High-affinity WT1 T cell receptors and their uses

Abstract

The present disclosure provides T cell receptors (TCRs) and related binding proteins having high functional affinity for the tumor-associated antigen p37 from Wilms tumor protein 1 (WT1), T cells expressing such high-affinity WT1-specific TCRs, nucleic acids encoding the same, and compositions for treating diseases or disorders in which cells overexpress WT1 and / or produce the p37 antigen, such as in cancer.

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 the specification. The text file containing the sequence listing is named 360056_466WO_SEQUENCE_LISTING.txt. This text file is 243KB, was created on March 8, 2020, and is being submitted electronically via EFS-Web. Background Art

[0003] Adoptive T-cell immunotherapy using genetically engineered T cells has shown promise in several trials targeting tumor-associated antigens using antigen receptors with sufficient affinity, including antibody-based chimeric receptors. 1-3 and high-affinity TCR 4-8 Although the natural process of diversity generation in the thymus employs RAG-mediated TCR gene rearrangement to generate highly diverse CDR3s of varying length and amino acid composition, the isolation of effective high-affinity TCRs within the affinity constraints imposed by central tolerance remains a substantial obstacle to the implementation of adoptive T cell immunotherapy for a variety of malignancies targeting identified candidate intracellular self / tumor antigens. 9,10 In addition, TCR adoptive immunotherapy has the ability to detect intracellular antigens presented on the cell surface by MHC class I.

[0004] WT1 protein is an attractive target for clinical development due to its immunological properties (Cheever et al., Clin. Cancer Res. 15:5323, 2009) and its expression in many aggressive tumor types associated with poor prognosis. WT1 is involved in regulating the expression of genes that promote proliferation and oncogenicity (Oji et al., Jpn. J. Cancer Res. 90:194, 1999). It is overexpressed in the majority of high-risk leukemias (Menssen et al., Leukemia 9:1060, 1995), up to 80% of NSCLC (Oji et al., Int. J. Cancer 100:297, 2002), 100% of mesotheliomas (Tsuta et al., App. Immunohistochem. Mol. Morphol. 17:126, 2009), and ≥80% of gynecological malignancies (Coosemans and Van Gool, Expert Rev. Clin. Immunol. 10:705, 2014). Several peptides of the WT1 protein are known to be tumor-associated antigens and are HLA-A*0201-restricted.

[0005] There is a clear need for alternative high WT1 antigen-specific TCR immunotherapies for various cancers (e.g., leukemias and tumors). The presently disclosed embodiments address these needs and provide other related advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1A and 1B Demonstrates how to identify WT1 using a high-throughput sequencing-based strategy 37 Specific TCR. (A) used to recognize high WT1 37-45 Schematic diagram of the initial sequencing-based strategy for peptide / MHC tetramer binding-associated TCR clonotypes. (B) The enrichment of the sorted population relative to the total population is shown, with selected TCRs highlighted. All TCRs, represented by black circles, were synthesized and evaluated for antigen specificity (27 in total).

[0007] Figure 2 The results of the functional assessment of TCR in conjunction with high-level CD8 independent (CD8i) tetramers are shown. TCR constructs are expressed in Jurkat cells lacking endogenous TCR α / β chains. The relationship between tetramer staining and CD3 expression of each TCR is shown (CD3 expression is directly related to transgenic TCR surface expression).

[0008] Figures 3A-3C Additional WT1 is shown 37 Specific TCRs were identified using a modified high-throughput sequencing-based strategy using CD8-independent (CD8i) tetramers. (A) TCRs were used to identify TCRs with high CD8-independence. 37 Schematic diagram of the improved sequencing-based strategy for peptide / MHC tetramer binding-associated TCR clonotypes. (B) Compared to a similar analysis in (C), the percentage enrichment of the original sorted population relative to the total population is shown when CD8i tetramers are used. An additional 14 TCRs were selected based on reduced surface CD3 levels and CD8i tetramer binding. All TCRs represented by shaded (diagonal pattern) circles were synthesized and evaluated for antigen specificity.

[0009] Figure 4 Shown are selected WT1 37 CD8i tetramer binding of TCRs. TCR constructs were expressed in Jurkat cells lacking endogenous TCR α / β chains. Tetramer staining is shown relative to CD3 expression for each TCR (CD3 expression directly correlates with transgenic TCR surface expression).

[0010] Figure 5A and 5B shows that the transduction of primary CD8 +PBMCs, selected TCR peptide EC in IFNγ assay 50 (A) Selected TCRs were transduced into CD8 isolated from donor PMBCs. + T cells. After one week, cells were sorted into tetramers + CD8 + The expanded antigen-specific cells were cultured with peptide-pulsed T2 target cells for 4 to 6 hours, and IFNγ production was determined by flow cytometry. (B) The percentage of IFNγ-producing cells was fitted to the dose-response curve by nonlinear regression to calculate the peptide EC for each TCR. 50 .

[0011] Figure 6 Shown is the expression of WT1 37 -specific TCR of primary CD8 + T cells efficiently kill WT1 + HLA-A2 + Breast cancer cell line MDA-MB-468. Sorted and purified for high tetramer binding, CD8 + Primary T cells were transduced with TCR and mixed with used A pool of breast cancer cell lines, MDA-MB-468, stained with Rapid Red dye. The total area of ​​red objects (correlated with the total number of viable target cells) was calculated for each TCR-transduced T cell population at designated time points over 72 hours. To assess the sustained reactivity of TCR-transduced T cells to persistent antigens, additional MDA-MB-468 cells were added at 48 hours.

[0012] Figure 7 CD4 expressing TCR10.1 is shown + and CD8 + T cells can eliminate WT1 after repeated attacks in vitro + A2 + Pancreatic cancer cell line PANC-1. CD4 + and CD8 + T cells were transduced to express WT1 37 TCR10.1.CD4 + T cells were further transduced to express CD8α and CD8β genes. After 8 days, the transduced cells were sorted to purify CD8 + Tetramer + and CD4 + / CD8 + Tetramer +T cells. CD4+ / CD8+, CD8+, or a mixture of antigen-specific cells of both populations (CD4 and CD8) were mixed at a ratio of 8:1 (in triplicate) with cells that had been previously transduced to express The pancreatic cancer cell line PANC-1 was mixed with TCR Red dye. The total area of ​​red objects (relative to the total number of viable target cells) was calculated at the indicated time points for each TCR-transduced T cell population. To assess the sustained responsiveness of TCR-transduced T cells to persistent antigens, additional PANC-1 cells were added at 48 hours.

[0013] Figures 8A-8D The killing of T cells transduced with the WT1 p37 peptide-specific TCR of the present disclosure (WT1 37-45 Comparison of tumor cell line killing by T cells transduced with the WT1 p126 peptide-specific C4 TCR from Schmitt et al. (Nat. Biotechnol. 35:1188, 2017) compared to the WT1 p137 peptide-specific TCR of the present disclosure. Note that the C4 TCR has a lower affinity for its peptide:MHC complex than the WT1 p137 peptide-specific TCR of the present disclosure. DETAILED DESCRIPTION

[0014] The present disclosure provides a method for treating an antigenic peptide consisting of amino acids 37-45 from WT1 (also referred to as WT1 37-45 A T cell receptor (TCR) that has high functional affinity for a peptide or p37 peptide antigen (e.g., VLDFAPPGA, SEQ ID NO: 59) associated with a major histocompatibility complex (MHC) (e.g., human leukocyte antigen, HLA). This p37 peptide antigen-specific TCR is useful, for example, for adoptive immunotherapy to treat cancers (e.g., cancers that overexpress WT1).

[0015] As background, because tumors arise from previously normal tissues, most tumor targets based on T cell immunotherapy are self-antigens. For example, this tumor-associated antigen (TAA) can be expressed at high levels in cancer cells, but may not be expressed or may be expressed minimally in other cells. During the development of T cells in the thymus, T cells that are weak in binding to self-antigens can survive in the thymus and may undergo further development and mutation, while T cells that are strong in binding to self-antigens are eliminated by the immune system because such cells can cause adverse autoimmune responses. Therefore, according to the relative ability of T cells to bind antigens, T cells are classified to prepare the immune system to respond to foreign invaders (i.e., identification of non-self antigens) while preventing autoimmune responses (i.e., identification of self antigens). This tolerance mechanism limits the naturally occurring T cells that can recognize tumor (self) antigens with high affinity, and therefore eliminates T cells that can effectively eliminate tumor cells. Therefore, it is difficult to separate T cells with TCRs specific for tumor antigens with high affinity, so most of these cells are basically eliminated by the immune system.

[0016] In the present disclosure, a high-throughput sequencing-based approach was applied to immune cells from approximately 15 healthy donors to identify TCRs with high functional affinity for the p37:MHC complex. This strategy also allows the selection of TCRs even when the TCR expression level on the T cell surface is low. The enrichment of the sorted population as a percentage of the total population was used to select p37-specific TCRs with high affinity and high functional avidity (i.e., those with the greatest anti-tumor effect) and their compositions in the present disclosure. Such high functional affinity p37-specific TCRs were identified in T cells that: (a) bind p37 peptide / MHC tetramers independent of CD8, (b) undergo less in vitro peptide-driven expansion, and (c) in some cases, are expressed at lower levels on the T cell surface than other TCRs in T cells that do not have this property. A total of 27 TCRs were synthesized and evaluated for their specificity for the p37 antigen (see Figure 1B ).

[0017] In certain embodiments, the specific T cell receptor (TCR) for the WT1 peptide includes a TCR α chain and a TCR β chain, wherein the TCR α chain includes a V containing the amino acid sequence shown in any one of SEQ ID NOs: 253-263 and 34-44. α domain and an α chain constant domain having the amino acid sequence of SEQ ID NO: 47, and a TCR β chain including a V domain having the amino acid sequence shown in any one of SEQ ID NOs: 253-263 and 23-33. βdomain and a β chain constant domain having an amino acid sequence of SEQ ID NO: 45 or 46, such a TCR specifically binds to a VLDFAPPGA (SEQ ID NO: 59): human leukocyte antigen complex on the surface of T cells and promotes IFNγ production, pEC 50 In certain embodiments, the selected TCR specifically binds to the VLDFAPPGA (SEQ ID NO: 59): human leukocyte antigen (HLA) complex, K D Less than or equal to approximately 10 -8 M, or wherein the TCR is reduced in K compared to the TCR disclosed in Schmitt et al., Nat. Biotechnol. 35: 1188, 2017 off High affinity TCRs were isolated from the VLDFAPPGA (SEQ ID NO: 59): human leukocyte antigen (HLA) complex at a ratio of 1:1.

[0018] The compositions and methods described herein will, in certain embodiments, have therapeutic utility in treating diseases or conditions associated with WT1 expression or overexpression (e.g., detectable WT1 expression levels that are statistically significantly higher than the WT1 expression levels detectable in normal or disease-free cells). Such diseases include hyperproliferative disorders or various forms of proliferative disorders, such as hematological malignancies or solid cancers. Non-limiting examples of these and related uses are described herein, including in vitro, ex vivo, and in vivo stimulation of WT1 antigen-specific T cell responses, such as by using expression of WT1 peptides (e.g., VLDFAPPGA, SEQ ID NO: 59, also known as WT1 37-45 recombinant T cells expressing an enhanced affinity TCR specific for p37 peptide or p37 peptide.

[0019] Before describing the present disclosure in more detail, it may be helpful to provide definitions of certain terms used herein. Additional definitions are set forth throughout this disclosure.

[0020] In this specification, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the listed range, and, where appropriate, its fractions (such as integer tenths and hundredths), unless otherwise stated. In addition, any physical properties related to any numerical ranges listed herein, such as polymer subunits, size or thickness, should be understood to include any integer within the listed range, unless otherwise stated. As used herein, the term "approximately" refers to ±10% of the specified range, value or structure, unless otherwise stated. It should be understood that the terms "a" or "an" used herein refer to "one or more" of the listed components. The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination thereof. As used herein, the terms "include," "have," and "comprise" are used synonymously, where the terms and their variations are intended to be interpreted as non-restrictive.

[0021] In addition, it should be understood that individual compounds or groups of compounds derived from the various combinations of structures and substituents described herein are disclosed in this application to the same extent as if each compound or group of compounds were shown individually. Therefore, the selection of a particular structure or a particular substituent is within the scope of this disclosure.

[0022] The term "consisting essentially of" is not equivalent to "comprising" but refers to the specific materials or steps of the claim, or those materials or steps that do not materially affect the essential characteristics of the claimed subject matter. For example, a protein domain, region or module (e.g., a binding domain, hinge region, connecting module) or a protein (which may have one or more domains, regions or modules) "consists essentially of" a specific amino acid sequence, and when the amino acid sequence of the domain, region, module or protein includes extensions, deletions, mutations or any combination thereof (e.g., amino acids at the amino or carboxyl termini or between domains), the combination of these amino acids accounts for up to 20% (e.g., up to 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of the domain, region, module or protein and does not materially affect (e.g., reduce the activity by no more than 50%, e.g., no more than 40%, 30%, 25%, 20%, 15%, 10%, 5% or 1%) the activity of the domain, region, module or protein (e.g., the binding affinity of the protein for the target).

[0023] As used herein, in some aspects, "immune system cell" refers to any cell of the immune system derived from hematopoietic stem cells in the bone marrow, which produces two main lineages: myeloid progenitor cells (which produce myeloid cells, such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and lymphoid progenitor cells (which produce lymphocytes, 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, γδT cells, regulatory T cells, stem cell memory T cells, natural killer cells (such as NK cells or NK-T cells), B cells and dendritic cells. Macrophages and dendritic cells can be referred to as "antigen presenting cells" or "APCs", which are professional cells that can activate T cells when the major histocompatibility complex (MHC) receptors compounded with peptides on the APC surface interact with the TCR on the T cell surface.

[0024] In some aspects, "major histocompatibility complex (MHC)" can refer 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 a non-covalently linked β2 microglobulin. MHC class II molecules are composed of transmembrane glycoproteins α and β, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides derived from the cytoplasmic matrix to the cell surface where they are expressed by CD8 + T cells recognize peptide:MHC complexes. MHC class II molecules deliver peptides from the vesicle system to the cell surface where they are transported by CD4 + T cells recognize MHC class II molecules. In humans, MHC is called human leukocyte antigen (HLA).

[0025] "T cells" or "T lymphocytes" are cells of the immune system that mature in the thymus and produce T cell receptors (TCRs). T cells may exhibit similarities to naive T cells (e.g., not exposed to antigen; T cells may be infected with antigens that are not exposed to antigens). CM Compared with normal T cells, the expression of CD62L, CCR7, CD28, CD3, CD127 and CD45RA increased, and the expression of CD45RO decreased), memory T cells (T M Phenotypes or markers associated with T cells (e.g., antigen experienced and long-lived) and effector cells (e.g., antigen experienced, cytotoxic). M can be further divided into central memory T cells (T CM , for example, compared with naive T cells, the expression of CD62L, CCR7, CD28, CD127, CD45RO and CD95 is increased, and the expression of CD54RA is decreased) and effector memory T cells (T EM , such as with naive T cells or T CMCompared with T cells, the expression of CD62L, CCR7, CD28, and CD45RA is reduced, and the expression of CD127 is increased. E ) can refer to antigen-experienced CD8 + Cytotoxic T lymphocytes, T CM In contrast, it has reduced expression of CD62L, CCR7, CD28 and is positive for granzymes and perforin. H ) may include CD4 + CD4 cells, which influence the activity of other immune cells by releasing cytokines. + T cells can activate and suppress adaptive immune responses, and which of the two functions is induced will depend on the presence of other cells or signals. T cells can be collected using known techniques and various subpopulations or combinations thereof can be enriched or eliminated using known techniques, such as binding affinity with antibodies, flow cytometry, or immunomagnetic selection. Other exemplary T cells include regulatory T cells, such as CD4+CD25+(Foxp3+) regulatory T cells and Treg17 cells, as well as Tr1, Th3, CD8+CD28-, and Qa-1 restricted T cells.

[0026] "T cell receptor" (TCR) refers in certain aspects 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) can specifically bind to an antigenic peptide bound to an MHC receptor. In some aspects, TCR refers to a binding protein comprising two TCR variable domains (Vα and Vβ) of the present disclosure. In some aspects, TCR includes a single-chain TCR (i.e., a single-chain fusion protein comprising a TCR variable domain of the present disclosure, or a CAR comprising a TCR variable domain of the present disclosure (discussed herein). In some aspects, TCR can exist on the cell surface or in a soluble form, generally consisting of a heterodimer having α and β chains (also referred to as TCR and TCRβ, respectively), or having γ and δ chains (also referred to as TCRγ and TCR, respectively).

[0027] Like immunoglobulins, the extracellular portion of a TCR chain (e.g., α chain, β chain) comprises two immunoglobulin domains, a variable domain (e.g., α chain variable domain or Vα, β chain variable domain or Vβ; typically 1 to 116 amino acids based on Kabat numbering Kabat et al., "Sequences of Proteins of Immunological Interest," USDept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.) at the N-terminus, and a constant domain (e.g., α-chain constant domain or Cα, typically 81 to 259 amino acids based on Kabat, β-chain constant domain or Cβ, typically 81 to 295 amino acids based on Kabat) adjacent to the cell membrane. Like immunoglobulins, the variable domains also comprise 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, the TCR is found on the surface of a T cell (or T lymphocyte) and binds to the CD3 complex. The TCR used in this statement can be derived from various animal species, such as humans, mice, rats, rabbits, or other mammals.

[0028] The term "variable region" or "variable domain" refers to the domain of an immunoglobulin superfamily binding protein, such as the α or β chain of a TCR (or the γ and δ chains of a γδTCR), that is involved in antigen binding by an immunoglobulin superfamily binding protein, such as a TCR. The variable structures of the α and β chains (Vα and Vβ, respectively) of a native TCR typically have similar structures, with each domain comprising four universally 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 may be sufficient to confer specificity for antigen binding. Furthermore, the Vα or Vβ domain of an antigen-bound TCR can be used to screen for libraries of complementary Vα or Vβ domains, respectively, to isolate a TCR that binds to a specific antigen.

[0029] The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and, in the art, 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 within the primary amino acid sequence. Generally, there are three CDRs per TCR α chain variable region (αCDR1, αCDR2, αCDR3) and three CDRs per TCR β chain variable region (βCDR1, βCDR2, βCDR3). In TCRs, CDR3 is considered the primary CDR responsible for recognizing processed antigens. Generally, CDR1 and CDR2 interact primarily or exclusively with the MHC.

[0030] CDR1 and CDR2 are encoded within the variable gene segment of the TCR variable region encoding sequence, while CDR3 is encoded by a spanning region of the variable and joining segments of Vα, or the variable, diversity, and joining segments of Vβ. Therefore, if the identity of the variable gene segments of Vα or Vβ is known, the sequences of their corresponding CDR1 and CDR2 can be deduced; for example, according to the numbering scheme described herein. Compared to CDR1 and CDR2, CDR3 is generally significantly more diverse due to the addition and loss of nucleotides during recombination.

[0031] TCR variable domain sequences can be aligned with a numbering scheme (such as Kabat, Chothia, EU, IMGT, Enhanced Chothia, and Aho), allowing annotation of equivalent residue positions and comparison of different molecules using, for example, the ANARCI software tool (2016, Bioinformatics 15:298-300). A numbering scheme provides a standardized division for the framework regions and CDRs in the TCR variable domain. In certain embodiments, the CDRs of the present disclosure are identified according to the IMGT numbering scheme (Lefranc et al., Dev. Comp. Immunol. 27:55, 2003; imgt.org / IMGTindex / V-QUEST.php). In certain embodiments, the CDR3 amino acid sequence of the present disclosure includes one or more junction amino acids; for example, as discussed herein, these may be generated during (RAG)-mediated rearrangement.

[0032] As used herein, the term "CD8 co-receptor" or "CD8" refers to the cell surface glycoprotein CD8, whether as an α-α homodimer or an α-β heterodimer. The CD8 co-receptor assists the function of cytotoxic T cells (CD8+) and exerts its effect through signaling through its cytoplasmic tyrosine phosphorylation pathway (Gao and Jakobsen, Immunol. Today 21:630-636, 2000; Cole and Gao, Cell. Mol. Immunol. 1:81-88, 2004). There are five (5) known human CD8 β chain isoforms (see UniProtKB identifier P10966) and one known human CD8 α chain isoform (see UniProtKB identifier P01732).

[0033] "CD4" is an immunoglobulin co-receptor glycoprotein that assists TCR in communicating with antigen-presenting cells (see Campbell & Reece, Biology 909 (Benjamin Cummings, Sixth Ed., 2002); UniProtKB identifier P01730. CD4 is present on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells and consists of four immunoglobulin domains (D1 to D4) expressed on the cell surface. During antigen presentation, CD4, along with the TCR complex, is recruited to different regions of the MHC II molecule (CD4 binds to MHC II β2, while the TCR complex binds to MHC II α1 / β1). Without wishing to be bound by theory, it is believed that proximity to the TCR complex enables CD4-associated kinase molecules to phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) present on the cytoplasmic domain of CD3. This activity is thought to amplify the signal generated by the activated TCR to generate or recruit various types of immune system cells, including T helper cells, and to initiate an immune response.

[0034] As used herein, "D / N / P region" in certain aspects refers to nucleotides or amino acids predicted to be located within the diversity (D) gene segment, which may include non-templated (N) nucleotides and palindromic (P) nucleotides inserted (or deleted) during the V(D)J recombination process that leads to T cell receptor diversity. The rearrangement of the variable (V), diversity (D), and joining (J) gene segments mediated by the recombination activation gene (RAG) is an imprecise process that results in the variable addition and subtraction of nucleotides (referred to as palindromic or P nucleotides). Subsequently, terminal deoxynucleotidyl transferase (TdT) activity further adds random non-templated (N) nucleotides. Finally, exonucleases remove unpaired nucleotides, and gaps are filled by DNA synthesis and repair enzymes. This trimming and repair mechanism leads to junction diversity, which is the basis for the efficient and specific recognition of different antigens by different TCRs. D gene segments can be identified using the annotation system of the International ImMunoGeneTics Information System (IMGT; imgt.org).

[0035] In certain aspects, "CD3" is a multiprotein complex composed of six chains (see Abbas and Lichtman, 2003; Janeway et al., pp. 172 and 178, 1999). In mammals, this complex includes a homodimer of one CD3γ chain, one CD3δ chain, two CD3ε chains, and a CD3ζ chain. The CD3γ, CD3δ, and CD3ε chains are highly related cell surface proteins of the immunoglobulin superfamily and contain a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a property that enables these chains to bind to the positively charged regions of T cell receptor chains. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a conserved motif known as 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 ITAMs are important for the signaling ability of the TCR complex. The CD3 used in the present disclosure can be derived from various animal species, including humans, mice, rats, or other mammals.

[0036] As used herein, "TCR complex" refers in certain aspects to a complex formed by the association of CD3 and 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.

[0037] In certain aspects, "component of a TCR complex," as used herein, 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).

[0038] As used herein, "antigen" or "Ag" refers to an immunogenic molecule that can cause an immune response. This immune response may involve the production of antibodies, the activation of specific immune-competent cells (such as T cells), or both. Antigens (immunogenic molecules) can be, for example, peptides, glycopeptides, polypeptides, glycopolypeptides, polynucleotides, polysaccharides, lipids or the like. Obviously, antigens can be synthesized, recombinantly produced, or extracted from biological samples. Exemplary biological samples that can contain one or more antigens include tissue samples, tumor samples, cells, biological fluids or combinations thereof. Antigens can be produced by cells that have been modified or genetically engineered to express the antigen, or by cells that endogenously (for example, without modification or genetic engineering involving human intervention) express immunogenic mutations or polymorphisms.

[0039] As used herein, "neoantigen" refers to a host cell product containing a structural change, alteration or mutation that creates a new antigen or antigenic epitope that has not been previously observed in the subject's genome (i.e., in a healthy tissue sample of the subject) and has not been "seen" or recognized by the host's immune system, which: (a) is processed by the cell's antigen processing and trafficking machinery and presented on the cell surface together with MHC (e.g., HLA) molecules; and (b) elicits an immune response (e.g., a cellular (T cell) response). Neoantigens can arise from, for example, changes (substitutions, additions, deletions) in encoding polynucleotides, resulting in altered or mutated products, or from the insertion of exogenous nucleic acid molecules or proteins into cells, or from genetic changes caused by exposure to environmental factors (e.g., chemicals, radioactivity). Neoantigens can be produced separately from tumor antigens, or they can be produced from tumor antigens or associated with tumor antigens. "Tumor neoantigens" (or "tumor-specific neoantigens") refer to proteins that contain neoantigenic determinants associated with, produced by, or produced in tumor cells or multiple cells within a tumor. Tumor neoantigenic determinants are found, for example, on antigenic tumor proteins or peptides that contain one or more somatic mutations or chromosomal rearrangements encoded by the DNA of tumor cells (e.g., pancreatic cancer, lung cancer, colorectal cancer), as well as on proteins or peptides from viral open reading frames of virus-associated tumors.

[0040] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, T cell receptor (TCR), chimeric antigen receptor, or other binding molecule, domain, or protein. Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and may have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0041] As used herein, "specific binding" or "specificity" refers in certain aspects to the binding or association of a T cell receptor (TCR) or its binding domain (such as scTCR or its fusion protein) with a target molecule, with an apparent affinity or K A (i.e., the equilibrium binding constant for a specific binding interaction expressed in 1 / M) equal to or greater than 10 9 M -1 (This is equal to the on-rate [k on ] and the dissociation rate [k off ] ratio), or functional affinity or EC 50 Equal to or greater than 10 -9 M, while not significantly binding or associating with any other molecule or component in the sample. TCRs 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). "High affinity" TCRs or binding domains are those that have 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 K A "Low affinity" binding proteins or binding domains are those with a maximum affinity of 10 7 M -1 , 10 6 M -1 , 10 5 M -1 K A Alternatively, affinity can be defined in units of M (e.g., 10 -9 M to 10 -13 The equilibrium dissociation constant (K) of a specific binding interaction of D ).

[0042] The term "functional avidity" refers to the biometric or activation threshold of a T cell's response to a given concentration of ligand in vitro, where the biometric may include cytokine production (e.g., IFNγ production, IL-2 production, etc.), cytotoxic activity, and proliferation. For example, T cells that respond biologically (immunologically) to very low antigen doses in vitro through cytokine production, cytotoxicity, or proliferation are considered to have high functional avidity, while T cells with lower functional avidity require higher amounts of antigen to elicit an immune response, similar to high-avidity T cells. It will be appreciated that functional avidity is distinct from affinity and avidity. Avidity refers to the strength of any specific binding between a binding protein and its antigen / ligand. Some binding proteins are multivalent and bind to multiple antigens—in this case, the overall strength of the binding is the avidity.

[0043] As used herein, "functional avidity" refers to a quantitative determinant of the activation threshold of a TCR expressed by a T cell. In vivo, T cells are exposed to similar antigen doses regardless of TCR avidity (high or low), but 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 (such as 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 that influence 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 levels 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 levels of molecules that impair T cell function and TCR signaling.

[0044] 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 "EC50". 50 The value is usually expressed in moles (mol / L), but it is usually converted to a logarithmic value as follows - log 10 (EC 50 ) - it provides a sigmoidal diagram (see, for example, Figure 5A ). For example, if EC 50 Equal to 1μM (10 -6 M), then log 10 (EC50 ) value is -6. Another value used is pEC 50 , which is defined as EC 50 The negative logarithm (-log 10 (EC 50 )). In the above example, this corresponds to an EC of 1 μM 50 The pEC50 value is 6. In certain embodiments, the functional affinity of the TCR of the present disclosure will be a measure of its ability to promote IFNγ production by T cells, which can be measured using the assays described herein. A "high functional affinity" TCR or its binding domain is one that has an EC50 of 6. 50 At least 10 -9 M, at least about 10 -10 M, at least about 10 - 11 M, at least about 10 -12 M or at least about 10 -13 In some embodiments, the response comprises the production of IFN-γ; for example, IFN-γ is produced by an immune cell expressing a TCR (e.g., a T cell, a NK cell, or a NK-T cell) in response to an antigen.

[0045] In some ways, "WT1 37-45 Antigen" or "WT1 37-45 Peptide" or "WT1 37-45 "Peptide antigen" or "p37 peptide" or "p37 antigen" or "p37 peptide antigen" each refers to a natural or synthetic portion of a WT1 protein ranging from about 9 amino acids to about 15 amino acids in length, including the amino acid sequence of VLDFAPPGA (SEQ ID NO: 59), which can form a complex with an MHC (e.g., HLA) molecule, such that the complex can bind to a specific TCR for the WT1 peptide:MHC (e.g., HLA) complex. Since WT1 is an internal host protein, the WT1 antigenic peptide will be presented in the context of class I MHC. In a specific embodiment, the WT1 polypeptide VLDFAPPGA (SEQ ID NO: 59) can associate with the human class I HLA allele HLA-A*201.

[0046] In some aspects, the phrase "WT1 37-45 Peptide-specific binding protein" or "WT1 37-45 Peptide-specific TCR" or "WT1 37-45 Antigen-specific TCR" or "WT1 37-45"Peptide antigen-specific TCR" or "WT1 p37 peptide-specific binding protein" or "WT1 p37 peptide-specific TCR" or "WT1 p37 antigen-specific TCR" or "WT1 p37 peptide antigen-specific TCR", interchangeably herein, refers to a protein or polypeptide that specifically binds to a WT1 p37 peptide in complex with an MHC or HLA molecule (e.g., on the surface of a cell) with about or at least about a specific affinity or functional avidity, preferably high functional avidity, as defined herein. Such binding proteins or polypeptides include a TCR variable domain as provided herein. In certain embodiments, the functional avidity of the WT1-specific binding protein to a WT1-derived peptide:HLA complex (or WT1-derived peptide:MHC complex) is log [EC 50 ] ranged from about -2.5 μM to about -3.75 μM (equivalent to -8.5 M to about -9.8 M). These values ​​are EC 50 The range is from approximately 3.16×10 -9 M to approximately 1.58×10 -10 M is measured, for example, by the detection method described in the following paragraphs and in Example 1 herein.

