58, wherein the host cell comprises a CD4+ T cell.

62. An isolated polynucleotide encoding a binding protein, wherein: The encoded binding protein comprises a TCR Vα domain and a TCR Vβ domain, and is capable of binding to the RMFPNAPYL:HLA-A*0201 complex shown in SEQ ID NO.: 94, wherein: (i) the encoded Vα domain comprises the CDR3 amino acid sequence shown in SEQ ID NO.: 28; (ii) the encoded Vβ domain comprises the CDR3 amino acid sequence shown in SEQ ID NO.: 40; (iii) the encoded Vα domain comprises the CDR1 amino acid sequence shown in SEQ ID NO.: 26; (iv) the encoded Vβ domain comprises the CDR1 amino acid sequence shown in SEQ ID NO.: 38; (v) the encoded Vα domain comprises the CDR2 amino acid sequence shown in SEQ ID NO.: 27; and (vi) The encoded Vβ domain comprises the CDR2 amino acid sequence shown in SEQ ID NO.:

39.

63. The isolated polynucleotide of claim 62, wherein: (i) the encoded Vα domain comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence shown in SEQ ID NO.: 4 or 12; and (ii) the encoded Vβ domain comprises or consists of an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO.: 8 or 16.

64. The isolated polynucleotide of claim 62, wherein: (i) the encoded TCR Vα is contained in a TCR α chain, wherein the TCR α chain comprises or consists of an amino acid sequence having at least 90% identity with the amino acid sequence shown in SEQ ID NO.: 53 or 49; and (ii) the encoded TCR Vβ is contained in a TCR β chain, wherein the TCR β chain comprises or consists of an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO.: 61 or 57.

65. The isolated polynucleotide of claim 62, wherein The polynucleotides are codon-optimized for expression in a host cell.

66. The isolated polynucleotide of claim 62, wherein the host cell is a human T cell, NK cell or NK-T cell.

67. The isolated polynucleotide of claim 66, wherein the host cell is a human T cell.

68. The isolated polynucleotide of claim 62, wherein The polynucleotide comprises a polynucleotide having at least 75% identity to the polynucleotide sequence shown in any one of SEQ ID NOs: 65, 69, 73 and 77.

69. The isolated polynucleotide of claim 62, wherein the encoded binding protein is a TCR.

70. The isolated polynucleotide of claim 63, wherein the encoded binding protein is a TCR.

71. An isolated polynucleotide according to claim 64, wherein the encoded TCR Vα is contained in a TCRα chain, wherein the TCRα chain comprises or consists of the amino acid sequence shown in SEQ ID NO.: 53; and the encoded TCR Vβ is contained in a TCRβ chain, wherein the TCRβ chain comprises or consists of the amino acid sequence shown in SEQ ID NO.:

61.

72. A vector comprising the polynucleotide of claim 62.

73. The vector according to claim 72, wherein The vector is a lentiviral vector or a retroviral vector.

74. A vector comprising the polynucleotide of any one of claims 63-71.

75. A host cell comprising a polynucleotide according to any one of claims 63-71 or a vector according to claim 72 or 73 and capable of expressing the encoded binding protein.

76. A host cell comprising the polynucleotide of claim 62.

77. A composition comprising: (i) the modified immune cell according to any one of claims 1-61; (ii) a polynucleotide according to any one of claims 62 to 71; (iii) a vector according to claim 72 or 73; and / or (iv) the host cell according to claim 76; and a pharmaceutically acceptable carrier, excipient or diluent.

78. Use of the modified immune cell of any one of claims 1-61, the polynucleotide of any one of claims 62-71, the vector of claim 72 or 73, and / or the host cell of claim 76 in the preparation of a medicament for treating a disease or condition associated with WT-1 expression in an HLA-A*0201-positive human subject, wherein the disease or condition is selected from breast cancer, pancreatic cancer, and acute myeloid leukemia, and wherein the modified immune cell is a human T cell.

79. The use according to claim 78, wherein The disease or condition is breast cancer.

80. The use according to claim 78, wherein The disease or condition is pancreatic cancer.

81. The use according to claim 78, wherein The disease or condition is acute myeloid leukemia.

82. The use according to claim 78, wherein The modified immune cell is modified ex vivo to contain the heterologous polynucleotide.

83. The use according to claim 78, wherein The modified immune cells are allogeneic, syngeneic or autologous to the subject.

84. The use according to claim 78, wherein The T cells are CD4 + T cells, CD8 + T cells, CD4 - CD8 - Double negative T cells, γδ T cells, or any combination thereof.

85. The use according to claim 78, wherein The T cells are naive T cells, central memory T cells, effector memory T cells or any combination thereof.

86. The use according to claim 78, wherein The medicament is intended to be administered to a subject in multiple doses.

87. The use according to claim 86, wherein The multiple doses are administered at administration intervals of 2 to 4 weeks.

88. The use according to claim 78, wherein The drug was added at 10 4 Modified immune cells / kg to 10 11 A dose of modified immune cells / kg is administered to the subject.

89. The use according to claim 78, wherein The medicament is for administration to a subject together with a cytokine.

90. The use according to claim 89, wherein The cytokine is IL-2, IL-15, IL-21 or any combination thereof.

91. The use according to claim 90, wherein The cytokine is IL-2 and is for simultaneous or sequential administration with the drug.

