WO2019055862A1 - High affinity t cell receptors and uses thereof - Google Patents

Abstract

The present disclosure provides binding proteins, including TCRs and CARs, with high or enhanced affinity against tumor associated antigen Wilms tumor protein 1 (WT1), T cells expressing such high affinity WT1 specific binding proteins, nucleic acids encoding the same, and compositions for use in treating diseases or disorders in which cells overexpress WT1, such as in cancer.

Description

HIGH AFFINITY T CELL RECEPTORS AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Serial No. 62/558,550, filed September 14, 2017, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

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 name of the text file containing the Sequence Listing is

360056_453WO_SEQUENCE_LISTING.txt. The text file is 226 KB, was created on September 13, 2018, and is being submitted electronically via EFS-Web to the U.S. PCT Receiving Office, concurrent with the filing of this specification.

BACKGROUND

Adoptive T cell immunotherapy with genetically engineered T cells has shown promise in multiple trials in which an antigen receptor of sufficient affinity was used to target a tumor-associated antigen, including antibody -based chimeric receptors^1"3 and high affinity TCRs^4"8. However, isolating an effective TCR within the affinity limits imposed by central tolerance remains a substantive roadblock to implementing this approach for the diversity of malignancies in which candidate intracellular self/tumor antigens have been identified^{9 10}.

Methods have been developed to enhance the affinity of TCRs intended for use in TCR gene therapy^{10 12"14}. These approaches generally employ saturation mutagenesis targeting the complementarity determining regions (CDRs) that interact predominantly with peptide (CDR3) and/or MHC (CDRl/2)¹⁵. Current methodologies targeting specific CDR3 residues for amino acid substitution generally yield libraries with diversity constrained by the unaltered length of the parental CDR3 sequence. By contrast, the natural process of diversity generation in the thymus employs RAG- mediated TCR gene rearrangements to generate highly diverse CDR3s varying in length as well as amino acid composition.

BRIEF SUMMARY

The present disclosure provides, according to certain aspects, a binding protein (e.g., an immunoglobulin superfamily binding protein, TCR or the like) having (a) a T cell receptor (TCR) a-chain variable (V_a) domain and a TCR β-chain variable having a CDR3 amino acid sequence set forth in any one of SEQ ID NOS: 1-1 1 ; (b) a TCR V_a domain having a CDR3 amino acid sequence of SEQ ID NO: 13, and a TCR νβ domain having a CDR3 amino acid sequence set forth in any one of SEQ ID NOS: 1-1 1 ; or (c) a TCR V_a domain and a TCR ν_β domain comprising a CDR3 having a mutated SEQ ID NO: 12 amino acid sequence, wherein D/N/P region amino acids of SEQ ID NO: 12 comprise up to five amino acid substitutions, up to a contiguous three amino acid additions, or a combination thereof, provided that the CDR3 does not comprise the amino acid sequence of SEQ ID NO: 12; wherein the binding protein is capable of specifically binding to a WT1 peptide :HLA complex on a cell surface, optionally independent of CD8 or in the absence of CD8.

In further aspects there are provided methods for treating a hyperproliferative disorder, comprising administering to human subject in need thereof a composition comprising a binding protein of this disclosure specific for human Wilms tumor protein 1 (WT1). In yet another aspect there is provided an adoptive immunotherapy method for treating a condition characterized by WT1 overexpression in cells of a subject having a hyperproliferative disorder, comprising administering to the subject an effective amount of a host cell expressing a binding protein of this disclosure.

In certain embodiments the methods provided are for treating a

hyperproliferative disorder that is a hematological malignancy or a solid cancer. For example, the hematological malignancy to be treated may be acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), non- Hodgkin's lymphoma (NHL), or multiple myeloma (MM). Exemplary solid cancer to be treated may be biliary cancer, bladder cancer, bone and soft tissue carcinoma, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonal cancer, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumor, head and neck squamous cell carcinoma, hepatic cancer, lung cancer, mesothelioma, malignant melanoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, renal cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ-cell tumor, urothelial cancer, uterine sarcoma, or uterine cancer. BRIEF DESCRIPTION OF THE FIGURES

Figures 1A-1E show that agonist signaling drives CD47CD8^" double negative (DN) TCRaP⁺ γδ-like T cell development in vitro and does not affect pre-selected CD4⁺/CD8⁺ double positive (DP) CD69^" thymocytes. Thymocytes from OTl mice were sorted for TCRp^" TCR γδ^" CD4^" CD 8^" CD81⁺ CD44⁺ DN1 and DN2 progenitor cells and cultured on OP9-DL1 cells transduced to express H-2K^b (OP9-K^bD^bDLl) for 20 days in the presence of various concentrations of SIINFEKL peptide (SEQ ID NO: 79) as indicated. (A, B, C) Cultures were analyzed by flow cytometry for CD4, CD8, TCRp, and CD24 on the days indicated. (D) The total number of DN TCRp+ cells from day 14 triplicate cultures was calculated for each concentration of added SIINFEKL (SEQ ID NO: 79) and for 1 μΜ was of the low affinity, positively selecting peptide EIINFEKL (SEQ ID NO:80), denoted as El . (E) Thymocytes from B6 or OTl transgenic mice were pre-selected for CD4⁺/CD8⁺ DP CD69^" and cultured on OP9- K^bD^bDLl for 3 days in the presence of IL-7 and FLT3L, and in the presence or absence of 1 μΜ WT1 peptide SIINFEKL (SEQ ID NO: 79) as indicated. Cultures were analyzed by flow cytometry for CD4, CD8, and TCR on day 3. Data were

representative of at least three independent experiments.

Figures 2A-2G show that ectopic expression of an antigen-specific TCRa chain prior to β-selection results in DN TCRaP⁺ cell development when cognate peptide is present. B6 thymocytes were sorted for CD4^" CD8^" CD81⁺ CD44⁺ cells, which include both DN1 and DN2 progenitor cells, transduced with a retroviral vector containing the TCRa chain of the affinity enhanced WTl -specific TCR (3D-PYY) and hCD2, and cultured on OP9-DL1 cells expressing H-2D^b in the presence or absence of 1.0 μΜ of WTl peptide RMFPNAPYL (SEQ ID NO: 82). (A) On day 16 of culture, transduced (hCD2⁺) and untransduced (hCD2^") cells were gated on CD4-CD8- DN progenitor cells and analyzed for CD24 and TCRp expression. (B) On day 21, the B6 DN1/DN2 thymocytes transduced with the 3D-PYY TCRa chain (hCD2⁺) and cultured on OP9- K^bD^bDLl cells in the presence of WTl peptide were sorted for DN TCRap⁺ cells. PCR was performed using a νβΐθ-specific forward primer and Cp2-specific reverse primer to isolate TCRP chains for library construction. The TCRP library was transduced into 58^_/" cells and TCRP chains conferring high affinity for WTl were isolated by cell sorting for expansion of transduced GFP⁺ cells, followed by two additional rounds of sorting and expansion based on high tetramer binding. (C) Isolated TCRP chains were submitted to IMGT V-Quest⁵³'⁵⁴ for sequence analysis for comparison with the parental 3ϋβ chain. (D) 58^"/_3D-PYYa cells were transduced with the retroviral TCR libraries and first sorted for transduced GFP⁺ cells (data not shown). This population then underwent two rounds of expansion and tetramer-based sorting to enrich for GFP⁺ signal and high WTl tetramer binding. Sorted 58^"/_3D-PYYa cells were also analyzed for staining with a non-specific H-2D^b tetramer containing GP33 peptide as a control for non-specific tetramer binding. (E) νβΐθ-θβΐ and νβ10-Οβ2 libraries were generated from agonist selected DN TCRo ⁺ cells purified from in vitro cultures of

3D-PYYa -transduced HPCs. Cells were sorted to enrich for GFP signal and analyzed for WTl tetramer binding and νβΐθ staining by flow cytometry. (F) CDR3β sequences for the parental 3ϋβ, νβΐθ clone#l and Τηγβ#1 were aligned to show conserved mutation site at position 108. (G) Four candidate TCRβ chains were detected at high frequency (of 12 unique TCRβ sequences identified in total). These 4 TCRβ constructs were transferred back into MigRl-attR, transduced into CD8⁺ 3Da⁺ 58^_/" cells, and assessed for relative tetramer binding.

Figures 3A-3C shows an analysis of enhanced-affinity TCRs recovered from the agonist-selected TCRβ library screen. (A) The relative affinity of the three TCRs exhibiting the highest tetramer binding was determined by staining 58^"/_3D-PYYa cells transduced with the candidate TCRs with titrated amounts of peptide/MHC tetramer and analyzing by flow cytometry. K_D measurements were performed using six 2-fold dilutions of PE-conjugated tetramers, and derived relative K_D values determined from binding curves by non-linear regression, based on the concentration of peptide/MHC tetramer that yielded half-maximal binding. (B) The parental νβΐθ TCR and a codon- optimized version of Οοηε#1αβ were transduced into 58^_/" cells and stained with WT1 tetramer and νβΐθ. After gating on cells expressing equivalent levels of νβΐθ TCR, tetramer binding was compared. (C) 58^_/" cells transduced with Οοηε#1αβ paired with 3Da were stained with WT1 specific tetramer, as well as several non-specific H-2D^b tetramers to assess potential peptide-independent reactivity towards MHC (GP33 :

LCMV glycoprotein; E4: Yellow fever virus 17D; MSLN: mouse Mesothelin; SQV: HIV-gag epitope).

Figures 4A-4I show that high affinity WT1 -specific T cells develop in 3D- ΡΥΥβ retrogenic mice. Lineage negative HPCs were purified from the BM of B6 mice, transduced with Mig2-3D-PYYa, and transferred into irradiated B6 recipient mice. (A) Eight weeks after BM transfer, thymus and spleen were analyzed by flow cytometry. Plots were gated on TCRβ⁺ T cells (Data representative of three independent experiments with two, two, and four mice per condition, respectively). (B) νβΐθ- restricted TCRβ libraries were generated from the DN TCRo ⁺ thymocyte population isolated from 3D-PYYa-rom 3D-PYY mice. The libraries were expressed in 58^{" "}3D- PYYa cells, from which WT1 tetramer⁺ cells were sorted and expanded. (C) TCRβ chains were recovered from sorted populations and sequenced. The three most highly enriched TCRβ chains were shown for comparison with the parental 3ϋβ. (D) Each of the isolated TCRβ chains were expressed in 58^"/_3D-PYYa cells and analyzed for relative WT1 tetramer staining with increasing surface νβΐθ expression. Cells expressing candidate library-derived TCRβ chains (as indicated in figure) were overlaid with 58^_/" cells expressing the parental 3D-PYYa 3ϋ-β TCR (as indicated in figure). (E) The WT1 tetramer binding kinetics of the 3ϋ-ΡΥΥα-Τϊτγβ#1 TCR was compared with the parental 3D-PYYa 3ϋ-β TCR. K_D measurements were performed using six 2- fold dilutions of PE-conjugated tetramers, and derived relative K_D values determined from binding curves by non-linear regression. (F) Peripheral CD8 T cells from P14 mice that were transduced to express the 3D-PYYa 3ϋ-β TCR or the 3D-PYYa- Τηγβ#1 TCR were incubated with target cells pulsed with decreasing concentrations WT1 peptide as indicated, and the percentage of IFNy producing cells determined at each peptide concentration. (G) The mean fluorescent intensity of IFNy staining within the transduced (νβ10⁺) population at each peptide concentration for the experiment is shown in (F). (H) Peripheral CD8⁺ T cells from P14 mice that were transduced to express the 3D-PYYa 3ϋ-β TCR (circles) or the 3D-PYYa-Thyp#l TCR (squares) were incubated with target cells pulsed with decreasing concentrations WT1 peptide as indicated, and the percentage of IFNy producing cells determined at each peptide concentration. Data were representative of at least three independent experiments. (I) The mean fluorescent intensity of IFNy staining within the transduced (νβ10⁺) population at each peptide concentration for the experiment shown in (H). Data were representative of at least three independent experiments.

Figure 5 illustrates the high-throughput sequencing and repertoire analysis performed on DN TCRaP⁺ thymocytes from 3DaPYY-retrogenic mice. DN TCRaP⁺ thymocytes from a 3DaPYY-retrogenic mouse were identified as indicated in Figure 4 and sorted by flow cytometric cell sorting. DNA was isolated from the sorted cells, and CDR3P regions were amplified and sequenced by Adaptive Biotechnologies Corp (Seattle, WA) using the immunoSEQ™ assay. Briefly, a multiplex PCR system was used to amplify CDR3P sequences from DNA samples using 35 forward primers for the νβ gene segment and 14 reverse primers for the Ιβ segment. This approach generates a 60 base-pair fragment capable of identifying the VDJ region spanning each unique CDR3p^1-3. Amplicons were sequenced using the Illumina HiSeq platform and data was analyzed using the ImmunoSEQ analyzer toolset. The percentage of in-frame rearrangements, and the relative percent usage of each TCR νβ chain is shown for the retrogenic thymus and compared to a normal B6 thymus. Furthermore, the absolute number of reads for the top 50 clones within the DN TCRaP ⁺ retrogenic thymocyte population is shown, highlighting the lack of distinct clone-specific expansion. The top 10 most abundant clones are shown, and TCR β chains utilizing νβ17 are highlighted to show the prevalence of νβ17 usage.

Figure 6 shows expression of CD27 by agonist-selected human T progenitors expressing the parental \Υ 1₃₇αβ TCR. CD34⁺ FIPCs were purified from umbilical cord blood, lentivirally transduced with WTl₃₇aP and co-cultured with the OP9-A2- DL1 cell line in the presence or absence of lug/ml WT1 peptide. Cultures were analyzed on day 22, gated on CD3⁺ cells, and assessed for CD4 and CD27 expression. CD3⁺ cells lacking CD4 expression were electronically gated and analyzed for CD8 expression to show that the majority of these cells were DN, with a smaller fraction of cells expressing CD8.