[0047] Assays for assessing affinity, apparent affinity, relative affinity, or functional affinity are known. As described herein, the apparent affinity or functional affinity of the TCRs of the present disclosure is measured by assessing binding of various concentrations of tetramers associated with the p37 peptide, for example, by flow cytometry using labeled tetramers. In some examples, the apparent K of the TCR is D or EC 50 is measured using a 2-fold dilution of the labeled tetramer over a range of concentrations, and the binding curve is then determined by nonlinear regression. For example, the apparent K D is determined as the ligand concentration that produces half-maximal binding, while EC 50 The concentration of ligand, eg, a cytokine (eg, IFNγ, IL-2), is determined to produce half-maximal production.

[0048] "MHC-peptide tetramer staining" in certain aspects refers to an assay for detecting antigen-specific T cells, characterized by a tetramer of MHC molecules, each comprising an identical peptide whose amino acid sequence is homologous (e.g., identical or related) to at least one antigen (e.g., WTI), 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 the addition of streptavidin, which can be fluorescently labeled. The tetramer can be detected by flow cytometry using fluorescent labeling. In certain embodiments, MHC-peptide tetramer assays are used to detect or select high-affinity or high-functional avidity TCRs of the present disclosure.

[0049] The level of cytokines can be determined according to the methods described herein and the practice of 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 resulting from antigen-specific stimulation or immune response stimulation can be determined by isolating lymphocytes, such as circulating lymphocytes in a peripheral blood cell sample or cells from lymph nodes, stimulating the cells with antigens, and measuring cytokine production, cell proliferation and / or cell viability, such as by adding tritiated thymidine or non-radioactive detection, such as MTT assays, etc. The effect of the immunogens described herein on the balance between Th1 and Th2 immune responses can be examined, for example, by determining the levels of Th1 cytokines, such as IFN-γ, IL-12, IL-2, and TNF-β, and type 2 cytokines, such as IL-4, IL-5, IL-9, IL-10, and IL-13.

[0050] In certain aspects, the term "WT1 p37 specific binding domain" or "WT1 37-45 Specific binding domain" or "WT1p37 specific binding fragment" or "WT1 37-45 The term "specific binding fragment" refers to a domain or portion of a WT1-specific TCR that is responsible for specific binding to the WT1 p37 antigen in complex with an MHC or HLA molecule. The WT1 p37 antigen-specific binding domain from the TCR alone (i.e., without any other portion of the WT1-specific TCR) can be soluble and can bind to the WT1 p37 peptide:MHC complex. D Less than 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M, or less than about 10 -13 In other embodiments, the WT1 p37 peptide-specific TCR has high functional affinity and specifically binds to the VLDFAPPGA (SEQ ID NO: 59): human leukocyte antigen (HLA) complex on the surface of T cells, promoting the production of IFNγ, pEC 508.5 or higher (e.g., up to about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, or about 13). Exemplary WT1 p37 peptide-specific binding domains include WT1 p37 peptide-specific scTCRs (e.g., single-chain αβTCR 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), which are anti-WT1 p37 peptide TCRs of the present disclosure or can be derived from the anti-WT1 p37 peptide TCRs of the present disclosure.

[0051] The principles of antigen processing by antigen-presenting cells (APCs) (e.g., dendritic cells, macrophages, lymphocytes, or other cell types) and presentation of antigens by APCs to T cells, including major histocompatibility complex (MHC)-restricted presentation between immunocompatible (e.g., sharing at least one allelic form of an MHC gene involved in antigen presentation) APCs and T cells, are well established (see, e.g., Murphy, Janeway's Immunobiology (8)). th Ed.) 2011 Garland Science, NY; chapters 6, 9, and 16. For example, processed antigenic peptides derived from cell membranes (e.g., tumor antigens, intracellular pathogens) are generally about 7 to about 11 amino acids in length and bind to class I MHC molecules, whereas peptides processed in vesicle systems (e.g., bacteria, viruses) are about 10 to 25 amino acids in length and bind to class II MHC molecules.

[0052] As used herein, a "transmembrane domain" refers to any amino acid sequence having a three-dimensional structure that is thermodynamically stable in a cell membrane and is generally about 15 to about 30 amino acids in length. The structure of a hydrophobic transmembrane domain may include an α-helix, a β-barrel, a β-sheet, a β-helix, or any combination thereof. Exemplary transmembrane domains are those from CD4, CD8, CD28, or CD27.

[0053] As used herein, "immune effector domain" is the intracellular portion of a scTCR or CAR fusion protein that can directly or indirectly promote the immune response of a cell when receiving an appropriate signal. In certain embodiments, the immune effector domain is part of a protein or protein complex that receives a signal when bound, or it binds directly to a target molecule to trigger a signal of the immune effector domain. When the immune effector domain comprises one or more signal domains or motifs, such as an immunoreceptor tyrosine-based activation motif (ITAM), it can directly promote immune cell responses. In other embodiments, the effector domain will indirectly promote cellular responses by binding to one or more other proteins that directly promote cellular responses. Exemplary immune effector domains include intracellular signaling domains from: 4-1BB, CD3ε, CD3δ, CD3ζ, CD27, CD28, CD79A, CD79B, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, Wnt, NKG2D, OX40, ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combination of two or three of the foregoing domains.

[0054] In some aspects, a "connector" refers to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs. An exemplary connector is a "variable domain connector," specifically a 5 to about 35 amino acid sequence that connects the T cell receptor Vα / β and Cα / β chains (e.g., Vα-Cα, Vβ-Cβ, Vα-Vβ) or connects each Vα-Cα, Vβ-Cβ, Vα-Vβ pair to a hinge or transmembrane domain, which provides a spacing function and flexibility sufficient to allow the two sub-binding domains to interact so that the resulting single-chain polypeptide maintains a specific binding affinity or functional affinity to the same target molecule as the T cell receptor. In certain embodiments, the variable domain connector comprises about 10 to about 30 amino acids or about 15 to about 25 amino acids. In specific embodiments, the variable domain connecting peptide comprises Gly x Ser y 1 to 10 repeats of wherein x and y are independently an integer from 0 to 10, as long as x and y are not both 0 (e.g., Gly4Ser (SEQ ID NO: 171), Gly3Ser (SEQ ID NO: 172), Gly2Ser or (Gly3Ser) n (Gly4Ser)1 (SEQ ID NO: 173), (Gly3Ser) n (Gly2Ser) n 、(SEQ ID NO: 174) (Gly3Ser) n(Gly4Ser) n (SEQ ID NO: 175), or (Gly4Ser) n (SEQ ID NO: 171), wherein n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10), wherein the linked variable domains form a functional binding domain (e.g., scTCR).

[0055] In certain aspects, "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, such as between a binding domain and an adjacent constant domain or between a TCR chain and an adjacent self-cleaving peptide. The linking amino acids may arise from the construction design of the fusion protein (e.g., amino acid residues generated by the use of restriction enzyme sites when constructing a nucleic acid molecule encoding the fusion protein) or during genetic recombination or rearrangement events (e.g., RAG-mediated rearrangement).

[0056] In certain aspects, an "altered domain" or "altered protein" refers to a motif, region, domain, peptide, polypeptide or protein that is at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%) identical 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), preferably wherein or wherein the CDR3 from each of the TCR alpha and beta variable domains is not altered.

[0057] In any of the embodiments disclosed herein, the TCR constant domains can be modified to enhance the pairing of desired TCR chains. For example, the pairing between the heterologous TCR α chain and the heterologous TCR β chain is enhanced in the host T cell due to the modification, resulting in a TCR composed of two heterologous chains being preferentially assembled, rather than an undesirable mispairing of the heterologous TCR chain with the endogenous TCR chain (see, e.g., Govers et al., Trends Mol. Med. 16(2):77(2010), the TCR modifications of which are incorporated herein by reference). Exemplary modifications that enhance the pairing of heterologous TCR chains include introducing complementary cysteine ​​residues into each of the heterologous TCR α chain and β chain. In some embodiments, the polynucleotide encoding the heterologous TCR α-chain encodes a cysteine ​​at amino acid position 48 (corresponding to the full-length mature human TCR α-chain sequence), and the polynucleotide encoding the heterologous TCR β-chain encodes a cysteine ​​at amino acid position 57 (corresponding to the full-length mature human TCR β-chain sequence).

[0058] A "chimeric antigen receptor" (CAR) is a fusion protein that is engineered to contain two or more naturally occurring amino acid sequences, domains, or motifs, linked together in a manner that does not occur naturally or naturally in the host cell, and that functions as a receptor when present on the cell surface. A CAR can include an extracellular portion comprising an antigen binding domain (e.g., obtained or derived from an immunoglobulin or immunoglobulin-like molecule, such as a TCR binding domain derived or derived from a TCR specific for a cancer antigen, a scFv derived or derived from an antibody, or an antigen binding domain derived or derived 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) (e.g., see, 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 CAR of the present disclosure that specifically binds to the WT1 antigen (e.g., in the context of a peptide:HLA complex) includes a TCR Vα domain and a Vβ domain.

[0059] As used herein, "nucleic acid" or "nucleic acid molecule" or "polynucleotide" refers in certain aspects to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments produced, for example, by polymerase chain reaction (PCR) or in vitro translation, and fragments produced by any of ligation, cleavage, endonuclease action, or exonuclease action. In certain embodiments, the nucleic acids of the present disclosure are produced by PCR. Nucleic acids can be composed of monomers that are naturally occurring nucleotides (such as deoxynucleotides and ribonucleotides), analogs of naturally occurring nucleotides (such as α-enantiomeric forms of naturally occurring nucleotides), or a combination of both. Modified nucleotides can have modifications or replacements of sugar moieties, pyrimidine or purine base moieties. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioates, phosphorodithioates, selenophosphates, diselenosenophosphates, phosphoroanilothioates, phosphoranilidates, phosphoramidates, and the like. Nucleic acid molecules can be single-stranded or double-stranded.

[0060] In certain aspects, the term "isolated" refers to removing material from its original environment (e.g., its natural environment if naturally occurring). 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 of the coexisting materials in the natural system, is isolated. Such a nucleic acid can be part of a vector and / or such a nucleic acid or polypeptide can be part of a composition (such as a cell lysate) and still be isolated because such a vector or composition is not part of the natural environment of the nucleic acid or polypeptide. The term "gene" refers to a segment of DNA involved in producing a polypeptide chain; it includes the regions "leader and trailer" before and after the coding region, as well as intervening sequences (introns) between individual coding segments (exons).

[0061] As used herein, the term "recombinant" refers in certain aspects to a cell, microorganism, nucleic acid molecule, or vector that has been genetically engineered by human intervention—that is, modified by the introduction of exogenous or heterologous nucleic acid molecules, or to a cell or microorganism that has been altered so that the expression of an endogenous nucleic acid molecule or gene is controlled, deregulated, or constitutive. Human-induced genetic alterations can include, for example, modifications that introduce nucleic acid molecules encoding one or more proteins or enzymes (which may include expression control elements, such as promoters), or additions, deletions, substitutions, or other functional disruptions or additions to the cellular genetic material. Exemplary modifications include modifications of the coding region of a heterologous or homologous polypeptide from a reference or parent molecule, or functional fragments thereof.

[0062] As used herein, "mutation" or "variant" in certain aspects 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 nucleotide or amino acid substitutions, insertions, or deletions. 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.

[0063] In some aspects, "conservative substitutions" are considered in the art to be substitutions of one amino acid for another amino acid with similar properties. Exemplary conservative substitutions are well known in the art (see, e.g., WO 97 / 09433 at page 10; Lehninger, Biochemistry, 2 nd Edition; Worth Publishers, Inc. NY, NY, pp. 71-77, 1975; Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA, p. 8, 1990).

[0064] The term "construct" refers to any polynucleotide containing a recombinant nucleic acid molecule in some aspects. The construct can be present in a vector (such as a bacterial vector, a viral vector) or can be integrated into a genome. A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid molecule. For example, a vector can be a plasmid, a cosmid, a virus, an RNA vector, or a linear or circular DNA or RNA molecule, and can include chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acid molecules. Exemplary vectors are those that can autonomously replicate (episomal vectors) or express a nucleic acid molecule connected thereto (expression vectors).

[0065] Exemplary viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated virus), coronaviruses, negative-strand RNA viruses such as influenza myxoviruses (e.g., influenza virus), rhabdoviruses (e.g., rabies and vesicular stomatitis virus), paramyxoviruses (e.g., measles and Sendai), positive-strand RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses including adenoviruses, herpesviruses (e.g., herpes simplex virus type 1 and type 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, noroviruses, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses include avian leukosis-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).

[0066] In certain aspects, the "lentiviral vector" used herein refers to an HIV-based lentiviral vector for gene delivery, which can be integrating or non-integrating, has a relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are typically produced after transient transfection of three (packaging, envelope, and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter target cells through the interaction of viral surface glycoproteins with receptors on the cell surface. After entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.

[0067] The term "operably linked" in certain aspects refers to the association of two or more nucleic acid molecules on a nucleic acid segment such that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence when it is capable of affecting the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Non-linked" refers to the lack of close association between related genetic elements, such that the function of one does not affect the other.

[0068] As used herein, "expression vector" refers in certain aspects to a DNA construct containing a nucleic acid molecule that is operably linked to appropriate control sequences and is capable of achieving expression of the nucleic acid molecule in an appropriate host. Such control sequences include a promoter for achieving transcription, an optional operator sequence for controlling such transcription, a sequence encoding a suitable mRNA ribosome binding site, and sequences for controlling transcription and translation termination. A vector can be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and operate independently of the host genome, or in some cases, can be integrated into the genome itself. In this specification, "plasmid", "expression plasmid", "virus" and "vector" are generally used interchangeably.

[0069] As used herein, the term "expression" refers in certain aspects to the process by which a polypeptide is produced according to the coding sequence of a nucleic acid molecule (e.g., a gene). This process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

[0070] In the context of inserting a nucleic acid molecule into a cell, the term "introduced" refers in certain aspects to "transfection," or "transformation," or "transduction," including reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell, where the nucleic acid molecule can be incorporated into the genome of the cell (e.g., a chromosome, plasmid, or mitochondrial DNA), converted to autonomous replication, or transiently expressed (e.g., transfected mRNA).

[0071] As used herein, "heterologous" or "exogenous" nucleic acid molecules, constructs or sequences refer in some aspects to nucleic acid molecules or portions of nucleic acid molecules that are not native to the host cell, but may be homologous to nucleic acid molecules or portions of nucleic acid molecules of the host cell. The source of a heterologous or exogenous nucleic acid molecule, construct or sequence may be from a different genus or species. In certain embodiments, a heterologous or exogenous nucleic acid molecule is added to a host cell or host genome by, for example, conjugation, transformation, transfection, electroporation, etc., wherein the added molecule 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 a non-natural enzyme, protein or other activity encoded by an exogenous nucleic acid molecule introduced into a host cell, even if the host cell encodes a homologous protein or activity. In addition, cells comprising a "modified" or "heterologous" polynucleotide or binding protein include progeny of the cell, regardless of whether the progeny itself is transduced, transfected, or otherwise manipulated or altered.

[0072] As described herein, more than one heterologous or exogenous nucleic acid molecule can be introduced into the host cell as a separate nucleic acid molecule, as multiple individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof. For example, as disclosed herein, the host cell can be modified to express two or more heterologous or exogenous nucleic acid molecules, encoding the desired TCR (e.g., TCRα and TCRβ) specific for the WT1 antigen peptide. When two or more exogenous nucleic acid molecules are introduced into the host cell, it can be understood that the two or more exogenous nucleic acid molecules can be used as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into a single site or multiple sites on the host chromosome, or any combination thereof. The number of heterologous nucleic acid molecules or protein activities of reference 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.

[0073] As used herein, the terms "endogenous" or "native" in certain aspects refer to a gene, protein, or activity that is normally present in a host cell. Furthermore, a gene, protein, or activity that is mutated, overexpressed, mobilized, 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, endogenous control sequences (e.g., promoters, translation attenuation sequences) 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 or regulation of the second native gene or nucleic acid molecule differs from the normal expression or regulation in the parent cell.

[0074] In certain aspects, the term "homologous" or "homology" refers to a molecule or activity found in or derived from 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 be selected to have altered expression levels, a different sequence, an altered activity, or any combination thereof.

[0075] In some aspects, "sequence identity" as used herein refers to the percentage of amino acid residues in one sequence that are identical to the amino acid residues in another reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity. Percent sequence identity values ​​can be generated using NCBI BLAST 2.0 software, such as defined in Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402, with parameters set to default values.

[0076] As used herein, "hematopoietic progenitor cells" in certain aspects can be cells that can be obtained from hematopoietic stem cells or fetal tissue and are capable of further differentiating into mature cell types (such as immune system cells). Exemplary hematopoietic progenitor cells include those with CD24 Lo Lin - CD81 + Cells with a phenotype of thymic origin or cells found in the thymus (called thymic progenitor cells).

[0077] As used herein, the term "host" refers in some aspects to cells (such as T cells) or microorganisms that are genetically modified with heterologous or exogenous nucleic acid molecules to produce polypeptides of interest (such as anti-WT1 TCRs with high or enhanced affinity). In certain embodiments, host cells can be selected to already have or be modified to include other genetic modifications to confer desired properties related to or unrelated to the biosynthesis of heterologous or exogenous proteins (e.g., including detectable markers; endogenous TCRs that are deleted, altered, or truncated; increasing the expression of costimulatory factors). In some embodiments, host cells are genetically modified to express proteins or fusion proteins that regulate immune signals in host cells, for example, to promote the survival and / or amplification advantages of modified cells (e.g., see the immunomodulatory fusion proteins of WO 2016 / 141357, the contents of which are incorporated herein by reference in their entirety). In other embodiments, host cells are genetically modified to introduce TCRs provided herein, or to knock out or minimize immunosuppressive signals (such as checkpoint inhibitors) in cells, which modifications can be performed using, for example, CRISPR / Cas systems (see, for example, US2014 / 0068797, U.S. Patent Application No. 8,697,359; WO 2015 / 071474). In certain embodiments, host cells are human hematopoietic progenitor cells transduced with heterologous or exogenous nucleic acid molecules encoding TCR α chains specific for WT1 antigen peptides.

[0078] As used herein, "hyperproliferative disease" refers in certain aspects to excessive growth or proliferation compared to normal or disease-free cells. Exemplary proliferative diseases include tumors, cancers, neoplastic tissues, carcinomas, sarcomas, malignant cells, pre-malignant cells, and non-neoplastic or non-malignant proliferative diseases (e.g., adenomas, fibromas, lipomas, leiomyomas, hemangiomas, fibrosis, restenosis, and autoimmune diseases such as rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, etc.). Certain diseases involving abnormal or excessive growth, which occur more slowly than proliferative diseases, may be referred to as "proliferative diseases," and include certain tumors, cancers, neoplastic tissues, carcinomas, sarcomas, malignant cells, pre-malignant cells, and non-neoplastic or non-malignant diseases.

[0079] In addition, "cancer" may refer to any accelerated proliferation of cells, including solid tumors, ascites tumors, hematological or lymphoid or other malignancies; connective tissue malignancies; metastatic disease; minimal residual disease after organ or stem cell transplantation; multidrug-resistant cancers, primary or secondary malignancies, angiogenesis-related or other forms of cancer.

[0080] Specific TCR for WT1 p37 antigen peptide

[0081] In certain aspects, the present invention discloses a WT1 p37 peptide-specific T cell receptor (TCR), comprising (a) a T cell receptor (TCR) α chain variable (V α ) domain, and a TCRβ chain variable (V ) domain having a CDR3 amino acid sequence shown in any one of SEQ ID NOs: 1-11, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, and 241. β (b) a TCR V having a CDR3 amino acid sequence shown in any one of SEQ ID NOs: 12-22, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, and 238 α domain, and TCR V β or (c) a TCR V domain having a CDR3 amino acid sequence as shown in any one of SEQ ID NOs: 12-22, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, and 238. α domain, and a TCR V domain having a CDR3 amino acid sequence shown in any one of SEQ ID NOs: 1-11, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, and 241. β For example, any TCR or its binding domain disclosed herein can specifically bind to the WT1 p37 peptide:HLA complex on the surface of a cell (e.g., a T cell), and / or can promote the production of IFNγ, pEC 505, or more (e.g., up to about 8.6, up to about 8.65, up to about 8.7, up to about 8.72, up to about 8.75, up to about 8.8, up to about 9, up to about 9.1, up to about 9.2, up to about 9.3, up to about 9.4, up to about 9.5, up to about 9.6, up to about 9.68, up to about 9.7, up to about 9.75, up to about 10, up to about 10.5, up to about 11, up to about 11.5, up to about 12, up to about 12.5, or up to about 13). In certain embodiments, the TCRs of the present disclosure can specifically bind to the VLDFAPPGA (SEQ ID NO: 59): human leukocyte antigen (HLA) complex, IFNγ-producing pEC 50 9.0 or higher, or IFNγ production pEC 50 In certain embodiments, the TCR or its binding domain (such as scTCR or its fusion protein) disclosed herein can specifically bind to the WT1p37 peptide:HLA complex and promote the production of IFNγ, and its pEC 50 The range is 8.5 to about 9.9, or 8.6 to about 9.8, or 8.7 to about 9.7, or 8.75 to about 9.65, etc. EC 50 can range from approximately 1.1×10 -9 M to approximately 3.0×10 -10 M, or any value in between. In a further example, any TCR disclosed herein can specifically bind to a WT1 peptide:HLA complex on the cell surface, independent of or in the absence of CD8. In a further embodiment, the TCR specifically binds to a VLDFAPPGA (SEQ ID NO: 59):human leukocyte antigen (HLA) complex with a K of D Less than or equal to approximately 10 -9 M, in certain embodiments, HLA includes HLA-A*201. The peptide antigen VLDFAPPGA (SEQ ID NO: 59) is a WT1 peptide antigen corresponding to amino acids 37-45 of the WT1 protein.

[0082] In any of the embodiments described herein, the present disclosure provides a T cell receptor (TCR) comprising an α chain and a β chain, wherein the TCR binds to a WT1:HLA-A*201 complex on the surface of a T cell and promotes (a) IFNγ production by pEC 50 to 8.5 or more (e.g., to about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, or about 13); or (b) binds to the cell surface independently of or in the absence of CD8.

[0083] In certain embodiments, V βThe domain comprises or is derived from TRBV7-6*01 / TRBJ2-7*01, TRBV20-1*02 / TRBJ2-7*01, TRBV15*02 / TRBJ1-5*01, TRBV13*01 / TRBJ2-5*01, TRBV50*01 / TRBJ2-7*01, TRBV11-3*01 / TRBJ1-1*01, TRBV19*01 / TRBJ1-6*02, TRBV27*01 / TRBJ2-7*01, TRBV13*01 / TRBJ2-7*01, TRBV11-1*01 / TRBJ14*01, or TRBV4-3*01 / TRBJ1-3*01. In further embodiments, V α The domain comprises or is derived from TRAV21*02 / TRAJ58*01, TRAV38-1*01 / TRAJ40*01, TRAV29 / DV5*01 / TRAJ6*01, TRAV29 / DV5*01 / TRAJ20*01, TRAV41*01 / TRAJ50*01, TRAV12-2*01 / TRAJ11*01, TRAV1-2*01 / TRAJ20*01, TRAV20*02 / TRAJ8*01, TRAV26-1*02 / TRAJ26*01, TRAV24*01 / TRAJ48*01, or TRAV20*02 / TRAJ37*02. In specific embodiments, the TCR comprises (a) V β domain (including or derived from TRBV7-6*01 / TRBJ2-7*01) and V α domain (including or derived from TRAV21*02 / TRAJ58*01); (b) V β domain (including or derived from TRBV27*01 / TRBJ2-7*01) and V α domain (comprising or derived from TRAV20*02 / TRJ8*01); or (c) V β Domain (including or derived from TRBV13*01 / TRBJ2-5*01), V α domain (comprising or derived from TRAV29 / DV5*01 / TRAJ20*01).

[0084] In certain embodiments, the TCR of the present disclosure further comprises: (i) a CDR1α amino acid sequence as set forth in any one of SEQ ID NOs: 194, 176, 182, 188, 200, 206, 212, 218, 224, 230, and 236, or a variant thereof comprising one or two amino acid substitutions, wherein, optionally, the one or two amino acid substitutions comprise conservative amino acid substitutions; and / or (ii) a CDR2α amino acid sequence as set forth in any one of SEQ ID NOs: 195, 177, 183, 189, 201, 207, 213, 219, 225, 231, and 237, or a variant thereof comprising one or two amino acid substitutions, wherein, optionally, the one or two amino acid substitutions comprise conservative amino acid substitutions.

[0085] In certain embodiments, the TCR of the present disclosure further comprises: (i) a CDR1β amino acid sequence as set forth in any one of SEQ ID NOs. 197, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233 and 239, or a variant thereof comprising one or two amino acid substitutions, wherein, optionally, the one or two amino acid substitutions comprise conservative amino acid substitutions; and / or (ii) a CDR2β amino acid sequence as set forth in any one of SEQ ID NOs: 198, 180, 186, 192, 204, 210, 216, 222, 228, 234 and 240, or a variant thereof comprising one or two amino acid substitutions, wherein, optionally, the one or two amino acid substitutions comprise conservative amino acid substitutions.

[0086] In certain embodiments, the TCRs of the present disclosure comprise canonical amino acid sequences of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β set forth in: (i) SEQ ID NOs: 194, 195, 196 or 12, 197, 198, and 199 or 1, respectively; (ii) SEQ ID NOs: 176, 177, 178 or 18, 179, 180, and 181 or 7, respectively; (iii) SEQ ID NOs: 182, 183, 184 or 20, 185, 186, and 187 or 9, respectively; (iv)

[0087] SEQ ID NO: 188, 189, 190 or 21, 191, 192, and 193 or 10, respectively; (v) SEQ ID NO: 200, 201, 202 or 13, 203, 204, and 205 or 2, respectively; (vi) SEQ ID NO: 206, 207, 208 or 14, 209, 210, and 211 or 3, respectively; (vii) SEQ ID NO: 212, 213, 214 or 15, 215, 216, and 217 or 4, respectively; (viii) SEQ ID NO: 218, 219, 220 or 17, 221, 222, and 223 or 6, respectively; (ix) SEQ ID NO: 224, 225, 226 or 19, 227, 228, and 229 or 8, respectively; (x) SEQ ID NO: 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, NO: 230, 231, 232 or 22, 233, 234, and 235 or 11; or (xi) SEQ ID NO: 236, 237, 238 or 16, 238, 240, and 241 or 5, respectively.

[0088] Any polypeptide disclosed herein, as encoded by a polynucleotide sequence, may include a "signal peptide" (also referred to as a leader sequence, leader peptide, or transit peptide). The signal peptide targets the newly synthesized polypeptide to an appropriate location inside or outside the cell. The signal peptide can be removed from the polypeptide during or once localization or secretion is complete. A polypeptide with a signal peptide is referred to herein as a "proprotein," and a polypeptide without the signal peptide is referred to herein as a "mature" protein or polypeptide. In any embodiment disclosed herein, the binding protein or fusion protein comprises or is a mature protein, or is or includes a proprotein.

[0089] In certain embodiments, amino acid residues 1-19 of SEQ ID NO: 23 are or include a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23, with amino acid residues 1-19 of SEQ ID NO: 23 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 242).

[0090] In certain embodiments, amino acid residues 1-15 of SEQ ID NO: 24 are or comprise a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain comprising or consisting of the amino acid sequence of SEQ ID NO: 23, with amino acid residues 1-15 of SEQ ID NO: 24 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 243).

[0091] In certain embodiments, amino acid residues 1-19 of SEQ ID NO: 25 are or include a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain comprising or consisting of the amino acid sequence of SEQ ID NO: 25, with amino acid residues 1-15 of SEQ ID NO: 25 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 244).

[0092] In certain embodiments, amino acid residues 1-29 of SEQ ID NO: 26 are or include a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain comprising or consisting of the amino acid sequence of SEQ ID NO: 26, with amino acid residues 1-29 of SEQ ID NO: 26 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 245).

[0093] In certain embodiments, amino acid residues 1-19 of SEQ ID NO: 27 are or include a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain comprising or consisting of the amino acid sequence of SEQ ID NO: 27 with amino acid residues 1-19 of SEQ ID NO: 27 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 246).

[0094] In certain embodiments, amino acid residues 1-19 of SEQ ID NO: 28 are or include a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain comprising or consisting of the amino acid sequence of SEQ ID NO: 28, with amino acid residues 1-19 of SEQ ID NO: 28 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 247).

[0095] In certain embodiments, amino acid residues 1-19 of SEQ ID NO: 29 are or include a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain comprising or consisting of the amino acid sequence of SEQ ID NO: 29 with amino acid residues 1-19 of SEQ ID NO: 29 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 248).

[0096] In certain embodiments, amino acid residues 1-19 of SEQ ID NO: 30 are or include a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain comprising or consisting of the amino acid sequence of SEQ ID NO: 30, with amino acid residues 1-19 of SEQ ID NO: 30 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 249).

[0097] In certain embodiments, amino acid residues 1-29 of SEQ ID NO: 31 are or include a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain comprising or consisting of the amino acid sequence of SEQ ID NO: 31 with amino acid residues 1-29 of SEQ ID NO: 31 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 250).