92. The use according to claim 91, wherein The cytokine is intended for sequential administration, provided that the subject is administered the drug at least three or four times prior to administration of the cytokine.

93. The use according to claim 89, wherein The cytokine was IL-2 and was administered subcutaneously.

94. The use according to claim 78, wherein The subject is further receiving immunosuppressive therapy.

95. The use according to claim 94, wherein The immunosuppressive therapy is selected from the group consisting of calcineurin inhibitors, corticosteroids, microtubule inhibitors, low-dose mycophenolic acid prodrugs, and any combination thereof.

96. The use according to claim 78, wherein The subject has received a non-myeloablative or myeloablative hematopoietic cell transplant.

97. The use according to claim 96, wherein the medicament is for administration to a subject at least three months after the non-myeloablative hematopoietic cell transplantation.

98. The use according to claim 97, wherein the medicament is for administration to a subject at least two months after the myeloablative hematopoietic cell transplantation.

99. The use according to claim 78, wherein The subject has previously received treatment for acute myeloid leukemia.

100. Use of the composition of claim 77 in the preparation of a medicament for treating a disease or condition associated with WT-1 expression in an HLA-A*0201-positive human subject, wherein the disease or condition is selected from breast cancer, pancreatic cancer, and acute myeloid leukemia, and wherein the modified immune cell is a human T cell.

101. The use according to claim 100, wherein the modified immune cells are selected from CD8+ T cells and CD4+ T cells.

102. A binding protein comprising a T cell receptor TCR α chain variable Vα domain and a TCR β chain variable Vβ domain, wherein: The Vα domain contains: (i) the CDR3 amino acid sequence CDR3α of SEQ ID NO.: 28; (ii) the CDR1 amino acid sequence CDR1α of SEQ ID NO.: 26; (iii) the CDR2 amino acid sequence CDR2α of SEQ ID NO.: 27; and The Vβ domain contains: (iv) the CDR3 amino acid sequence CDR3β of SEQ ID NO: 40; (v) the CDR1 amino acid sequence CDR1β of SEQ ID NO.: 38; (vi) the CDR2 amino acid sequence CDR2β of SEQ ID NO.: 39, The binding protein is capable of binding to the RMFPNAPYL:HLA-A*0201 complex shown in SEQ ID NO:

94.

103. The binding protein of claim 102, wherein: (i) the Vα domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO.: 4 or 12; and (ii) the Vβ domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO.: 8 or 16.

104. The binding protein of claim 102, wherein the Vα domain comprises the amino acid sequence shown in SEQ ID NO.: 12, and the Vβ domain comprises the amino acid sequence shown in SEQ ID NO.:

16.

105. The binding protein according to claim 102, wherein the Vα domain consists of the amino acid sequence shown in SEQ ID NO.: 12, and the Vβ domain consists of the amino acid sequence shown in SEQ ID NO.:

16.

106. The binding protein of claim 102, further comprising: (1) TCR α chain constant domain Cα or a fragment thereof; and / or (2) TCRβ chain constant domain Cβ or a fragment thereof.

107. The binding protein according to claim 106, wherein Ca comprises or consists of the amino acid sequence of any one of SEQ ID NOs.: 41-44.

108. The binding protein according to claim 106, wherein Cβ comprises or consists of the amino acid sequence of SEQ ID NO.:

45.

109. A binding protein according to claim 106, wherein the binding protein comprises a TCRβ chain and a TCRα chain, wherein the TCRβ chain has at least 90% identity with the amino acid sequence shown in SEQ ID NO.: 61 or 57, comprises or consists of the amino acid sequence shown in SEQ ID NO.: 61 or 57, and the TCRα chain has at least 90% identity with the amino acid sequence shown in SEQ ID NO.: 53 or 49, comprises or consists of the amino acid sequence shown in SEQ ID NO.: 53 or 49.

110. The binding protein according to claim 106, wherein the binding protein comprises a TCRβ chain and a TCRα chain, the TCRβ chain comprises the amino acid sequence shown in SEQ ID NO.: 61, and the TCRα chain comprises the amino acid sequence shown in SEQ ID NO.:

53.

111. The binding protein according to claim 106, wherein the binding protein comprises a TCRβ chain and a TCRα chain, wherein the TCRβ chain consists of the amino acid sequence shown in SEQ ID NO.: 61, and the TCRα chain consists of the amino acid sequence shown in SEQ ID NO.:

53.

112. A method for preparing a modified immune cell according to any one of claims 1-61, comprising transfecting or transducing an immune cell using a polynucleotide according to any one of claims 62-71 or a vector according to claim 72 or 73.

113. Use of the binding protein according to any one of claims 102-111 in the preparation of a medicament for treating a disease or condition associated with WT-1 expression in an HLA-A*0201 positive subject, wherein the disease or condition is selected from breast cancer, pancreatic cancer and acute myeloid leukemia.

114. The use according to claim 113, wherein the disease or condition is breast cancer.

115. The use according to claim 113, wherein the disease or condition is pancreatic cancer.

116. The use according to claim 113, wherein the disease or disorder is acute myeloid leukemia.

Images