Figures 7A-7C show that ectopic expression of a human antigen-specific TCRP chain in human HPCs differentiated in the presence of cognate antigen in vitro can result in agonist-selected T cells with enhanced affinity for antigen. CD34⁺ HPCs were purified from umbilical cord blood, lentivirally transduced with WTl₃₇a-IRES-GFP and co-cultured with the OP9-A2-DL1 cell line in the presence of ^g/ml WT1 peptide. (A) untransduced GFP^" or (B) WTl₃₇a-expressing GFP⁺ cells were analyzed on day 31 of culture for expression of CD3, TCRp, CD8, and CD27. (C) TCRp libraries were generated from CD3⁺ TCRp ⁺ CD27⁺DN cells as well as CD3⁺ TCRp ⁺ CD8⁺ cells expressing high levels of CD27 present after day 20 of culture. Cpi and Cp2 libraries were generated using a 5' Vpi7 primer or using a 5' universal RACE primer and transduced into H9 cells transduced to express WTl₃₇a (H9.WTl₃₇aP). H9.WTl₃₇a cells transduced with Vpi7 libraries were first sorted for Vpi7. All library-transduced cells underwent two rounds of low stringency tetramer⁺ sorts followed by cell expansion, and then sorted for tetramer^M cells from which antigen-specific TCRP chain genes were cloned for further analysis.

Figures 8A- 8B show human WTl₃₇-specific TCRP chains isolated through screening of agonist-selected TCRP libraries. (A) Analysis of selected TCRP chains co-expressed with WTl₃₇a in TCRaP negative Jurkat76 cells. CD3 surface expression correlates with transgenic TCR expression in this cell line since Jurkat76 cells lack endogenous TCR chains. (B) Comparison of CDR3 amino acid and nucleotide sequences of the identified TCRP chains using IMGT/V-Quest (Brochet et al, Nucleic Acids Res 3<5:W503, 2008; Giudicelli et al., Cold Spring Harb. Protoc, pdb.prot5633- pdb.prot5633, 2011). Non-germline-encoded residues flanking (non-tempi ated (N) nucleotides and palindromic (P) nucleotides) and including the D-gene segment (collectively referred to herein as D/N/P region nucleotides, which encode D/N/P region amino acids) are boxed for each TCR.

Figures 9A-9E show analysis of a subset of human enhanced-affinity TCRs. (A) Comparison of with the parental \ΥΤ1₃₇β. Amino acids predicted to be encoded by the TCRP D gene segment, N nucleotides and P nucleotides (D/N/P region) are enclosed within the boxed region. (B) Polynucleotides encoding codon-optimized candidate TCRP chains each linked to WTl₃₇a by a P2A were incorporated into a lentiviral expression construct and expressed in TCRaP negative Jurkat76 cells.

WT1 -specific tetramer staining relative to TCR surface expression for each candidate TCR (as indicated in figure) was compared to the wildtype \ΥΤ1₃₇αβ TCR (as indicated in figure). (C) The relative affinity of each of the candidate TCRs was determined by staining TCR-transduced cells with six 2-fold dilutions of PE-conjugated tetramers, and the derived relative K_D values calculated from binding curves by non-linear regression, based on the concentration of tetramer that yielded half-maximal binding. (D)

Duplicate samples of sort-purified TCR-transduced human T cells were incubated 1 : 1 with T2 cells alone, or pulsed with WT1₃₇ peptide at the indicated concentrations for 6 hours in the presence of GolgiPlug™ and GolgiStop™ (BD biosciences, San Jose, CA); and then permeabilized, stained with anti-IFNy antibody, and analyzed by flow cytometry. Each data point represents the average percentage ± s.d. of IFNy⁺ cells within the CD8⁺ T cell population. (E) The relative affinity of each of the candidate TCRs was determined by staining TCR-transduced T cells with titrated concentrations of WT1 tetramer, and MFI values were fit to equilibrium binding curves and the derived relative K_D values calculated by nonlinear regression. Error bars represent 95% confidence limits for each calculated K_D value

Figures 10A and 10B show in vivo safety of enhanced-affinity murine TCRs.

CD8⁺ P14 T cells were retrovirally transduced with the TCR 3ϋαβ; or 3DaPYY paired with either 3ϋβ or enhanced affinity TCR chains νβ10#1 and Τηγβ#1. B6 mice were individually co-injected with 6 x 10⁶ T cells and 6 x 10⁶ irradiated (3000rad) WT1 peptide-pulsed B6 splenocytes and administered IL-2 (10⁴ units/mouse) for 10 days post-injection. (A) Animals were weighed on the days indicated. (B) Blood was collected on days 6 and 126 to analyze T cell expansion and persistence. Figure 11A and 11B show similar CD8 and TCR transgene expression and the lack of antigen-independent IFNy production in TCR-gene modified primary human CD8⁺ cells. In order to evaluate antigen-specific T cell function, donor human CD8 T cells were lentivirally transduced to express TCR \Υ 1₃₇αβ (which is a TCR specific for WT1 peptide VLDFAPPGA (SEQ ID NO: 81) and referred to as WT1₃₇ peptide), or selected library-derived TCR chains paired with WTl₃₇a. (A) The TCR-transduced human T cells were stained with WT1₃₇ tetramer to show uniform expression and functional pairing of each TCR (first row). The cells were further analyzed for νβ17 (second row) and CD8 (third row) expression to confirm that each transgenic TCR (for νβ17⁺ TCRs) and CD8 was expressed at equivalent levels. (B) The TCR-transduced human T cells were incubated 1 : 1 with T2 cells alone , or pulsed with ΙΟΟμΜ WT1₃₇ peptide for 6 hours in the presence of GolgiPlug™ (BD biosciences, San Jose, CA); and then permeabilized, stained with anti-IFNy antibody, and analyzed by flow cytometry. IFNy production by the TCR-transduced human T cells in the presence of WT1₃₇ peptide is shown in the top row and in the absence of WT1₃₇ peptide is shown in the bottom row.

DETAILED DESCRIPTION

The present disclosure provides binding proteins, such as T cell receptors (TCRs), having high affinity for WT1 peptide antigens associated with a major histocompatibility complex (MHC) (e.g., human leukocyte antigen, ULA) for use in, for example, adoptive immunotherapy to treat cancer. In addition, the binding domains from the TCRs can be used to construct and chimeric antigen receptors (CARs), which could bind a WT1 antigen in the absence of an MHC molecule.

The binding proteins and compositions of the present disclosure were generated and identified by using methods that access CDR3 diversity with retained target antigen specificity by harnessing the V(D)J recombination machinery active during T cell development. Briefly, the present disclosure provides methods for enhancing TCR affinity by introducing and expressing a heterologous polynucleotide encoding a TCRa chain from a WTl-specifc TCR into hematopoietic progenitor cells (HPCs). The transgenic HPCs were expanded and differentiated in the presence of a WT1 peptide antigen to generate a large pool of progenitor T cells with unique, naturally occurring Tcrb gene rearrangements. This pool of cells expressed diverse set of TCRP chains that, when paired with the parental transgenic WTl -specific TCRa chain, conferred WTl -specificity and enhanced affinity for WTl as compared to the original, parental TCR. An advantage of the instant disclosure is to provide a high affinity binding proteins {e.g., TCRs) specific for a WTl peptide, wherein a cell expressing such a binding protein is capable of binding to a WTl :HLA complex independent of CD8 or in the absence of CD8. In addition, such TCRs may optionally be capable of more efficiently associating with a CD3 protein as compared to an endogenous TCR.

In certain embodiments, a binding protein {e.g., TCR) for a WTl peptide comprises a T cell receptor (TCR) a-chain having an amino acid sequence of SEQ ID NO: 39, and a TCR β-chain variable domain as set forth in any one of SEQ ID NOS:27-37. In certain embodiments, such high affinity TCRs are capable of binding to a VLDFAPPGA (SEQ ID NO:81):HLA complex with a K_D of less than or equal to about 10^"8 M, or wherein the high affinity TCR dissociates from a VLDFAPPGA (SEQ ID NO:81):HLA complex at a reduced k_0ff rate as compared to a parental TCR composed of an a-chain of SEQ ID NO:39 and a β-chain of SEQ ID NO:38.

The compositions and methods described herein will in certain embodiments have therapeutic utility for the treatment of diseases and conditions associated with WTl overexpression {e.g., detectable WTl expression at a level that is greater in magnitude, in a statistically significant manner, than the level of WTl expression that is detectable in a normal or disease-free cell). Such diseases include various forms of hyperproliferative disorders, such as hematological malignancies and solid cancers. Non-limiting examples of these and related uses are described herein and include in vitro, ex vivo and in vivo stimulation of WTl antigen-specific T cell responses, such as by the use of recombinant T cells expressing an enhanced affinity TCR specific for a WTl peptide (e.g., VLDFAPPGA, SEQ ID NO:81, also known as WT1₃₇ peptide).

Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein.

Additional definitions are set forth throughout this disclosure. In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term "about" means ± 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include," "have" and "comprise" are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

In addition, it should be understood that the individual compounds, or groups of compounds, derived from the various combinations of the structures and substituents described herein, are disclosed by the present application to the same extent as if each compound or group of compounds was set forth individually. Thus, selection of particular structures or particular substituents is within the scope of the present disclosure.

The term "consisting essentially of is not equivalent to "comprising" and refers to the specified materials or steps of a claim, or to those that do not materially affect the basic characteristics of the claimed subject matter. For example, a protein domain, region, or module (e.g., a binding domain, hinge region, linker module) or a protein (which may have one or more domains, regions, or modules) "consists essentially of a particular amino acid sequence when the amino acid sequence of a domain, region, module, or protein includes extensions, deletions, mutations, or a combination thereof (e.g., amino acids at the amino- or carboxy -terminus or between domains) that, in combination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of a domain, region, module, or protein and do not

substantially affect (i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s), region(s), module(s), or protein (e.g., the target binding affinity of a binding protein).

As used herein, an "immune system cell" in some aspects means any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, meagakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells). Exemplary immune system cells include a CD4+ T cell, a CD8+ T cell, a CD4- CD8- double negative T cell, a γδ T cell, a regulatory T cell, a stem cell memory T cell, a natural killer cell (e.g., a NK cell or a NK-T cell), a B cell, and a dendritic cell. Macrophages and dendritic cells may be referred to as "antigen presenting cells" or "APCs," which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell.

"Major histocompatibility complex" (MHC) in some aspects can refer to glycoproteins that deliver peptide antigens to a cell surface. MHC class I molecules are heterodimers having a membrane spanning a chain (with three a domains) and a non- covalently associated β2 microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, a and β, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where a peptide:MHC complex is recognized by CD8⁺ T cells. MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are recognized by CD4⁺ T cells. Human MHC is referred to as human leukocyte antigen (HLA).

A "T cell" or "T lymphocyte" is an immune system cell that matures in the thymus and produces T cell receptors (TCRs). T cells can exhibit phenotypes or markers associated with naive T cells (e.g., not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO as compared to TCM), memory T cells (TM) (e.g., antigen- experienced and long-lived), and effector cells (antigen-experienced, cytotoxic). T_M can be further divided into subsets exhibiting phenotypes or markers associated with of central memory T cells (TC_M, e.g., increased expression of CD62L, CCR7, CD28, CD 127, CD45RO, and CD95, and decreased expression of CD54RA as compared to naive T cells) and effector memory T cells (T_EM, e.g., decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD 127 as compared to naive T cells or TC_M)- Effector T cells (T_E) can refer to antigen-experienced CD8⁺ cytotoxic T lymphocytes that has decreased expression of CD62L ,CCR7, CD28, and are positive for granzyme and perforin as compared to TC_M- Helper T cells (T_H) can include CD4⁺ cells that influence the activity of other immune cells by releasing cytokines. CD4⁺ T cells can activate and suppress an adaptive immune response, and which of those two functions is induced will depend on presence of other cells and signals. T cells can be collected using known techniques, and the various subpopulations or combinations thereof can be enriched or depleted by known techniques, such as by affinity binding to antibodies, flow cytometry, or immunomagnetic selection. Other exemplary T cells include regulatory T cells, such as CD4+ CD25+ (Foxp3+) regulatory T cells and Tregl7 cells, as well as Trl, Th3, CD8+CD28-, and Qa-1 restricted T cells.

"T cell receptor" (TCR) in some aspects refers to an immunoglobulin superfamily member (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) capable of specifically binding to an antigen peptide bound to a MHC receptor. A TCR can be found on the surface of a cell or in soluble form and generally is comprised of a heterodimer having a and β chains (also known as TCRa and TCRP, respectively), or γ and δ chains (also known as TCRy and TCR6, respectively). Like immunoglobulins, the extracellular portion of TCR chains {e.g., a-chain, β-chain) contain two immunoglobulin domains, a variable domain {e.g., a-chain variable domain or V_a, β-chain variable domain or \¾ typically amino acids 1 to 116 based on Kabat numbering Kabat et al., "Sequences of Proteins of

Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5^th ed.) at the N-terminus, and one constant domain {e.g., a-chain constant domain or C_a, typically amino acids 81 to 259 based on Kabat, β-chain constant domain or C_β, typically amino acids 81 to 295 based on Kabat) adjacent to the cell membrane. Also like immunoglobulins, the variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs) (see, e.g., Jores et al., Proc. Natl Acad. Sci. U.S.A. 57:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al, Dev. Comp. Immunol. 27:55, 2003). In certain embodiments, a TCR is found on the surface of T cells (or T lymphocytes) and associates with the CD3 complex. The source of a TCR as used in the present disclosure may be from various animal species, such as a human, mouse, rat, rabbit or other mammal.