[0098] In certain embodiments, amino acid residues 1-19 of SEQ ID NO: 32 are or include a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain comprising or consisting of the amino acid sequence of SEQ ID NO: 32, with amino acid residues 1-19 of SEQ ID NO: 32 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 251).

[0099] In certain embodiments, amino acid residues 1-19 of SEQ ID NO: 33 are or include a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain comprising or consisting of the amino acid sequence of SEQ ID NO: 33, with amino acid residues 1-19 of SEQ ID NO: 33 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 252).

[0100] In certain embodiments, amino acid residues 1-19 of SEQ ID NO: 34 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain comprising or consisting of the amino acid sequence of SEQ ID NO: 34, but with amino acid residues 1-19 of SEQ ID NO: 34 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 253).

[0101] In certain embodiments, amino acid residues 1-20 of SEQ ID NO: 35 are or include a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain comprising or consisting of the amino acid sequence of SEQ ID NO: 35, but with amino acid residues 1-20 of SEQ ID NO: 35 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 254).

[0102] In certain embodiments, amino acid residues 1-26 of SEQ ID NO: 36 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain comprising or consisting of the amino acid sequence of SEQ ID NO: 36, but with amino acid residues 1-26 of SEQ ID NO: 36 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 255).

[0103] In certain embodiments, amino acid residues 1-26 of SEQ ID NO: 37 are or include a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain comprising or consisting of the amino acid sequence of SEQ ID NO: 37, but with amino acid residues 1-26 of SEQ ID NO: 37 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 256).

[0104] In certain embodiments, amino acid residues 1-22 of SEQ ID NO: 38 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain comprising or consisting of the amino acid sequence of SEQ ID NO: 38, but with amino acid residues 1-22 of SEQ ID NO: 38 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 257).

[0105] In certain embodiments, amino acid residues 1-21 of SEQ ID NO: 39 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain comprising or consisting of the amino acid sequence of SEQ ID NO: 39, but with amino acid residues 1-21 of SEQ ID NO: 39 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 258).

[0106] In certain embodiments, amino acid residues 1-17 of SEQ ID NO: 40 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain comprising or consisting of the amino acid sequence of SEQ ID NO: 40, but with amino acid residues 1-17 of SEQ ID NO: 40 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 259).

[0107] In certain embodiments, amino acid residues 1-21 of SEQ ID NO: 41 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain comprising or consisting of the amino acid sequence of SEQ ID NO: 41, but with amino acid residues 1-21 of SEQ ID NO: 41 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 260).

[0108] In certain embodiments, amino acid residues 1-17 of SEQ ID NO: 42 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain comprising or consisting of the amino acid sequence of SEQ ID NO: 42, but with amino acid residues 1-17 of SEQ ID NO: 42 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 261).

[0109] In certain embodiments, amino acid residues 1-22 of SEQ ID NO: 43 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain comprising or consisting of the amino acid sequence of SEQ ID NO: 43, but with amino acid residues 1-22 of SEQ ID NO: 43 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 262).

[0110] In certain embodiments, amino acid residues 1-21 of SEQ ID NO: 44 are or include a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain comprising or consisting of the amino acid sequence of SEQ ID NO: 44, but with amino acid residues 1-21 of SEQ ID NO: 44 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 263).

[0111] In certain embodiments, the specific T cell receptor (TCR) for the WT1 peptide:HLA complex has a V α domain, the Vα The domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 253-263 and 34-33, having V β domain, the V β The domain comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 242-252 and 23-33 or any combination thereof. α The domain comprises or consists of the amino acid sequence of SEQ ID NO: 34, V β The domain comprises or consists of the amino acid sequence of SEQ ID NO: 23. In further specific embodiments, (a) V α The domain comprises or consists of the amino acid sequence of SEQ ID NO: 41, V β The domain comprises or consists of the amino acid sequence of SEQ ID NO: 30; (b) V α The domain comprises or consists of the amino acid sequence of SEQ ID NO: 37, V β The domain comprises or consists of the amino acid sequence of SEQ ID NO: 26; or (c) V α The domain comprises or consists of the amino acid sequence of SEQ ID NO: 42, V β The domain comprises or consists of the amino acid sequence of SEQ ID NO: 31. In further specific embodiments, V α The domain comprises or consists of the amino acid sequence of SEQ ID NO: 24, V β The domain comprises or consists of the amino acid sequence of SEQ ID NO:35.

[0112] In some embodiments, the Vα domain and the Vβ domain comprise or consist of the amino acid sequences set forth in the following SEQ ID NOs: (i) 253 and 242, respectively; (ii) 259 and 248, respectively; (iii) 261 and 250, respectively; (iv) 262 and 251, respectively; (v) 257 and 246, respectively; (vi) 254 and 243, respectively; (vii) 255 and 244, respectively; (viii) 256 and 245, respectively; (ix) 258 and 247, respectively; (x) 260 and 249, respectively; (xi) 263 and 252, respectively; (xii) 34 and 23, respectively; (xiii) 40 and 29, respectively; (xiv) 42 and 31, respectively; (xv) 43 and 32, respectively; (xvi) 35 and 24, respectively; (xvii) 36 and 25, respectively; (xviii) 37 and 26, respectively; (xix) 39 and 28, respectively; (xx) 41 and 30, respectively; (xxi) 44 and 33, respectively; or (xxii) 38 and 27, respectively.

[0113] In certain embodiments, the high-functional affinity recombinant TCRs for WT1 p37 peptide as described herein include variant polypeptide species whose amino acid sequences have one or more amino acid substitutions, insertions, or deletions relative to the amino acid sequences of any one or more of SEQ ID NOs: 48-58 set forth herein, provided that the CDR3 is not altered and the TCR retains or substantially retains its specific WT1 p37 binding function.

[0114] Conservative substitutions of amino acids are well known and may occur naturally or be introduced during recombinant TCRs. Amino acid substitutions, deletions, and additions can be introduced into proteins using mutagenesis methods known in the art (see, for example, 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 that have changed specific codons according to the desired replacement, deletion, or insertion. In addition, random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error-prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis, can be used to prepare immunogenic polypeptide variants (see, for example, Sambrook et al., supra).

[0115] Various criteria known to those skilled in the art indicate whether a substituted amino acid at a particular position in a peptide or polypeptide is conservative (or similar). For example, a similar amino acid or conservative amino acid substitution refers to a substitution of an amino acid residue with an amino acid residue having a similar side chain. 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 branches (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline, which is considered more difficult to classify, shares properties with amino acids having aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine). In some cases, replacing glutamic acid with glutamine or aspartic acid with asparagine can be considered a similar substitution, as glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. As is understood in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence of the polypeptide and its conservative 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).

[0116] Variants of wild-type TCRs specific for the WT1 p37 antigen:MHC complex, or their binding domains, may include TCRs having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% amino acid sequence identity to any of the exemplary amino acid sequences disclosed herein (e.g., SEQ ID NOs: 23-58), with the proviso that V β CDR3 and V αThe CDR3 of the domain does not contain any changes, and the changes in other parts do not reduce the functional affinity (or relative affinity) by more than 10%, 15% or 20% compared to the wild-type TCR. In some optional embodiments, the variant TCR further includes any one of SEQ ID NOs: 34-44 (parent Vα domain) or any one of SEQ ID NOs: 23-33 (parent Vβ domain) Vα domain CDR1, Vα domain CDR2, Vβ domain CDR1, Vβ domain CDR2 or any combination thereof, and the amino acid sequence is unchanged. In each of these embodiments, the TCR retains its ability to specifically induce IFNγ production, pEC 50 is 8.5, 8.6, 8.7, 8.8, 8.9 or higher, or the TCR retains its ability to specifically bind to a peptide antigen:HLA complex (e.g., VLDFAPPGA (SEQ ID NO: 59):HLA complex), K D Less than or equal to approximately 10 -9 M, and specifically binds 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.3-fold, 3.5-fold, up to 5-fold better than the wild-type TCR consisting of any one of SEQ ID NOs: 48-58.

[0117] In a further embodiment, the present disclosure provides a p37-specific TCR or binding domain thereof, comprising (a) a TCR α chain variable (Vα) domain having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 34-35 and 38-44, and a TCR β chain variable (Vβ) domain having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 23-25, 27, 28, 30, 32, and 33; (b) a TCR Vα domain having at least 92% sequence identity to the amino acid sequence of SEQ ID NOs: 36 or 37, and a TCR Vβ domain having at least 92% sequence identity to the amino acid sequence of SEQ ID NOs: NOs: 23-25, 27, 28, 30, 32 and 33 having at least 90%, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity; or (c) a TCR Vα domain comprising or consisting of the amino acid sequence of SEQ ID NOs: 34-44, and a TCR Vβ domain having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 23-25, 27, 28, 30, 32 and 33.

[0118] In further embodiments, the present disclosure provides a p37-specific TCR or binding domain thereof comprising (a) a TCR Va domain having at least 90% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 34-35 and 38-44, and a V beta domain having at least 92% (e.g., 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 29; (b) a TCR Va domain having at least 92% sequence identity to the amino acid sequence of SEQ ID NOs: 36 or 37, and a TCR V beta domain having at least 92% sequence identity to the amino acid sequence of SEQ ID NO: 29; or (c) a TCR Va domain comprising or consisting of the amino acid sequence of SEQ ID NOs: 34-44, and a TCR V beta domain having at least 92% sequence identity to the amino acid sequence of SEQ ID NO: 29.

[0119] In yet a further embodiment, the present disclosure provides a p37-specific TCR, or a binding domain thereof, comprising (a) a TCR Vα domain having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 34-35 and 38-44, and a TCR Vβ domain having at least 93% sequence identity to the amino acid sequence of SEQ ID NO: 31; (b) a TCR Vα domain having at least 92% sequence identity to the amino acid sequence of SEQ ID NO: 36 or 37, and a TCR Vβ domain having at least 93% sequence identity to the amino acid sequence of SEQ ID NO: 31; or (c) a TCR Vα domain comprising or consisting of the amino acid sequence of SEQ ID NOs: 34-44, and a TCR Vβ domain having at least 93% sequence identity to the amino acid sequence of SEQ ID NO: 31.

[0120] In further embodiments, the present disclosure provides a p37-specific TCR, or a binding domain thereof, comprising (a) a TCR Vα domain having at least 90% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 34-35 and 38-44, and a Vβ domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 26; (b) a TCR Vα domain having at least 92% sequence identity to the amino acid sequence of SEQ ID NO: 36 or 37, and a TCR Vβ domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 26; or (c) a TCR Vα domain comprising or consisting of the amino acid sequence of SEQ ID NOs: 34-44, and a TCR Vβ domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 26.

[0121] In further embodiments, the present disclosure provides a p37-specific TCR, or a binding domain thereof, comprising (a) a TCR Vα domain having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 34-35 and 38-44, and a Vβ domain comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 23-33; (b) a TCR Vα domain having at least 92% sequence identity to the amino acid sequence of SEQ ID NOs: 36 or 37, and a TCR Vβ domain comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 23-33; or (c) a TCR Vα domain comprising or consisting of the amino acid sequence of SEQ ID NOs: 34-44, and a TCR Vβ domain comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 23-33.

[0122] In any of the above embodiments, the TCR is capable of binding to the WT1 p37 peptide VLDFAPPGA (SEQ ID NO: 59):HLA complex on the surface of cells (eg, T cells) and specifically inducing the production of IFNγ, pEC 50 is 8.5, 8.6, 8.7, 8.8, 8.9 or higher, and / or the TCR is capable of specific binding to the WT1 peptide VLDFAPPGA (SEQ ID NO: 59): HLA cell surface complex independent of or in the absence of CD8. In any of the above embodiments, the amino acid sequence of the Vβ domain, including CDR1 and / or CDR2, is unchanged from the CDR1 and / or CDR2 present in any of SEQ ID NOs: 23-33, respectively.

[0123] In certain embodiments, any of the above-mentioned WT1 p37 peptide-specific T cell receptors (TCRs) can be an antigen-binding fragment of a TCR. In further implementations, the antigen-binding fragment of a TCR includes a single-chain TCR (scTCR), which can be contained in a chimeric antigen receptor (CAR). In some embodiments, the WT1 p37 peptide-specific TCR is a multi-chain binding protein, e.g., comprising a TCR α chain comprising a Vα domain and an α chain constant domain, wherein the TCR α chain constant domain has at least about 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of SEQ ID NO: 47; and a TCR β chain comprising a Vβ domain and a β chain constant domain, wherein the TCR β chain constant domain has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the amino acid sequence of SEQ ID NO: 45 or 46. In a further embodiment, the present disclosure provides a WT1 p37 peptide-specific TCR comprising or consisting of an α chain constant domain having the amino acid sequence of SEQ ID NO: 47, and / or comprising or consisting of a β chain constant domain having the amino acid sequence of SEQ ID NO: 45 or 46.

[0124] In a further embodiment, the present disclosure provides a WT1 p37 peptide-specific TCR comprising a TCR α chain comprising a Vα domain and an α chain constant domain, wherein: (a) the Vα domain has at least 90% sequence identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to the amino acid sequence set forth in any one of SEQ ID NOs: 34-35 and 38-44, and the α chain constant domain has at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 47; or (b) the Vα domain has at least 92% sequence identity to the amino acid sequence of SEQ ID NOs: 36 or 37, and the α chain constant domain has at least 98% sequence identity to the amino acid sequence of SEQ ID NO: 47.

[0125] In some embodiments, the TCR comprises a TCR alpha chain comprising a Valpha domain and an alpha chain constant domain, wherein: (a) the Valpha domain comprises the amino acid sequence set forth in any one of SEQ ID NOs: 242-252 and 34-44, and the alpha chain constant domain comprises the amino acid sequence of SEQ ID NO: 47; or (b) the Valpha domain consists of the amino acid sequence of any one of SEQ ID NOs: 242-252 and 34-44, and the alpha chain constant domain has at least 90% identity (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) to, comprises, or consists of the amino acid sequence of SEQ ID NO: 47.

[0126] In some embodiments, the α chain constant domain is present, and the Vα domain and the α chain constant domain together constitute the TCRα chain. In some embodiments, the β chain constant domain is present, and the Vβ domain and the β chain constant domain together constitute the TCRβ chain.

[0127] In some embodiments, TCR includes scTCR, or scTCR is provided, which is derived from TCR of the present disclosure. In some embodiments, TCR includes CAR, or CAR is provided, which is derived from (e.g., including one or more variable domains, which are derived from) TCR currently disclosed.

[0128] In further embodiments, a composition is provided, comprising a WT1-specific high-functional affinity recombinant TCR or its binding domain according to any one of the above embodiments and a pharmaceutically acceptable carrier, diluent or excipient.

[0129] For example, a method for isolating and purifying a recombinantly produced soluble TCR may comprise 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 commercially available filters. 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 immunogens from their natural environment. Methods for large-scale production of one or more isolated / recombinant soluble TCRs described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of the soluble TCR can be carried out according to the methods described herein and known in the art, and in compliance with the laws and guidelines of domestic and foreign regulatory agencies.

[0130] In certain embodiments, nucleic acid molecules encoding high-affinity or high-functional avidity TCRs specific for the WT1 p37 peptide complexed with MHC 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., Blood 114:4099, 2009; Robins et al., Sci. Translat. Med. 2:47ra64, 2010; Robins et al., (Sept. 10) J. Imm. Meth. Epub ahead of print, 2011; Warren et al., Genome Res. 21:790, 2011) and can be employed in the practice of embodiments according to the present disclosure. Likewise, 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 well as adoptive transfer procedures using T cells with 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 2011 / 0243972; US 2011 / 0189141; Leen 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., Hum. Gen. 20:1240, 2009; et al., Ann. Rev. Immunol. 25:243, 2007), therefore, according to the teachings herein, high affinity TCRs, including those specific for WT1 peptide antigens complexed with HLA receptors, can be considered to be adapted to the presently disclosed embodiments.

[0131] The WT1-specific TCRs or their binding domains described herein (e.g., SEQ ID NOs: 23-58 and non-CDR3 variants thereof) can be functionally characterized by any of a number of well-established methods for measuring T cell activity, including determining T cell binding, activation, or induction, as well as determining antigen-specific responses of T cells. Examples include determining T cell proliferation, T cell cytokine release, antigen-specific T cell stimulation, MHC-restricted T cell stimulation, cytotoxic T lymphocyte (CTL) activity (e.g., by detecting pre-loaded target cells). 51Cr release), 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). See also 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.

[0132] Polynucleotide encoding WT1 p37 antigen peptide-specific TCR

[0133] Heterologous, isolated, or recombinant nucleic acid molecules encoding high-affinity or high-functional avidity recombinant T cell receptors (TCRs) or their binding domains (e.g., scTCRs or fusion proteins thereof) specific for the WT1 p37 peptide can be produced and prepared according to the various methods and techniques described herein (see Examples). Construction of expression vectors for recombinantly producing high-affinity or high-functional avidity engineered TCRs or their binding domains specific for the target WT1 p37 peptide can be accomplished using any suitable molecular biology engineering technique known in the art, including restriction enzyme digestion, ligation, transformation, plasmid purification, and DNA sequencing, such as those described in Sambrook et al. (1989 and 2001 editions; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY) and Ausubel et al. (Current Protocols in Molecular Biology, 2003). In order to obtain efficient transcription and translation, the polynucleotide in each recombinant expression construct includes at least one appropriate expression control sequence (also called regulatory sequence), such as a leader sequence, in particular a promoter that is operably (i.e., operatively) linked to the nucleotide sequence encoding the immunogen.

[0134] Certain embodiments relate to nucleic acids encoding the polypeptides contemplated herein, for example, engineered TCRs or binding domains thereof that are specific for the WT1 p37 peptide:MHC complex with high affinity or high functional affinity. As will be appreciated by those skilled in the art, nucleic acids can refer to any form of single-stranded or double-stranded DNA, cDNA, or RNA, and can include both positive and negative strands of complementary nucleic acids, including antisense DNA, cDNA, and RNA. Also included are siRNAs, microRNAs, RNA-DNA hybrids, ribozymes, and various other naturally occurring or synthetic forms of DNA or RNA.

[0135] In certain embodiments, provided herein are isolated polynucleotides encoding engineered (e.g., codon-optimized) WT1 p37 peptide-specific high-functional affinity TCRs or their binding domains, wherein the Vα domain is encoded by a polynucleotide that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 97, 98, and 101-107. In specific embodiments, the Vα domain encoded by the polynucleotide comprises or consists of a nucleotide sequence set forth in any one of SEQ ID NOs: 97-107. In further embodiments, the polynucleotides provided herein encode the high functional affinity engineered TCRs of the present disclosure that are specific for the WT1 p37 peptide, or binding domains thereof, wherein the Vβ domain is encoded by a polynucleotide that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% identical to the nucleotide sequence set forth in any one of SEQ ID NOs: 75-77, 79, 82, 84 and 85. In specific embodiments, the Vβ domain is encoded by a polynucleotide comprising or consisting of the nucleotide sequence set forth in any one of SEQ ID NOs: 75-85.

[0136] In some embodiments, a TCR or binding domain thereof provided herein comprises a Vα domain encoded by a polynucleotide having at least 75% (75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100%) sequence identity to any one of SEQ ID NOs: 97, 98, and 101-107, or a Vα domain encoded by a polynucleotide having at least 94% sequence identity to a polynucleotide sequence of SEQ ID NOs: 99 or 100, or a Vα domain encoded by a polynucleotide comprising or consisting of SEQ ID NOs: NO: 97-107; and a Vβ domain encoded by the following polynucleotide: the polynucleotide has at least 75% sequence identity with the polynucleotide sequence listed in any one of SEQ ID NO: 75-77, 79, 82, 84 and 85, or the Vβ domain encoded by the following polynucleotide: the polynucleotide has at least 95% identity with the polynucleotide sequence listed in any one of SEQ ID NO: 78, 80, 81 and 83, or the Vβ domain encoded by the following polynucleotide: the polynucleotide includes or consists of the nucleotide sequence listed in any one of SEQ ID NO: 75-85.

[0137] In any of the above embodiments, the polynucleotide encoding the Vα domain, the Vβ domain, or both may further encode an α chain constant domain or a β chain constant domain, respectively. In certain embodiments, the TCRs of the present disclosure include a TCR α chain constant domain, wherein the α chain constant domain is encoded by a polynucleotide comprising at least 98% to 100% sequence identity to SEQ ID NO: 110. In specific embodiments, the α-chain constant domain is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 110. In further embodiments, provided herein is a β-chain constant domain encoded by a polynucleotide having at least 99.9% to 100% sequence identity to SEQ ID NO: 108 or 109. In specific embodiments, the β-chain constant domain is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 108 or 109.

[0138] In any of the above embodiments, the polynucleotide encoding the TCR includes a TCR α chain, a TCR β chain, or both. In certain embodiments, the TCR of the present disclosure is encoded by a polynucleotide comprising a nucleotide sequence encoding a self-cleaving peptide, which is arranged between a polynucleotide sequence encoding the TCR α chain and a polynucleotide sequence encoding the TCR β chain. Exemplary self-cleaving peptides include the amino acid sequence of any one of SEQ ID NOs: 60-63; or consist of the amino acid sequence of any one of SEQ ID NOs: 60-63. Such self-cleaving peptides can be encoded by a polynucleotide comprising the polynucleotide sequence of any one of SEQ ID NOs: 166-170; or a polynucleotide consisting of the polynucleotide sequence of any one of SEQ ID NOs: 166-170.

[0139] In certain embodiments, the TCR α chain, the self-cleaving peptide, and the TCR β chain are encoded by a polynucleotide that is at least 95% (e.g., 95%, 96%, 97%, 98%, 99%, or 100%) identical to any one of SEQ ID NOs: 155-165. In further embodiments, the TCR α chain, the self-cleaving peptide, and the TCR β chain are encoded by a polynucleotide that includes or consists of the sequence of any one of SEQ ID NOs: 155-165. In further embodiments, the encoded TCR alpha chain, self-cleaving peptide and TCR beta chain comprise at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) identity to any one of SEQ ID NOs: 48-58, or the encoded TCR alpha chain, self-cleaving peptide and TCR beta chain comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 48-58.

[0140] In any of the embodiments disclosed herein, the polynucleotide encoding the binding protein may further include: (i) a polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor α chain, wherein, optionally, the encoded polypeptide is or includes a CD8 co-receptor α chain; (ii) a polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor β chain, wherein, optionally, the encoded polypeptide is or includes a CD8 co-receptor β chain; or (iii) the polynucleotide of (i) and the polynucleotide of (ii). Without being bound by theory, in certain embodiments, co-expression or simultaneous expression of the binding protein and a CD8 co-receptor protein or a portion thereof having a function of binding to an HLA molecule can increase the expression of host cells (e.g., immune cells, such as T cells, optionally CD4 T cells) compared to expression of the binding protein alone. +It is understood that the binding protein encoding polynucleotide and the CD8 co-receptor polypeptide encoding polynucleotide can be present in a single nucleic acid molecule (e.g., in the same expression vector) or in different nucleic acid molecules in the host cell.

[0141] In certain further embodiments, the polynucleotide comprises: (a) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain; (b) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor β chain; and (c) a polynucleotide encoding a self-cleaving peptide positioned between the polynucleotide of (a) and the polynucleotide of (b). In further embodiments, the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide positioned between: (1) a polynucleotide encoding a binding protein (such as a TCR of the present disclosure) and a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain; and / or (2) a polynucleotide encoding a binding protein and a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor β chain.

[0142] In further embodiments, the polynucleotide may comprise, operably linked in frame: (i) (pnCD8α)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnTCR); (ii) (pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnTCR); (iii) (pnTCR)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnCD8β); (iv) (pnTCR)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnCD8α); (v) (pnCD8α)-(pnSCP1)-( or (vi) (pnCD8β)-(pnSCP1)-(pnTCR)-(pnSCP2)-(pnCD8α), wherein pnCD8α is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, wherein pnCD8β is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, wherein pnTCR is a polynucleotide encoding a TCR, and wherein pnSCP1 and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides can be selected to be the same or different (e.g., P2A, T2A, F2A, E2A).

[0143] In some embodiments, the encoded TCR comprises a TCR alpha chain and a TCR beta chain, wherein the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between a polynucleotide encoding the TCR alpha chain and a polynucleotide encoding the TCR beta chain. In certain embodiments, the polynucleotide comprises, operably linked in frame: (i) (pnCD8alpha)-(pnSCP1)-(pnCD8beta)-(pnSCP2)-(pnTCRbeta)-(pnSCP3)-(pnTCRalpha);

[0144] (ii) (pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnTCRβ)-(pnSCP3)-(pnTCRα); (iii) (pnCD8α)-( pnSCP1)-(pnCD8β)-(pnSCP2)-(pnTCRα)-(pnSCP3)-(pnTCRβ); (iv)(pnCD8β)-(pnSCP1)-(pnCD8α )-(pnSCP2)-(pnTCRα)-(pnSCP3)-(pnTCRβ); (v)(pnTCRβ)-(pnSCP1)-(pnTCRα)-(pnSCP2)-(pnCD 8α)-(pnSCP3)-(pnCD8β); (vi)(pnTCRβ)-(pnSCP1)-(pnTCRα)-(pnSCP2)-(pnCD8β)-(pnSCP3)-(p or (viii) (pnTCRα)-(pnSCP1)-(pnTCRβ)-(pnSCP2)-(pnCD8β)-(pnSCP3)-(pnCD8α), wherein pnCD8α is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, wherein pnCD8β is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, wherein pnTCRα is a polynucleotide encoding a TCRα chain, wherein pnTCRβ is a polynucleotide encoding a TCRβ chain, and wherein pnSCP1, pnSCP2 and pnSCP3 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides may be selected to be the same or different.

[0145] In a further embodiment, the binding protein is expressed as part of a transgenic construct that encodes, and / or the host cell of the present disclosure can encode: one or more additional auxiliary proteins, such as a safety switch protein; a tag, a selection marker; a CD8 co-receptor β chain; a CD8 co-receptor α chain or both; or any combination thereof. PCT application PCT / US2017 / 053112 describes polynucleotides and transgenic constructs for encoding and expressing 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), which polynucleotides, transgenic constructs and auxiliary components, including their nucleotide and amino acid sequences, are incorporated herein by reference. It will be understood that any or all of the binding proteins, safety switch proteins, tags, selection markers, CD8 co-receptor β chains or CD8 co-receptor α chains of the present disclosure can be encoded by a single nucleic acid molecule or by multiple polynucleotide sequences that are or are present on separate nucleic acid molecules.

[0146] 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 of 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 from the 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).

[0147] Other auxiliary components useful for the modified host cells of the present disclosure include tags or selectable markers that allow cells to be identified, sorted, isolated, enriched, or tracked. For example, labeled host cells with desired characteristics (such as antigen-specific TCRs and safety switch proteins) can be sorted from unlabeled cells in a sample and more efficiently activated and expanded for inclusion in a product with a desired purity.

[0148] As used herein, the term "selective marker" includes nucleic acid constructs (and encoded gene products) that confer recognizable changes to cells, allowing detection and positive selection of immune cells transduced with polynucleotides comprising selectable markers. RQR is a selectable marker comprising the major extracellular loop of CD20 and two minimal CD34 binding sites. In some embodiments, the RQR encoding polynucleotide comprises a polynucleotide encoding a 16-amino acid CD34 minimal epitope. In some embodiments, the CD34 minimal epitope is integrated into the amino-terminal position of the CD8 co-receptor stalk domain (Q8). In further implementations, the CD34 minimal binding site sequence can be combined with the targeting epitope of CD20 to form a complete marker / suicide gene (RQR8) for T cells (Philip et al., 2014, incorporated herein by reference). This construct allows selection of host cells expressing the construct, for example, using a CD34-specific antibody bound to magnetic beads (Miltenyi), and allows selective deletion of the transgene expressing engineered T cells using the clinically accepted drug antibody rituximab (Philip et al., 2014).

[0149] Further 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 feature of CD19 and CD34 is that they are readily available in the Miltenyi CliniMACs. TMSelection system is available, and clinical grade sorting can be performed for these markers. However, CD19 and CD34 are relatively large surface proteins, which may have an impact on the transcription efficiency of vector packaging capacity and integration vectors. Surface markers containing extracellular, non-signaling domains or various proteins (such as CD19, CD34, LNGFR) can also be used. Any selection marker can be used, and should be acceptable to good manufacturing practices (Good Manufacturing Practices). In certain embodiments, the selection marker is expressed together with the polynucleotide encoding the gene product of interest (for example, the binding protein of the present disclosure, such as TCR or CAR). Further examples of selection markers include, for example, reporters, such as GFP, EGFP, β-gal or chloramphenicol acetyltransferase (CAT). In certain embodiments, selection markers, for example, CD34 is expressed by cells, and CD34 can be used to select enrichment, or separate (for example, by immunomagnetic selection) transduced cells of interest for use in methods described herein. As used herein, CD34 markers are distinguished from anti-CD34 antibodies, or for example scFv, TCR or other antigen recognition moieties combined with CD34.

[0150] 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.

[0151] With respect to RQR polypeptides, without wishing to be bound by theory, the distance of the epitope or target sequence from the host cell surface may be 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 within an encoded CD8 co-receptor β chain, α chain, or both, or a fragment or variant thereof of one or both. In specific embodiments, the modified host cell comprises a heterologous polynucleotide encoding iCasp9 and a heterologous polynucleotide encoding a recombinant CD8 co-receptor protein comprising a β chain comprising an RQR polypeptide and further comprising a CD8 α chain.

[0152] In any of the above embodiments, the polynucleotides encoding, for example, a TCR, or its binding domain, or a CD8 co-receptor or its extracellular portion, are codon-optimized for efficient expression in target host cells. In some embodiments, the host cells include human immune system cells, such as T cells, NK cells, or NK-T cells (Scholten et al., Clin. Immunol. 119:135, 2006). Codon optimization can be performed using known techniques and tools, such as using OptimumGeneTM Tools or GeneArt (Life Technologies). Codon-optimized sequences include partially codon-optimized sequences (i.e., one or more codons, but less than all codons, are optimized for expression in a host cell) and fully codon-optimized sequences. It will be appreciated that in embodiments where a polynucleotide encodes more than one polypeptide (e.g., a TCR α chain, a TCR β chain, a CD8 co-receptor α chain, a CD8 co-receptor β chain, and one or more self-cleaving peptides), each polypeptide may be independently fully codon-optimized, partially codon-optimized, or not codon-optimized.

[0153] In certain embodiments, the present disclosure provides a host cell comprising a heterologous polynucleotide encoding any one or more TCRs or binding domains thereof of the present disclosure, wherein the modified or recombinant host cell expresses the TCR or binding domain thereof encoded by the heterologous polynucleotide on its cell surface.

[0154] Various techniques can be used for recombinant (i.e., engineered) DNA, polypeptide 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 methods commonly 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, as well as those described in various general and more specific references to microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology techniques cited and discussed in 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 2001John 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,2008Wiley-VCH);PCR Protocols(Methods in Molecular Biology)(Park,Ed.,3rd Edition,2010Humana 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 BruceA.Bunnell Eds., 2008); Hematopoietic Stem Cell Protocols (Methods in Molecular Medicine) (Christopher A. Klug, and Craig T. Jordan Eds., 2001); Molecular Biology) (Leslie P. Weiner Ed., 2008). .

[0155] In any of the above embodiments, the polynucleotides of the present invention are contained in a host cell, or in certain embodiments, are contained in a vector, and the vector comprising the polynucleotides may be in a host cell. Therefore, vectors comprising the polynucleotides provided herein are provided herein. In certain embodiments, the polynucleotides are operably linked to expression control sequences. Suitable vectors for certain embodiments disclosed herein are known and can be selected for specific purposes or cells. Exemplary vectors may include a nucleic acid molecule capable of transporting another nucleic acid molecule connected thereto, or a nucleic acid molecule capable of replicating in a host organism. Some examples of vectors include plasmids, viral vectors, cosmids, and others. Some vectors may be capable of autonomous replication in the introduced host cell (such as bacterial vectors and episomal mammalian vectors with bacterial replication origins), while other vectors may be integrated into the host cell's genome, or promote the integration of the polynucleotide insertion after introduction into the host cell, thereby replicating together with the host genome (such as lentiviral vectors). In addition, some vectors are capable of directing the expression of genes to which they are operatively connected (these vectors may be referred to as "expression vectors"). According to related embodiments, it will be further understood that if one or more agents (e.g., polynucleotides encoding high-affinity or high-functional-avidity recombinant TCRs or binding domains thereof, as described herein, specific for WT1 p37) are co-administered to a subject, each agent can reside in a separate or identical vector, and multiple vectors (each containing a different agent or the same agent) can be introduced into a cell or cell population or administered to a subject.

[0156] In certain embodiments, the polynucleotide encoding the recombinant TCR or its binding domain with high affinity or high functional affinity for WT1 p37 peptide:MHC disclosed herein can be operably linked to certain expression control elements of a vector. For example, a polynucleotide sequence required for expression and processing of the coding sequence to which it is linked can be operably linked. The expression control sequence may 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 improve protein stability; and sequences that may increase protein secretion. If the expression control sequence is adjacent to the gene of interest and the expression control sequence, they can be operably linked together, and these expression control sequences act in trans or remotely on the gene of interest. In certain embodiments, the polynucleotide encoding the TCR or its binding domain disclosed herein is contained in an expression vector, which is a viral vector, such as a lentiviral vector, a γ-retroviral vector, or an adenoviral vector.

[0157] In a specific embodiment, the recombinant expression vector is delivered to appropriate cells, for example, T cells or antigen presenting cells, i.e. cells (e.g., dendritic cells) that display peptide / MHC complexes on their cell surfaces and lack CD8 cells. In certain embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell. For example, the immune system cell can be a CD4+T cell, a CD8+T cell, a CD4-CD8- double negative T cell, a γδT cell, a natural killer cell, a dendritic cell, or any combination thereof, wherein, optionally, if there is a combination, the combination includes CD4+T cells and CD8+T cells. In certain embodiments, wherein the T cell is a host, the T cell can be naive, a central memory T cell, an effector memory T cell, or any combination thereof. Therefore, the recombinant expression vector can also include, for example, a lymphoid tissue-specific transcriptional regulatory element (TRE), such as a B lymphocyte, a T lymphocyte, or a dendritic cell-specific TRE. Lymphoid tissue-specific TREs are known in the art (see, e.g., Thompson et al., Mol. Cell. Biol. 12: 1043, 1992); Todd et al., J. Exp. Med. 177: 1663, 1993); Penix et al., J. Exp. Med. 178: 1483, 1993).

[0158] In addition to vectors, certain embodiments also relate to host cells comprising the currently disclosed heterologous polynucleotides or vectors. In certain embodiments, the host cell expresses a TCR encoded by a polynucleotide on its cell surface, and wherein the polynucleotide is heterologous to the host cell. It will be readily understood by those skilled in the art that many suitable host cells are available in the art. Host cells can include any single cell or cell culture that can accept the binding of a vector or nucleic acid and / or protein, as well as any progeny cells. The term also includes the progeny of the host cell, whether genetically or phenotypically identical or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, monkey cells (simian cells), insect cells, yeast cells, and bacterial cells. These cells can be induced to incorporate vectors or other materials using viral vectors, transformation by calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory, 1989).

[0159] In certain embodiments, the Vα domain of a TCR expressed by a host cell is encoded by a polynucleotide having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 97, 98, and 101-107, or at least 94% sequence identity to SEQ ID NOs: 99 or 100. In certain embodiments, the Vα domain is encoded by a polynucleotide that: (a) includes the sequence of any one of SEQ ID NOs: 97-107; or (b) consists of the sequence of any one of SEQ ID NOs: 97-107.

[0160] In certain embodiments, the Vβ domain of the host cell is encoded by a polynucleotide comprising at least 75% sequence identity to any one of the polynucleotides of SEQ ID NOs: 75-77, 79, 82, 84, and 85, or at least 95% sequence identity to any one of the polynucleotides of SEQ ID NOs: 78, 80, 81, and 83. In certain embodiments, the Vβ domain is encoded by a polynucleotide that: (a) comprises the sequence of any one of the polynucleotides of SEQ ID NOs: 75-85; or (b) consists of the sequence of any one of the polynucleotides of SEQ ID NOs: 75-85.

[0161] In certain embodiments, the TCR α chain comprises an α chain constant domain encoded by a polynucleotide that is at least 98% identical to SEQ ID NO: 110. In certain embodiments, the TCR α chain comprises an α chain constant domain encoded by a polynucleotide that: (a) comprises a polynucleotide sequence of SEQ ID NO: 110; or (b) consists of a polynucleotide sequence of SEQ ID NO: 110. In certain embodiments, the TCR β chain comprises a β chain constant domain encoded by a polynucleotide that has at least 99.9% sequence identity to SEQ ID NO: 108 or 109. In certain embodiments, the TCR β chain comprises a β chain constant domain encoded by a polynucleotide that: (a) comprises a polynucleotide sequence of SEQ ID NO: 108 or 109; or (b) consists of a polynucleotide sequence of SEQ ID NO: 108 or 109.

[0162] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a self-cleaving peptide, the nucleotide sequence being disposed between a polynucleotide sequence encoding a TCR α chain and a polynucleotide sequence encoding a TCR β chain.

[0163] In some embodiments, the encoded self-cleaving peptide: (a) comprises the amino acid sequence of any one of SEQ ID NOs: 60-63; or (b) consists of the sequence of any one of SEQ ID NOs: 60-63.

[0164] In some embodiments, the polynucleotide encoding the self-cleaving peptide: (a) comprises the sequence of any one of the polynucleotides of SEQ ID NOs: 166-170; or (b) consists of the sequence of any one of the polynucleotides of SEQ ID NOs: 166-170.

[0165] In some embodiments, the TCR alpha chain, the self-cleaving peptide, and the TCR beta chain are encoded by a polynucleotide that is at least 95% identical to any one of SEQ ID NOs: 155-165.

[0166] In some embodiments, the TCR alpha chain, self-cleaving peptide, and TCR beta chain are encoded by the following polynucleotides: (a) comprising the sequence of any one of the polynucleotides of SEQ ID NOs: 155-165; or (b) consisting of the sequence of any one of the polynucleotides of SEQ ID NOs: 155-165.

[0167] In some embodiments, the encoded TCR alpha chain, self-cleaving peptide, and TCR beta chain comprise an amino acid sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9%, or 100% identical to any one of the polypeptides of SEQ ID NOs: 48-58. In some embodiments, the encoded TCR alpha chain, self-cleaving peptide, and TCR beta chain: (a) comprise the amino acid sequence of any one of the polypeptides of SEQ ID NOs: 48-58; or (b) consist of the amino acid sequence of any one of the polypeptides of SEQ ID NOs: 48-58.

[0168] In some embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell. In some embodiments, the immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double negative T cell, a γδ T cell, a natural killer cell, a natural killer T cell, a dendritic cell, or any combination thereof, wherein, optionally, the combination includes CD4+ T cells and CD8+ T cells.

[0169] In some embodiments, wherein the host immune system cell is a T cell. In some embodiments, the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

[0170] In certain embodiments, the TCR has higher surface expression on a T cell compared to an endogenous TCR (e.g., when the endogenous TCR is not artificially inhibited or prevented from expression).

[0171] In certain embodiments, the host cell 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.

[0172] In some embodiments, the host cell comprises: (a) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 co-receptor α chain; (b) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 co-receptor β chain; and (c) a polynucleotide encoding a self-cleaving peptide, which is disposed between the polynucleotide of (a) and the polynucleotide of (b).

[0173] In any of the presently disclosed embodiments, when both host cells and tumor cells are present in a sample, the host cells (e.g., immune cells, such as human T cells) are capable of killing: (i) tumor cells of the breast cancer cell line MDA-MB-468; (ii) tumor cells of the pancreatic cancer cell line PANC-1; (iii) tumor cells of the breast cancer cell line MDA-MB-231; (iv) tumor cells of the myeloid leukemia cell line K562 expressing HLA-A2, wherein, optionally, HLA-A2 includes HLA-A*201; (v) tumor cells of the colon cancer cell line RKO expressing HLA-A2, wherein, optionally, HLA-A2 includes HLA-A*201; or (vi) any combination of tumor cells of (i)-(v). In some embodiments,

[0174] In specific embodiments, host cells are capable of killing tumor cells when the host cells and tumor cells are present in the sample at a ratio of 32:1 (host cells: tumor cells), 16:1, 8:1, 4:1, 2:1, or 1.5:1. Killing of target cells can be determined, for example, Bioimaging platform (Essen Bioscience). In certain embodiments, the platform uses activated caspase and labeled (e.g., RapidRed or NucRed) tumor cell signals, where overlap is measured and an increase in the overlap area equates to tumor cell death due to apoptosis. Killing can also be determined using a 4-hour assay where target cells are labeled with chromium ( 51 Cr) loading, after 4 hours of co-culture with immune cells expressing the binding protein of the present disclosure, the supernatant was measured. 51 Cr.

[0175] In any of the above embodiments, host cells (e.g., immune cells) can be modified to reduce or eliminate the expression of one or more endogenous genes that encode polypeptides involved in immune signaling or other related activities. Exemplary gene knockouts include those encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, HLA molecules, TCR molecules, etc. Without wishing to be bound by theory, certain endogenously expressed immune cell proteins may be recognized as foreign by the allogeneic host receiving the modified immune cells, which may result in the modified immune cells being cleared (e.g., HLA alleles), or may downregulate the immune activity of the modified immune cells (e.g., PD-1, LAG-3, CTLA4, FasL, TIGIT, TIM3), or may interfere with the binding activity of the heterologously expressed binding proteins disclosed herein (e.g., the endogenous TCR of the modified T cells binds to non-Ras antigens, thereby interfering with the modified immune cells binding to cells expressing Ras antigens).

[0176] Thus, reducing or eliminating the expression or activity of such an endogenous gene or protein can improve the activity, tolerance, or persistence of the modified 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 cells are donor cells (e.g., allogeneic) or autologous cells. In certain embodiments, the host cells of the present disclosure include a chromosomal knockout of one or more genes encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, 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 gene editing techniques, compositions, and adoptive cell therapies of which are hereby incorporated by reference in their entirety).

[0177] As used herein, the term "chromosomal gene knockout" refers to a genetic alteration or introduction of an inhibitor in a host cell to prevent (e.g., reduce, delay, inhibit, or abolish) the production of a functionally active endogenous polypeptide product by the host cell. Alterations resulting in chromosomal gene knockout can include, for example, the introduction of nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletions, and chain breaks, as well as heterologous expression of inhibitory nucleic acid molecules that inhibit endogenous gene expression in the host cell.

[0178] In certain embodiments, chromosomal gene knockout or gene knock-in is achieved by editing the chromosome of the host cell. Chromosome editing can be performed using, for example, endonucleases. "Endonucleases" herein refer to enzymes that can catalyze the cleavage of phosphodiester bonds within a polynucleotide chain. In certain embodiments, endonucleases can cleave target genes, thereby inactivating or "knocking out" the target genes. Endonucleases can be naturally occurring, recombinant, genetically modified, or fused endonucleases. The breaks in the nucleic acid chains 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" and target gene "knocking out," or alternatively, the target gene can be inactivated by donor gene knock-in or target gene knock-out events. NHEJ is an error-prone repair process that typically results in changes in the cleavage site of the DNA sequence, such as replacement, deletion, or addition of at least one nucleotide. NHEJ can be used to "knock out" the target gene. Examples of endonucleases include zinc finger nucleases, TALE-nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.

[0179] 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 (e.g., Fok1 endonuclease). Each zinc finger motif of approximately 30 amino acids binds to approximately 3 base pairs of DNA, and the amino acids of certain residues can be changed to alter the sequence specificity of the triplet (see, e.g., 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 linked in series to create binding specificity for a desired DNA sequence, such as a region between 9 and 18 base pairs in length. As background, ZFNs mediate genome editing by catalyzing the formation of site-specific DNA double-strand breaks (DSBs) in the genome and promote the directed integration of transgenes that include flanking sequences homologous to the genome at the DSB site through homology-directed repair. Alternatively, the DSBs generated by ZFNs can be repaired by non-homologous end joining (NHEJ), which is an error-prone intracellular repair pathway that results in the insertion or deletion of nucleotides at the cleavage site. In certain embodiments, gene knockout comprises insertions, deletions, mutations, or a combination thereof performed using ZFN molecules.

[0180] 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, such as a FokI endonuclease. A "TALE DNA binding domain" or "TALE" is composed of one or more TALE repeat domains / units, each of which typically has a highly conserved 33-35 amino acid sequence with differences at the 12th and 13th amino acids. The TALE repeat domain is involved in the binding of the TALE to the target DNA sequence. The different amino acid residues are called repeat variable regions (RVDs) and are associated with specific nucleotide recognition. The natural (typical) code for DNA recognition of these TALEs has been determined, and the HD (histidine-aspartic acid) sequence at positions 12 and 13 of the TALE causes the TALE to bind to cytosine (C), NG (asparagine-glycine) to bind to T nucleotides, NI (asparagine-isoleucine) to bind to A, NN (asparagine-asparagine) to bind to G or A nucleotides, and NG (asparagine-glycine) to bind to T nucleotides. Non-classical (atypical) RVDs are also known (e.g., see U.S. Patent Publication No. US 2011 / 0301073, which is incorporated herein by reference in its entirety). TALEN can be used to direct double-strand breaks (DSBs) at specific sites in the T cell genome. Non-homologous end joining (NHEJ) connects DNA from both sides of the double-strand break, with almost no annealing of sequence overlap, thereby introducing errors in knockout gene expression. In addition, if there are homologous flanking sequences in the transgene, homology-directed repair can introduce the transgene at the position of the DSB. In certain embodiments, gene knockout includes insertions, deletions, mutations, or a combination thereof, and is made using TALEN molecules.

[0181] As used herein, the "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 (called protospacers) within the genome through base pairing complementarity, and then cleaves the DNA if a short, conserved protospacer-associated motif (PAM) is immediately 3' to the complementary target sequence. Based on the sequence and structure of the Cas nuclease, the CRISPR / Cas system is divided into three types (i.e., type I, type II, and type III). The surveillance complexes guided by type I and type III crRNA require multiple Cas subunits. The type II system is the most studied and includes at least three components: an RNA-guided Cas9 nuclease, crRNA, and a trans-acting crRNA (tracrRNA). The tracrRNA includes a double-stranded forming region. crRNA and tracrRNA form a double chain, can interact with Cas9 nuclease, and by the Watson-Crick base pairing between the spacer on crRNA and the pre-spacer sequence on the target DNA upstream of PAM, the Cas9 / crRNA:tracrRNA complex is guided to a specific site on the target DNA. Cas9 nuclease cleaves double-strand breaks in the region defined by the crRNA spacer. Insertions and / or deletions are repaired by NHEJ, destroying the expression of the target locus. In addition, a transgene with a homologous flanking sequence can be introduced at the DSB site by homology-directed repair. crRNA and tracrRNA can be designed 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 that is 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 Publication No. US2014 / 0068797, U.S. Patent Application Publication No. US 2014 / 0186843; U.S. Patent Application No. 8,697,359 and PCT Publication 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 made using the CRISPR / Cas nuclease system. Exemplary gRNA sequences and methods using the same 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 gRNA, CAS9 DNA, vectors, and gene knockout techniques of which are incorporated herein by reference in their entirety.

[0182] As used herein, "meganuclease," also known as "homing endonuclease," refers to an endodeoxynuclease characterized by a large recognition site (approximately 12 to 40 base pairs of double-stranded DNA sequence). Based on sequence and structural motifs, meganucleases can be divided into five families: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK. Exemplary meganucleases include I-SceI, I-Ceul, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, 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).

[0183] 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, FasL, 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 a further embodiment, chromosomal gene knockout is generated using a homing endonuclease modified with the modular DNA-binding domain of a TALEN to create a fusion protein termed megaTAL. MegaTAL 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 conjunction with an exogenous donor template encoding a polypeptide of interest.

[0184] In certain embodiments, the chromosomal gene knockout comprises an inhibitory nucleic acid molecule that is introduced into a host cell (e.g., an immune cell), the molecule comprising a heterologous polynucleotide encoding an antigen-specific receptor that specifically binds to a tumor-associated antigen, wherein the inhibitory nucleic acid molecule encodes a target-specific inhibitor, wherein the encoded target-specific inhibitor inhibits endogenous gene expression in the host cell (e.g., PD-1, TIM3, LAG3, CTLA4, TIGIT, FasL, an HLA component, or a TCR component, or any combination thereof).

[0185] Chromosomal gene knockout can be directly confirmed by DNA sequencing of host immune cells after the knockout procedure or preparation. Chromosomal gene knockout can also be inferred from the absence of gene expression (e.g., absence of gene-encoded mRNA or polypeptide product) after knockout.

[0186] In certain embodiments, the chromosomal gene knockout comprises a knockout of an HLA component gene selected from the group consisting of an α1 macroglobulin gene, an α2 macroglobulin gene, an α3 macroglobulin gene, a β1 microglobulin gene, or a β2 microglobulin gene.

[0187] In certain embodiments, the chromosomal gene knockout comprises a knockout of a TCR component gene selected from a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, or a combination thereof.

[0188] Furthermore, it will be appreciated that any currently disclosed gene editing techniques and tools can be used to introduce the TCR-encoding and / or CD8 co-receptor-encoding polynucleotides of the present disclosure into the host cell genome.

[0189] In another aspect, provided herein are compositions and unit dosages comprising the modified host cells of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient.

[0190] In certain embodiments, the host cell composition or 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 number of naive T cells 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).

[0191] In some embodiments, the host cell composition or 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 in a ratio of about 1:1, wherein the host cell composition or unit dose comprises a reduced amount of or substantially no naive T cells. In further embodiments, the host cell composition or 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 in a ratio of about 1:1, wherein the unit dose comprises a reduced amount of or substantially no naive T cells. In still further embodiments, the host cell composition or unit dose comprises (i) a composition comprising at least about 70% engineered CD4+ T cells and (ii) a composition comprising at least about 70% engineered CD8+ T cells in a ratio of about 1:1, wherein the unit dose comprises a reduced amount of or substantially no naive T cells. In some embodiments, the host cell composition or 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 host cell composition or unit dose comprises a reduced amount of or substantially no naive T cells. In some embodiments, the host cell composition or 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 host cell composition or unit dose comprises a reduced amount of or substantially no naive T cells. In some embodiments, the host cell composition or 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 host cell composition or unit dose comprises a reduced amount of or substantially no naive T cells.

[0192] It is understood that the host cell compositions or unit doses of the present disclosure can include any host cell or any combination of host cells described herein. In certain embodiments, for example, the host cell compositions or unit doses include modified CD8+ T cells, modified CD4+ T cells, or both, wherein these T cells are modified to encode a specific binding protein for the Ras peptide:HLA-A*02:01 complex, and further include modified CD8+ T cells, modified CD4+ T cells, or both, wherein these T cells are modified to encode a specific binding protein for the WT1 peptide:HLA-A*02:01 complex. Additionally or alternatively, the host cell compositions or unit doses of the present disclosure can include any host cell or host cell combination described herein, and can further include expressing antibodies against different antigens (e.g., different WT1 antigens or antigens from different proteins or targets, e.g., 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 B, 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, WT-1 peptides bound to HLA, LTβR, LIFRβ, LRP5, MUC1, The invention relates to modification of cells (e.g., immune cells, such as T cells) by binding proteins specific to proteins such as OSMRβ, TCRα, TCRβ, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD79a, CD79b, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, TLR7, TLR9, PTCH1, WT-1, HA1-H, Robo1, α-fetoprotein (AFP), Frizzled, OX40, PRAME and SSX-2.For example, a unit dose may include modified CD8+T cells expressing a binding protein that specifically binds to the WT1-HLA complex and modified CD4+T cells (and / or improved CD8+T cells) expressing a binding protein (such as CAR) that specifically binds to the HER2 antigen. It will also be understood that any host cell disclosed herein can be administered in combination therapy.

[0193] In any of the embodiments described herein, the host cell composition or unit dose comprises equal or approximately equal amounts of engineered CD45RA-CD3+CD8+ and modified CD45RA-CD3+CD4+ TM cells.

[0194] Uses and treatments

[0195] In certain aspects, the present disclosure relates to a method for treating a hyperproliferative or proliferative disease characterized by expression or overexpression of Wilms tumor protein 1 (WT1), wherein a recombinant TCR or its binding domain comprising high affinity or high functional affinity is administered to a human subject in need thereof, and is specific to human WT1 according to any of the above-mentioned TCRs or any binding domains described herein, or is designed to express a host cell thereof, such as a T cell, or a composition comprising any TCR, or its binding domain, or a host cell described herein. In some embodiments, the TCR is expressed by a host cell, such as a hematopoietic progenitor cell or a human immune system cell. In some embodiments, the immune system cell is a CD4+T cell, a CD8+T cell, a CD4-CD8-double negative T cell, a γδT cell, a natural killer cell, a natural killer T cell, a dendritic cell, or any combination thereof.

[0196] The presence of a hyperproliferative disease or a proliferative disease or a malignant condition in a subject refers to the presence of dysplastic, cancerous and / or transformed cells in the subject, including, for example, neoplasms, tumors, non-contact inhibitory or oncogenically transformed cells, etc. (e.g., solid cancers; hematological cancers including lymphomas and leukemias, such as acute myeloid leukemia, chronic myeloid leukemia, etc.), which are all known in the art and have established diagnostic and classification criteria (e.g., Hanahan and Weinberg, Cell 144:646, 2011; Hanahan and Weinberg, Cell 100:57, 2000; Cavallo et al., Canc. Immunol. Immunother. 60:319, 2011; Kyrigideis et al., J. Carcinog. 9:3, 2010). In certain embodiments, the cancer cell can be a cell of acute myeloid leukemia, B-cell lymphocytic leukemia, T-cell lymphocytic leukemia, or myeloma, including cancer stem cells that are capable of initiating and serially engrafting any of these types of cancer (see, e.g., Park et al., Molec. Therap. 17:219, 2009).

[0197] In certain embodiments, methods of treating hyperproliferative or proliferative diseases, such as hematological malignancies or solid cancers, are provided (see, e.g., Nakatsuka et al., Modern Pathology 19:804-714 (2006). Exemplary hematological malignancies include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or multiple myeloma (MM).

[0198] In further embodiments, methods of treating a hyperproliferative or proliferative disease, such as a solid cancer, are provided, which is selected from biliary cancer, bladder cancer, bone and soft tissue cancer, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, dermatoid tumor, embryonal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumors, head and neck squamous cell carcinoma, liver cancer, lung cancer, mesothelioma, malignant melanoma, osteosarcoma, ovarian cancer (see, e.g., Hylander et al., Gynecologic Oncology 101: 12-17 (2006)), pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytoma, primary thyroid cancer, prostate cancer, kidney cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ cell tumor, urethral cancer, uterine sarcoma, or uterine cancer.

[0199] In some embodiments, the TCR is capable of promoting antigen-specific T cell responses against human WT1 in a class I HLA-restricted manner. In some embodiments, the first class HLA-restricted response is independent of the transporter associated with antigen processing (TAP). In some embodiments, the antigen-specific T cell response includes at least one of a CD4+ helper T lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response. In some embodiments, the CTL response is directed against cells that overexpress WT1.

[0200] Also provided herein are any of the TCRs, polynucleotides, compositions, vectors, and host cells (including any combination) for use in methods of treating a proliferative or hyperproliferative disease associated with expression or overexpression of Wilms tumor protein 1 (WT1).

[0201] Also provided herein are any TCR, polynucleotide, composition, vector, and host cell (including any combination) for use in a method for the manufacture of a medicament for treating a proliferative or hyperproliferative disease associated with expression or overexpression of Wilms tumor protein 1 (WT1).

[0202] As understood by those skilled in the medical field, the terms "treat" and "treatment" refer to the medical management of a disease, disorder, or condition in a subject (i.e., a patient, host, which can be human or non-human animal) (see, for example, Stedman's Medical Dictionary). In general, appropriate dosages and treatment regimens provide one or more high-functional affinity recombinant TCRs or binding domains thereof specific for human WT1 (e.g., SEQ ID NOs: 23-58, and variants provided herein), or host cells expressing the same, and optional adjunctive therapies (e.g., cytokines, such as IL-2, IL-15, IL-21, or any combination thereof), in amounts sufficient to provide a therapeutic or prophylactic benefit. The therapeutic or prophylactic benefits resulting from therapeutic treatments or prophylactic or preventative methods include, for example, improved clinical outcomes, where the goal is to prevent, slow, or otherwise reduce (e.g., in a statistically significant manner relative to an untreated control) an undesirable physiological change or disorder, or to prevent, slow, or otherwise reduce the extent or severity of such a disease or disorder. Beneficial or desired clinical results of treating a subject include reduction, alleviation or relief of symptoms caused by or associated with the disease or disorder being treated; reduction in the occurrence of symptoms; improvement in quality of life; prolongation of the disease-free state (i.e., reducing the likelihood or propensity of a subject to develop the symptoms on which the disease is diagnosed); reduction in the extent of the disease; stabilization (i.e., non-worsening) of the disease state; delay or slowing of disease progression; improvement or palliation of the disease state; and remission (whether partial or complete), whether detectable or undetectable; or overall survival.

[0203] "Treatment" can also refer to prolonging survival compared to the expected survival rate if the subject does not receive treatment. Subjects in need of the methods and compositions described herein include those already suffering from the disease or disorder, as well as subjects susceptible to or at risk of developing the disease or disorder. Subjects in need of prophylactic treatment include those in whom the disease, condition or disorder is to be prevented (i.e., the likelihood of the disease or disorder occurring or recurring is reduced). The clinical benefit provided by the compositions (and formulations comprising the compositions) and methods described herein can be assessed by designing and performing in vitro assays, preclinical studies, and clinical studies, as described in the Examples, for which the compositions are intended to be beneficial.

[0204] In another aspect, the present disclosure relates to methods for treating hyperproliferative diseases or proliferative diseases or conditions characterized by overexpression or expression of Wilms tumor protein 1 (WT1) by administering to a human subject in need thereof a composition comprising an isolated polynucleotide encoding a high-affinity or high-functional avidity recombinant TCR or its binding domain, any of the above-encoded TCRs or binding domains having specificity for human WT1, or a host cell (e.g., a T cell) comprising the same, or a composition comprising any of the TCRs or binding domains or host cells described herein. In certain embodiments, the polynucleotide encoding the TCR or its binding domain specific for human WT1 p37 peptide:MHC is codon-optimized for the host cell of interest. In further embodiments, any of the above-described polynucleotides is operably linked to an expression control sequence and optionally contained in an expression vector, such as a viral vector. Exemplary viral vectors include lentiviral vectors and gamma-retroviral vectors. In related embodiments, the vector is capable of delivering the polynucleotide to a host cell, such as a hematopoietic progenitor cell or an immune system cell (e.g., a human hematopoietic progenitor cell or a human immune system cell). Exemplary immune system cells include CD4+ T cells, CD8+ T cells, CD4-CD8- double negative T cells, γδ T cells, natural killer cells, dendritic cells, or any combination thereof (e.g., human). In certain embodiments, the immune system cells are T cells, such as naive T cells, central memory T cells, effector memory T cells, or any combination thereof, all of which are optionally human.