As used herein, "D/N/P region" in some aspects refers to nucleotides, or amino acids encoded by the nucleotides, predicted to be located within diversity (D) gene segment, which can include non-templated (N) nucleotides and palindromic (P) nucleotides that are inserted (or deleted) during the V(D)J recombination process that leads to diversity of T cell receptors. Recombination activating gene (RAG)-mediated rearrangement of variable (V), diversity (D) and joining (J) gene segments is an inaccurate process that results in the variable addition or subtraction of nucleotides (referred to as palindromic or P nucleotides), which is followed by terminal

deoxynucleotidyl transferase (TdT) activity that adds further adds random

non-templated (N) nucleotides. Finally, exonculeases remove unpaired nucleotides and gaps are filled by DNA synthesis and repair enzymes. Such a trim and repair mechanism leads to the junctional diversity that underpins the efficient and specific recognition of different antigens by different TCRs. D gene segments can be identified using the annotation system from the international ImMunoGeneTics information system (IMGT; at www.imgt.org).

In some aspects, "CD3" is known in the art as a multi-protein complex of six chains (see, Abbas and Lichtman, 2003; Janeway et al, pi 72 and 178, 1999). In mammals, the complex comprises a CD3y chain, a CD35 chain, two CD3s chains, and a homodimer of 0)3ζ chains. The CD3y, CD35, and CD3s chains are highly related cell surface proteins of the immunoglobulin superfamily containing a single

immunoglobulin domain. The transmembrane regions of the CD3y, CD35, and CD3s chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T cell receptor chains. The intracellular tails of the CD3y, CD35, and CD3s chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or IT AM, whereas each CD3ζ chain has three. Without wishing to be bound by theory, it is believed the ITAMs are important for the signaling capacity of a TCR compelx. CD3 as used in the present disclosure may be from various animal species, including human, mouse, rat, or other mammals.

As used herein, "TCR complex" in some aspects refers to a complex formed by the association of CD3 with TCR. For example, a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3s chains, a homodimer of CD3ζ chains, a TCRa chain, and a TCRP chain. Alternatively, a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3s chains, a homodimer of CD3ζ chains, a TCRy chain, and a TCR5 chain.

In some aspects, a "component of a TCR complex," as used herein, refers to a TCR chain (i.e., TCRa, TCRp, TCRy or TCR5), a CD3 chain (i.e., CO3j, CD35, CD3s or CD3ζ), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRa and TCRP, a complex of TCRy and TCR5, a complex of CD3s and CD35, a complex of CD3y and CD3s, or a sub-TCR complex of TCRa, TCRp, CD3y, CD35, and two CD3s chains).

In some aspects, a "binding domain" (also referred to as a "binding region" or

"binding moiety"), as used herein, refers to a molecule or portion thereof (e.g., peptide, oligopeptide, polypeptide, protein) that possesses the ability to specifically and non- covalently associate, unite, or combine with a target (e.g., WT1, WT1 peptide:MHC complex). A binding domain includes any naturally occurring, synthetic, semi- synthetic, or recombinantly produced binding partner for a biological molecule, a molecular complex (i.e., complex comprising two or more biological molecules), or other target of interest. Exemplary binding domains include single chain

immunoglobulin variable regions (e.g., scTCR, scFv), receptor ectodomains, ligands (e.g., cytokines, chemokines), or synthetic polypeptides selected for their specific ability to bind to a biological molecule, a molecular complex or other target of interest. As used herein, "specifically binds" or "specific for" in some aspects refers to an association or union of a binding protein (e.g., TCR receptor) or a binding domain (or fusion protein thereof) to a target molecule with an affinity or KA (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10⁵ M^"1 (which equals the ratio of the on-rate [k_on] to the off-rate [k_0ff] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample. Binding proteins or binding domains (or fusion proteins thereof) may be classified as "high affinity" binding proteins or binding domains (or fusion proteins thereof) or as "low affinity" binding proteins or binding domains (or fusion proteins thereof). "High affinity" binding proteins or binding domains refer to those binding proteins or binding domains having a KA of at least 10⁷ M^"1, at least 10⁸ M^"1, at least 10⁹ M^"1, at least 10¹⁰ M^"1, at least 10¹¹ M^"1, at least 10¹² M^{" l}, or at least 10¹³ M^"1. "Low affinity" binding proteins or binding domains refer to those binding proteins or binding domains having a K_A of up to 10⁷ M^"1, up to 10⁶ M^"1, up to 10⁵ M^"1. Alternatively, affinity may be defined as an equilibrium dissociation constant (K_D) of a particular binding interaction with units of M (e.g., 10^"5 M to 10^"13 M or less).

In certain embodiments, a receptor or binding domain may have "enhanced affinity," which refers to selected or engineered receptors or binding domains with stronger binding to a target antigen than a wild type (or parent) binding domain. For example, enhanced affinity may be due to a KA (equilibrium association constant) for the target antigen that is higher than the wild type binding domain, due to a K_D

(equilibrium dissociation constant) for the target antigen that is less than that of the wild type binding domain, due to an off-rate (koff) for the target antigen that is less than that of the wild type binding domain, or a combination thereof. In certain embodiments, enhanced affinity TCRs may be codon-optimized to enhance expression in a particular host cell, such as a T cell (Scholten et al., Clin. Immunol. 779: 135, 2006).

A variety of assays are known for identifying binding domains of the present disclosure that specifically bind a particular target, as well as determining binding domain or fusion protein affinities, such as Western blot, ELISA, analytical

ultracentrifugation, spectroscopy and surface plasmon resonance (Biacore®) analysis (see, e.g., Scatchard et al., Ann. N. Y. Acad. Sci. 57:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Patent Nos.

5,283,173, 5,468,614, or the equivalent).

In some aspects, the term "WTl -specific binding protein" refers to a protein or polypeptide that specifically binds to WTl or a peptide or fragment thereof. In some embodiments, a protein or polypeptide binds to WTl or a peptide thereof, such as a WTl peptide in complexed with an MHC or HLA molecule, e.g., on a cell surface, with at or at least about a particular affinity. In certain embodiments, a WTl -specific binding protein binds a WTl -derived peptide:HLA complex (or WTl -derived peptide:MHC complex) with a K_D of less than about 10^"8 M, less than about 10^"9 M, less than about 10^"10 M, less than about 10^"11 M, less than about 10^"12 M, or less than about 10^"13 M, or with an affinity that is about the same as, at least about the same as, or is greater than at or about the affinity exhibited by an exemplary WTl specific binding protein provided herein, such as any of the WTl -specific TCRs provided herein, for example, as measured by the same assay. In certain embodiments, a WTl-specific binding protein comprises a WTl -specific immunoglobulin superfamily binding protein or binding portion thereof.

Assays for assessing affinity or apparent affinity or relative affinity are known. In certain examples, apparent affinity for a TCR is measured by assessing binding to various concentrations of tetramers, for example, by flow cytometry using labeled tetramers. In some examples, apparent K_D of a TCR is measured using 2-fold dilutions of labeled tetramers at a range of concentrations, followed by determination of binding curves by non-linear regression, apparent K_D being determined as the concentration of ligand that yielded half-maximal binding.

In some aspects, the term "WTl binding domain" or "WTl binding fragment" refer to a domain or portion of a WTl -specific binding protein responsible for the specific WTl binding. A WTl-specific binding domain alone (i.e., without any other portion of a WTl -specific binding protein) can be soluble and can bind to WTl with a K_D of less than about 10^"8 M, less than about 10^"9 M, less than about 10^"10 M, less than about 10^"11 M, less than about 10^"12 M, or less than about 10^"13 M. Exemplary WT1- specific binding domains include WTl -specific scTCR (e.g., single chain aPTCR proteins such as Va-L-Υβ, Υβ-L-Va, Va-Ca-L-Va, or Va-L-VP-Cp, wherein Va and νβ are TCRa and β variable domains respectively, Ca and CP are TCRa and β constant domains, respectively, and L is a linker) and scFv fragments as described herein, which can be derived from an anti-WTl TCR or antibody.

Principles of antigen processing by antigen presenting cells (APC) (such as dendritic cells, macrophages, lymphocytes or other cell types), and of antigen presentation by APC to T cells, including major histocompatibility complex (MHC)- restricted presentation between immunocompatible (e.g., sharing at least one allelic form of an MHC gene that is relevant for antigen presentation) APC and T cells, are well established (see, e.g., Murphy, Janeway' s Immunobiology (8^th Ed.) 201 1 Garland Science, NY; chapters 6, 9 and 16). For example, processed antigen peptides originating in the cytosol (e.g., tumor antigen, intracellular pathogen) are generally from about 7 amino acids to about 1 1 amino acids in length and will associate with class I MHC molecules, whereas peptides processed in the vesicular system (e.g., bacterial, viral) will vary in length from about 10 amino acids to about 25 amino acids and associate with class II MHC molecules.

In some aspects, "WTl antigen" or "WTl peptide antigen" refer to a naturally or synthetically produced portion of a WTl protein ranging in length from about 7 amino acids to about 15 amino acids, which can form a complex with a MHC (e.g., HLA) molecule and such a complex can bind with a TCR specific for a WTl peptide:MHC (e.g., HLA) complex. Since WTl is an internal host protein, WTl antigen peptides will be presented in the context of class I MHC. In particular embodiments, a WTl peptide is VLDFAPPGA (SEQ ID NO:81), which is known to associate with human class I HLA (and, more specifically, associates with allele HLA-A*201).

A "linker" in some aspects refers to an amino acid sequence that connects two proteins, polypeptides, peptides, domains, regions, or motifs and may provide a spacer function compatible with interaction of the two sub-binding domains so that the resulting polypeptide retains a specific binding affinity (e.g., scTCR) to a target molecule or retains signaling activity (e.g., TCR complex). In certain embodiments, a linker is comprised of about two to about 35 amino acids, for instance, or about four to about 20 amino acids or about eight to about 15 amino acids or about 15 to about 25 amino acids. In some aspects, "junction amino acids" or "junction 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.

Junction amino acids may result from the construct design of a fusion protein (e.g., amino acid residues resulting from the use of a restriction enzyme site during the construction of a nucleic acid molecule encoding a fusion protein).

In some aspects, an "altered domain" or "altered protein" refers to a motif, region, domain, peptide, polypeptide, or protein with a non-identical sequence identity to a wild type motif, region, domain, peptide, polypeptide, or protein (e.g., a wild type TCRa chain, TCRP chain, TCRa constant domain, TCRP constant domain) of 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%).

As used herein, "nucleic acid" or "nucleic acid molecule" in some aspects refer to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments generated, for example, by the polymerase chain reaction (PCR) or by in vitro translation, and fragments generated by any of ligation, scission, endonuclease action, or exonuclease action. In certain embodiments, the nucleic acids of the present disclosure are produced by PCR. Nucleic acids may be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., a-enantiomeric forms of naturally- occurring nucleotides), or a combination of both. Modified nucleotides can have modifications in or replacement of sugar moieties, or pyrimidine or purine base moieties. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages. Analogs of phosphodiester linkages include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like. Nucleic acid molecules can be either single stranded or double stranded.

In some aspects, the term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is 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 co-existing materials in the natural system, is isolated. Such nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide. The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region "leader and trailer" as well as intervening sequences (introns) between individual coding segments (exons).

As used herein, the term "recombinant" in some aspects refers to a cell, microorganism, nucleic acid molecule, or vector that has been genetically engineered by human intervention - that is, modified by introduction of an exogenous or heterologous nucleic acid molecule, or refers to a cell or microorganism that has been altered such that expression of an endogenous nucleic acid molecule or gene is controlled, deregulated or constitutive. Human generated genetic alterations may include, for example, modifications that introduce nucleic acid molecules (which may include an expression control element, such as a promoter) that encode one or more proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of or addition to a cell's genetic material. Exemplary modifications include those in coding regions or functional fragments thereof of heterologous or homologous polypeptides from a reference or parent molecule.

As used herein, "mutation" or "mutated" in some aspects refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. A mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s). In certain embodiments, a mutation is a substitution of one or three codons or amino acids, a deletion of one to about 5 codons or amino acids, or a combination thereof.

A "conservative substitution" in some aspects is recognized in the art as a substitution of one amino acid for another amino acid that has 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).

The term "construct" in some aspects refers to any polynucleotide that contains a recombinant nucleic acid molecule. A construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome. A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid molecule. Vectors may be, for example, plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi -synthetic or synthetic nucleic acid molecules. Exemplary vectors are those capable of autonomous replication (episomal vector) or expression of nucleic acid molecules to which they are linked (expression vectors).

Exemplary viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno- associated viruses), coronavims, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In

Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

In some aspects, "lentiviral vector," as used herein, means HIV-based lentiviral vectors for gene delivery, which can be integrative or non-integrative, have relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.

The term "operably-linked" in some aspects refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably-linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Unlinked" means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.

As used herein, "expression vector" in some aspects refers to a DNA construct containing a nucleic acid molecule that is operably-linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself. In the present specification, "plasmid," "expression plasmid," "virus" and "vector" are often used interchangeably.

The term "expression", as used herein, in some aspects refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post- translational modification, or any combination thereof.

The term "introduced" in the context of inserting a nucleic acid molecule into a cell, in some aspects means "transfection", or 'transformation" or "transduction" and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g., chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

As used herein, "heterologous" or "exogenous" nucleic acid molecule, construct or sequence in some aspects refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to a host cell, but may be homologous to a nucleic acid molecule or portion of a nucleic acid molecule from the host cell. The source of the 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 (i.e., not endogenous or native) to a host cell or host genome by, for example, conjugation, transformation, transfection, electroporation, or the like, wherein the added molecule may integrate into the host genome or exist as extra-chromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and may be present in multiple copies. In addition, "heterologous" refers to a non-native enzyme, protein or other activity encoded by an exogenous nucleic acid molecule introduced into the host cell, even if the host cell encodes a homologous protein or activity.