[0205] In another aspect, the present disclosure relates to a method for treating a hyperproliferative disease or a proliferative disease or a condition characterized by overexpression of Wilms tumor protein 1 (WT1), by administering to a human subject in need thereof an effective amount of a host cell comprising a heterologous polynucleotide or expression vector according to any of the above embodiments or as described herein, wherein the engineered or recombinant host cell expresses on its cell surface a TCR encoded by the heterologous polynucleotide, the TCR being specific for human WT1 p37:MHC. In certain embodiments, the present disclosure relates to a method for treating a hyperproliferative disease or a proliferative disease or a condition characterized by production of Wilms tumor protein 1 (WT1) p37 peptide or the presence of a WT1 p37 peptide:MHC complex, by administering to a human subject in need thereof an effective amount of a host cell comprising a heterologous polynucleotide or expression vector according to any of the above embodiments or as described herein, wherein the engineered or recombinant host cell expresses on its cell surface a TCR encoded by the heterologous polynucleotide, the TCR being specific for human WT1 p37:MHC.

[0206] Also provided is a method of adoptive immunotherapy for treating a disease characterized by overexpression of WT1 in cells of a subject having a hyperproliferative or proliferative disease, comprising administering to the subject an effective amount of the host cell or composition of the present disclosure.

[0207] In some embodiments, the host cell is modified in vitro. In some embodiments, the host cell is an allogeneic cell, a homologous cell, or an autologous cell of the subject. In some embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell. In some embodiments, the immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double negative T cell, a γδ T cell, a natural killer cell, a dendritic cell, or any combination thereof.

[0208] In some embodiments, the T cells are naive T cells, central memory T cells, effector memory T cells, or any combination thereof.

[0209] In some embodiments, the hyperproliferative or proliferative disorder is a hematological malignancy or a solid cancer.

[0210] In some embodiments, the hematological malignancy is selected from acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), or multiple myeloma (MM).

[0211] In some embodiments, the solid cancer is selected from breast cancer, ovarian cancer, lung cancer, biliary tract cancer, bladder cancer, bone and soft tissue cancer, brain tumor, cervical cancer, colon cancer, large intestinal adenocarcinoma, colorectal cancer, ligamentous tumor, embryonal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological cancer, head and neck squamous cell carcinoma, liver cancer, mesothelioma, malignant melanoma, osteosarcoma, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, kidney cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ cell tumor, urethral cancer, uterine sarcoma, or uterine cancer.

[0212] In some embodiments, the host cells are administered parenterally.

[0213] In some embodiments, the method comprises administering multiple doses of the host cell to the subject. In some embodiments, the multiple doses are administered at intervals of about two weeks to about four weeks.

[0214] The cells described herein that express a recombinant TCR (e.g., high affinity or high functional avidity) specific for the human WT1 p37 peptide, or a binding domain thereof, can be administered to a subject in a pharmaceutically or physiologically acceptable or suitable excipient or carrier. A pharmaceutically acceptable excipient is a biocompatible carrier, such as physiological saline, which is described in more detail herein, and is suitable for administration to a human or other non-human mammalian subject.

[0215] A therapeutically effective dose is an amount of adoptively transferred host cells (expressing a high affinity or high functional avidity recombinant TCR, or its binding domain, specific for human WT1 p37 peptide:MHC) in a human or non-human animal for treatment that produces a clinically desirable outcome (i.e., a sufficient amount to induce or enhance a specific T cell immune response (e.g., a cytotoxic T cell response) against cells that overexpress WT1 or produce WT1 p37 peptide, with statistical significance). As is well known, the dose for any one patient depends on many factors, including the patient's size, weight, body surface area, age, the specific therapy to be administered, sex, time and route of administration, general health, and other drugs administered concurrently. Doses will vary, but a preferred dose for host cells comprising a recombinant expression vector as described herein is about 10 4 cells / m 2 , about 5×10 4 cells / m 2 , about 10 5 cells / m 2 , about 5×10 5 cells / m 2 , about 10 6 cells / m 2 , about 5×10 6 cells / m 2 , about 10 7 cells / m 2 , about 5×10 7 cells / m 2 , about 5×10 8 cells / m 2 , about 10 9 cells / m 2 , about 5×10 9 cells / m 2 , about 10 10 cells / m 2 , about 5×10 10 cells / m 2 , or about 10 11 cells / m 2 In some embodiments, the dosage comprises about 10 7 cells / m 2 , about 5×10 7cells / m 2 , about 10 8 cells / m 2 , about 5×10 8 cells / m 2 , about 10 9 cells / m 2 , about 5×10 9 cells / m 2 , about 10 10 cells / m 2 , about 5×10 10 cells / m 2 , or about 10 11 cells / m 2 .

[0216] The pharmaceutical composition can be administered in a manner consistent with the disease or condition to be treated (or prevented) as determined by a person skilled in the art of medicine. The appropriate dosage and the appropriate duration and frequency of administration will be determined by factors such as the patient's health, the patient's size (i.e., weight, mass, or body area), the type and severity of the patient's disease, the specific form of the active ingredient, and the method of administration. In general, the amount of the composition provided by the appropriate dosage and treatment regimen is sufficient to provide therapeutic and / or preventive benefits (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). For prophylactic use, the dosage should be sufficient to prevent, delay the onset of, or reduce the severity of the disease associated with the disease or disorder. The preventive effect of the immunogenic composition 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 implemented by those skilled in the art.

[0217] Conditions associated with overexpression (or, in some embodiments, expression) of WT1 include any disorder or condition in which there is insufficient, excessive, or inappropriate activity of WT1 cellular or molecular events, typically due to abnormally high (statistically significant) expression levels of WT1 in afflicted cells (e.g., leukemia cells) relative to normal cells. Subjects with such diseases or conditions will benefit from treatment with the compositions or methods of the embodiments described herein. Thus, some conditions associated with overexpression of WT1 may include acute as well as chronic disorders and diseases, such as those pathological conditions that predispose a subject to a particular disease.

[0218] Some examples of conditions associated with WT1 overexpression include hyperproliferative disorders, which in some aspects refer to a state in which cells in a subject are activated and / or proliferating (and may also be transcriptionally overactive), including tumors, neoplasms, cancers, malignancies, and the like. In addition to activated or proliferating cells, proliferative disorders may also involve abnormalities or dysregulation of cell death processes, whether through necrosis or apoptosis. Abnormalities in such cell death processes may be associated with various conditions, including cancer (including primary and secondary malignancies, and metastases) or other conditions.

[0219] According to certain embodiments, almost any type of cancer characterized by overexpression of WT1 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, myelomas, and others). In addition, "cancer" can refer to any cell with accelerated proliferation, including solid tumors, ascites tumors, blood or lymphatic or other malignancies; connective tissue malignancies; metastatic diseases; minimal residual disease after organ or stem cell transplantation; multidrug-resistant cancers, primary or secondary malignancies, angiogenesis associated with malignant tumors, or other forms of cancer. In the embodiments disclosed herein, specific embodiments are also contemplated in which only one of the above-mentioned disease types is included, or specific conditions can be excluded, regardless of whether they are characterized by overexpression of WT1.

[0220] Certain therapeutic or preventive methods contemplated herein include administering host cells (which may be autologous, allogeneic, or homologous) comprising a desired nucleic acid molecule as described herein, which is stably integrated into the chromosomes of the cells. For example, such cell compositions can be generated in vitro using autologous, allogeneic, or homologous immune system cells (e.g., T cells, antigen-presenting cells, natural killer cells) so that the desired WT1-targeted T cell composition can be administered to a subject as adoptive immunotherapy.

[0221] As used herein, in certain aspects, the administration of a composition or therapy refers to the delivery of the composition or therapy to a subject, without regard to the route or manner of delivery. Administration may be continuous or intermittent, and may be parenteral. Administration may be used to treat subjects who have been confirmed to have a recognized disorder, disease, or morbidity, or to treat subjects who are susceptible to such a disorder, disease, or morbidity or are at risk. Administration in combination with adjuvant therapy may include multiple agents (such as WT1-specific modified (i.e., recombinant or engineered) host cells with one or more cytokines; immunosuppressive therapies, such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low doses of mycophenolic acid prodrugs, or any combination thereof) administered simultaneously and / or sequentially in any order and on any dosing schedule. For example, the therapies of the present disclosure can be combined with specific inhibitors or modulators of immunosuppressive components, such as inhibitors or modulators of immune checkpoint molecules (e.g., anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies; see, e.g., Pardol, Nature Rev. Cancer 12:252, 2012; Chen and Mellman, Immunity 39:1, 2013).

[0222] In some embodiments, the host cell is at about 10 7 cells / m 2 to about 10 11 cells / m 2 In some embodiments, the method further comprises administering a cytokine. In some embodiments, the cytokine is IL-2, IL-15, IL-21, or any combination thereof. In some embodiments, the cytokine is IL-2 and is administered simultaneously or sequentially with the host cells. In some embodiments, the cytokine is administered sequentially, provided that the subject has been administered at least three or four times with the host cells prior to administration of the cytokine.

[0223] In some embodiments, the cytokine is IL-2 and is administered subcutaneously.

[0224] In some embodiments, the subject is further receiving immunosuppressive therapy.

[0225] In some embodiments, the immunosuppressive therapy is selected from a calcineurin inhibitor, a corticosteroid, a microtubule inhibitor, a low dose of a mycophenolic acid prodrug, or any combination thereof.

[0226] In some embodiments, the subject has received a non-myeloablative or myeloablative hematopoietic cell transplant.

[0227] In some embodiments, the subject is administered the host cells at least three months after the non-myeloablative hematopoietic cell transplant.

[0228] In certain embodiments, the subject is given host cells at least two months after the bone marrow hematopoietic cell transplantation. The technology and scheme of carrying out HCT are known in the art and can include transplanting any suitable donor cell, such as the cell from cord blood, bone marrow or peripheral blood, hematopoietic stem cell, activated stem cell or the cell from amniotic fluid. Therefore, in certain embodiments, the modified immune cell of the present disclosure can be used together with hematopoietic stem cell or soon after hematopoietic stem cell in the HCT treatment of modification. In certain embodiments, HCT includes donor hematopoietic cells, which include the chromosome knockout of the gene encoding HLA components, the chromosome knockout of the gene encoding TCR components or both.

[0229] In further embodiments, the subject has received lymphodepleting chemotherapy prior to receiving the composition or HCT. In certain embodiments, the lymphodepleting chemotherapy comprises a conditioning regimen consisting of cyclophosphamide, fludarabine, antithymocyte globulin, or a combination thereof.

[0230] In certain embodiments, multiple doses of the recombinant host cells described herein are administered to a subject at intervals of about 2 to about 4 weeks. In further embodiments, cytokines are administered sequentially, provided that the subject has been administered at least three or four recombinant host cells prior to administration of the cytokines. In certain embodiments, cytokines are administered subcutaneously (e.g., IL-2, IL-15, IL-21).

[0231] In further embodiments, the treated subject is further receiving immunosuppressive therapy, such as a specific antibody against PD-1 (e.g., pidilizumab, nivolumab, or pembrolizumab), a specific antibody against PD-L1 (e.g., MDX-1105, BMS-936559, MEDI4736, MPDL3280A, or MSB0010718C), a specific antibody against CTLA4 (e.g., tremelimumab or ipilimumab), a calcineurin inhibitor, a corticosteroid, a microtubule inhibitor, a low-dose mycophenolic acid prodrug, or any combination thereof. In further embodiments, the treated subject has received a non-myeloablative or myeloablative hematopoietic cell transplant, wherein the treatment may be performed at least two months to at least three months after the non-myeloablative hematopoietic cell transplant.

[0232] In some aspects, an effective amount for treatment or pharmaceutical composition refers to an amount sufficient to achieve the desired clinical effect or beneficial treatment as described herein within the desired dosage and 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 morbid state, the term "therapeutic amount" can be used to refer to treatment, while a "prophylactic effective amount" can be used to describe an effective amount administered to a subject susceptible to or at risk of a disease or morbid state (e.g., recurrence) as a preventive process.

[0233] The level of cytotoxic T lymphocyte (CTL) immune response can be determined by any of the numerous immunological methods described herein and conventional in the art. The level of CTL immune response can be determined before and after administering any of the WT1 specific TCRs expressed by, for example, T cells as described herein. Cytotoxicity assays to determine CTL activity can be performed using any of several techniques and methods conventional in the art (see, for example, Henkart et al., "Cytotoxic T-Lymphocytes" in Fundamental Immunology, Paul (ed.) (2003 Lippincott Williams & Wilkins, Philadelphia, PA), pages 1127-50, and references cited therein).

[0234] Antigen-specific T cell responses are typically determined by comparing observed T cell responses, based on any of the T cell functional parameters described herein (e.g., proliferation, cytokine release, CTL activity, altered cell surface marker phenotype, etc.), between T cells exposed to a cognate antigen (i.e., an antigen used to stimulate or activate T cells when presented by an immunocompatible antigen-presenting cell) under appropriate circumstances and T cells from the same source population exposed to a structurally different or unrelated control antigen. A response to the cognate antigen that is greater than the response to the control antigen and that is statistically significant indicates antigen specificity.

[0235] A biological sample can be obtained from a subject to determine the presence and level of an immune response to the WT1-derived antigenic peptides described herein. As used herein, a "biological sample" can be a blood sample (from which serum or plasma can be prepared), a biopsy specimen, a body fluid (such as lung lavage fluid, ascites, mucosal washes, synovial fluid), bone marrow, lymph nodes, tissue transplants, organ cultures, or any other tissue or cell preparation from a subject or biological source. A biological sample can also be obtained from a subject prior to receiving any immunogenic composition, and the biological sample can serve as a control for establishing baseline (i.e., pre-immunization) data.

[0236] The pharmaceutical compositions described herein may be presented in unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to maintain the stability of the formulation. In certain embodiments, a unit dose comprises a recombinant host cell described herein in a dose of about 10 7 cells / m 2 to about 10 11 cells / m 2 Appropriate dosages and treatment regimens are developed and specific compositions described herein are used in various treatment regimens, including, for example, parenteral or intravenous administration or formulations.

[0237] If the subject composition is administered parenterally, the composition may also include a sterile aqueous or oily solution or suspension. Suitable non-toxic parenterally 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 include 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 essentially non-toxic in the amount used. In addition, the active compound can be incorporated into sustained-release preparations and formulations. The dosage unit form used herein refers to a physically discrete unit suitable as a unit dose for the subject to be treated; each unit may contain a predetermined number of recombinant cells or active compounds calculated to produce the desired therapeutic effect together with an appropriate pharmaceutical carrier.

[0238] In general, an appropriate dosage and treatment regimen provides an amount of active molecules or cells sufficient to provide a therapeutic or preventive benefit. This response can be monitored by establishing better clinical outcomes (e.g., more frequent remissions, complete or partial remissions, or longer disease-free survival) in treated subjects compared to untreated 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.

[0239] The method according to the present disclosure may further include administering one or more additional agents in combination therapy to treat a disease or disorder. For example, in certain embodiments, combination therapy includes administering a composition of the present disclosure together with (simultaneously, synchronously or successively) an immune checkpoint inhibitor. In certain embodiments, combination therapy includes administering a composition of the present disclosure (e.g., TCR, polynucleotides, vectors or host cells or a combination thereof) together with an agonist of a stimulating immune checkpoint agent. In a further embodiment, combination therapy includes administering a composition of the present disclosure together with an adjuvant therapy, such as a chemotherapeutic agent, radiotherapy, surgery, an antibody or any combination thereof.

[0240] As used herein, the term "immune suppression agent" or "immunosuppression agent" refers to one or more cells, proteins, molecules, compounds, or complexes that provide inhibitory signals to help control or suppress an immune response. For example, immunosuppressants include those that partially or completely block immune stimulation; reduce, prevent, or delay immune activation; or increase, activate, or upregulate immune suppression. Exemplary immunosuppressive agents for targets (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.

[0241] The technology and scheme of carrying out HCT are known in the art and can include transplanting any suitable donor cell, such as the cell from umbilical cord blood, bone marrow or peripheral blood, hematopoietic stem cell, activated stem cell or the cell from amniotic fluid.Therefore, in certain embodiments, the immune cell of the modification of the present disclosure can be used together with hematopoietic stem cell or soon after hematopoietic stem cell in the HCT treatment of modification.In certain embodiments, HCT includes donor hematopoietic cells, which include chromosome knockout of the gene encoding HLA components, chromosome knockout of the gene encoding TCR components or both.

[0242] In further embodiments, the subject has received lymphodepleting chemotherapy prior to receiving the composition or HCT. In certain embodiments, the lymphodepleting chemotherapy comprises a conditioning regimen consisting of cyclophosphamide, fludarabine, antithymocyte globulin, or a combination thereof.

[0243] The method according to the present disclosure may further include administering one or more additional agents in the combination therapy to treat a disease or disorder. For example, in certain embodiments, the combination therapy includes administering the composition of the present disclosure together with (simultaneously, synchronously or successively) an immune checkpoint inhibitor. In certain embodiments, the combination therapy includes administering the composition of the present disclosure together with an agonist of a stimulating immune checkpoint agent. In a further embodiment, the combination therapy includes administering the composition of the present disclosure together with an adjuvant therapy, such as a chemotherapeutic agent, radiotherapy, surgery, an antibody or any combination thereof.

[0244] As used herein, the term "immune suppression agent" or "immunosuppression agent" refers to one or more cells, proteins, molecules, compounds, or complexes that provide inhibitory signals to help control or suppress an immune response. For example, immunosuppressants include those that partially or completely block immune stimulation; reduce, prevent, or delay immune activation; or increase, activate, or upregulate immune suppression. Exemplary immunosuppressive agents for targets (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.

[0245] Immunosuppressants Inhibitors (also known as immune checkpoint inhibitors) can be compounds, antibodies, antibody fragments or fusion polypeptides (such as Fc fusions, such as CTLA4-Fc or LAG3-Fc), antisense molecules, ribozymes or RNAi molecules or low molecular weight organic molecules. In any of the embodiments disclosed herein, the method may include a composition of the present disclosure with one or more inhibitors of any of the following immunosuppressive components, alone or in any combination.

[0246] In certain embodiments, the compositions of the present invention are used in combination with a PD-1 inhibitor, e.g., a PD-1 specific antibody or binding fragment thereof, such as pidilizumab, nivolumab, pembrolizumab, MEDI0680 (formerly AMP-514), AMP-224, BMS-936558, or any combination thereof. In further embodiments, the compositions 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. Also considered: cemiplimab; IBI-308; nivolumab+relatlimab; BCD-100; camrelizumab; JS-001; spartalizumab; tislelizumab; AGEN-2034; BGBA-333+tislelizumab; CBT-501; dostarlimab; durvalumab+MEDI-0680; JNJ-3283; pazopanib hydrochloride+pembrolizumab; pidilizumab; REGN-1979+cemiplimab; ABBV-181; ADUS-100+spartalizumab; AK-104; AK-105; AMP-224; BAT-1306; BI-754091; CC-90006; cemiplimab+REGN-3767; CS-1003; GLS-010; LZM-009; MEDI-5752; MGD-013; PF-06801591; Sym-021; tislelizumab+pamiparib; XmAb-20717; AK-112; A LPN-202; AM-0001; antibodies antagonizing PD-1 for Alzheimer's disease; BH-2922; BH-2941; BH-2950; BH-2954; biologics antagonizing CTLA-4 and PD-1 for solid tumors; bispecific monoclonal antibodies targeting PD-1 and LAG-3 for oncology; BLSM-101; CB-201; CB-213; CBT-103; CBT-107; cellular immunotherapy plus PD-1 inhibitor; CX-188; HAB-21; HEISCOIII-003; IKT-202; JTX-4014; MCLA-134; MD-402; mDX-400; MGD-019; monoclonal antibodies antagonizing PDCD1 for oncology;Monoclonal antibodies that antagonize PD-1 for oncology; anti-PD-1 antiviral agents for oncology; OT-2; PD-1 antagonist + ropeginterferon α-2b; PEGMP-7; PRS-332; RXI-762; STIA-1110; TSR-075; vaccines targeting HER2 and PD-1 for oncology; vaccines targeting PD-1 for oncology and autoimmune diseases; XmAb-23104; antisense oligonucleotides that inhibit PD-1 for oncology; AT-16201; Bispecific monoclonal antibodies targeting PD-1 for oncology; IMM-1802; monoclonal antibodies that antagonize PD-1 and CTLA-4 for solid tumors and hematologic malignancies; nivolumab biosimilar; recombinant proteins that agonize CD278 and CD28 and antagonize PD-1 for oncology; recombinant proteins that agonize PD-1 for autoimmune and inflammatory diseases; SNA-01; SSI-361; YBL-006; AK-103; JY-034; AUR-012; BGB-108; inhibitors of PD-1 and Gal-9 and TIM-3 for solid tumors; ENUM-244C8; ENUM-388D4; MEDI-0680; monoclonal antibodies that antagonize PD-1 for metastatic melanoma and metastatic lung cancer; monoclonal antibodies that inhibit PD-1 for oncology; monoclonal antibodies that target CTLA-4 and PD-1 for oncology; monoclonal antibodies that antagonize PD-1 for NSCLC; monoclonal antibodies that inhibit PD-1 and TIM-3 for oncology; monoclonal antibodies that inhibit PD-1 for oncology; recombinant proteins that inhibit PD-1 and VEGF-A White, for hematological malignancies and solid tumors; small molecules that antagonize PD-1, for oncology; Sym-016; inebilizumab + MEDI-0680; vaccines targeting PDL-1 and IDO, for metastatic melanoma; anti-PD-1 monoclonal antibodies and cellular immunotherapy, for glioblastoma; antibodies that antagonize PD-1, for oncology; monoclonal antibodies that inhibit PD-1 / PD-L1, for hematological malignancies and bacterial infections; monoclonal antibodies that inhibit PD-1, for HIV; or small molecules that inhibit PD-1, for solid tumors.

[0247] In certain embodiments, the compositions of the present disclosure are used with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof.

[0248] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of CTLA4. In specific embodiments, the compositions of the present disclosure are used in combination with a CTLA4-specific antibody or a binding fragment thereof, such as ipilimumab, tremelimumab, a CTLA4-Ig fusion protein (such as abatacept, belatacept), or any combination thereof.

[0249] In certain embodiments, the compositions of the present disclosure are used in combination with a B7-H3 specific antibody or binding fragment thereof, such as enoblituzumab (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 Dangaj et al., Cancer Res. 73:4820, 2013, and U.S. Patent No. 9,574,000 and PCT Patent Publication Nos. WO / 201640724A1 and WO 2013 / 025779A1.

[0250] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of CD244.

[0251] In certain embodiments, the compositions of the present disclosure 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.

[0252] In certain embodiments, the cellular compositions of the present disclosure are used in combination with an inhibitor of TIM3.

[0253] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of Gal9.

[0254] In certain embodiments, the compositions of the present disclosure are used in conjunction with inhibitors of adenosine signaling, such as decoy adenosine receptors.

[0255] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of A2aR.

[0256] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of KIR, such as lirilumab (BMS-986015). In certain embodiments, the compositions of the present disclosure are used in combination with inhibitory cytokines (typically cytokines other than TGFβ) or inhibitors of Treg development or activity.

[0257] In certain embodiments, the compositions of the present disclosure 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-methyltryptophan (1-MT)-tira-pazamine, or any combination thereof.

[0258] In certain embodiments, the compositions of the present disclosure are used in combination with an arginase inhibitor, such as N(ω)-nitro-L-arginine methyl ester (L-NAME), N-ω-hydroxy-nor-1-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-borohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine ​​(BEC), or any combination thereof.

[0259] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.).

[0260] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of TIGIT (eg, COM902 (Compugen, Toronto, Ontario Canada)), an inhibitor of CD155 (eg, COM701 (Compugen)), or both.

[0261] In certain embodiments, the compositions of the present disclosure 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.

[0262] In certain embodiments, the compositions of the present disclosure are used in combination with a LAIR1 inhibitor.

[0263] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.

[0264] In certain embodiments, the compositions of the present disclosure are used in combination with an agent that increases the activity of a stimulatory immune checkpoint molecule (ie, is an agonist). For example, the compositions of the present disclosure can be combined with CD137 (4-1BB) agonists (e.g., urelumab), 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), GITR agonists (e.g., humanized monoclonal antibodies described in 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 of the embodiments disclosed herein, the method may include administering the composition of the present disclosure with one or more agonists of stimulatory immune checkpoint molecules, including any of the above, alone or in any combination.

[0265] In certain embodiments, the combination therapy comprises a composition of the present disclosure and a secondary 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.

[0266] In certain embodiments, a combination therapy comprises administering a composition of the present disclosure and further administering radiation therapy or surgery. Radiation therapy is well known in the art and includes X-ray therapy, such as gamma ray therapy, and radiopharmaceutical therapy. Surgery and surgical techniques suitable for treating a subject's specific cancer are well known to those of ordinary skill in the art.

[0267] In certain embodiments, the combined treatment method comprises administering a composition of the present disclosure 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 (such as folic acid antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), DNA synthesis inhibitors, DNA interactors (such as intercalating agents), and DNA repair inhibitors. Illustrative chemotherapeutic agents include, but are not limited to, the following classes: 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 (clarithromycin)); antiproliferative / antiapoptotic agents, including natural products, such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disrupting agents, such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and nabendazim, epipodophyllum Toxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, Cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, ifosfamide, melphalan, nitrogen mustard, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, paclitaxel, taxotere, temozolomide, teniposide, triethylenethiophosphate, and etoposide (VP 16)); antibiotics, such as dactinomycin (actinomycin D)), daunorubicin, doxorubicin (Adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; the enzyme (L-asparaginase, which metabolizes L-asparagine systemically and deprives cells that do not have the ability to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents, such as nitrogen mustard (mechlorethamine ), cyclophosphamide and analogs, melphalan, chlorambucil), ethyleneimine and methylmelamine (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 complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminophenidone;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). agents, 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 (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin D, teniposide, 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 toxin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheria toxin, and caspase activators; and chromatin disrupting agents.

[0268] Cytokines can be used to manipulate the host's immune response for anti-cancer activity. See, for example, Floros & Tarhini, Semin. Oncol. 42(4): 539-548, 2015. Cytokines useful for promoting 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, alone or in any combination with the compositions of the present disclosure.

[0269] Also provided herein are methods of modulating adoptive immunotherapy, wherein the methods comprise administering to a subject that has previously received a modified host cell comprising a heterologous polynucleotide encoding a safety switch protein of the present disclosure a homologous compound of a safety switch protein in an amount effective to deplete the previously administered modified host cell in the subject.

[0270] In certain embodiments, the safety switch protein comprises tEGFR and the homologous compound is cetuximab, or the safety switch protein comprises iCasp9 and the homologous compound is AP1903 (e.g., dimeric AP1903), or the safety switch protein comprises an RQR polypeptide and the homologous compound is rituximab, or the safety switch protein comprises a myc binding domain and the homologous compound is an antibody specific for the myc binding domain.

[0271] In a further aspect, methods for making compositions or unit doses of the present disclosure are provided. In certain embodiments, these methods comprise combining (i) aliquots 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).

[0272] In certain embodiments, the method further includes, before packaging, cultivating the transduced host cells, and selecting the transduced cells as binding (i.e., expressing) the vector. In a further embodiment, the method includes, after cultivation and selection and before packaging, amplifying the transduced host cells. In any one embodiment of the present method, the composition or unit dose of manufacture can be frozen for future use. Any suitable host cell can be used for manufacturing a composition or unit dose according to the present method, for example, including hematopoietic stem cells, T cells, primary T cells, T cell lines, NK cells or NK-T cells. In a specific embodiment, the method includes a host cell, which is a CD8+T cell, a CD4+T cell or both.

[0273] In certain embodiments, the compositions of the present disclosure are used with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof.

[0274] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of CTLA4. In specific embodiments, the compositions of the present disclosure are used in combination with a CTLA4-specific antibody or a binding fragment thereof, such as ipilimumab, tremelimumab, a CTLA4-Ig fusion protein (such as abatacept, belatacept), or any combination thereof.

[0275] In certain embodiments, the compositions of the present disclosure are used in combination with a B7-H3 specific antibody or binding fragment thereof, such as enoblituzumab (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 Dangaj et al., Cancer Res. 73:4820, 2013, and U.S. Patent No. 9,574,000 and PCT Patent Publication Nos. WO / 201640724A1 and WO 2013 / 025779A1.

[0276] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of CD244.

[0277] In certain embodiments, the compositions of the present disclosure 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.

[0278] In certain embodiments, the cellular compositions of the present disclosure are used in combination with an inhibitor of TIM3.

[0279] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of Gal9.

[0280] In certain embodiments, the compositions of the present disclosure are used in conjunction with inhibitors of adenosine signaling, such as decoy adenosine receptors.

[0281] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of A2aR.

[0282] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of KIR, such as lirilumab (BMS-986015). In certain embodiments, the compositions of the present disclosure are used in combination with inhibitory cytokines (typically cytokines other than TGFβ) or inhibitors of Treg development or activity.

[0283] In certain embodiments, the compositions of the present disclosure 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-methyltryptophan (1-MT)-tira-pazamine, or any combination thereof.

[0284] In certain embodiments, the compositions of the present disclosure are used in combination with an arginase inhibitor, such as N(ω)-nitro-L-arginine methyl ester (L-NAME), N-ω-hydroxy-nor-1-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-borohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine ​​(BEC), or any combination thereof.

[0285] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.).

[0286] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitor of TIGIT (eg, COM902 (Compugen, Toronto, Ontario Canada)), an inhibitor of CD155 (eg, COM701 (Compugen)), or both.

[0287] In certain embodiments, the compositions of the present disclosure 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.

[0288] In certain embodiments, the compositions of the present disclosure are used in combination with a LAIR1 inhibitor.

[0289] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.