As described herein, more than one heterologous or exogenous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of 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, a host cell can be modified to express two or more heterologous or exogenous nucleic acid molecules encoding desired TCR specific for a WT1 antigen peptide (e.g., TCRa and TCRP). When two or more exogenous nucleic acid molecules are introduced into a host cell, it is understood that the two or more exogenous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof. The number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cell. As used herein, the term "endogenous" or "native" in some aspects refers to a gene, protein, or activity that is normally present in a host cell. Moreover, a gene, protein or activity that is mutated, overexpressed, shuffled, duplicated or otherwise altered as compared to a parent gene, protein or activity is still considered to be endogenous or native to that particular host cell. For example, an endogenous control sequence from a first gene (e.g., promoter, translational attenuation sequences) may be used to alter or regulate expression of a second native gene or nucleic acid molecule, wherein the expression or regulation of the second native gene or nucleic acid molecule differs from normal expression or regulation in a parent cell.

In some aspects, the term "homologous" or "homolog" 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 may be homologous to a native host cell gene, and may optionally have an altered expression level, a different sequence, an altered activity, or any combination thereof.

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

As used herein, a "hematopoietic progenitor cell" in some aspects can be a cell that can be derived from hematopoietic stem cells or fetal tissue and is capable of further differentiation into mature cells types {e.g., immune system cells). Exemplary hematopoietic progenitor cells include those with a CD24^Lo Lin^" CD81⁺ phenotype or those found in the thymus (referred to as progenitor thymocytes).

As used herein, the term "host" in some aspects refers to a cell {e.g., T cell) or microorganism targeted for genetic modification with a heterologous or exogenous nucleic acid molecule to produce a polypeptide of interest {e.g., high or enhanced affinity anti-WTl TCR). In certain embodiments, a host cell may optionally already possess or be modified to include other genetic modifications that confer desired properties related or unrelated to biosynthesis of the heterologous or exogenous protein (e.g., inclusion of a detectable marker; deleted, altered or truncated endogenous TCR; increased co-stimulatory factor expression). In some embodiments, host cells are genetically modified to express a protein or fusion protein that modulates immune signaling in a host cell to, for example, promote survival and/or expansion advantage to the modified cell (e.g., see immunomodulatory fusion proteins of WO 2016/141357, which are herein incorporated by reference in their entirety). In other embodiments, host cells are genetically modified to knock-down or minimize immunosuppressive signals in a cell (e.g., a checkpoint inhibitor), which modification may be made using, for example, a CRISPR/Cas system (see, e.g., US 2014/0068797, U.S. Pat. No.

8,697,359; WO 2015/071474). In certain embodiments, a host cell is a human hematopoietic progenitor cell transduced with a heterologous or exogenous nucleic acid molecule encoding a TCRa chain specific for a WT1 antigen peptide.

As used herein, "hyperproliferative disorder" in some aspects refers to excessive growth or proliferation as compared to a normal or undiseased cell. Exemplary hyperproliferative disorders include tumors, cancers, neoplastic tissue, carcinoma, sarcoma, malignant cells, pre-malignant cells, as well as non-neoplastic or non- malignant hyperproliferative disorders (e.g., adenoma, fibroma, lipoma, leiomyoma, hemangioma, fibrosis, restenosis, as well as autoimmune diseases such as rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, or the like).

Binding Proteins Specific for WT1 Antigen Peptides

Several peptides of the WT1 protein are known to be tumor-associated antigen peptides that are HLA A*0201 -restricted antigens. The WT1 protein is an attractive target for clinical development due to its immune characteristics (Cheever et al., Clin. Cancer Res. 75:5323, 2009), and its expression in many aggressive tumor-types having associated poor-prognoses. WT1 is involved in the regulation of gene expression that promotes proliferation and oncogenicity (Oji et al., Jpn. J. Cancer Res. 90: 194, 1999), is over-expressed in most high-risk leukemias (Menssen et al., Leukemia 9: 1060, 1995), up to 80% of NSCLCs (Oji et al, Int. J. Cancer 100:291, 2002), 100% of mesotheliomas (Tsuta et αΙ., Αρρ. Immunohistochem. Mol. Morphol. 17 126, 2009), and >80% of gynecological malignancies (Coosemans and Van Gool, Expert Rev. Clin. Immunol. 70:705, 2014).

In certain aspects, the instant disclosure provides a WTl specific binding protein (e.g., an immunoglobulin superfamily binding protein or portion thereof), comprising (a) a T cell receptor (TCR) a-chain variable (V_a) domain and a TCR β-chain variable (ν_β) having a CDR3 amino acid sequence shown in any one of SEQ ID NOS: 1-11; (b) a TCR V_a domain having a CDR3 amino acid sequence of SEQ ID NO: 13, and a TCR νβ domain having a CDR3 amino acid sequence shown in any one of SEQ ID NOS: 1-11; or (c) a TCR V_a domain and a TCR ν_β domain comprising a CDR3 of 12-16 amino acids that is a variant of the amino acid sequence of SEQ ID NO: 12, wherein D/N/P region amino acids of SEQ ID NO: 12 comprise up to five amino acid substitutions, up to a contiguous three amino acid additions, or a combination thereof, provided that the CDR3 does not comprise the amino acid sequence of SEQ ID NO: 12. For example, any of these binding proteins of this disclosure can specifically bind to a WTl peptide:HLA complex on a cell surface independent of CD8 or in the absence of CD8. In further embodiments, a binding protein specifically binds to a VLDFAPPGA (SEQ ID NO:81):human leukocyte antigen (HLA) complex with a K_D of less than or equal to about 10^"8 M. In certain embodiments, the HLA comprises HLA-A*201. The peptide antigen VLDFAPPGA (SEQ ID NO:81) is a WTl peptide antigen and corresponds to amino acids 37-45 of the WTl protein.

In any of the embodiments described herein, the present disclosure provides a binding protein or high affinity engineered T cell receptor (TCR), comprising an a-chain and a β-chain, wherein the TCR binds to a WTl :HLA-A*201 complex on a cell surface independent or in the absence of CD8. In certain embodiments, a ν_β chain comprises or is derived from a TRBV19, TRBVlO-3, or TRBV24-1 gene. In further embodiments, a V_a chain comprises or is derived from a TRAV29/DV5 gene. In particular embodiments, a binding protein comprises (a) a ν_β chain comprising or derived from a TRBV19 gene and a V_a chain comprises or is derived from a

TRAV29/DV5 gene; (b) a νβ chain comprises or is derived from a TRBV10-3 gene and a V_a chain comprises or is derived from a TRAV29/DV5 gene; or (c) a ν_β chain comprises or is derived from a TRBV24-1 gene and a V_a chain comprises or is derived from a TRAV29/DV5 gene. In any of the aforementioned embodiments, a binding protein or TCR comprises amino acids encoded by a TRBJ1-5, TRBJ2-7, TRBJ1-2, or TRBJ2-6 gene.

In certain embodiments, a binding protein specific for a WTl peptide:HLA complex has a V_a domain that comprises or consists of the amino acid sequence of SEQ ID NO:26, has a ν_β domain comprises or consists of the amino acid sequence as set forth in any one of SEQ ID NOS: 14-24, or any combination thereof. In particular embodiments, a V_a domain comprises or consists of the amino acid sequence of SEQ ID NO:26 and a νβ domain comprises or consists of the amino acid sequence as set forth in any one of SEQ ID NOS: 14-16, and 24. In further particular embodiments, a V_a domain comprises or consists of the amino acid sequence of SEQ ID NO:26 and a νβ domain comprises or consists of the amino acid sequence as set forth in any one of SEQ ID NOS: 17-19, and 23. In further particular embodiments, a V_a domain comprises or consists of the amino acid sequence of SEQ ID NO:26 and a νβ domain comprises or consists of the amino acid sequence as set forth in any one of SEQ ID NOS:20-22.

In certain embodiments, a binding protein (e.g., an immunoglobulin superfamily binding protein or portion thereof) or high affinity recombinant T cell receptor (TCR) specific for WTl as described herein includes variant polypeptide species that have one or more amino acid substitutions, insertions, or deletions in the amino acid sequence relative to the amino acid sequences of any one or more of SEQ ID NOS:25, 26, 38-42 and 54, as presented herein, provided that the binding protein retains or substantially retains its specific WTl binding function.

Conservative substitutions of amino acids are well known and may occur naturally or may be introduced when the binding protein or TCR is recombinantly produced. Amino acid substitutions, deletions, and additions may be introduced into a protein using mutagenesis methods known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY, 2001). Oligonucleotide-directed site-specific (or segment specific) mutagenesis procedures may be employed to provide an altered polynucleotide that has particular codons altered according to the substitution, deletion, or insertion desired. Alternatively, random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error prone polymerase chain reaction mutagenesis, and oligonucleotide- directed mutagenesis may be used to prepare immunogen polypeptide variants (see, e.g., Sambrook et al., supra).

A variety of criteria known to persons skilled in the art indicate whether an amino acid that is substituted at a particular position in a peptide or polypeptide is conservative (or similar). For example, a similar amino acid or a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Similar amino acids may 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 nonpolar side chains {e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with beta-branched side chains {e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains {e.g., tyrosine, phenylalanine, tryptophan).

Proline, which is considered more difficult to classify, shares properties with amino acids that have aliphatic side chains {e.g., leucine, valine, isoleucine, and alanine). In certain circumstances, substitution of glutamine for glutamic acid or asparagine for aspartic acid may be considered a similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively. As understood in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and conserved amino acid substitutes thereto of the polypeptide to the sequence of a second polypeptide {e.g., using GENEWORKS, Align, the BLAST algorithm, or other algorithms described herein and practiced in the art).

Variants of a parent binding protein or parent TCR specific for WT1 may include a protein that has at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to any of the exemplary amino acid sequences disclosed herein {e.g., SEQ ID NOS: 1-54), provided that the CDR3 of the V_p domain does not comprise the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the CDR3 amino acid sequence of SEQ ID NO: 12 from the parent protein or parent TCR comprises mutations in D/N/P region amino acids, wherein the D/N/P region mutations comprise (a) an addition of one, two or three contiguous amino acids; (b) substitutions of three, four, five, six, seven, or eight amino acids; or (c) any combination thereof, thereby resulting in a binding protein or high affinity engineered TCR of this disclosure. In further embodiments, a J gene segment comprises a TRBJ1-5, TRBJ2-7, TRBJl-2, or TRBJ2-7 gene. In any of the aforementioned embodiments, the CDR3 amino acid sequence of SEQ ID NO: 12 may optionally further comprise J gene segment variants that total from two up to 12 amino acid substitutions (i.e., including substitutions in both the D/N/P region and J gene segment relative to SEQ ID NO: 12). In still further optional embodiments, a variant binding protein or TCR comprises no change in amino acid sequence of the V_a domain CDR1, the V_a domain CDR2, the ν_β domain CDR1, the ν_β domain CDR2, or any combination thereof, found in SEQ ID NO:26 (parental V_a domain) or 25 (parental ν_β domain). In each of these embodiments, the binding protein retains its ability to specifically bind to a peptide antigen:HLA complex (e.g., VLDFAPPGA (SEQ ID NO:81):HLA complex) with a K_D of less than or equal to about 10^"8 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 parent binding protein or TCR comprised of SEQ ID NO:25 and 26, or comprised of SEQ ID NO:38 and 39.

In further embodiments, the present disclosure provides a binding protein comprising (a) a T cell receptor (TCR) a-chain variable (V_a) domain having at least 90% sequence identity to an amino acid sequence of SEQ ID NO:26, and a TCR β-chain variable (\¾) domain having at least 90% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOS: 14-24; (b) a TCR V_a domain comprising or consisting of an amino acid sequence of SEQ ID NO:26, and a TCR ν_β domain having at least 90% sequence identity to an amino acid sequence as set forth in any one of SEQ ID NOS: 14-24; or (c) a V_a domain having at least 90% sequence identity to an amino acid sequence of SEQ ID NO:26, and a ν_β domain comprising or consisting of an amino acid sequence as set forth in any one of SEQ ID NOS: 14-24; wherein the binding protein is capable of specifically binding to a WT1 peptide:HLA cell surface complex independent, or in the absence, of CD8, such as a VLDFAPPGA (SEQ ID NO:81):HLA complex.

In particular embodiments, a binding protein comprising a TCR V_a domain having at least 90% sequence identity to an amino acid sequence of SEQ ID NO:26, and a TCR νβ domain comprising an amino acid sequence that is at least: (a) 98% identical to the amino acid sequence of SEQ ID NO: 14; (b) 97% identical to the amino acid sequence of SEQ ID NO: 15; (c) 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 16-19; (d) 93.5% identical to the amino acid sequence of SEQ ID NO:20 or 21; or (e) 91.5% identical to the amino acid sequence of SEQ ID NO:22.

In certain embodiments, any of the aforementioned WTl specific binding proteins are each a T cell receptor (TCR) or a chain, region, fragment or portion thereof, a chimeric antigen receptor or an antigen-binding fragment of a TCR, any of which can be chimeric, humanized or human. In further embodiments, an antigen-binding fragment of the TCR comprises a single chain TCR (scTCR) or a chimeric antigen receptor (CAR). In certain embodiments, a WTl specific binding protein is a TCR or a chain, region, fragment or portion thereof. In some embodiments, the WTl specific binding protein can be a multi-chain binding protein, for example, comprising an a-chain and a β-chain.

In any of the aforementioned embodiments, the present disclosure provides a WTl -specific binding protein comprising an a-chain constant domain having at least 90% sequence identity to the amino acid sequence SEQ ID NO:42, a β-chain constant domain having at least 90% sequence identity to an amino acid sequence of SEQ ID NO:40 or 41, or a combination thereof. In further embodiments, the present disclosure provides a WTl-specific binding protein comprising or consisting of an a-chain constant domain having an amino acid sequence of SEQ ID NO:42, comprising or consisting of a β-chain constant domain having an amino acid sequence of SEQ ID NO: 40 or 41, or a combination thereof.