[0290] In certain embodiments, the compositions of the present disclosure are used in combination with an agent that increases the activity of a stimulatory immune checkpoint molecule (ie, is an agonist). For example, the compositions of the present disclosure can be combined with CD137 (4-1BB) agonists (e.g., urelumab), 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), GITR agonists (e.g., humanized monoclonal antibodies described in 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 of the embodiments disclosed herein, the method may include administering the composition of the present disclosure with one or more agonists of stimulatory immune checkpoint molecules, including any of the above, alone or in any combination.

[0291] In certain embodiments, the combination therapy comprises a composition of the present disclosure and a secondary 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.

[0292] In certain embodiments, a combination therapy comprises administering a composition of the present disclosure and further administering radiation therapy or surgery. Radiation therapy is well known in the art and includes X-ray therapy, such as gamma ray therapy, and radiopharmaceutical therapy. Surgery and surgical techniques suitable for treating a subject's specific cancer are well known to those of ordinary skill in the art.

[0293] In certain embodiments, the combined treatment method comprises administering a composition of the present disclosure 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 (such as folic acid antagonists, pyrimidine analogs, purine analogs, and sugar-modified analogs), DNA synthesis inhibitors, DNA interactors (such as intercalating agents), and DNA repair inhibitors. Illustrative chemotherapeutic agents include, but are not limited to, the following classes: 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 (clarithromycin)); antiproliferative / antiapoptotic agents, including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disrupting agents, such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and vinorelbine; oxaliplatin, epipodophyllotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cyclophosphamide (Cytoxan), dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, ifosfamide, melphalan, nitrogen mustard, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, paclitaxel (taxol), taxotere, temozolomide, teniposide, triethylenethiophosphate, 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 systemically metabolizes L-asparagine and deprives cells that do not have the ability to synthesize their own asparagine); antiplatelet agents; 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 complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminophenidone;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 (brefedin); 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 (ARBs); antagonists; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab, rituximab); chimeric antigen receptors; cell cycle inhibitors and differentiation inducers (retinoic acid); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, actinomycin D, teniposide, 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 toxin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheria toxin, and caspase activators; and chromatin disruptors.

[0294] Cytokines can be used to manipulate the host's immune response for anti-cancer activity. See, for example, Floros & Tarhini, Semin. Oncol. 42(4): 539-548, 2015. Cytokines useful for promoting 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, alone or in any combination with the compositions of the present disclosure.

[0295] Also provided herein are methods of modulating adoptive immunotherapy, wherein the methods comprise administering to a subject that has previously received a modified host cell comprising a heterologous polynucleotide encoding a safety switch protein of the present disclosure a homologous compound of a safety switch protein in an amount effective to deplete the previously administered modified host cell in the subject.

[0296] In certain embodiments, the safety switch protein comprises tEGFR and the homologous compound is cetuximab, or the safety switch protein comprises iCasp9 and the homologous compound is AP1903 (e.g., dimeric AP1903), or the safety switch protein comprises an RQR polypeptide and the homologous compound is rituximab, or the safety switch protein comprises a myc binding domain and the homologous compound is an antibody specific for the myc binding domain.

[0297] In a further aspect, methods for making compositions or unit doses of the present disclosure are provided. In certain embodiments, these methods comprise combining (i) aliquots 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).

[0298] In certain embodiments, the method further includes, before packaging, cultivating the transduced host cells, and selecting the transduced cells as binding (i.e., expressing) the vector. In a further embodiment, the method includes, after cultivation and selection and before packaging, amplifying the transduced host cells. In any one embodiment of the present method, the composition or unit dose of manufacture can be frozen for future use. Any suitable host cell can be used for manufacturing a composition or unit dose according to the present method, for example, including hematopoietic stem cells, T cells, primary T cells, T cell lines, NK cells or NK-T cells. In a specific embodiment, the method includes a host cell, which is a CD8+T cell, a CD4+T cell or both.

[0299] Example

[0300] Example 1

[0301] method

[0302] cell lines

[0303] T2 is a TAP-deficient T-cell leukemia / B-LCL hybrid cell line that exclusively expresses HLA A*02:01. 11 293T / 17 is a highly transfectable cell line purchased from ATCC. Jurkat76 cells are a TCRα / TCRβ-deficient derivative of the parental Jurkat cell line and do not naturally express CD8 12Jurkat76 cells were previously transduced to express CD8αβ (Jurkat-CD8). The cell line was maintained in RPMI 1640 medium supplemented with HEPES (Invitrogen, GIBCO) and 10% heat-inactivated FBS (Hyclone, GE Healthcare Life Sciences), 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0304] Human T cell culture:

[0305] Leukocyte-depleted samples were collected from healthy donors at the Seattle Cancer Care Alliance under protocol 868.01 with written informed consent in accordance with the Declaration of Helsinki and approved by the Institutional Review Board. PBMCs were isolated from HLA-typed donors as previously described. 13,14 , 10 HLA-A*02:01 restricted T cell lines were generated for each donor, which were responsive to peptide WT1 37-45 、VLDFAPPGA has specificity (a total of 10 donors). In short, using EasySep TM CD8+ T cells were purified using a human CD8+ T cell isolation kit (StemCell Technologies) by adherence to plastic and cultured with 1000 U / ml IL-4 and 800 U / ml GMCSF for 2 days. A maturation cytokine cocktail was added on the last day before harvest. DCs were generated from autologous PBMCs. DCs were loaded with 1 μg / ml of peptide for 90 minutes, then washed to remove excess peptide and irradiated at 4000 Rad. Approximately 5 × 10 6 CD8+ T cells were co-cultured with peptide-pulsed DCs plus 30 ng / ml IL-21 at a ratio of 2.5:1. T cells were maintained in RPMI1640 medium containing HEPES (Invitrogen, GIBCO) supplemented with 5% heat-inactivated pooled human serum (Bloodworks Northwest), 100 U / mL penicillin, 100 μg / mL streptomycin, and 55 μM 2-β-mercaptoethanol. Cultures were cultured every 2-3 days with regular medium and supplemented with 12.5 U / ml IL-2, 2250 U / ml IL-7, and IL-15. Every 10 days, T cells were restimulated by culturing with irradiated, peptide-pulsed autologous PBMCs at a ratio of 1:2.

[0306] Flow cytometry-based cell sorting

[0307] At the end of antigen-specific amplification, T cell lines from all donors were pooled on ice. Pooled samples were split and stained with peptide / HLA-A2 tetramers under three conditions: (1) a wild-type tetramer concentration empirically determined to maximize separation of positive and negative populations, as described in the "Tetramer Binding and Affinity Measurements" section; (2) a 100-fold dilution of the optimal tetramer dose; and 3) a separate modified tetramer made by mutating the HLA-A2 molecule at positions D227K and T228A in the α3 domain, which interacts with CD8 15 This tetramer has been shown to selectively bind high-affinity CD8-independent TCRs. 16,17 For each tetramer-stained sample, cells with the highest level of tetramer binding (top approximately 2% of labeled cells) were flow cytometrically sorted. The sorted populations were analyzed using Adaptive Biotechnologies' immunosequencing assay to quantify the relative abundance of each clonotype. Additional samples containing the entire tetramer-positive population were also sorted from the best tetramer-stained sample, and TCRαβ pairing information was determined using the Adaptive Biotechnologies pairSeq Assay. 18 Sure.

[0308] Data Analysis

[0309] Enrichment calculation

[0310] The enrichment score for each clonotype was calculated as follows: (sorted tetramers + The frequency in the pooled sample was calculated as (frequency in the unsorted pooled sample) / (frequency in the unsorted pooled sample). Clonotypes not detected in the pooled sample were assigned a frequency corresponding to 1 cell in the pooled sample for the calculation of enrichment.

[0311] TCR Sequencing and Alpha / Beta Pairing:

[0312] TCR profiling was performed using immunosequencing from Adaptive Biotechnologies, and single-cell V(D)J profiling (TCRα / β pairing) was performed using Chromium Single Cell Immune Profiling from 10xgenomics.

[0313] TCR transduction

[0314] In BioXp TMA codon-optimized TCR construct in the TCRβ-p2a-TCR orientation was synthesized on 3200 (SGI-DNA) and cloned into the pRRLSIN.cPPT.MSCV.WPRE lentiviral expression plasmid (a gift from Dr. Richard Morgan at NCI) by Gibson assembly. This expression vector was then packaged into 293T cells using a third-generation lentiviral packaging system. Lentiviral supernatant was harvested 48 hours later and filtered to remove cell debris. Approximately 5×10 5 Jurkat76 cells were combined with 2 ml of lentiviral supernatant plus 5 μg / ml polybrene. The cells were centrifuged at 1000 g for 90 minutes at 30°C to facilitate transduction. For TCR transduction of primary CD8+ T cells, EasySep was used. TM Human CD8+ T cell isolation kit (StemCell Technologies) enriched for HLA-A2 + PBMCs were used for CD8+ T cells and Dynabeads TM Human T-Expander CD3 / CD28 (Gibco) was activated for 4 hours. Approximately 2×10 6 CD8+ T cells were combined with 2 ml of lentiviral supernatant plus 5 μg / ml protamine and 50 U / ml IL-2. Transgenic TCR+ cells were sorted by flow cytometry using peptide / HLA-A*02:01 tetramers to obtain a pure population of antigen-specific cells for downstream analysis.

[0315] TCR binding data

[0316] Assessment of correct TCR pairing

[0317] Jurkat76 cells were transduced with each TCR construct and analyzed for tetramer binding relative to CD3 surface expression, which reflects the total amount of transgenic TCR surface expression in these cells lacking endogenous TCR.

[0318] Binding and affinity measurements of tetramers

[0319] The optimal tetramer dose is determined by titrating the tetramer against the positive T cell population and selecting the concentration that best separates the positive and negative cell populations without increasing background staining of the negative cell population.

[0320] TCR functional data

[0321] IFN-γ production

[0322] Primary CD8+ T cells were lentivirally transduced with each TCR expression construct and sorted to generate a homogeneous population of tetramer-positive cells, which were then incubated at a 1:1 ratio with decreasing doses of peptide (1-10 -5 M)-pulsed T2 target cells were mixed. Where indicated, autologous PBMCs were also used as APCs. After 4 hours of culture in the presence of Golgi inhibitors (BD GolgiPlug and GolgiStop), cells were surface stained with anti-CD8, fixed (BD Cytofix / Cytoperm), and intracellularly labeled with anti-IFN-γ in BD Perm / Wash buffer. Cells were analyzed by flow cytometry to determine the percentage of IFN-γ+ cells in each sample. These data were fitted to dose-response curves using nonlinear regression in Graphpad Prism (four parameters - variable slope, with the bottom and top of the curve constrained to 0 and 100, respectively).

[0323] Figures 1(A) and 1(B) show how WT1 was identified using a high-throughput sequencing-based strategy. 37-45 Peptide-specific TCRs. TCR clonotypes enriched in high tetramer binding sorts compared to the total tetramer-positive population are identified as likely to have high affinity or high functional avidity for peptide / HLA-A2 ligands. (A) TCR clonotypes enriched in high tetramer binding sorts compared to the total tetramer-positive population are identified as likely to have high affinity or high functional avidity for peptide / HLA-A2 ligands. 37-45 Schematic diagram of the initial sequencing-based strategy for peptide / MHC tetramer binding-associated TCR clonotypes. (B) The enrichment of the sorted population relative to the total population is shown, with selected TCRs highlighted. All TCRs, represented by black circles, were synthesized and evaluated for antigen specificity (27 in total).

[0324] Figure 2 Results of tetramer binding studies are shown, assessing the specificity and relative tetramer binding affinity of selected TCRs. TCR constructs were expressed in Jurkat cells lacking endogenous TCR α / β chains. Tetramer staining is shown in relation to CD3 expression for each TCR (CD3 expression is directly correlated with transgenic TCR surface expression).

[0325] Example 2

[0326] Identification of high-functional affinity TCRs

[0327] Since some high-affinity TCRs have been shown to bind tetramers independent of CD8, a second experiment was performed to identify additional TCRs that were specifically enriched in the high tetramer binding sorted population when tetramers independent of CD8 (CD8i) were used. Figures 3A-3C Shown how to identify additional WT1 by a modified high-throughput sequencing-based strategy using CD8-independent (CD8i) tetramers37-45 Peptide-specific TCR. Figure 3A Schematic diagram of a modified sequencing strategy for identifying WT1 that is highly independent of CD8 37 TCR clonotypes associated with peptide / MHC tetramer binding. Figure 3B and 3C The enrichment of the original sorted population relative to the total population is shown compared to a similar analysis using CD8i tetramers. Fourteen additional TCRs were selected based on surface CD3 levels and CD8i tetramer binding. Figure 3B and 3C All named TCR clonotypes in the were synthesized and assessed for antigen specificity. Figure 3C All TCRs represented by medium shaded (diagonal pattern) circles represent additional TCRs identified using CD8i tetramers.

[0328] Example 3

[0329] Relationship between tetramer staining and CD3 expression

[0330] Figure 4 Additional CD8i tetramer selection of WT1 is shown 37-45 CD8i tetramer binding of peptide-specific TCRs. TCR constructs were expressed in Jurkat cells lacking endogenous TCR chains (and lacking CD8 expression). Figure 4 Tetramer staining for each TCR is shown relative to CD3 expression (CD3 expression directly correlates with transgenic TCR surface expression). TCRs that bound the most strongly to tetramers, resulting in high levels of tetramer staining relative to anti-CD3 staining, were selected for further analysis.

[0331] Example 4

[0332] IFNγ assay to measure functional avidity (EC 50 )

[0333] The ability of TCR to signal T cell activation at limiting antigen concentrations is mediated by peptide ECs. 50 To measure, EC 50 The EC value is the amount of peptide that target cells need to be pulsed with to elicit a response (e.g., IFNγ production) in 50% of T cells transduced with the TCR of the present invention. This value is directly related to the ability of T cells expressing a specific TCR to kill target cells expressing the antigen. To determine the peptide EC value of a selected TCR, 50 , each TCR was transduced into CD8+ T cells isolated from donor PMBCs ( Figure 5A ). After one week, cells were sorted into tetramers + CD8 +The expanded antigen-specific cells were cultured with peptide-pulsed T2 target cells for 4-6 hours, and the production of IFNγ was measured by flow cytometry ( Figure 5A The percentage of cells producing IFNγ was fitted to the dose-response curve by nonlinear regression to calculate the peptide EC for each TCR. 50 ( Figure 5B ).

[0334] Example 5

[0335] Expression of WT1 37-45 Primary CD8+ T cells expressing peptide-specific TCR kill HLA-A2 in vitro + WT1 + MDA-MB-468 cells

[0336] To directly assess TCR-transduced CD8+ T cell-mediated lysis of tumor cells naturally expressing and presenting the WT1p37 antigen on HLA-A2, donor-derived CD8+ T cells were transduced with one of each of the selected TCRs and sorted for high tetramer binding. TCR-transduced T cells were then mixed (in triplicate) at an 8:1 ratio with the breast cancer cell line MDA-MB-468, which had been treated with Rapid Red dye staining. The total area of ​​red objects (correlated with the total number of viable target cells) was calculated at designated time points over a 72-hour period for each TCR-transduced T cell population. The most potent tumor-reactive T cells will maintain long-term reactivity to tumor antigens after in vivo transfer into the patient. Therefore, to assess the sustained reactivity of TCR-transduced T cells to persistent antigens, additional MDA-MB-468 cells were added over a 48-hour period. Figure 6 .

[0337] Example 6

[0338] Expression of WT1 37-45 Peptide-specific TCRs of primary CD4+ and CD8+ T cells against HLA-A2 + WT1 + Killing of PANC-1 cells in vitro

[0339] Both CD4+ and CD8+ T cells can play a role in clearing tumors in vivo. Therefore, an MHC class I restricted TCR that can also signal an antigen-specific response in CD4+ T cells is better than a TCR that can only activate CD8+ T cells. The ability of an MHC class I restricted TCR to function in CD4+ T cells appears to depend in part on the affinity of the TCR for peptide MHC. In many cases, transduction of CD4+ T cells with genes encoding CD8α and CD8β helps to effectively elicit antigen-specific responses. Therefore, to evaluate the efficacy of CD4 (transduced with CD8α / CD8β) versus CD8 T cells expressing TCR10.1 targeting HLA-A2 + WT1 + The ability of tumor cells, CD4+ and CD8+ T cells were transduced to express WT1 37-45 TCR10.1.CD4 + T cells were further transduced to express CD8α and CD8β genes. After 8 days, the transduced cells were sorted to purify CD8 + Tetramer + and CD4 + / CD8 + Tetramer + Antigen-specific cells of CD4+, CD8+, or a mixture of these two populations (CD4 and CD8) were mixed at an 8:1 ratio (in triplicate) with the pancreatic cancer cell line PANC-1, which had been previously transduced to express Red dye. The total area of ​​red objects (relative to the total number of viable target cells) was calculated at the indicated time points for each TCR-transduced T cell population. To assess the sustained responsiveness of TCR-transduced T cells to long-term antigens, additional PANC-1 cells were added at 48 hours. Figure 7 Shown, expression of WT1 37-45 Both CD4+ and CD8+ T cells expressing TCR10.1 can eliminate WT1 after repeated in vitro challenge + A2 + Pancreatic cancer cell line PANC-1.

[0340] The WT1 p126 epitope is not always efficiently processed / presented by cells expressing WT1 and HLA-A2 (Jaigirdar et al., J. Immunother. 39:105, 2017). In particular, several solid tumor-derived cell lines expressing WT1 and HLA-A2 were not effectively targeted by WT1-p126-specific TCRs, regardless of whether they were pre-cultured with IFNγ to upregulate immunoproteasome expression. In certain aspects, the present disclosure relates, in part, to the discovery that the WT1-p37 epitope is more widely processed and presented by various tumor types than the WT1-p126 epitope. Figures 8A-8DLysis of various WT1+A2+ tumor cell lines by a WT1-p126 peptide-specific TCR compared to a WT1 p37 peptide-specific TCR is shown. These data highlight the fact that WT1 p-37 peptide-specific TCRs appear to be generally more reliably able to target a broad range of WT1+A2+ tumors.

[0341] The various embodiments described herein can be combined to provide further embodiments. All patents, patent application publications, patent applications, and non-patent publications mentioned in this specification and / or listed in the application data sheet, including but not limited to U.S. patent application No. 62 / 816,746 filed on March 11, 2019, are incorporated herein by reference in their entirety. In general, the terms used in the following claims should not be construed as limited to the specific embodiments disclosed herein, but should be construed to include all possible embodiments, along with the full scope of equivalents to which such claims are entitled.

[0342] References

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[0347] 5.Chapuis,A.G.et al.Transferred melanoma-specific CD8+T cellspersist,mediate tumor regression,and acquire central memory phenotype.ProcNatl Acad S ci U S A(2012).doi:10.1073 / pnas.1113748109

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[0356] 14.Chapuis,A.G.et al.Transferred WT1-reactive CD8+T cells can mediatea ntileukemic activity and persist in post-transplant patients.Sci Transl Med5,174ra1 27(2013).