In any of the aforementioned embodiments, the present disclosure provides a WTl -specific binding protein comprising an a-chain having at least 90% sequence identity to the amino acid sequence SEQ ID NO: 39, a β-chain having at least 90% sequence identity to the amino acid sequence as set forth in any one of SEQ ID NOS:27-37, or a combination thereof. In further embodiments, a binding protein (e.g., TCR) specific for a WTl peptide (e.g., a WTl peptide in an MHC/HLA complex) comprises a TCR a-chain comprising or consisting of the amino acid sequence of SEQ ID NO:39, a TCR β-chain comprising or consisting of the amino acid sequence as set forth in any one of SEQ ID NOS:27-37, or any combination thereof. In certain embodiments, such high affinity TCRs are capable of binding to a VLDFAPPGA (SEQ ID NO:81):HLA complex with a K_D of less than or equal to about 10^"8 M, or wherein the high affinity TCR dissociates from a VLDFAPPGA (SEQ ID NO:81):HLA complex at a reduced k_0ff rate as compared to a parental TCR composed of an a-chain of SEQ ID NO:39 and a β-chain of SEQ ID NO:38.

In more embodiments, there is provided a composition comprising a

WTl -specific binding protein or high affinity recombinant TCR according to any one of the aforementioned embodiments and a pharmaceutically acceptable carrier, diluent, or excipient.

Methods useful for isolating and purifying recombinantly produced soluble

TCR, by way of example, may include obtaining supernatants from suitable host cell/vector systems that secrete a recombinant soluble TCR into culture media and then concentrating the media using a commercially available filter. Following concentration, the concentrate may be applied to a single suitable purification matrix or to a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse phase UPLC steps may be employed to further purify a recombinant

polypeptide. These purification methods may also be employed when isolating an immunogen from its natural environment. Methods for large scale production of one or more of the isolated/recombinant soluble TCR described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of the soluble TCR may be performed according to methods described herein and known in the art and that comport with laws and guidelines of domestic and foreign regulatory agencies.

In certain embodiments, nucleic acid molecules encoding an immunoglobulin superfamily binding protein or high affinity TCR specific for WTl are used to transfect/transduce a host cell (e.g., T cells) for use in adoptive transfer therapy. Advances in TCR sequencing have been described (e.g., Robins et al, Blood 114:4099, 2009; Robins et a/., Sci. Translat. Med. 2:47ra64, 2010; Robins et al, (Sept. 10) J. 1mm. Meth. Epub ahead of print, 2011; Warren et al, Genome Res. 21 :790, 2011) and may be employed in the course of practicing the embodiments according to the present disclosure. Similarly, methods for transfecting/transducing T cells with desired nucleic acids have been described {e.g., U.S. Patent Application Pub. No. US 2004/0087025) as have adoptive transfer procedures using T-cells of desired antigen-specificity {e.g., SchmM et al, Hum. Gen. 20: 1240, 2009; Dossett et al, Mol. Ther. 77:742, 2009; Till et al, Blood 772:2261, 2008; Wang et al, Hum. Gene Ther. 75:712, 2007; Kuball et al, Blood 709:2331, 2007; US 2011/0243972; US 2011/0189141; Leen et al, Ann. Rev. Immunol. 25:243, 2007), such that adaptation of these methodologies to the presently disclosed embodiments is contemplated, based on the teachings herein, including those directed to high affinity TCRs specific for WT1 peptide antigens complexed with an HLA receptor.

The WT1 -specific binding proteins or domains as described herein (e.g., SEQ

ID NOS: 1-54, and variants thereof), may be functionally characterized according to any of a large number of art accepted methodologies for assaying T cell activity, including determination of T cell binding, activation or induction and also including

determination of T cell responses that are antigen-specific. Examples include determination of 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 ⁵¹Cr release from pre-loaded target cells), changes in T cell phenotypic marker expression, and other measures of T-cell functions. Procedures for performing these and similar assays are may 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.

"MHC-peptide tetramer staining" in some aspects refers to an assay used to detect antigen-specific T cells, which features a tetramer of MHC molecules, each comprising an identical peptide having an amino acid sequence that is cognate (e.g., identical or related to) at least one antigen (e.g., WT1), wherein the complex is capable of binding T cell receptors specific for the cognate antigen. Each of the MHC molecules may be tagged with a biotin molecule. Biotinylated MHC/peptides are tetramerized by the addition of streptavidin, which can be fluorescently labeled. The tetramer may be detected by flow cytometry via the fluorescent label. In certain embodiments, an MHC-peptide tetramer assay is used to detect or select enhanced affinity TCRs of the instant disclosure.

Levels of cytokines may be determined according to methods described herein and practiced in the art, including for example, ELISA, ELISPOT, intracellular cytokine staining, and flow cytometry and combinations thereof (e.g., intracellular cytokine staining and flow cytometry). Immune cell proliferation and clonal expansion resulting from an antigen-specific elicitation or stimulation of an immune response may be determined by isolating lymphocytes, such as circulating lymphocytes in samples of peripheral blood cells or cells from lymph nodes, stimulating the cells with antigen, and measuring cytokine production, cell proliferation and/or cell viability, such as by incorporation of tritiated thymidine or non-radioactive assays, such as MTT assays and the like. The effect of an immunogen described herein on the balance between a Thl immune response and a Th2 immune response may be examined, for example, by determining levels of Thl 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.

Polynucleotides Encoding Binding Proteins Speci fic for WT1 Antigen Peptides

Heterologous, isolated or recombinant nucleic acid molecules encoding a binding protein (e.g., immunoglobulin superfamily binding protein) or high affinity recombinant T cell receptor (TCR) specific for WT1 as described herein may be produced and prepared according to various methods and techniques described herein (see Examples). Construction of an expression vector that is used for recombinantly producing a binding protein or high affinity engineered TCR specific for a WT1 peptide of interest can be accomplished by using any suitable molecular biology engineering techniques known in the art, including the use of restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing as described in, for example, 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). To obtain efficient transcription and translation, a polynucleotide in each recombinant expression construct includes at least one appropriate expression control sequence (also called a regulatory sequence), such as a leader sequence and particularly a promoter operably {i.e., operatively) linked to the nucleotide sequence encoding the immunogen.

Certain embodiments relate to nucleic acids that encode the polypeptides contemplated herein, for instance, binding proteins or high affinity engineered TCRs specific for WTl . As one of skill in the art will recognize, a nucleic acid may refer to a single- or a double-stranded DNA, cDNA or RNA in any form, and may include a positive and a negative strand of the nucleic acid which complement each other, including anti-sense DNA, cDNA and RNA. Also included are siRNA, microRNA, RNA— DNA hybrids, ribozymes, and other various naturally occurring or synthetic forms of DNA or RNA.

In certain embodiments, provided herein are isolated polynucleotides that encode a binding protein or high affinity engineered TCR of this disclosure specific for a WTl peptide, wherein a V_a domain is encoded by a polynucleotide that is at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 100. In particular embodiments, a polynucleotide encodes a V_a domain that comprises or consists of the nucleotide sequence of SEQ ID NO: 113. In further embodiments, provided herein are polynucleotides that encode a binding protein or high affinity engineered TCR of this disclosure specific for a WTl peptide, wherein a νβ domain is encoded by a polynucleotide that is at least 75% or 77% identical to the nucleotide sequence of SEQ ID NO: 99, provided that the polynucleotide encodes a CDR3 that does not have the amino acid sequence of SEQ ID NO: 12. In particular embodiments, a νβ domain is encoded by a polynucleotide that comprises or consists of the nucleotide sequence as set forth in any one of SEQ ID NOS:88-98. In some embodiments, a binding protein or TCR provided herein comprises a V_a domain encoded by a polynucleotide that is at least 75% or 78% identical to the nucleotide sequence of SEQ ID NO: 100, and a ν_β domain encoded by the polynucleotide that comprises or consists of the nucleotide sequence as set forth in any one of SEQ ID NOS:88-98. In still more embodiments, a binding protein or TCR provided herein comprises a

V_a domain encoded by a polynucleotide that comprises or consists of the nucleotide sequence of SEQ ID NO: 113, and a νβ domain encoded by a polynucleotide that comprises or consists of the nucleotide sequence as set forth in any one of SEQ ID NOS:88-98.

In any of the aforementioned embodiments, a polynucleotide encoding a V_a domain, ν_β domain, or both, may further encode an a-chain constant domain or a β-chain constant domain, respectively. In certain embodiments, a binding protein or TCR of this disclosure comprises a TCR a-chain constant domain, wherein the a-chain constant domain is encoded by a polynucleotide comprising a nucleotide sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 116. In particular embodiments, an a-chain constant domain is encoded by a polynucleotide that comprises or consists of the nucleotide sequence of SEQ ID NO: 119. In further embodiments, provided herein a β-chain constant domain encoded by a polynucleotide that is at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 114 or 115, provided that the polynucleotide encodes a CDR3 that does not have the amino acid sequence of SEQ ID NO: 12. In particular embodiments, a β-chain constant domain is encoded by a polynucleotide that comprises or consists of the nucleotide sequence of SEQ ID NO: 117 or 118. In some embodiments, a binding protein or TCR provided herein comprises an a-chain constant domain encoded by a polynucleotide that is at least 75% or 79% identical to the nucleotide sequence of SEQ ID NO: 116, and a β-chain constant domain encoded by the polynucleotide that comprises or consists of the nucleotide sequence of SEQ ID NO: 117 or 118. In still more embodiments, a binding protein or TCR provided herein comprises an a-chain constant domain encoded by a polynucleotide that comprises or consists of the nucleotide sequence of SEQ ID NO: 119, and a β-chain constant domain encoded by a polynucleotide that comprises or consists of the nucleotide sequence of SEQ ID NO: 117 or 118. In any of the aforementioned embodiments, a polynucleotide encoding a binding protein or TCR comprises a TCR a-chain, a TCR β-chain, or both. In certain embodiments, a binding protein or TCR of this disclosure comprises a TCR a-chain, wherein the a-chain is encoded by a polynucleotide comprising a nucleotide sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 132. In particular embodiments, an a-chain is encoded by a polynucleotide that comprises or consists of the nucleotide sequence of SEQ ID NO: 145. In further embodiments, provided herein a β-chain encoded by a polynucleotide that is at least 75%, 76%, 77%, 78%, 79%, 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence as set forth in any one of SEQ ID

NOS: 120-130, provided that the polynucleotide encodes a CDR3 that does not have the amino acid sequence of SEQ ID NO: 12. In particular embodiments, a β-chain is encoded by a polynucleotide that comprises or consists of the nucleotide sequence as set forth in any one of SEQ ID NOS: 133-143. In some embodiments, a binding protein or TCR provided herein comprises an a-chain encoded by a polynucleotide that is at least 75%) or 78%) identical to the nucleotide sequence of SEQ ID NO: 132, and a β-chain encoded by the polynucleotide that comprises or consists of the nucleotide sequence as set forth in any one of SEQ ID NOS: 133-143. In still more embodiments, a binding protein or TCR provided herein comprises an a-chain encoded by a polynucleotide that comprises or consists of the nucleotide sequence of SEQ ID NO: 145, and a β-chain encoded by a polynucleotide that comprises or consists of the nucleotide sequence as set forth in any one of SEQ ID NOS: 133-143.

In any of the aforementioned embodiments, a polynucleotide encoding a binding protein of the instant disclosure is codon optimized for efficient expression in a target host cell. In certain embodiments, the present disclosure provides a host cell comprising a heterologous polynucleotide encoding any one or more of the binding proteins and TCRs of this disclosure, wherein the modified or recombinant host cell expresses on its cell surface the binding protein or TCR encoded by the heterologous polynucleotide.

Standard techniques may be used for recombinant (i.e., engineered) DNA, peptide and oligonucleotide synthesis, immunoassays and tissue culture and

transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may be generally performed according to conventional methods well- known in the art and as described in various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology and immunology techniques that are cited and discussed throughout the present specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;

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: A Practical Approach, vol. I & II (IRL Press, Oxford Univ. Press USA, 1985); Current Protocols in Immunology (Edited by: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober 2001 John Wiley & Sons, NY, NY); Real-Time PCR: Current Technology and Applications, Edited by Julie Logan, Kirstin Edwards 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 (Academic Press, New York, 1991); Oligonucleotide Synthesis (N. Gait, Ed., 1984); Nucleic Acid Hybridization (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 Genome Sequencing (Janitz, 2008 Wiley- VCH); PCR

Protocols β/lethods in Molecular Biology) (Park, Ed., 3^rd Edition, 2010 Humana Press); Immobilized Cells And Enzymes (IRL Press, 1986); the treatise, Methods In

Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1998); Immunochemical Methods In Cell And 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 Scientific Publications, Oxford, 1988); Embryonic Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2002); Embryonic Stem Cell Protocols: Volume I: Isolation and Characterization (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2006); Embryonic Stem Cell Protocols: Volume II: Differentiation Models (Methods in Molecular Biology) (Kurstad Turksen, Ed., 2006); Human Embryonic Stem Cell Protocols (Methods in Molecular Biology) (Kursad Turksen Ed., 2006); Mesenchymal Stem Cells: Methods and

Protocols (Methods in Molecular Biology) (Darwin J. Prockop, Donald G. Phinney, and Bruce A. Bunnell Eds., 2008); Hematopoietic Stem Cell Protocols (Methods in Molecular Medicine) (Christopher A. Klug, and Craig T. Jordan Eds., 2001);

Hematopoietic Stem Cell Protocols (Methods in Molecular Biology) (Kevin D. Bunting Ed., 2008) Neural Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Leslie P. Weiner Ed., 2008).

Certain embodiments include nucleic acid molecules contained in a vector. One of skill in the art can readily ascertain suitable vectors for use with certain embodiments disclosed herein. An exemplary vector may comprise a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked, or which is capable of replication in a host organism. Some examples of vectors include plasmids, viral vectors, cosmids, and others. Some vectors may be capable of autonomous replication in a host cell into which they are introduced {e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), whereas other vectors may be integrated into the genome of a host cell or promote integration of the polynucleotide insert upon introduction into the host cell and thereby replicate along with the host genome {e.g., lentiviral vector)). Additionally, some vectors are capable of directing the expression of genes to which they are operatively linked (these vectors may be referred to as "expression vectors"). According to related embodiments, it is further understood that, if one or more agents {e.g., polynucleotides encoding binding proteins or high affinity recombinant TCRs specific for WT1, or variants thereof, as described herein) is co-administered to a subject, that each agent may reside in separate or the same vectors, and multiple vectors (each containing a different agent the same agent) may be introduced to a cell or cell population or administered to a subject. In certain embodiments, a polynucleotide encoding a binding protein or high affinity recombinant TCR specific for WTl of this disclosure may be operatively linked to certain expression control elements of a vector. For example, polynucleotide sequences that are needed to effect the expression and processing of coding sequences to which they are ligated may be operatively linked. Expression control sequences 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 enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion. Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest. In certain embodiments, polynucleotides encoding binding proteins of the instant disclosure are contained in an expression vector that is a viral vector, such as a lentiviral vector or a γ-retroviral vector.

In particular embodiments, the recombinant expression vector is delivered to an appropriate cell, for example, a T cell or an antigen-presenting cell, i.e., a cell that displays a peptide/MHC complex on its cell surface (e.g., a dendritic cell) and lacks CD8. 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 γδ T cell, a natural killer cell, a dendritic cell, or any combination thereof . In certain embodiments, wherein a T cell is the host, the T cell can be naive, a central memory T cell, an effector memory T cell, or any combination thereof. The recombinant expression vectors may therefore also include, for example, lymphoid tissue-specific transcriptional regulatory elements (TREs), such as a B lymphocyte, T lymphocyte, or dendritic cell specific TREs.

Lymphoid tissue specific TREs are known in the art (see, e.g., Thompson et al., Mol. Cell. Biol. 72: 1043, 1992); Todd et al, J. Exp. Med. 777: 1663, 1993); Penix et al, J. Exp. Med. 775: 1483, 1993).

In addition to vectors, certain embodiments relate to host cells that comprise the vectors that are presently disclosed. One of skill in the art readily understands that many suitable host cells are available in the art. A host cell may include any individual cell or cell culture which may receive a vector or the incorporation of nucleic acids and/or proteins, as well as any progeny cells. The term also encompasses progeny of the host cell, whether genetically or phenotypically the same or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells. These cells may be induced to incorporate the vector or other material by use of a viral vector,

transformation via 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).

Methods of Treatment

In certain aspects, the instant disclosure is directed to methods for treating a hyperproliferative disorder or a condition characterized by Wilms tumor protein 1 (WTl) overexpression by administering to human subject in need thereof a composition comprising a binding protein or high affinity recombinant TCR specific for human WTl according to any of the aforementioned binding proteins or TCRs or any binding proteins or TCRs described herein, or a host cell, such as a T cell, engineered to express the same, or compositions comprising any of the binding proteins or TCRs or host cells described herein.

The presence of a hyperproliferative disorder or malignant condition in a subject refers to the presence of dysplastic, cancerous and/or transformed cells in the subject, including, for example neoplastic, tumor, non-contact inhibited or oncogenically transformed cells, or the like {e.g., solid cancers; hematologic cancers including lymphomas and leukemias, such as acute myeloid leukemia, chronic myeloid leukemia, etc.), which are known in the art and for which criteria for diagnosis and classification are established {e.g., Hanahan and Weinberg, Cell 144:646, 2011; Hanahan and Weinberg, Cell 100:57, 2000; Cavallo et al., Cane. Immunol. Immunother. 60:319, 2011; Kyrigideis et al., J. Carcinog. 9:3, 2010). In certain embodiments, such cancer cells may be cells of acute myeloid leukemia, B-cell lymphoblastic leukemia, T-cell lymphoblastic leukemia, or myeloma, including cancer stem cells that are capable of initiating and serially transplanting any of these types of cancer (see, e.g., Park et al., Molec. Therap. 17:219, 2009).

In certain embodiments, there are provided methods for treating a

hyperproliferative disorder, such as a hematological malignancy or a solid cancer.

Exemplary hematological malignancies include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or multiple myeloma (MM).

In further embodiments, there are provided methods for treating a

hyperproliferative disorder, such as a solid cancer is selected from biliary cancer, bladder cancer, bone and soft tissue carcinoma, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonal cancer, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumor, head and neck squamous cell carcinoma, hepatic cancer, lung cancer, mesothelioma, malignant melanoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, renal cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ-cell tumor, urothelial cancer, uterine sarcoma, or uterine cancer.

As understood by a person skilled in the medical art, the terms, "treat" and

"treatment," refer to medical management of a disease, disorder, or condition of a subject (i.e., patient, host, who may be a human or non-human animal) (see, e.g., Stedman's Medical Dictionary). In general, an appropriate dose and treatment regimen provide one or more of a binding protein or high affinity recombinant TCR specific for human WTl (e.g., SEQ ID NOS: 1-54, and variants thereof) or a host cell expressing the same, and optionally an adjunctive therapy (e.g., a cytokine such as IL-2, IL-15, IL-21 or any combination thereof), in an amount sufficient to provide therapeutic or prophylactic benefit. Therapeutic or prophylactic benefit resulting from therapeutic treatment or prophylactic or preventative methods include, for example an improved clinical outcome, wherein the object is to prevent or retard or otherwise reduce (e.g., decrease in a statistically significant manner relative to an untreated control) an undesired physiological change or disorder, or to prevent, retard or otherwise reduce the expansion or severity of such a disease or disorder. Beneficial or desired clinical results from treating a subject include abatement, lessening, or alleviation of symptoms that result from or are associated the disease or disorder to be treated; decreased occurrence of symptoms; improved quality of life; longer disease-free status (i.e., decreasing the likelihood or the propensity that a subject will present symptoms on the basis of which a diagnosis of a disease is made); diminishment of extent of disease; stabilized (i.e., not worsening) state of disease; delay or slowing of disease progression; amelioration or palliation of the disease state; and remission (whether partial or total), whether detectable or undetectable; or overall survival.

"Treatment" can also mean prolonging survival when compared to expected survival if a subject were not receiving treatment. Subjects in need of the methods and compositions described herein include those who already have the disease or disorder, as well as subjects prone to have or at risk of developing the disease or disorder.

Subjects in need of prophylactic treatment include subjects in whom the disease, condition, or disorder is to be prevented (i.e., decreasing the likelihood of occurrence or recurrence of the disease or disorder). The clinical benefit provided by the

compositions (and preparations comprising the compositions) and methods described herein can be evaluated by design and execution of in vitro assays, preclinical studies, and clinical studies in subjects to whom administration of the compositions is intended to benefit, as described in the examples.

In another aspect, the instant disclosure is directed to methods for treating a hyperproliferative disorder or a condition characterized by Wilms tumor protein 1 (WTl) overexpression by administering to human subject in need thereof a composition comprising an isolated polynucleotide encoding a binding protein or high affinity recombinant TCR specific for human WTl according to any the aforementioned encoded binding proteins or TCRs or any described herein, or a host cell, such as a T cell, comprising the same, or a composition comprising any of the binding proteins, TCRs or host cells described herein. In certain embodiments, the polynucleotide encoding a binding protein or TCR specific for human WTl is codon optimized for a host cell of interest. In further embodiments, any of the aforementioned polynucleotides are operably linked to an expression control sequence and is optionally contained in an expression vector, such as a viral vector. Exemplary viral vectors include lentiviral vectors and γ-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., human hematopoietic progenitor cell or a human immune system cell). Exemplary immune system cells include 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 (e.g., human). In certain embodiments, the immune system cell is a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof, all of which are optionally human.

In still another aspect, the instant disclosure is directed to methods for treating a hyperproliferative disorder or a condition characterized by Wilms tumor protein 1 (WT1) overexpression by administering to human subject in need thereof a host cell comprising a heterologous polynucleotide or an expression vector according to any of the aforementioned embodiments, or any described herein, wherein the engineered or recombinant host cell expresses on its cell surface a binding protein or TCR specific for human WT1 encoded by the heterologous polynucleotide.

Cells expressing the binding protein or recombinant TCR (e.g., high affinity) specific for human WT1 as described herein may be administered to a subject in a pharmaceutically or physiologically acceptable or suitable excipient or carrier.

Pharmaceutically acceptable excipients are biologically compatible vehicles, e.g., physiological saline, which are described in greater detail herein, that are suitable for administration to a human or other non-human mammalian subject.

A therapeutically effective dose is an amount of host cells (expressing a binding protein or high affinity recombinant TCR specific for human WT1) used in adoptive transfer that is capable of producing a clinically desirable result (i.e., a sufficient amount to induce or enhance a specific T cell immune response against cells overexpressing WT1 (e.g., a cytotoxic T cell response) in a statistically significant manner) in a treated human or non-human mammal. As is well known in the medical arts, the dosage for any one patient depends upon many factors, including the patient's size, weight, body surface area, age, the particular therapy to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Doses will vary, but a preferred dose for administration of a host cell comprising a recombinant expression vector as described herein is about 10⁷ cells/m², about 5 x 10 7 cells/m 2 , about 108 cells/m2 , about 5 x 108 cells/m2 , about 109 cells/m2 , about 5 x 10⁹ cells/m², about 10¹⁰ cells/m², about 5 x 10¹⁰ cells/m², or about 10¹¹ cells/m².

Pharmaceutical compositions may be administered in a manner appropriate to the disease or condition to be treated (or prevented) as determined by persons skilled in the medical art. An appropriate dose and a suitable duration and frequency of administration of the compositions will be determined by such factors as the health condition of the patient, size of the patient (i.e., weight, mass, or body area), the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. In general, an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome, such as more frequent complete or partial remissions, or longer disease-free and/or overall survival, or a lessening of symptom severity). For prophylactic use, a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder. Prophylactic benefit of the immunogenic compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.

A condition associated with WTl overexpression includes any disorder or condition in which underactivity, over-activity or improper activity of a WTl cellular or molecular event is present, and typically results from unusually high (with statistical significance) levels of WTl expression in afflicted cells (e.g., leukemic cells), relative to normal cells. A subject having such a disorder or condition would benefit from treatment with a composition or method of the presently described embodiments. Some conditions associated with WTl overexpression thus may include acute as well as chronic disorders and diseases, such as those pathological conditions that predispose the subject to a particular disorder.

Some examples of conditions associated with WTl overexpression include hyperproliferative disorders, which in some aspects refer to states of activated and/or proliferating cells (which may also be transcriptionally overactive) in a subject including tumors, neoplasms, cancer, malignancy, etc. In addition to activated or proliferating cells, the hyperproliferative disorder may also include an aberration or dysregulation of cell death processes, whether by necrosis or apoptosis. Such aberration of cell death processes may be associated with a variety of conditions, including cancer (including primary, secondary malignancies as well as metastasis), or other conditions.

According to certain embodiments, virtually any type of cancer that is characterized by WTl overexpression may be treated through the use of compositions and methods disclosed herein, including hematological cancers (e.g., leukemia including acute myeloid leukemia (AML), T or B cell lymphomas, myeloma, and others). Furthermore, "cancer" may refer to any accelerated proliferation of cells, including solid tumors, ascites tumors, blood or lymph or other malignancies;

connective tissue malignancies; metastatic disease; minimal residual disease following transplantation of organs or stem cells; multi-drug resistant cancers, primary or secondary malignancies, angiogenesis related to malignancy, or other forms of cancer. Also contemplated within the presently disclosed embodiments are specific

embodiments wherein only one of the above types of disease is included, or where specific conditions may be excluded regardless of whether or not they are characterized by WTl overexpression.

Certain methods of treatment or prevention contemplated herein include administering a host cell (which may be autologous, allogeneic or syngeneic) comprising a desired nucleic acid molecule as described herein that is stably integrated into the chromosome of the cell. For example, such a cellular composition may be generated ex vivo using autologous, allogeneic or syngeneic immune system cells (e.g., T cells, antigen-presenting cells, natural killer cells) in order to administer a desired,

WTl -targeted T-cell composition to a subject as an adoptive immunotherapy. As used herein, administration of a composition or therapy in some aspects refers to delivering the same to a subject, regardless of the route or mode of delivery. Administration may be effected continuously or intermittently, and parenterally.

Administration may be for treating a subject already confirmed as having a recognized condition, disease or disease state, or for treating a subject susceptible to or at risk of developing such a condition, disease or disease state. Co-administration with an adjunctive therapy may include simultaneous and/or sequential delivery of multiple agents in any order and on any dosing schedule (e.g., WT1 specific modified (i.e., recombinant or engineered) host cells with one or more cytokines; immunosuppressive therapy such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof). For example, a therapy of this disclosure can be combined with specific inhibitors or modulators of

immunosuppression components, such as inhibitors or modulators of immune checkpoint molecules (e.g., anti-PD-1, anti-PD-Ll, or anti-CTLA-4 antibodies; see, e.g., Pardol, Nature Rev. Cancer 12:252, 2012; Chen and Mellman, Immunity 39.1, 2013).

In certain embodiments, a plurality of doses of a recombinant host cell as described herein is administered to the subject, which may be administered at intervals between administrations of about two to about four weeks. In further embodiments, a cytokine is administered sequentially, provided that the subject was administered the recombinant host cell at least three or four times before cytokine administration. In certain embodiments, the cytokine is administered subcutaneously (e.g., JL-2, IL-15, IL-21). In still further embodiments, the subject being treated is further receiving immunosuppressive therapy, such as an antibody specific for PD-1 (e.g., pidilizumab, nivolumab, or pembrolizumab), an antibody specific for PD-L1 (e.g., MDX-1105,

BMS-936559, MEDI4736, MPDL3280A, or MSB0010718C), an antibody specific for CTLA4 (e.g., tremelimumab or ipilimumab), calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof. In yet further embodiments, the subject being treated has received a non- myeloablative or a myeloablative hematopoietic cell transplant, wherein the treatment may be administered at least two to at least three months after the non-myeloablative hematopoietic cell transplant.