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[0360] 18. Howie, B. et al. High-throughput pairing of T cell receptor alpha and beta sequences. Sci Transl Med 7, 301ra131 (2015). Sequence Listing <110> Fred Hutchinson Cancer Research Center T.M. Schmidt A.G. Chapiy P.D. Greenberg <120> High-affinity WT1 T cell receptor and its use <130> 360056.466WO <140> PCT <141> 2020-03-10 <150> US 62 / 816,746 <151> 2019-03-11 <160> 263 <170> FastSEQ for Windows version 4.0 <210> 1 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR10.1-β CDR3 (IMGT connection) <400> 1 Cys Ala Ser Ser Leu Thr Gly Ser Tyr Glu Gln Tyr Phe 1 5 10 <210> 2 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR11.2-β CDR3 (IMGT connection) <400> 2 Cys Ser Ala Thr Pro Glu Ala Ser Ser Pro Tyr Glu Gln Tyr Phe 1 5 10 15 <210> 3 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR12.1-β CDR3 (IMGT connection) <400> 3 Cys Ala Thr Ser Asn Leu Gln Gly Arg Gln Pro Gln His Phe 1 5 10 <210> 4 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR13.1-β CDR3 (IMGT connection) <400> 4 Cys Ala Ser Ser Leu Arg Leu Gly Arg Glu Thr Gln Tyr Phe 1 5 10 <210> 5 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR13.2-β CDR3 (IMGT connection) <400> 5 Cys Ala Ser Ser Leu Gly Gln Ala Tyr Glu Gln Tyr Phe 1 5 10 <210> 6 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR14.1-β CDR3 (IMGT connection) <400> 6 Cys Ala Ser Ser Leu Thr Arg Gly Ala Glu Ala Phe Phe 1 5 10 <210> 7 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR15.1-β CDR3 (IMGT connection) <400> 7 Cys Ala Ser Ser Arg Asp Arg Glu Gln Glu Ser Pro Leu His Phe 1 5 10 15 <210> 8 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR16.1-β CDR3 (IMGT connection) <400> 8 Cys Ala Ser Ser Phe Ser Gly Gly Thr Tyr Glu Gln Tyr Phe 1 5 10 <210> 9 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR16.2-β CDR3 (IMGT connection) <400> 9 Cys Ala Ser Ser Tyr Arg Gly Gly Ser Thr Tyr Glu Gln Tyr Phe 1 5 10 15 <210> 10 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR18.1-β CDR3 (IMGT connection) <400> 10 Cys Ala Ser Ser Gln Arg Asp Ser Pro Asn Glu Lys Leu Phe Phe 1 5 10 15 <210> 11 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR19.1-β CDR3 (IMGT connection) <400> 11 Cys Ala Ser Ser Gln Asp Pro Tyr Lys Leu Ser Gly Asn Thr Ile Tyr 1 5 10 15 Phe <210> 12 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR10.1-α CDR3 (IMGT connection) <400> 12 Cys Ala Val Lys Glu Thr Ser Gly Ser Arg Leu Thr Phe 1 5 10 <210> 13 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR11.2-α CDR3 (IMGT connection) <400> 13 Cys Ala Phe Ile Tyr Pro Ser Tyr Thr Ser Gly Thr Tyr Lys Tyr Ile 1 5 10 15 Phe <210> 14 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR12.1-α CDR3 (IMGT connection) <400> 14 Cys Ala Ala Ser Gly Thr Gly Gly Ser Tyr Ile Pro Thr Phe 1 5 10 <210> 15 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR13.1-α CDR3 (IMGT connection) <400> 15 Cys Ala Ala Ser Gly Ile Gly Asp Tyr Lys Leu Ser Phe 1 5 10 <210> 16 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR13.2-α CDR3 (IMGT connection) <400> 16 Cys Ala Val Arg Thr Ser Tyr Asp Lys Val Ile Phe 1 5 10 <210> 17 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR14.1-α CDR3 (IMGT connection) <400> 17 Cys Ala Val Asn Leu Leu Gly Ala Thr Gly Tyr Ser Thr Leu Thr Phe 1 5 10 15 <210> 18 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR15.1-α CDR3 (IMGT connection) <400> 18 Cys Ala Val Arg Gly Ile Asn Asp Tyr Lys Leu Ser Phe 1 5 10 <210> 19 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR16.1-α CDR3 (IMGT connection) <400> 19 Cys Ala Val Ile Thr Gly Phe Gln Lys Leu Val Phe 1 5 10 <210> 20 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR16.2-α CDR3 (IMGT connection) <400> 20 Cys Ile Ala Gly Val Gly Arg Gly Gln Asn Phe Val Phe 1 5 10 <210> twenty one <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR18.1-α CDR3 (IMGT connection) <400> twenty one Cys Ala Phe His Pro Asn Phe Gly Asn Glu Lys Leu Thr Phe 1 5 10 <210> twenty two <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR19.1 - αa CDR3 (IMGT connection) <400> twenty two Cys Ala Val Gln Pro Arg Gly Asp Gly Ser Ser Asn Thr Gly Lys Leu 1 5 10 15 Ile Phe <210> twenty three <211> 133 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR10.1 - Vβ <400> 23 Met Gly Thr Ser Leu Leu Cys Trp Val Val Leu Gly Phe Leu Gly Thr 1 5 10 15 Asp His Thr Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr 20 25 30 Lys Arg Gly Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His 35 40 45 Val Ser Leu Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe 50 55 60 Leu Thr Tyr Phe Asn Tyr Glu Ala Gln Gln Asp Lys Ser Gly Leu Pro​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<223> Synthetic sequence WT137-45 TCR11.2 - Vβ <400> 24 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 Thr 100 105 110 Pro Glu Ala Ser Ser Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Thr Glu 130 <210> 25 <211> 133 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR12.1 - Vβ <400> 25 Met Gly Pro Gly Leu Leu His Trp Met Ala Leu Cys Leu Leu Gly Thr 1 5 10 15 Gly His Gly Asp Ala Met Val Ile Gln Asn Pro Arg Tyr Gln Val Thr 20 25 30 Gln Phe Gly Lys Pro Val Thr Leu Ser Cys Ser Gln Thr Leu Asn His 35 40 45 Asn Val Met Tyr Trp Tyr Gln Gln Lys Ser Ser Gln Ala Pro Lys Leu 50 55 60 Leu Phe His Tyr Tyr Asp Lys Asp Phe Asn Asn Glu Ala Asp Thr Pro 65 70 75 80 Asp Asn Phe Gln Ser Arg Arg Pro Asn Thr Ser Phe Cys Phe Leu Asp 85 90 95 Ile Arg Ser Pro Gly Leu Gly Asp Ala Ala Met Tyr Leu Cys Ala Thr 100 105 110 Ser Asn Leu Gln Gly Arg Gln Pro Gln His Phe Gly Asp Gly Thr Arg 115 120 125 Leu Ser Ile Leu Glu 130 <210> 26 <211> 143 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR13.1 - Vβ <400> 26 Met Leu Ser Pro Asp Leu Pro Asp Ser Ala Trp Asn Thr Arg Leu Leu 1 5 10 15 Cys His Val Met Leu Cys Leu Leu Gly Ala Val Ser Val Ala Ala Gly 20 25 30 Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg Glu Thr Ala 35 40 45 Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val Tyr Trp Tyr 50 55 60 Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser Phe Tyr Glu 65 70 75 80 Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe Ser Ala Gln 85 90 95 Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser Leu Glu Leu 100 105 110 Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Leu Arg Leu Gly Arg 115 120 125 Glu Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Leu Val Leu Glu 130 135 140 <210> 27 <211> 133 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR13.2 - Vβ <400> 27 Met Gly Thr Arg Leu Leu Cys Trp Val Val Leu Gly Phe Leu Gly Thr 1 5 10 15 Asp His Thr Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Ala 20 25 30 Lys Arg Gly Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His 35 40 45 Val Ser Leu Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe 50 55 60 Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro 65 70 75 80 ​​​​​​​​​​​​​​​​​​<211> 133 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence WT137-45 TCR14.1 - Vβ <400> 28 Met Gly Thr Arg Leu Leu Cys Trp Val Ala Phe Cys Leu Leu Val Glu [[ID=!4]]1 5 10 15 Glu Leu Ile Glu Ala Gly Val Val Gln Ser Pro Arg Tyr Lys Ile Ile 20 25 30 Glu Lys Lys Gln Pro Val Ala Phe Trp Cys Asn Pro Ile Ser Gly His 35 40 45 Asn Thr Leu Tyr Trp Tyr Leu Gln Asn Leu Gly Gln Gly Pro Glu Leu 50 55 60 Leu Ile Arg Tyr Glu Asn Glu Glu Ala Val Asp Asp Ser Gln Leu Pro 65 70 75 80 Lys Asp Arg Phe Ser Ala Glu Arg Leu Lys Gly Val Asp Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ala Glu Leu Gly Asp Ser Ala Val Tyr Leu Cys Ala 100 105 110 Ser Ser Leu Thr Arg Gly Ala Glu Ala Phe Phe Gly Gln Gly Thr Arg 115 120 125 Leu Thr Val Val Glu 130 <210> 29 <211> 134 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137 - 45 TCR15.1 - Vβ <400> 29 Met Ser Asn Gln Val Leu Cys Cys Val Val Leu Cys Phe Leu Gly Ala 1 5 10 15 Asn Thr Val Asp Gly Gly Ile Thr Gln Ser Pro Lys Tyr Leu Phe Arg 20 25 30 Lys Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His 35 40 45 Asp Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Gln Ile Val Asn Asp Phe Gln Lys Gly Asp Ile Ala 65 70 75 80 Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr 85 90 95 Val Thr Ser Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser 100 105 110 Ser Arg Asp Arg Glu Gln Glu Ser Pro Leu His Phe Gly Asn Gly Thr 115 120 125 Arg Leu Thr Val Thr Glu 130 <210> 30 <211> 133 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR16.1 - Vβ <400> 30 Met Gly Pro Gln Leu Leu Gly Tyr Val Val Leu Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Leu Glu Ala Gln Val Thr Gln Asn Pro Arg Tyr Leu Ile Thr 20 25 30 Val Thr Gly Lys Lys Leu Thr Val Thr Cys Ser Gln Asn Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Gln 50 55 60 Ile Tyr Tyr Ser Met Asn Val Glu Val Thr Asp Lys Gly Asp Val Pro 65 70 75 80 Glu Gly Tyr Lys Val Ser Arg Lys Glu Lys Arg Asn Phe Pro Leu Ile 85 90 95 Leu Glu Ser Pro Ser Pro Asn Gln Thr Ser Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Phe Ser Gly Gly Thr Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Thr Glu 130 <210> 31 <211> 144 <212> PRT <213> artificial sequence <220> <223> synthetic sequence WT137-45 TCR16.2 - Vβ <400> 31 Met Leu Ser Pro Asp Leu Pro Asp Ser Ala Trp Asn Thr Arg Leu Leu 1 5 10 15 Cys His Val Met Leu Cys Leu Leu Gly Ala Val Ser Val Ala Ala Gly 20 25 30 Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg Glu Thr Ala 35 40 45 Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val Tyr Trp Tyr 50 55 60 Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser Phe Tyr Glu 65 70 75 80 Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe Ser Ala Gln 85 90 95 Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser Leu Glu Leu 100 105 110 Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Tyr Arg Gly Gly Ser 115 120 125 Thr Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr Glu 130 135 140 <210> 32 <211> 135 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence WT137 - 45 TCR18.1 - Vβ <400> 32 Met Ser Thr Arg Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala 1 5 10 15 Glu Leu Ser Glu Ala Glu Val Ala Gln Ser Pro Arg Tyr Lys Ile Thr 20 2​​​​​​​​​​​​​​​​​​​​​Ser Ser Gln Arg Asp Ser Pro Asn Glu Lys Leu Phe Phe Gly Ser Gly 115 120 125 Thr Gln Leu Ser Val Leu Glu 130 135 <210> 33 <211> 136 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR19.1 - Vβ <400> 33 Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala 1 5 10 15 Val Pro Met Glu Thr Gly Val Thr Gln Thr Pro Arg His Leu Val Met 20 25 30 Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His Leu Gly His 35 40 45 Asn Ala Met Tyr Trp Tyr Lys Gln Ser Ala Lys Lys Pro Leu Glu Leu 50 55 60 Met Phe Val Tyr Ser Leu Glu Glu Arg Val Glu Asn Asn Ser Val Pro 65 70 75 80 Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser His Leu Phe Leu His 85 90 95 Leu His Thr Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser 100 105 110 Ser Gln Asp Pro Tyr Lys Leu Ser Gly Asn Thr Ile Tyr Phe Gly Glu 115 120 125 Gly Ser Trp Leu Thr Val Val Glu 130 135 <210> 34 <211> 131 <212> PRT <213> Synthetic sequence <220> <223> Synthetic sequence WT137 - 45 TCR10.1 Vα <400> 34 Met Glu Thr Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln Trp 1 5 10 15 Val Ser Ser Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val 20 25 30 Pro Glu Gly Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala 35 40 45 Ile Tyr Asn Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr 50 55 60 Ser Leu Leu Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg 65 70 75 80 Leu Asn Ala Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile 85 90 95 Ala Ala Ser Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Lys 100 105 110 Glu Thr Ser Gly Ser Arg Leu Thr Phe Gly Glu Gly Thr Gln Leu Thr 115 120 125 Val Asn Pro 130 <210> 35 <211> 139 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence WT137-45 TCR11.2 Vα <400> 35 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 Asn 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 Ile Tyr Pro Ser Tyr Thr Ser Gly Thr Tyr Lys Tyr Ile Phe 115 120 125 Gly Thr Gly Thr Arg Leu Lys Val Leu Ala Asn 130 135<00016​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ 85 90 95 Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala 100 105 110 Val Tyr Phe Cys Ala Ala Ser Gly Thr Gly Gly Ser Tyr Ile Pro Thr 115 120 125 Phe Gly Arg Gly Thr Ser Leu Ile Val His Pro Tyr 130 135 140 <210> 37 <211> 139 <212> PRT <213> artificial sequence <220> <223> synthetic sequence WT137-45 TCR13.1 Vα <400> 37 Met Ala Met Leu Leu Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro 1 5 10 15 Asp Trp Val Asn Ser Gln Gln Light Asp Asp Asp Gln Gln Val Light Gln 20 25 30 Asn Ser Pro Ser Leu Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn 35 40 45 Cys Asp Tyr Thr Asn Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys 50 55 60 Tyr Pro Ala Glu Gly Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys 65 70 75 80 Asp Lys Asn Glu Asp Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala 85 90 95 Lys His Leu Ser Leu His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala 100 105 110 Val Tyr Phe Cys Ala Ala Ser Gly Ile Gly Asp Tyr Lys Leu Ser Phe 115 120 125 Gly Ala Gly Thr Thr Val Thr Val Arg Ala Asn 130 135 <210> 38 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence WT137-45 TCR13.2 Vα <400> 38 Met Val Lys Ile Arg Gln Phe Leu Leu Ala Ile Leu Trp Leu Gln Leu 1 5 10 15 Ser Cys Val Ser Ala Ala Lys Asn Glu Val Glu Gln Ser Pro Gln Asn 20 25 30 Leu Thr Ala Gln Glu Gly Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser 35 40 45 Val Gly Ile Ser Ala Leu His Trp Leu Gln Gln His Pro Gly Gly Gly 50 55 60 Ile Val Ser Leu Phe Met Leu Ser Ser Gly Lys Lys Lys His Gly Arg 65 70 75 80 Leu Ile Ala Thr Ile Asn Ile Gln Glu Lys His Ser Ser Leu His Ile 85 90 95 Thr Ala Ser His Pro Arg Asp Ser Ala Val Tyr Ile Cys Ala Val Arg 100 105 110 Thr Ser Tyr Asp Lys Val Ile Phe Gly Pro Gly Thr Ser Leu Ser Val 115 120 125 Ile Pro Asn 130 <210> 39 <211> 135 <212> PRT <213> artificial sequence <220> <223> synthetic sequence WT137-45 TCR14.1 Vα <400> 39 Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15 Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu 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 Leu Leu Gly Ala Thr Gly Tyr Ser Thr Leu Thr Phe Gly Lys Gly 115 120 125 Thr Met Leu Leu Val Ser Pro 130 135 <210> 40 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence WT137 - 45 TCR15.1 Vα <400> 40 Met Trp Gly Val Phe Leu Leu Tyr Val Ser Met Lys Met Gly Gly Thr 1 5 10 15 Thr Gly Gln Asn Ile Asp Gln Pro Thr Glu Met Thr Ala Thr Glu Gly 20 25 30 Ala Ile Val Gln Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Asn Gly 35 40 45 Leu Phe Trp Tyr Gln Gln His Ala Gly Glu Ala Pro Thr Phe Leu Ser 50 55 60 Tyr Asn Val Leu Asp Gly Leu Glu Glu Lys Gly Arg Phe Ser Ser Phe 65 70 75 80 Leu Ser Arg Ser Lys Gly Tyr Ser Tyr Leu Leu Leu Lys Glu Leu Gln 85 90 95 Met Lys Asp Ser Ala Ser Tyr Leu Cys Ala Val Arg Gly Ile Asn Asp 100 105 110 Tyr Lys Leu Ser Phe Gly Ala Gly Thr Thr Val Thr Val Arg Ala Asn 115 120 125 <210> 41 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence WT137 - 45 TCR16.1 Vα <400> 41 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[[ID=4I]] 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 Ile 100 105 110 Thr Gly Phe Gln Lys Leu Val Phe Gly Thr Gly Thr Arg Leu Leu Val 115 120 125 Ser Pro Asn 130 <210> 42 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence WT137-45 TCR16.2 Vα <400> 42 Met Arg Leu Val Ala Arg Val Thr Val Phe Leu Thr Phe Gly Thr Ile 1 5 10 15 Ile Asp Ala Lys Thr Thr Gln Pro Thr Ser Met Asp Cys Ala Glu Gly 20 25 30 Arg Ala Ala Asn Leu Pro Cys Asn His Ser Thr Ile Ser Gly Asn Glu 35 40 45 Tyr Val Tyr Trp Tyr Arg Gln Ile His Ser Gln Gly Pro Gln Tyr Ile 50 55 60 Ile His Gly Leu Lys Asn Asn Glu Thr Asn Glu Met Ala Ser Leu Ile 65 70 75 80 Ile Thr Glu Asp Arg Lys Ser Ser Thr Leu Ile Leu Pro His Ala Thr 85 90 95 Leu Arg Asp Thr Ala Val Tyr Tyr Cys Ile Ala Gly Val Gly Arg Gly 100 105 110 Gln Asn Phe Val Phe Gly Pro Gly Thr Arg Leu Ser Val Leu Pro Tyr 115 120 125 <210> 43 <211> 136 <212> PRT <213> artificial sequence <220> <223> Synthesis of WT137-45 TCR18.1 Vα <400> 43 Met Glu Lys Asn Pro Leu Ala Ala Pro Leu Leu Ile Leu Trp Phe His 1 5 10 15 Leu Asp Cys Val Ser Ser Ile Leu Asn Val Glu Gln Ser Pro Gln Ser 20 25 30 Leu His Val Gln Glu Gly Asp Ser Thr Asn Phe Thr Cys Ser Phe Pro 35 40 45 Ser Ser Asn Phe Tyr Ala Leu His Trp Tyr Arg Trp Glu Thr Ala Lys 50 55 60 Ser Pro Glu Ala Leu Phe Val Met Thr Leu Asn Gly Asp Glu Lys Lys 65 70 75 80 Lys Gly Arg Ile Ser Ala Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr 85 90 95 Leu Tyr Ile Lys Gly Ser Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys 100 105 110 Ala Phe His Pro Asn Phe Gly Asn Glu Lys Leu Thr Phe Gly Thr Gly 115 120 125 Thr Arg Leu Thr Ile Ile Pro Asn 130 135 <210> 44 <211> 136 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR19.1 Vα <400> 44 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 Pro Arg Gly Asp Gly Ser Ser Asn Thr Gly Lys Leu Ile Phe Gly Gln 115 120 125 Gly Thr Thr Leu Gln Val Lys Pro 130 135 <210> 45 <211> 176 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence WT137 - 45 TCR15.1 Cβ <400> 45 Asp Leu Asn Lys 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 Phe 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 Val 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 Phe 165 170 175 <{210}> 46 <{211}> 178 <{212}> PRT <{213}> Artificial Sequence [[ID=df40]]<{220}> <{223}> Synthetic Sequence WT137-45 TCR16.2 Cβ <{400}> 46 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> 47 <211> 140 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence WT137 - 45 TCR15.1 Cα <400> 47 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 ¼ 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> 48 <211> 605 <212> PRT <213> Synthetic sequence <220> <223> Synthetic sequence WT1 37 - 45 TCR10.1 TCRβ - P2A - TCRα <400> 48 Met Gly Thr Ser Leu Leu Cys Trp Val Val Leu Gly Phe Leu Gly Thr 1 5 10 15 Asp His Thr Gly Ala Gly Val Ser Gln Ser Pro Arg Tyr Lys Val Thr 20 25 30 Lys Arg Gly Gln Asp Val Ala Leu Arg Cys Asp Pro Ile Ser Gly His 35 40 45 Val Ser Leu Tyr Trp Tyr Arg Gln Ala Leu Gly Gln Gly Pro Glu Phe 50 55 60 Leu Thr Tyr Phe Asn Tyr Glu Ala Gln Gln Asp Lys Ser Gly Leu Pro 65 70 75 80 Asn Asp Arg Phe Ser Ala Glu Arg Pro Glu Gly Ser Ile Ser Thr Leu 85 90 95 Thr Ile Gln Arg Thr Glu Gln Arg Asp Ser Ala Met Tyr Arg Cys Ala 100 105 110 Ser Ser Leu Thr Gly Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly Gly Ser Gly Ala Thr Asn Phe Ser Leu 305 310 315 320 Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu Thr 325 330 335 Leu Leu Gly Leu Leu Ile Leu Trp Leu Gln Leu Gln Trp Val Ser Ser 340 345 350 Lys Gln Glu Val Thr Gln Ile Pro Ala Ala Leu Ser Val Pro Glu Gly 355 360 365 Glu Asn Leu Val Leu Asn Cys Ser Phe Thr Asp Ser Ala Ile Tyr Asn 370 375 380 Leu Gln Trp Phe Arg Gln Asp Pro Gly Lys Gly Leu Thr Ser Leu Leu 385 390 395 400 Leu Ile Gln Ser Ser Gln Arg Glu Gln Thr Ser Gly Arg Leu Asn Ala 405 410 415 Ser Leu Asp Lys Ser Ser Gly Arg Ser Thr Leu Tyr Ile Ala Ala Ser 420 425 430 Gln Pro Gly Asp Ser Ala Thr Tyr Leu Cys Ala Val Lys Glu Thr Ser 435 440 445 Gly Ser Arg Leu Thr Phe Gly Glu Gly Thr Gln Leu Thr Val Asn Pro 450 455 460 Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys 465 470 475 480 Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr 485 490 495 Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys 500 505 510 Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala 515 520 525 Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser 530 535 540 Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp 545 550 555 560 Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe 565 570 575 Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala 580 585 590 Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 595 600 605 <210> 49 <211> 612 <212> PRT <213> Artificial sequence <220> <223> Synthetic sequence WT137-45 TCR11.2 TCRβ-P2A-α <400> 49 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 Thr 100 105 110 Pro Glu Ala Ser Ser Pro Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly Gly Ser Gly Ala Thr Asn Phe Ser Leu 305 310 315 320 Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Thr Rec 325 330 335 Val Ser Leu Leu Trp Ala Val Val Val Ser Thr Cys Leu Glu Ser Gly 340 345 350 Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser Val Gln Glu 355 360 365 Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser Glu Asn Asn 370 375 380 Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln Met Ile Leu 385 390 395 400 Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr Glu Asn Arg 405 410 415 Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser Leu Lys Ile 420 425 430 Ser Asp Ser Gln Leu Gly Asp Thr Ala Met Tyr Phe Cys Ala Phe Ile 435 440 445 Tyr Pro Ser Tyr Thr Ser Gly Thr Tyr Lys Tyr Ile Phe Gly Thr Gly 450 455 460 Thr Arg Leu Lys Val Leu Ala Asn Ile Gln Asn Pro Asp Pro Ala Val 465 470 475 480 Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe 485 490 495 Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp 500 505 510 Val Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe 515 520 525 Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys 530 535 540 Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro 545 550 555 560 Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu 565 570 575 Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg 580 585 590 Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg 595 600 605 Leu Trp Ser Ser 610 <210> 50 <211> 611 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence WT137-45 TCR12.1 TCRβ-P2A-α <400> 50 Met Gly Pro Gly Leu Leu His Trp Met Ala Leu Cys Leu Leu Gly Thr 1 5 10 15 Gly His Gly Asp Ala Met Val Ile Gln Asn Pro Arg Tyr Gln Val Thr 20 25 30 Gln Phe Gly Lys Pro Val Thr Leu Ser Cys Ser Gln Thr Leu Asn His 35 40 45 Asn Val Met Tyr Trp Tyr Gln Gln Lys Ser Ser Gln Ala Pro Lys Leu 50 55 60 Leu Phe His Tyr Tyr Asp Lys Asp Phe Asn Asn Glu Ala Asp Thr Pro 65 70 75 80 Asp Asn Phe Gln Ser Arg Arg Pro Asn Thr Ser Phe Cys Phe Leu Asp 85 90 95 Ile Arg Ser Pro Gly Leu Gly Asp Ala Ala Met Tyr Leu Cys Ala Thr 100 105 110 Ser Asn Leu Gln Gly Arg Gln Pro Gln His Phe Gly Asp Gly Thr Arg 115 120 125 Leu Ser Ile Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Phe Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys 305 310 315 320 Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala Met Leu Leu 325 330 335 Gly Ala Ser Val Leu Ile Leu Trp Leu Gln Pro Asp Trp Val Asn Ser 340 345 350 Gln Gln Lys Asn Asp Asp Gln Gln Val Lys Gln Asn Ser Pro Ser Leu 355 360 365 Ser Val Gln Glu Gly Arg Ile Ser Ile Leu Asn Cys Asp Tyr Thr Asn 370 375 380 Ser Met Phe Asp Tyr Phe Leu Trp Tyr Lys Lys Tyr Pro Ala Glu Gly 385 390 395 400 Pro Thr Phe Leu Ile Ser Ile Ser Ser Ile Lys Asp Lys Asn Glu Asp 405 410 415 Gly Arg Phe Thr Val Phe Leu Asn Lys Ser Ala Lys His Leu Ser Leu 420 425 430 His Ile Val Pro Ser Gln Pro Gly Asp Ser Ala Val Tyr Phe Cys Ala 435 440 445 Ala Ser Gly Thr Gly Gly Ser Tyr Ile Pro Thr Phe Gly Arg Gly Thr 450 455 460 Ser Leu Ile Val His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr 465 470 475 480 Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr 485 490 495 Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val 500 505 510 Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys 515 520 525 Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala 530 535 540 Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser 545 550 555 560 Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr 565 570 575 Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile 580 585 590 Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu 595 600 605 Trp Serum Serum 610 <210> 51 <211> 622 <212> PRT <213> artificial sequence <220> <223> Synthesis of WT137-45 TCR13.1 TCRβ-P2A-TCRα <400> 51 Met Leu Ser Pro Asp Leu Pro Asp Ser Ala Trp Asn Thr Arg Leu Leu 1 5 10 15 Cys His Val Met Leu Cys Leu Leu Gly Ala Val Ser Val Ala Ala Gly 20 25 30 Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg Glu Thr Ala 35 40 45 Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val Tyr Trp Tyr 50 55 60 Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser Phe Tyr Glu 65 70 75 80 Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe Ser Ala Gln 85 90 95 Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser Leu Glu Leu 100 105 110 Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Leu Arg Leu Gly Arg 115 120 125 Glu Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Leu Val Leu Glu Asp 130 135 140 Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu 145 150 155 160 Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr 165 170 175 Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys 180 185 190 Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys Glu Gln 195 200 205 Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg Val 210 215 220 Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln Val 225 230 235 240 Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala 245 250 255 Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp 260 265 270 Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr 275 280 285 Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu 290 295 300 Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Ser Arg 305 310 315 320 Gly Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp 325 330 335 Val Glu Glu Asn Pro Gly Pro Met Ala Met Leu Leu Gly Ala Ser Val 340 345 350 Leu Ile Leu Trp Leu Gln Pro Asp Trp Val Asn Ser Gln Gln Lys Asn 355 360 365 Asp Asp Gln Gln Val Lys Gln Asn Ser Pro Ser Leu Ser Val Gln Glu 370 375 380 Gly Arg Ile Ser Ile Leu Asn Cys Asp Tyr Thr Asn Ser Met Phe Asp 385 390 395 400 Tyr Phe Leu Trp Tyr Lys Lys Tyr Pro Ala Glu Gly Pro Thr Phe Leu 405 410 415 Ile Ser Ile Ser Ser Ile Lys Asp Lys Asn Glu Asp Gly Arg Phe Thr 420 425 430 Val Phe Leu Asn Lys Ser Ala Lys His Leu Ser Leu His Ile Val Pro 435 440 445 Ser Gln Pro Gly Asp Ser Ala Val Tyr Phe Cys Ala Ala Ser Gly Ile 450 455 460 Gly Asp Tyr Lys Leu Ser Phe Gly Ala Gly Thr Thr Val Thr Val Arg 465 470 475 480 Ala Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser 485 490 495 Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln 500 505 510 Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys 515 520 525 Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val 530 535 540 Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn 545 550 555 560 Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys 565 570 575 Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn 580 585 590 Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val 595 600 605 Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 610 615 620<​​​​​​​​​​​​​​​​​​​​​​​​​​35 40 45 Val Ser Leu Phe Trp Tyr Gln Gln Ala Leu Gly Gln Gly Pro Glu Phe 50 55 60 Leu Thr Tyr Phe Gln Asn Glu Ala Gln Leu Asp Lys Ser Gly Leu Pro 65 70 75 80 Ser Asp Arg Phe Phe Ala Glu Arg Pro Glu Gly Ser Val Ser Thr Leu 85 90 95 Lys Ile Gln Arg Thr Gln Gln Glu Asp Ser Ala Val Tyr Leu Cys Ala 100 105 110 Ser Ser Leu Gly Gln Ala Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly Gly Ser Gly Ala Thr Asn Phe Ser Leu 305 310 315 320 Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Val Lys 325 330 335 Ile Arg Gln Phe Leu Leu Ala Ile Leu Trp Leu Gln Leu Ser Cys Val 340 345 350 Ser Ala Ala Lys Asn Glu Val Glu Gln Ser Pro Gln Asn Leu Thr Ala 355 360 365 Gln Glu Gly Glu Phe Ile Thr Ile Asn Cys Ser Tyr Ser Val Gly Ile 370 375 380 Ser Ala Leu His Trp Leu Gln Gln His Pro Gly Gly Gly Ile Val Ser 385 390 395 400 Leu Phe Met Leu Ser Ser Gly Lys Lys Lys His Gly Arg Leu Ile Ala 405 410 415 Thr Ile Asn Ile Gln Glu Lys His Ser Ser Leu His Ile Thr Ala Ser 420 425 430 His Pro Arg Asp Ser Ala Val Tyr Ile Cys Ala Val Arg Thr Ser Tyr 435 440 445 Asp Lys Val Ile Phe Gly Pro Gly Thr Ser Leu Ser Val Ile Pro Asn 450 455 460 Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser 465 470 475 480 Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn 485 490 495 Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val 500 505 510 Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp 515 520 525 Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile 530 535 540 Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val 545 550 555 560 Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln 565 570 575 Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly 580 585 590 Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 595 600 <210> 53 <211> 607 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence WT137-45 TCR14.1 TCRβ-P2A-TCRα <400> 53 Met Gly Thr Arg Leu Leu Cys Trp Val Ala Phe Cys Leu Leu Val Glu 1 5 10 15 Glu Leu Ile Glu Ala Gly Val Val Gln Ser Pro Arg Tyr Lys Ile Ile 20 25 30 Glu Lys Lys Gln Pro Val Ala Phe Trp Cys Asn Pro Ile Ser Gly His 35 40 45 Asn Thr Leu Tyr Trp Tyr Leu Gln Asn Leu Gly Gln Gly Pro Glu Leu 50 55 60 Leu Ile Arg Tyr Glu Asn Glu Glu Ala Val Asp Asp Ser Gln Leu Pro 65 70 75 80 Lys Asp Arg Phe Ser Ala Glu Arg Leu Lys Gly Val Asp Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ala Glu Leu Gly Asp Ser Ala Val Tyr Leu Cys Ala 100 105 110 Ser Ser Leu Thr Arg Gly Ala Glu Ala Phe Phe Gly Gln Gly Thr Arg 115 120 125 Leu Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Phe Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys 305 310 315 320 Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Lys Ser Leu Arg 325 330 335 Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser Trp Val Trp Ser Gln 340 345 350 Gln Lys Glu Val Glu Gln Asn Ser Gly Pro Leu Ser Val Pro Glu Gly 355 360 365 Ala Ile Ala Ser Leu Asn Cys Thr Tyr Ser Asp Arg Gly Ser Gln Ser 370 375 380 Phe Phe Trp Tyr Arg Gln Tyr Ser Gly Lys Ser Pro Glu Leu Ile Met 385 390 395 400 Phe Ile Tyr Ser Asn Gly Asp Lys Glu Asp Gly Arg Phe Thr Ala Gln 405 410 415 Leu Asn Lys Ala Ser Gln Tyr Val Ser Leu Leu Ile Arg Asp Ser Gln 420 425 430 Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala Val Asn Leu Leu Gly Ala 435 440 445 Thr Gly Tyr Ser Thr Leu Thr Phe Gly Lys Gly Thr Met Leu Leu Val 450 455 460 Ser Pro Asp Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp 465 470 475 480 Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser 485 490 495 Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp 500 505 510 Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala 515 520 525 Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn 530 535 540 Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser 545 550 555 560 Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu 565 570 575 Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys 580 585 590 Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser<0​​​​​​​​​​​​​​​​​​​​​​​​​ Lys Glu Gly Gln Asn Val Thr Leu Ser Cys Glu Gln Asn Leu Asn His 35 40 45 Asp Ala Met Tyr Trp Tyr Arg Gln Asp Pro Gly Gln Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr Ser Gln Ile Val Asn Asp Phe Gln Lys Gly Asp Ile Ala 65 70 75 80 Glu Gly Tyr Ser Val Ser Arg Glu Lys Lys Glu Ser Phe Pro Leu Thr 85 90 95 Val Thr Ser Ala Gln Lys Asn Pro Thr Ala Phe Tyr Leu Cys Ala Ser 100 105 110 Ser Arg Asp Arg Glu Gln Glu Ser Pro Leu His Phe Gly Asn Gly Thr 115 120 125 Arg Leu Thr Val Thr Glu Asp Leu Asn Lys Val Phe Pro Pro Glu Val 130 135 140 Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala 145 150 155 160 Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu Leu 165 170 175 Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp 180 185 190 Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys 195 200 205 Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg 210 215 220 Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp 225 230 235 240 Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala 245 250 255 Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln 260 265 270 Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys 275 280 285 Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met 290 295 300 Val Lys Arg Lys Asp Phe Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu 305 310 315 320 Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Trp Gly Val 325 330 335 Phe Leu Leu Tyr Val Ser Met Lys Met Gly Gly Thr Thr Gly Gln Asn 340 345 350 Ile Asp Gln Pro Thr Glu Met Thr Ala Thr Glu Gly Ala Ile Val Gln 355 360 365 Ile Asn Cys Thr Tyr Gln Thr Ser Gly Phe Asn Gly Leu Phe Trp Tyr 370 375 380 Gln Gln His Ala Gly Glu Ala Pro Thr Phe Leu Ser Tyr Asn Val Leu 385 390 395 400 Asp Gly Leu Glu Glu Lys Gly Arg Phe Ser Ser Phe Leu Ser Arg Ser 405 410 415 Lys Gly Tyr Ser Tyr Leu Leu Leu Lys Glu Leu Gln Met Lys Asp Ser 420 425 430 Ala Ser Tyr Leu Cys Ala Val Arg Gly Ile Asn Asp Tyr Lys Leu Ser 435 440 445 Phe Gly Ala Gly Thr Thr Val Thr Val Arg Ala Asn Ile Gln Asn Pro 450 455 460 Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser 465 470 475 480 Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser 485 490 495 Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg 500 505 510 Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser 515 520 525 Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp 530 535 540 Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu 545 550 555 560 Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val 565 570 575 Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu 580 585 590 Met Thr Leu Arg Leu Trp Ser Ser 595 600 <210> 55 <211> 604 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence WT137-45 TCR16.1 TCRβ-P2A-TCRα <400> 55 Met Gly Pro Gln Leu Leu Gly Tyr Val Val Leu Cys Leu Leu Gly Ala 1 5 10 15 Gly Pro Leu Glu Ala Gln Val Thr Gln Asn Pro Arg Tyr Leu Ile Thr 20 25 30 Val Thr Gly Lys Lys Leu Thr Val Thr Cys Ser Gln Asn Met Asn His 35 40 45 Glu Tyr Met Ser Trp Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Gln 50 55 60 Ile Tyr Tyr Ser Met Asn Val Glu Val Thr Asp Lys Gly Asp Val Pro 65 70 75 80 Glu Gly Tyr Lys Val Ser Arg Lys Glu Lys Arg Asn Phe Pro Leu Ile 85 90 95 Leu Glu Ser Pro Ser Pro Asn Gln Thr Ser Leu Tyr Phe Cys Ala Ser 100 105 110 Ser Phe Ser Gly Gly Thr Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro 180 185 190 Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215 220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu 225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser Arg Gly Gly Ser Gly Ala Thr Asn Phe Ser Leu 305 310 315 320 Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu Lys 325 330 335 Met Leu Glu Cys Ala Phe Ile Val Leu Trp Leu Gln Leu Gly Trp Leu 340 345 350 Ser Gly Glu Asp Gln Val Thr Gln Ser Pro Glu Ala Leu Arg Leu Gln 355 360 365 Glu Gly Glu Ser Ser Ser Leu Asn Cys Ser Tyr Thr Val Ser Gly Leu 370 375 380 Arg Gly Leu Phe Trp Tyr Arg Gln Asp Pro Gly Lys Gly Pro Glu Phe 385 390 395 400 Leu Phe Thr Leu Tyr Ser Ala Gly Glu Glu Lys Glu Lys Glu Arg Leu 405 410 415 Lys Ala Thr Leu Thr Lys Lys Glu Ser Phe Leu His Ile Thr Ala Pro 420 425 430 Lys Pro Glu Asp Ser Ala Thr Tyr Leu Cys Ala Val Ile Thr Gly Phe 435 440 445 Gln Lys Leu Val Phe Gly Thr Gly Thr Arg Leu Leu Val Ser Pro Asn 450 455 460 Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser 465 470 475 480 Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn 485 490 495 Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val 500 505 510 Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp 515 520 525 Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile 530 535 540 Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val 545 550 555 560 Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln 565 570 575 Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly 580 585 590 Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 595 600 <210> 56 <211> 612 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence WT137-45 TCR16.2 TCRβ-P2A-TCRα <400> 56 Met Leu Ser Pro Asp Leu Pro Asp Ser Ala Trp Asn Thr Arg Leu Leu 1 5 10 15 Cys His Val Met Leu Cys Leu Leu Gly Ala Val Ser Val Ala Ala Gly 20 25 30 Val Ile Gln Ser Pro Arg His Leu Ile Lys Glu Lys Arg Glu Thr Ala 35 40 45 Thr Leu Lys Cys Tyr Pro Ile Pro Arg His Asp Thr Val Tyr Trp Tyr 50 55 60 Gln Gln Gly Pro Gly Gln Asp Pro Gln Phe Leu Ile Ser Phe Tyr Glu 65 70 75 80 Lys Met Gln Ser Asp Lys Gly Ser Ile Pro Asp Arg Phe Ser Ala Gln 85 90 95 Gln Phe Ser Asp Tyr His Ser Glu Leu Asn Met Ser Ser Leu Glu Leu 100 105 110 Gly Asp Ser Ala Leu Tyr Phe Cys Ala Ser Ser Tyr Arg Gly Gly Ser 115 120 125 Thr Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr Glu 130 135 140 Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser 145 150 155 160 Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala 165 170 175 Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp Val Asn Gly 180 185 190 Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro Leu Lys Glu 195 200 205 Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg Leu Arg 210 215 220 Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg Cys Gln 225 230 235 240 Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg 245 250 255 Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala 260 265 270 Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala 275 280 285 Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val 290 295 300 Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg Lys Asp Ser 305 310 315 320 Arg Gly Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly 325 330 335 Asp Val Glu Glu Asn Pro Gly Pro Met Arg Leu Val Ala Arg Val Thr 340 345 350 Val Phe Leu Thr Phe Gly Thr Ile Ile Asp Ala Lys Thr Thr Gln Pro 355 360 365 Thr Ser Met Asp Cys Ala Glu Gly Arg Ala Ala Asn Leu Pro Cys Asn 370 375 380 His Ser Thr Ile Ser Gly Asn Glu Tyr Val Tyr Trp Tyr Arg Gln Ile 385 390 395 400 His Ser Gln Gly Pro Gln Tyr Ile Ile His Gly Leu Lys Asn Asn Glu 405 410 415 Thr Asn Glu Met Ala Ser Leu Ile Ile Thr Glu Asp Arg Lys Ser Ser 420 425 430 Thr Leu Ile Leu Pro His Ala Thr Leu Arg Asp Thr Ala Val Tyr Tyr 435 440 445 Cys Ile Ala Gly Val Gly Arg Gly Gln Asn Phe Val Phe Gly Pro Gly 450 455 460 Thr Arg Leu Ser Val Leu Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val 465 470 475 480 Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe 485 490 495 Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp 500 505 510 Val Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe 515 520 525 Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys 530 535 540 Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro 545 550 555 560 Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu 565 570 575 Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg 580 585 590 Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg 595 600 605 Leu Trp Ser Ser 610 <210> 57 <211> 609 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence WT137-45 TCR18.1 TCRβ-P2A-TCRα <400> 57 Met Ser Thr Arg Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala 1 5 10 15 Glu Leu Ser Glu Ala Glu Val Ala Gln Ser Pro Arg Tyr Lys Ile Thr 20 25 30 Glu Lys Ser Gln Ala Val Ala Phe Trp Cys Asp Pro Ile Ser Gly His 35 40 45 Ala Thr Leu Tyr Trp Tyr Arg Gln Ile Leu Gly Gln Gly Pro Glu Leu 50 55 60 Leu Val Gln Phe Gln Asp Glu Ser Val Val Asp Asp Ser Gln Leu Pro 65 70 75 80 Lys Asp Arg Phe Ser Ala Glu Arg Leu Lys Gly Val Asp Ser Thr Leu 85 90 95 Lys Ile Gln Pro Ala Glu Leu Gly Asp Ser Ala Met Tyr Leu Cys Ala 100 105 110 Ser Ser Gln Arg Asp Ser Pro Asn Glu Lys Leu Phe Phe Gly Ser Gly 115 120 125 Thr Gln Leu Ser Val Leu Glu Asp Leu Asn Lys Val Phe Pro Pro Glu 130 135 140 Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys 145 150 155 160 Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val Glu 165 170 175 Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr 180 185 190 Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr 195 200 205 Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro 210 215 220 Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn 225 230 235 240 Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser 245 250 255 Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr 260 265 270 Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly 275 280 285 Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala 290 295 300 Met Val Lys Arg Lys Asp Phe Gly Ser Gly Ala Thr Asn Phe Ser Leu 305 310 315 320 Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Glu Lys 325 330 335 Asn Pro Leu Ala Ala Pro Leu Leu Ile Leu Trp Phe His Leu Asp Cys 340 345 350 Val Ser Ser Ile Leu Asn Val Glu Gln Ser Pro Gln Ser Leu His Val 355 360 365 Gln Glu Gly Asp Ser Thr Asn Phe Thr Cys Ser Phe Pro Ser Ser Asn 370 375 380 Phe Tyr Ala Leu His Trp Tyr Arg Trp Glu Thr Ala Lys Ser Pro Glu 385 390 395 400 Ala Leu Phe Val Met Thr Leu Asn Gly Asp Glu Lys Lys Lys Gly Arg 405 410 415 Ile Ser Ala Thr Leu Asn Thr Lys Glu Gly Tyr Ser Tyr Leu Tyr Ile 420 425 430 Lys Gly Ser Gln Pro Glu Asp Ser Ala Thr Tyr Leu Cys Ala Phe His 435 440 445 Pro Asn Phe Gly Asn Glu Lys Leu Thr Phe Gly Thr Gly Thr Arg Leu 450 455 460 Thr Ile Ile Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu 465 470 475 480 Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe 485 490 495 Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile 500 505 510 Thr Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn 515 520 525 Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala 530 535 540 Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu 545 550 555 560 Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr 565 570 575 Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu 580 585 590 Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser 595 600 605 Ser <210> 58 <211> 611 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence WT137 - 45 TCR19.1 TCRβ - P2A - TCRα <400> 58 Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu Cys Leu Leu Gly Ala 1 5 10 15 Val Pro Met Glu Thr Gly Val Thr Gln Thr Pro Arg His Leu Val Met 20 25 30 Gly Met Thr Asn Lys Lys Ser Leu Lys Cys Glu Gln His Leu Gly His 35 40 45 Asn Ala Met Tyr Trp Tyr Lys Gln Ser Ala Lys Lys Pro Leu Glu Leu 50 55 60 Met Phe Val Tyr Ser Leu Glu Glu Arg Val Glu Asn Asn Ser Val Pro 65 70 75 80 Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser His Leu Phe Leu His 85 90 95 Leu His Thr Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser 100 105 110 Ser Gln Asp Pro Tyr Lys Leu Ser Gly Asn Thr Ile Tyr Phe Gly Glu 115 120 125 Gly Ser Trp Leu Thr Val Val Glu Asp Leu Asn Lys Val Phe Pro Pro 130 135 140 Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln 145 150 155 160 Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Phe Pro Asp His Val 165 170 175 Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser Gly Val Cys 180 185 190 Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg 195 200 205 Tyr Cys Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn 210 215 220 Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu 225 230 235 240 Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val 245 250 255 Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Val Ser 260 265 270 Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu ...