An effective amount of a therapeutic or pharmaceutical composition in some aspects refers to an amount sufficient, at dosages and for periods of time needed, to achieve the desired clinical results or beneficial treatment, as described herein. An effective amount may be delivered in one or more administrations. If the administration is to a subject already known or confirmed to have a disease or disease-state, the term "therapeutic amount" may be used in reference to treatment, whereas "prophylactically effective amount" may be used to describe administrating an effective amount to a subject that is susceptible or at risk of developing a disease or disease-state (e.g., recurrence) as a preventative course.

The level of a cytotoxic T lymphocyte (CTL) immune response may be determined by any one of numerous immunological methods described herein and routinely practiced in the art. The level of a CTL immune response may be determined prior to and following administration of any one of the herein described WT1 -specific binding proteins expressed by, for example, a T cell. Cytotoxicity assays for determining CTL activity may be performed using any one of several techniques and methods routinely practiced in the art (see, e.g., Henkart et al., "Cytotoxic

T-Lymphocytes" in Fundamental Immunology, Paul (ed.) (2003 Lippincott Williams & Wilkins, Philadelphia, PA), pages 1127-50, and references cited therein).

Antigen-specific T cell responses are typically determined by comparisons of observed T cell responses according to any of the herein described T cell functional parameters (e.g., proliferation, cytokine release, CTL activity, altered cell surface marker phenotype, etc.) that may be made between T cells that are exposed to a cognate antigen in an appropriate context (e.g., the antigen used to prime or activate the T cells, when presented by immunocompatible antigen-presenting cells) and T cells from the same source population that are exposed instead to a structurally distinct or irrelevant control antigen. A response to the cognate antigen that is greater, with statistical significance, than the response to the control antigen signifies antigen-specificity.

A biological sample may be obtained from a subject for determining the presence and level of an immune response to a WT1 -derived antigen peptide as described herein. A "biological sample" as used herein may be a blood sample (from which serum or plasma may be prepared), biopsy specimen, body fluids (e.g., lung lavage, ascites, mucosal washings, synovial fluid), bone marrow, lymph nodes, tissue explant, organ culture, or any other tissue or cell preparation from the subject or a biological source. Biological samples may also be obtained from the subject prior to receiving any immunogenic composition, which biological sample is useful as a control for establishing baseline (i.e., pre-immunization) data.

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 preserve the stability of the formulation until. In certain embodiments, a unit dose comprises a recombinant host cell as described herein at a dose of about 10⁷ cells/m² to about 10¹¹ cells/m². The development of suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens, including e.g., parenteral or intravenous administration or formulation.

If the subject composition is administered parenterally, the composition may also include sterile aqueous or oleaginous solution or suspension. Suitable non-toxic parenterally acceptable diluents or solvents include water, Ringer's solution, isotonic salt solution, 1,3-butanediol, ethanol, propylene glycol or polythethylene glycols in mixtures with water. Aqueous solutions or suspensions may further comprise one or more buffering agents, such as sodium acetate, sodium citrate, sodium borate or sodium tartrate. Of course, any material used in preparing any dosage unit formulation should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations. Dosage unit form, as used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit may contain a predetermined quantity of recombinant cells or active compound calculated to produce the desired therapeutic effect in association with an appropriate pharmaceutical carrier.

In general, an appropriate dosage and treatment regimen provides the active molecules or cells in an amount sufficient to provide therapeutic or prophylactic benefit. Such a response can be monitored by establishing an improved clinical outcome (e.g., more frequent remissions, complete or partial, or longer disease-free survival) in treated subjects as compared to non-treated subjects. Increases in preexisting immune responses to a tumor protein generally correlate with an improved clinical outcome. Such immune responses may generally be evaluated using standard proliferation, cytotoxicity or cytokine assays, which are routine in the art and may be performed using samples obtained from a subject before and after treatment.

EXAMPLES

EXAMPLE 1

METHODS

Mice

C57BL/6 (B6) mice were purchased from the Jackson Laboratory, OT1 mice were a gift from M. Bevan (University of Washington, Seattle, WA), and P14 TCR transgenic mice bred on the B6 background were a kind gift from Dr. Murali Krishna- Kaja. All animal research performed for this study was approved under University of Washington Institutional Animal Care and Use Committee protocol 2013-01. Generation of Retrogenic Mice

Retrogenic mice were generated as described previously³⁰'⁵¹. Briefly, bone marrow (BM) cells were isolated from the leg bones of B6 mice, and HPCs were enriched by magnetic cell sorting, using the mouse lineage cell depletion kit from Miltenyi. The HPCs were cultured in media containing IL-3 (20 ng/mL), IL-6 (50 ng/mL), stem cell factor (50 ng/mL), and FLT-3L (5 ng/mL) for 3 days. On the first 2 days after BM isolation, the purified progenitor cells were retrovirally transduced with the 3D-PYYa construct; on the third day the transduced HPCs were injected into sublethally irradiated B6 host mice. No blinding or randomization was performed for these experiments. Cell lines

The retroviral packaging line PlatE was obtained from Cell Biolabs (San Diego, CA), The OP9-K^bD^bDLl cell line was generated by transducing the OP9 cell line with a retroviral construct containing the Dll-1 gene followed by an IRES and H-2K^b (to generate OP9-K^bDLl cells), and separately transduced with H-2D^b. The OP9- K^bD^bDLl-WTl cell line was also then transduced to express murine WT1. The OP9- A2-DL1 cell line was generated by transducing OP9-K^bDLl cells with a retroviral construct encoding HLA-A2-IRES-human β2Μ. The OP9 cells and a retroviral construct containing the Dill gene followed by IRES-GFP were obtained from the lab of Juan Carlos Zuniga-Pflucker. The 58^"/_3D-PYYa cell line was generated by retrovirally tranducing a CD8a/CD8p expressing variant of the TCRa/TCRP -deficient cell line 58-/-²⁷'⁵¹ with Mig2-3D-PYYa. H9.WTl₃₇ cell line was generated by lentivirally transducing the human T cell line H9 with WTl₃₇a-IRES-GFP. The TCRap negative Jurkat76 cell line was obtained from David Kranz.

Flow Cytometric Analysis and Cell Sorting

Flow cytometric analysis data was collected using a BD Biosciences Canto I or Canto II running BD FACSDiva software, and Flow cytometric cell sorting was performed using a BD Biosciences Aria II running FACSDiva software. Data analysis was performed using FlowJo software (Tree Star, Ashland OR). The following antibody clones were used for analysis of murine cell antigens: CD4 (RM4-5), CD8a (53-6.7), TCRp (H57- 597), CD24 (Ml/69), and VplO (B21.5); and of human cell surface antigens: CD2 (RPA-2.10), Vpl7 (E17.SF3.15.13), and CD27 (M-T271). HLA-A2/WT1 and H- 2Db/WTl tetramers were generated by the Immune Monitoring Lab at the Fred

Hutchinson Cancer Research Center.

TCR Constructs and Plasmids

The murine WT1 -specific TCR 3D was isolated from the highest affinity T cell clone that could be detected from immunized B6 mice that was specific for the immunodominant H-2D^b epitope of WT1 RMFPNAPYL (WT1 i_26-i₃₄) (SEQ ID NO:82). The Mig2-3D-PYYa construct consists of a codon-optimized enhanced affinity variant of the 3D TCRa chain³⁰, followed by an IRES and the transmembrane and extracellular domains of human CD2. TCR gene expression is driven by the retroviral LTR. The Retroviral vector Mig-AttR and lentiviral vector pRRLSIN-AttR were generated by cloning the ccdb gene flanked by AttR sites (the EcoRV fragment) from the lentiviral vector pLenti (Invitrogen) into the Hpal site of the retroviral vector MigRl ¹⁰'⁵², or ASC I and Sal-I sites of pRRLSIN.cPPT.MSCV / GFP.WPRE, replacing GFP. The lentiviral vectors pRTLSIN-WTl₃₇aP and pRRLSIN-WTl₃₇a were generated from the vector pRRLSIN.cPPT.MSCV / GFP.WPRE by replacing GFP with a cassette encoding pRRLSIN-WTl₃₇a or both the TCRa and β chains of WT1₃₇ separated by a P2A element. The vector pRRLSIN.cPPT.MSCV / GFP.WPRE was a kind gift from Richard Morgan.

In Vitro T cell Differentiation

Thymocytes were isolated from B6 or OT1 mice, depleted of CD4⁺ and CD8⁺ cells by magnetic bead separation (Miltenyi Biotec), and sorted for CD81⁺ CD44⁺ CD4^" CD8^" double negative 1 and 2 (DN1/2) early progenitor thymocytes. DN1/2 cells were either directly transferred to OP9-DL1 cells, or retrovirally transduced with supernatant from PlatE cells transduced with Mig2-3D-PYYa prior to OP9-DL1 culture. Progenitor thymocytes were cultured in the presence of IL-7 (lng/ml) and Flt3L (5ng/ml) as previously described²⁷'⁵¹, and cognate antigen (peptide WT1₁₂₆.₁₃₄). For human T cell differentiation, CD34⁺ hematopoietic stem and progenitor cells (UPCs) were purified from cord blood by magnetic bead separation (Miltenyi Biotec), lentivirally transduced, and cultured on OP9-A2-DL1 cells in the presence of human cytokines SCF (20 ng/ml), TPO (20ng/ml), IL-7 (10 ng/ml) and Flt3L (5ng/ml) in the presence or absence of peptide WTl_37-45 as indicated. The de-identified cord blood samples were provided by Colleen Delaney (Fred Hutchinson Cancer Research Center, Seattle, WA) through a cord blood collection protocol at Swedish Medical Center, Swedish IR 3834S- 03/FHCRC IRB #5647, which requires informed consent be obtained from all subjects.

Generation and Screening of TCRs

RNA was isolated from In vitro cultured or retrogenic murine thymocytes or in v/Yro-differentiated human progenitors that were sorted as indicated, and RACE-ready cDNA was generated (SMARTer RACE kit, Clontech). Full-length Tcrb genes were PCR amplified using a modified RACE universal primer mix that contains a CACC at the 5'-end or a CACC-containing a murine νβΐθ or human νβ17 primer paired with a mouse or human Cpi or Cp2 specific reverse primer. The CACC was added to the 5'-end to facilitate directional cloning into the Topo vector pEntr/D-Topo (Invitrogen). Topo reactions were electroporated into Electromax DH10B Tl cells and 50,000- 500,000 colonies were recovered. The libraries were then combined and transferred into Mig-AttR (mouse) or pRRLSIN-AttR (human) by Gateway cloning (Invitrogen). 58^"/_3D-PYYa cells (H9.WTI₃₇01 cells for human libraries) were transduced with the TCRp retroviral libraries and subjected to multiple rounds of sorting based on high level WT1 -specific tetramer staining. Library-derived TCRP chains were recovered from transduced cells exhibiting the highest level of tetramer staining and analyzed by sequence analysis. Candidate TCRP chains were expressed in the relevant TCRa- expressing T cell line or co-expressed with TCRa in primary T cells and relative affinity was assessed by staining transduced cells with 2-fold serial dilutions of WT1 tetramer and fitting the MFI values for each positive population to a saturation binding curve by nonlinear regression using GraphPad Prism software.

EXAMPLE 2

AGONIST TCR SIGNALING PRIOR TO THYMIC P-SELECTION IN VITRO Agonist signals through an αβ-TCR expressed prior to β-selection in TCR transgenic mice results in the differentiation of "γδ like" DN TCRaP⁺ cells²³'²⁴. To determine if progenitor thymocytes from these mice also differentiate into DN TCRaP⁺ cells in vitro in response to cognate antigen, TCRap^" CD4^" CD8^" CD81⁺ CD44⁺

(DN1/DN2) progenitor thymocytes were sorted from ovalbumin (OVA)-specific TCR- transgenic OT1 mice and cultured on OP9-DL1 cells expressing H-2K^b and H-2D^b (OP9-K^bD^bDLl) in the absence of peptide, or with increasing concentrations of the OVA peptide SIINFEKL (SEQ ID NO:79). In the absence of peptide, DP T cells were detected at day 16, and constituted a major fraction of the cell culture by day 20, as previously reported²⁸'³¹. However, development and/or survival of DP T cells was diminished by even very low concentrations of SIINFEKL (SEQ ID NO:79) peptide (0.0001 μΜ), and DP cells were completely absent from cultures containing 0.01 μΜ or more of peptide (Figure 1 A), suggesting DP cells fail to develop or are negatively selected by strong agonist signaling in OP9-K^bD^bDLl cultures, as observed in vivo ². To evaluate for development of TCRaP⁺ DN cells as observed in TCR transgenic mice²³'²⁴, the DN population was analyzed for expression of TCRp and CD24, a molecule down-regulated as thymocytes mature ²⁵'³³. The majority of cells on day 5 displayed an immature TCRP^" CD24⁺ phenotype (Figure IB), but by day 16 most DN cells from all culture conditions expressed TCRp, with more TCRp CD24^" cells detected at the higher peptide concentrations. By day 20, -60% of all DN cells were TCRP⁺ CD24^" from cultures containing 0.01 μΜ, whereas in cultures that received no or very low concentration (0.0001 μΜ) only -20% of DN cells were TCRp⁺ CD24^" and -50% were TCRP^", and likely of NK lineage (Figures IB and 1C). Furthermore, DN TCRP⁺ cells that developed in response to high levels of peptide expressed higher levels of cell surface TCRP, consistent with a more mature phenotype (Figure 1C). High concentrations of SIINFEKL (SEQ ID NO:79), but not the positive-selecting antagonist peptide EIINFEKL (SEQ ID NO:80) (El)³², led to increased absolute numbers of DN TCRP⁺ T cells (Figure ID), indicating more avid TCR signaling enhances development of this population.