Claims

1. A T cell receptor (TCR), comprising: The variable (Vα) domain of the TCR α-chain and the variable (Vβ) domain of the TCR β-chain, wherein the Vα domain comprises the CDR1α, CDR2α, and CDR3α amino acid sequences set forth by SEQ ID NOs: 194, 195, and 196 or 12, respectively, wherein the Vβ domain comprises the CDR1β, CDR2β, and CDR3β amino acid sequences set forth by SEQ ID NOs: 197, 198, and 199 or 1, respectively, and wherein the TCR binds to the VLDFAPPGA (SEQ ID NO: 59): human leukocyte antigen (HLA) complex, wherein the HLA comprises HLA-A*201.

2. The TCR according to claim 1, wherein the TCR further specifically binds to the VLDFAPPGA (SEQ ID NO: 59): human leukocyte antigen (HLA) complex on the cell surface, the complex independent of CD8 or in the absence of CD8.

3. The TCR according to claim 1, wherein the Vα domain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth by SEQ ID NO:

34.

4. The TCR according to claim 1, wherein the Vβ domain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth by SEQ ID NO:

23.

5. The TCR according to claim 1, wherein the Vα domain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth by SEQ ID NO: 34, and wherein the Vβ domain comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence set forth by SEQ ID NO:

23.

6. The TCR according to claim 1, wherein the Vα domain comprises the amino acid sequence set forth by SEQ ID NO: 253 or 34.

7. The TCR according to claim 1, wherein the Vα domain consists of the amino acid sequence set forth by SEQ ID NO: 253 or 34.

8. The TCR according to claim 1, wherein the Vβ domain comprises the amino acid sequence set forth by SEQ ID NO: 242 or 23.

9. The TCR according to claim 1, wherein the Vβ domain consists of the amino acid sequence set forth by SEQ ID NO: 242 or 23.

10. The TCR according to claim 1, wherein the TCR comprises a TCR α-chain constant domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

47.

11. The TCR according to claim 1, wherein the TCR comprises a TCR β-chain constant domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 45 or 46.

12. The TCR according to claim 1, wherein the TCR comprises a TCR α-chain comprising a Vα domain and a constant domain of the α-chain, wherein the Vα domain has at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 34, and the constant domain of the α-chain has at least 98% sequence identity with the amino acid sequence of SEQ ID NO:

47.

13. The TCR according to claim 1, wherein the TCR comprises a TCR α-chain comprising a Vα domain and a constant domain of the α-chain, wherein: (a) the Vα domain comprises the amino acid sequence set forth in SEQ ID NO: 253 or 34, and the constant domain of the α-chain comprises the amino acid sequence of SEQ ID NO: 47; or (b) the Vα domain consists of the amino acid sequence set forth in SEQ ID NO: 253 or 34, and the constant domain of the α-chain consists of the amino acid sequence of SEQ ID NO:

47.

14. The TCR according to claim 1, wherein the TCR comprises a TCR β-chain comprising a Vβ domain and a constant domain of the β-chain, wherein the Vβ domain has at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 23, and the constant domain of the β-chain comprises the amino acid sequence of SEQ ID NO: 45 or has at least 95% sequence identity with the amino acid sequence of SEQ ID NO:

46.

15. The TCR according to claim 1, wherein the TCR comprises a TCR β-chain comprising a Vβ domain and a constant domain of the β-chain, wherein: (a) the Vβ domain comprises the amino acid sequence set forth in SEQ ID NO: 242 or 23, and the constant domain of the β-chain comprises the amino acid sequence of SEQ ID NO: 45 or 46; (b) the Vβ domain consists of the amino acid sequence set forth in SEQ ID NO: 242 or 23, and the constant domain of the β-chain consists of the amino acid sequence of SEQ ID NO: 45 or 46; (c) the Vβ domain comprises the amino acid sequence set forth in SEQ ID NO: 23, and the constant domain of the β-chain comprises the amino acid sequence of SEQ ID NO: 46; or (d) the Vβ domain consists of the amino acid sequence set forth in SEQ ID NO: 23, and the constant domain of the β-chain consists of the amino acid sequence of SEQ ID NO:

46.

16. The TCR according to claim 1, wherein the Vα domain and the Vβ domain comprise or consist of the amino acid sequences set forth in the following SEQ ID NOs: (i) 253 and 242, respectively; or (ii) 34 and 23, respectively.

17. The TCR according to claim 16, further comprising an alpha chain constant domain and / or a beta chain constant domain, wherein the alpha chain constant domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 47, and wherein the beta chain constant domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 45 or 46.

18. The TCR according to claim 17, wherein there is an alpha chain constant domain, and the Vα domain and the alpha chain constant domain together constitute the TCR alpha chain.

19. The TCR according to claim 17, wherein there is a beta chain constant domain, and the Vβ domain and the beta chain constant domain together constitute the TCR beta chain.

20. The TCR according to claim 1, comprising: (i) a TCR alpha chain containing a Vα domain and an alpha chain constant domain, the Vα domain comprising the amino acid sequence set forth in SEQ ID NO: 253, and the alpha chain constant domain comprising the amino acid sequence set forth in SEQ ID NO: 47; and (ii) a TCR beta chain containing a Vβ domain and a beta chain constant domain, the Vβ domain comprising the amino acid sequence set forth in SEQ ID NO: 242, and the beta chain constant domain comprising the amino acid sequence set forth in SEQ ID NO:

46.

21. The TCR according to claim 1, comprising: (i) a TCR alpha chain containing a Vα domain and an alpha chain constant domain, the Vα domain consisting of the amino acid sequence set forth in SEQ ID NO: 253, and the alpha chain constant domain consisting of the amino acid sequence set forth in SEQ ID NO: 47; and (ii) a TCR beta chain containing a Vβ domain and a beta chain constant domain, the Vβ domain consisting of the amino acid sequence set forth in SEQ ID NO: 242, and the beta chain constant domain consisting of the amino acid sequence set forth in SEQ ID NO:

46.

22. The TCR according to claim 1, wherein the TCR comprises a scTCR.

23. The TCR according to claim 1, comprising a polypeptide of the formula Vα-L-Vβ or Vβ-L-Vα, wherein: Vα is a Vα domain; Vβ is a Vβ domain; and L is a linker.

24. The TCR according to claim 16, comprising a polypeptide of the formula Vα-L-Vβ or Vβ-L-Vα, wherein: Vα is a Vα domain; Vβ is a Vβ domain; and L is a linker.

25. The TCR according to claim 1, wherein the TCR comprises a CAR.

26. An isolated polynucleotide encoding the TCR according to any one of claims 13-21.

27. An isolated polynucleotide encoding the TCR according to claim 1.

28. The polynucleotide according to claim 27, wherein the polynucleotide encoding the TCR is codon-optimized for a host cell of interest.

29. The polynucleotide according to claim 27, wherein the amino acid sequence encoded by the polynucleotide has at least 95% identity with the amino acid sequence set forth in SEQ ID NO: 48, which comprises or consists of the same.

30. The polynucleotide according to claim 27, comprising the polynucleotide sequence set forth in any one of SEQ ID NO: 64, 75, 86, 97, 108, 109, 110, 111, 122, 133, 144, and 155.

31. The polynucleotide according to claim 27, further comprising: (i) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha chain; (ii) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta chain; or (iii) the polynucleotide of (i) and the polynucleotide of (ii).

32. The polynucleotide according to claim 31, wherein, The polypeptide encoded by the polynucleotide of (i) is the CD8 co-receptor alpha chain.

33. The polynucleotide according to claim 31, wherein, The polypeptide encoded by the polynucleotide of (i) comprises the CD8 co-receptor alpha chain.

34. The polynucleotide according to claim 31, wherein, The polypeptide encoded by the polynucleotide of (ii) is the CD8 co-receptor beta chain.

35. The polynucleotide according to claim 31, wherein, The polypeptide encoded by the polynucleotide of (ii) comprises the CD8 co-receptor beta chain.

36. The polynucleotide according to claim 31, comprising: (a) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha chain; (b) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta chain; and (c) a polynucleotide encoding a self-cleaving peptide, disposed between the polynucleotide of (a) and the polynucleotide of (b).

37. The polynucleotide according to claim 31, further comprising a polynucleotide encoding a self-cleaving peptide and placed between: (1) a polynucleotide encoding a binding protein and a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor alpha chain; and / or (2) a polynucleotide encoding a binding protein and a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta chain.

38. The polynucleotide according to claim 31, comprising the following operably linked in-frame: (i) (pnCD8α)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnTCR); (ii) (pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnTCR); (iii) (pnTCR)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnCD8β); (iv) (pnTCR)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnCD8α); (v) (pnCD8α)-(pnSCP1)-(pnTCR)-(pnSCP2)-(pnCD8β); or (vi) (pnCD8β)-(pnSCP1)-(pnTCR)-(pnSCP2)-(pnCD8α), wherein pnCD8α is a polynucleotide encoding the following polypeptide: the polypeptide comprises the extracellular portion of the CD8 coreceptor α chain, wherein pnCD8β is a polynucleotide encoding the following polypeptide: the polypeptide comprises the extracellular portion of the CD8 coreceptor α chain, Among them, pnTCR is a polynucleotide encoding a TCR, and wherein pnSCP1 and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotide and / or the encoded self-cleaving peptide may be the same or different.

39. The polynucleotide according to claim 31, wherein the encoded TCR comprises a TCRα chain and a TCRβ chain, wherein the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide, and the polynucleotide is placed between the polynucleotide encoding the TCRα chain and the polynucleotide encoding the TCRβ chain.

40. The polynucleotide according to claim 39, comprising the following operably linked within a framework: (i) (pnCD8α)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnTCRβ)-(pnSCP3)-(pnTCRα); (ii) (pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnTCRβ)-(pnSCP3)-(pnTCRα); (iii) (pnCD8α)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnTCRα)-(pnSCP3)-(pnTCRβ); (iv) (pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnTCRα)-(pnSCP3)-(pnTCRβ); (v) (pnTCRβ)-(pnSCP1)-(pnTCRα)-(pnSCP2)-(pnCD8α)-(pnSCP3)-(pnCD8β); (vi) (pnTCRβ)-(pnSCP1)-(pnTCRα)-(pnSCP2)-(pnCD8β)-(pnSCP3)-(pnCD8α); (vii) (pnTCRα)-(pnSCP1)-(pnTCRβ)-(pnSCP2)-(pnCD8α)-(pnSCP3)-(pnCD8β); or (viii) (pnTCRα)-(pnSCP1)-(pnTCRβ)-(pnSCP2)-(pnCD8β)-(pnSCP3)-(pnCD8α), wherein pnCD8α is a polynucleotide encoding the following polypeptide: the polypeptide comprises the extracellular portion of the CD8 coreceptor α chain, wherein pnCD8β is a polynucleotide encoding the following polypeptide: the polypeptide comprises the extracellular portion of the CD8 coreceptor α chain, Among them, pnTCRα is a polynucleotide encoding the TCRα chain, wherein pnTCRβ is a polynucleotide encoding the TCRβ chain, and wherein pnSCP1, pnSCP2, and pnSCP3 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotide and / or the encoded self-cleaving peptide may be the same or different.

41. An expression vector comprising the polynucleotide of claim 27 operably linked to an expression control sequence.

42. The expression vector according to claim 41, wherein the expression vector is capable of delivering the polynucleotide to a host cell.

43. The expression vector according to claim 42, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.

44. The expression vector according to claim 43, wherein the human immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double negative T cell, a γδ T cell, a natural killer cell, a dendritic cell, or any combination thereof.

45. The expression vector according to claim 44, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

46. The expression vector according to claim 41, wherein the expression vector is a viral vector.

47. The expression vector according to claim 46, wherein the viral vector is an adenovirus vector, a lentivirus vector, or a γ-retrovirus vector.

48. An expression vector comprising the polynucleotide of claim 26 operably linked to an expression control sequence.

49. A host cell comprising the polynucleotide according to any one of claims 27-40 or the expression vector according to any one of claims 41-47, wherein the host cell expresses a TCR encoded by the polynucleotide on its cell surface, and wherein the polynucleotide is heterologous to the host cell.

50. A host cell comprising the polynucleotide of claim 26, wherein the host cell expresses a TCR encoded by the polynucleotide on its cell surface, and wherein the polynucleotide is heterologous to the host cell.

51. The host cell according to claim 50, wherein the Vα domain is encoded by a polynucleotide comprising at least 75% sequence identity to the polynucleotide of SEQ ID NO:

97.

52. The host cell according to claim 50, wherein the Vα domain is encoded by a polynucleotide: (a) comprising the sequence of the polynucleotide of SEQ ID NO: 97; or (b) consisting of the sequence of the polynucleotide of SEQ ID NO:

97.

53. The host cell according to claim 50, wherein the Vβ domain is encoded by a polynucleotide comprising at least 75% sequence identity to the polynucleotide of SEQ ID NO:

75.

54. The host cell according to claim 50, wherein the Vβ domain is encoded by a polynucleotide: (a) comprising the sequence of the polynucleotide of SEQ ID NO: 75; or (b) consisting of the sequence of the polynucleotide of SEQ ID NO:

75.

55. The host cell according to claim 50, wherein the TCR α-chain comprises an α-chain constant domain encoded by a polynucleotide that is at least 98% identical to SEQ ID NO:

110.

56. The host cell according to claim 50, wherein the TCR α-chain comprises an α-chain constant domain encoded by a polynucleotide: (a) comprising the polynucleotide sequence of SEQ ID NO: 110; or (b) consisting of the polynucleotide sequence of SEQ ID NO:

110.

57. The host cell according to claim 50, wherein the β-chain of the TCR comprises a β-chain constant domain encoded by a polynucleotide having at least 99.9% sequence identity to SEQ ID NO: 108 or 109.

58. The host cell according to claim 50, wherein the TCR β-chain comprises a β-chain constant domain encoded by a polynucleotide: (a) comprising the polynucleotide sequence of SEQ ID NO: 108 or 109; or (b) consisting of the polynucleotide sequence of SEQ ID NO: 108 or 109.

59. The host cell according to claim 50, wherein the polynucleotide comprises a nucleotide sequence encoding a self-cleaving peptide, which is placed between the polynucleotide sequence encoding the TCR α-chain and the polynucleotide sequence encoding the TCR β-chain.

60. The host cell according to claim 59, wherein the encoded self-cleaving peptide: (a) comprises the amino acid sequence of any one of the polypeptides of SEQ ID NOs: 60-63; or (b) consists of the sequence of any one of the polypeptides of SEQ ID NOs: 60-63.

61. The host cell according to claim 59, wherein the polynucleotide encoding the self-cleaving peptide: (a) comprises the sequence of any one of the polynucleotides of SEQ ID NOs: 166-170; or (b) consists of the sequence of any one of the polynucleotides of SEQ ID NOs: 166-170.

62. The host cell according to claim 59, wherein the TCR α-chain, the self-cleaving peptide, and the TCR β-chain are encoded by a polynucleotide that is at least 95% identical to SEQ ID NO:

155.

63. The host cell according to claim 59, wherein the TCR α-chain, the self-cleaving peptide, and the TCR β-chain are encoded by a polynucleotide: (a) comprising the sequence of the polynucleotide of SEQ ID NO: 155; or (b) consisting of the sequence of the polynucleotide of SEQ ID NO:

155.

64. The host cell according to claim 59, wherein the encoded TCR α-chain, self-cleaving peptide, and TCR β-chain comprise an amino acid sequence having at least 95% identity to the polypeptide of SEQ ID NO:

48.

65. The host cell according to claim 59, wherein the encoded TCR α-chain, self-cleaving peptide, and TCR β-chain: (a) comprise the amino acid sequence of the polypeptide of SEQ ID NO: 48; or (b) consist of the amino acid sequence of the polypeptide of SEQ ID NO:

48.

66. The host cell according to claim 50, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.

67. The host cell according to claim 66, wherein the human immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double negative T cell, a γδ T cell, a natural killer cell, a natural killer T cell, a dendritic cell, or any combination thereof.

68. The host cell according to claim 67, wherein, If such a combination exists, the human immune system cells include CD4+ T cells and CD8+ T cells.

69. The host cell according to claim 66, wherein the human immune system cell is a T cell.

70. The host cell according to claim 69, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

71. The host cell according to claim 50, wherein the TCR has a higher surface expression on the T cell as compared to the endogenous TCR.

72. The host cell according to claim 50, 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 beta chain; or (iii) the polynucleotide of (i) and the polynucleotide of (ii).

73. The host cell according to claim 72, wherein, The polypeptide encoded by the polynucleotide of (i) is the CD8 co-receptor alpha chain.

74. The host cell according to claim 72, wherein, The polypeptide encoded by the polynucleotide of (i) comprises the CD8 co-receptor alpha chain.

75. The host cell according to claim 72, wherein, The polypeptide encoded by the polynucleotide of (ii) is the CD8 co-receptor beta chain.

76. The host cell according to claim 72, wherein, The polypeptide encoded by the polynucleotide of (ii) comprises the CD8 co-receptor beta chain.

77. The host cell according to claim 72, wherein, The host cell comprises CD4+ T cells.

78. The host cell according to claim 72, 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 beta chain; and (c) a polynucleotide encoding a self-cleaving peptide, placed between the polynucleotide of (a) and the polynucleotide of (b).

79. The host cell according to claim 50, wherein when both host cells and tumor cells are present in a sample, the host cells are capable of killing: (i) tumor cells of the breast cancer cell line MDA-MB-468; (ii) tumor cells of the pancreatic cancer cell line PANC-1; (iii) tumor cells of the breast cancer cell line MDA-MB-231; (iv) tumor cells of the myeloid leukemia cell line K562 expressing HLA-A2; (v) tumor cells of the colon cancer cell line RKO expressing HLA-A2; or (vi) any combination of the tumor cells of (i)-(v).

80. The host cell according to claim 79, wherein, The HLA-A2 includes HLA-A*201.

81. The host cell according to claim 79, wherein the host cell is capable of killing tumor cells when the host cell and tumor cells are present in a sample at a host cell:tumor cell ratio of 32:1, 16:1, 8:1, 4:1, 2:1 or 1.5:

1.

82. A composition comprising the host cell according to any one of claims 50 - 81 and a pharmaceutically acceptable carrier, diluent or excipient.

83. The composition according to claim 82, comprising host CD4+ T cells and host CD8+ T cells.

84. The composition according to claim 82, wherein the dose of the host cells is 10 7 cells / m 2 to 10 11 cells / m 2 .

85. A composition comprising the TCR according to any one of claims 1 - 25, the polynucleotide according to any one of claims 27 - 40, or the expression vector according to any one of claims 41 - 47 and a pharmaceutically acceptable carrier, diluent or excipient.

86. Use of the TCR according to claim 1 in the preparation of a medicament for treating a proliferative or hyperproliferative disease in a subject associated with the expression or overexpression of Wilms tumor protein 1 (WT1), wherein the proliferative or hyperproliferative disease is selected from breast cancer and pancreatic cancer, wherein the TCR is expressed on the surface of a host cell, and the host cell comprises the host cell according to any one of claims 50 - 81.

87. The use according to claim 86, wherein the host cell is a human T cell.

88. The use according to claim 87, wherein the human T cell is a CD8+ T cell or a CD4+ T cell.

89. Use of the TCR according to any one of claims 1 - 23 in the preparation of a medicament for treating a proliferative or hyperproliferative disease in a subject associated with the expression or overexpression of Wilms tumor protein 1 (WT1), wherein the proliferative or hyperproliferative disease is selected from breast cancer and pancreatic cancer.

90. The use according to claim 89, wherein the TCR is expressed on the surface of a host cell.

91. The use according to claim 89, wherein the proliferative or hyperproliferative disease is breast cancer.

92. The use according to claim 89, wherein the proliferative or hyperproliferative disease is pancreatic cancer.

93. The use according to claim 89, wherein the TCR is capable of promoting an antigen - specific T cell response against human WT1 in an MHC class I - restricted manner.

94. The use according to claim 93, wherein the MHC class I - restricted response is transporter associated with antigen processing (TAP) - independent.

95. The use according to claim 93, wherein the antigen - specific T cell response comprises at least one of a CD4+ helper T lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response.

96. The use according to claim 95, wherein the CTL response is against WT1 - overexpressing cells.

97. The use according to claim 90, wherein the host cell is modified in vitro.

98. The use according to claim 97, wherein the host cell is an allogeneic cell, an autologous cell or a syngeneic cell of the subject.

99. The use according to claim 89, wherein the drug is administered parenterally.

100. The use according to claim 89, wherein the drug is administered to a subject in multiple doses.

101. The use according to claim 100, wherein the multiple doses are administered at intervals of 2 to 4 weeks.

102. The use according to claim 90, wherein the drug is administered to the subject at a dose of 10 7 cells / m 2 to 10 11 cells / m 2 .

103. The use according to claim 89, wherein the drug is used in combination with further comprising a cytokine.

104. The use according to claim 103, wherein the cytokine is IL-2, IL-15, IL-21 or any combination thereof.

105. The use according to claim 104, wherein the cytokine is IL-2 and is administered simultaneously or sequentially with the drug.

106. The use according to claim 105, wherein the cytokine is administered sequentially, and the drug is administered at least three or four times before the cytokine is administered.

107. The use according to claim 104, wherein the cytokine is IL-2 and is administered subcutaneously.

108. The use according to claim 89, wherein the drug is used in combination with further comprising immunosuppressive therapy.

109. The use according to claim 108, wherein the immunosuppressive therapy is selected from calcineurin inhibitors, corticosteroids, microtubule inhibitors, low-dose mycophenolic acid prodrugs or any combination thereof.

110. The use according to claim 89, wherein the drug is used in combination with further comprising non-myeloablative or myeloablative hematopoietic cell transplantation administered before the drug.

111. The use according to claim 110, wherein the drug is administered at least three months after non-myeloablative hematopoietic cell transplantation.

112. The use according to claim 110, wherein the drug is administered at least two months after myeloablative hematopoietic cell transplantation.

113. The use according to claim 89, wherein the drug is used in combination with further comprising an immune checkpoint inhibitor and / or an agonist of a stimulatory immune checkpoint agent.

114. Use of a polynucleotide according to any one of claims 27-40, an expression vector according to any one of claims 41-47, a host cell according to any one of claims 50-81, or any combination thereof, in a method for manufacturing a drug for treating a proliferative or hyperproliferative disease associated with the expression or overexpression of Wilms tumor protein 1 (WT1), wherein the proliferative or hyperproliferative disease is selected from breast cancer and pancreatic cancer.

115. The use according to claim 114, wherein the host cell is a human T cell.

116. The use according to claim 115, wherein the human T cell is a CD8+ T cell or a CD4+ T cell.

117. Use of the TCR according to claim 16 in the preparation of a drug for treating a proliferative or hyperproliferative disease associated with the expression or overexpression of Wilms tumor protein 1 (WT1) in a subject, wherein the proliferative or hyperproliferative disease is selected from breast cancer and pancreatic cancer, wherein the TCR is expressed on the surface of the host cell, and the host cell comprises the host cell according to any one of claims 50 - 81.

118. The use according to claim 117, wherein the host cell is a human T cell.

119. The use according to claim 118, wherein the human T cell is a CD8+ T cell or a CD4+ T cell.

120. The use of the TCR according to claim 20 in the preparation of a medicament for treating a proliferative or hyperproliferative disease associated with the expression or overexpression of Wilms tumor protein 1 (WT1) in a subject, wherein the proliferative or hyperproliferative disease is selected from breast cancer and pancreatic cancer, wherein the TCR is expressed on the surface of the host cell, and the host cell comprises the host cell according to any one of claims 50 - 81.

121. The use according to claim 120, wherein the host cell is a human T cell.

122. The use according to claim 121, wherein the human T cell is a CD8+ T cell or a CD4+ T cell.

123. The use of the TCR according to claim 21 in the preparation of a medicament for treating a proliferative or hyperproliferative disease associated with the expression or overexpression of Wilms tumor protein 1 (WT1) in a subject, wherein the proliferative or hyperproliferative disease is selected from breast cancer and pancreatic cancer, wherein the TCR is expressed on the surface of the host cell, and the host cell comprises the host cell according to any one of claims 50 - 81.

124. The use according to claim 123, wherein the host cell is a human T cell.

125. The use according to claim 124, wherein the human T cell is a CD8+ T cell or a CD4+ T cell.

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