To confirm that TCRaP⁺ DN cells observed in these cultures did not develop by passing through a DP stage, pre-selection CD69^" DP T cells were sorted from the thymus of wild type B6 or OTl TCR-transgenic mice and cultured on OP9-K^bD^bDLl cells in the presence or absence of SIINFEKL (SEQ ID NO:79) peptide. Whereas B6 DP cells were not impacted by the presence of SIINFEKL (SEQ ID NO:79) peptide, OTl DP thymocytes cultured in the presence of SIINFEKL (SEQ ID NO: 79) exhibited the hallmarks of negative selection, including CD4/CD8 co-receptor down-modulation and loss of cellularity³⁴, and DN TCRP⁺ cells were not detected (Figure IE).

EXAMPLE 3

GENERATION OF AGONIST-SELECTED TCRp CHAINS IN VITRO

IN THE PRESENCE OF AN ANTIGEN-SPECIFIC TCRa CHAIN

To determine whether a transgenic TCRa chain alone could redirect some thymocytes to a DN TCRp⁺ lineage, DN1 and DN2 thymocytes (TCRap^" CD4^" CD8^" CD81⁺ CD44⁺) were sorted from B6 mice and retrovirally transduced with a vector containing the extracellular domain of human CD2 (hCD2) to track transduced cells, and the TCRa chain 3D-PYYa from a previously described mouse H-2D^b-restricted TRBV4 (VplO)/TRAV9-l TCR specific for WTl tumor antigen peptide RMFPNAPYL (SEQ ID NO:82) and affinity-enhanced by saturation mutagenesis of CDR3a³⁰'³⁵. Transduced progenitor thymocytes were cultured on OP9-K^bD^bDLl cells in the presence or absence of 1.0M RMFPNAPYL (SEQ ID NO:82) peptide for 14 days. DN cells within the untransduced hCD2 negative fraction contained <2% TCRaP⁺ cells, regardless of the presence of the WTl peptide in the culture. In contrast, the DN fraction from the hCD2⁺ transduced population contained 6.8% TCRP⁺ cells on day 16 in the absence of peptide, and TCRaP⁺ cells increased to 16.6% if 1.0 μΜ WTl peptide was added (Figure 2A). Thus, a significant population of DN TCR αβ⁺ cells developed from DN1/2 thymocytes ectopically expressing a TCRa chain from a TCR of known specificity prior to the β-selection checkpoint.

If development of the DN TCRaP⁺ cells results from ligand binding, a fraction of these cells should recognize the WTl peptide added to the cultures. However, WTl tetramer⁺ DN cells could not be readily detected, either due to lack of CD8 expression, lower TCR surface levels compared to peripheral T cells, or rarity of positive cells. Therefore, a library of the agonist- selected TCRP chains from the DN TCRP⁺ population was generated to study the breadth and specificity of agonist- selected TCRP chains. 3D-PYYa-transduced DN1/DN2 thymocytes were differentiated on OP9-

K^bD^bDLl cells in the presence of WTl antigen, non-adherent cells collected for several days up to day 21, and hCD2⁺ DN TCRp⁺ cells sorted (Figure 2B). Sorts from individual days were pooled (-50,000 cells total), RNA purified, and cDNA generated. A retroviral TCRP library composed of only PCR-amplified νβΐθ sequences, in order to retain the parental CDRl/2 domains, linked by an IRES to GFP was generated (Figure 2C and Example 1). The TCRap^" and CD8-deficient cell line 58^" that had been previously transduced to express CD8a, CD8P, and 3D-PYYa was retrovirally transduced with the TCRP library. Transduced cells were sorted first on GFP positive cells, and then sorted and expanded two more times to enrich for νβ10⁺ cells binding high levels of WTl-tetramer (Figure 2D). Sorted cells were also stained with an unrelated H-2D^b/GP33 tetramer to affirm that WTl-tetramer⁺ cells were not binding MHC in a peptide-independent manner (Figure 2D). Sorted tetramer⁺ cells were lysed, and retroviral inserts recovered by PCR. Sequences from >30 clones were analyzed, and the top four TCRp chains by frequency (VplO clone#l, VplO clone#2, VplO clone#3, and VpiO clone#4) were advanced for further study. All these clones had CDR3P sequences that shared multiple residues with the parental 3DP chain (Figure 2C), with VplO clone#l almost identical to the original 3Dp (Figures 2C and 2F). The four candidate TCRP chains were retrovirally transduced into 58^"/_3D-PYYa cells and analyzed by flow cytometry (Figure 2G). All four TCRs bound tetramer, although VpiO clone#4 was not analyzed further because it exhibited the weakest homology to the original 3D CDR3P and bound tetramer at significantly lower levels.

To assess the relative affinity of each TCRP chain for WT1 peptide/MHC, 58^_/" 3D-PYYa D-PYY for WT1 peptide/parental 3Dp or the generated candidate TCRp chains were stained with six 2-fold serial dilutions of WT1 tetramer and mean fluorescence intensity (MFI) values fit to a saturation binding curve by non-linear regression to approximate relative affinity (Figure 3 A). The apparent affinity for tetramer of two of the three candidate TCRP chain clones, when paired with 3D-PYYa, was higher than the original 3DP, with VpiO clone#l having >3 fold higher affinity (Figure 3 A). To more accurately compare tetramer staining of 3D-PYYa, tetramer staining of TCRP was compared to the original 3Dp. The polynucleotide encoding VpiO clone#l was codon-optimized such that the only sequence differences between the original 3DP and VpiO clone#l were in the CDR3 region. Both constructs were transduced into 58^_/" PYYa cells and assessed for tetramer staining. Transduced T cells expressing VpiO clone#l TCRP chain at similar levels to the parental 3DP chain exhibited enhanced tetramer staining (Figure 3B). To determine if VpiO clone#l TCRP chain conferred an increased propensity to bind H-2D^b in a peptide-independent manner, VplO clone#l transduced 58^"/_3D-PYYa D-PYY were stained with a panel of H-2D^b-tetramers containing alternative peptides (GP33 : LCMV glycoprotein; E4: Yellow fever virus 17D; MSLN: mouse Mesothelin; SQV: HIV-gag epitope).

Compared with the WTl-specific tetramer, which bound at high levels, no staining could be detected using the other peptide/H-2D^b tetramers (Figure 3C), indicating the increased affinity observed for these receptors is due to specificity for the WTl peptide and not the result of increased affinity for MHC alone.

EXAMPLE 4

DEVELOPMENT OF TCRP+ DN CELLS IN VIVO IN THYMUS Since WTl expression is readily detected in the thymus³⁶'³⁷, we examined whether a similar selection process would occur during thymic development in vivo in mice ectopically expressing the TCRa at this stage of T cell development. In this experiment, agonist-selected T cells may accumulate throughout the life of the animal, allowing a broader repertoire of TCRs reactive with this self-antigen to be assessed. Therefore, TCRa retrogenic mice were generated by transferring 3D-PYYa-transduced hematopoietic stem cells (HSCs) into irradiated recipient mice³⁸. A substantially increased population of DN TCRP⁺ cells was detected among 3D-PYYa-transduced thymocytes compared to untransduced controls (Figure 4A), similar to what was seen in vitro {see Figure 2). Furthermore, these DN TCRP⁺ thymocytes contained an increased proportion of T cells expressing the νβΐθ gene utilized by the original 3ϋβ (Figure 4A), indicating at least some DN TCRP⁺ cells might be specific for the same WTl antigen. A substantial increase in DN TCRp⁺ cells was also evident among peripheral 3D-PYYa-expressing splenocytes as compared to the untransduced population (54.5% versus 7.25%) (Figure 4A).

To determine if the DN TCRaP⁺ cells developing in the thymus of 3D-PYYa mice express self-reactive TCRs that surpass the threshold of normal thymic selection for αβ⁺ T cells, VplO-Cpl and Vpl0-Cp2 libraries were generated from DN TCRap⁺ cells (Figure 4B), and screened for TCRP chains conferring high level WTl tetramer staining when expressed in 58^"/_3D-PYYa D-PYY. After three rounds of sorting to enrich for tetramer⁺ library-transduced 58^"/_3D-PYYa D-PYY (Figure 2E), TCRp chains were recovered (Figure 4C), and three dominant clones (ThyP#l, ThyP#2, and ThyP#3) that bound WTl -tetramer when co-expressed with 3D-PYYa were further analyzed. ThyP#l showed enhanced tetramer binding compared to 3ϋβ, while ThyP#2 and ThyP#3 appeared to have similar or slightly lower affinity compared to 3ϋβ, respectively (Figure 4D). To further characterize beta chain clone ThyP#l, TCR 3D-PYYa-ThyPl chains were expressed in primary P14 CD8⁺ T cells and compared to T cells expressing 3ϋ-ΡΥΥα-3ϋβ. T cells expressing each TCR were stained with titrated concentrations of tetramer and fit to a saturation binding curve as described above for Figure 3 A. TCR 3D-PYYa-ThyP#l exhibited enhanced affinity compared to T cells expressing 3ϋ-ΡΥΥα-3ϋβ (Figure 4E), and enhanced IFNy production in response to peptide-pulsed target cells (Figures 4F-4I). Τηγβ#1 differs from the original 3ϋβ chain by only one amino acid at position 108 (Figure 4B), which is one of the two positions that differed from 3ϋβ νβΐθ clone#l isolated from the OP9-DL1 derived TCRP library screen (see Figures 2B and 2F), indicating the proline at this position in the parental mouse 3ϋβ either does not optimally contribute to peptide binding or has a negative impact on peptide binding.

To assess the in vivo safety of the highest affinity murine TCRs generated, P14 CD8⁺ T cells were transduced to express codon-optimized, P2A-linked TCR chains from constructs containing wild type 3ϋαβ or 3D-PYYa paired with 3ϋβ in vitro derived νβΐθ clone#l or thymus-derived Τ1ινβ#1. TCR-transduced T cells were injected into recipient B6 animals, which were assessed for autoimmunity, including weight loss and transgenic T cell expansion. Somewhat decreased numbers were observed for T cells transduced with the enhanced affinity νβ at the late time point (day 126), perhaps reflecting a moderate increase in peripheral tolerance and/or deletion³⁹. Otherwise, no toxicities or differences were observed between these WT1 -specific T cells of differing affinity (Figures 10A and 10B).

To examine the full TCRβ repertoire of agonist- selected DN TCRo + cells, DN TCRo ⁺ thymocytes from 3D-PYYa retrogenic mice were analyzed by high-throughput sequencing and repertoire analysis⁴⁰ (Figure 5). A total of 141,113 unique TCRβ chain sequences were identified from 1,486,250 reads representing 288,000 sorted cells. The clonality was 0.05, with the most frequent clone representing 0.04% of the total, indicating this lineage-diverted population is highly diverse with limited clonal expansion. Only a small percentage of the TCRβ repertoire expressed νβΐθ (TRBV4), and substantial expansion of TRBV17 cells was observed compared to total B6 thymocytes, with 9 of the top 10 most common TCRβ sequences utilizing this νβ-gene. However, screening of diverse RACE PCR-based libraries revealed only νβΐθ TCRP chains were reactive with WTl (data not shown). Therefore, TCRs incorporating νβ17 chains are more likely specific for thymus-expressed antigens other than WTl .

Thus, restricting TCRP chain libraries to the parental TCRP gene should not only increase the frequency of TCRs that bind the target antigen but also minimize isolation of TCRs with alternative specificities.

EXAMPLE 5

IN VITRO GENERATION OF HUMAN AGONIST-SELECTED TCRp CHAINS

Agonist-mediated γδ-like lineage differentiations of human progenitors in OP9- DL1 cultures were examined. Cord blood-derived CD34⁺ hematopoietic progenitor cells (HPCs) were selected and transduced to express the P2A-linked TCRaP chains of a high affinity HLA-A2-restricted human TCR specific for WTl peptide VLDFAPPGA (SEQ ID NO: 81), which corresponds to WTl amino acids 37-45 (WTl₃₇-45, also referred to herein as WTl₃₇aP) and cultured for three weeks on OP9-DL1 cells expressing HLA-A2 (OP9-A2-DL1) in the presence or absence of WTl peptide VLDFAPPGA (SEQ ID NO:81) (Figure 6). Previous studies have shown that the majority of human γδ lineage cells developing in thymic cultures of CD34⁺ HPCs on OP9-DL1 cells express low levels of CD4, have heterogeneous CD8 expression, and are mostly CD27⁺ by day 28 of culture, and that human HPCs transduced to express a TCRaP similarly express CD27 following in vitro differentiation with cognate peptide⁴¹, whereas αβ lineage cells are mostly CD4⁺ CD8⁺ CD27^" at this timepoint⁴². Therefore, the expression of these markers by human HPCs transduced with WTl₃₇aP and cultured on OP9-A2-DL1 cells was assessed after 22 days. A substantial population of CD3⁺ CD4^" CD27⁺ cells heterogeneous for CD 8 (-75% CD 8^") that were mostly absent from untransduced controls were found (Figure 6). Furthermore, the addition of cognate WTl peptide VLDFAPPGA (SEQ ID NO: 81) to the cultures resulted in an increased proportion of these cells (from 29.6% to 40.2%), indicating an enrichment for agonist-selected T cells. Human CD34⁺ HPCs were then transduced with just the TCRa chain of TCR WTI37 (WTI370 , and cultured with OP9-A2-DL1 cells in the presence of cognate WT1 peptide VLDFAPPGA (SEQ ID NO:81). Cultures were analyzed on day 30 for CD3, TCRP and CD27 expression (Figures 7A and 7B). Compared to untransduced controls, WTl₃₇a expressing progenitors contained a greater proportion of CD3⁺ CD27⁺ cells, which included a unique population of TCRP⁺ CD8^" cells within the transduced CD3⁺ CD27⁺ subset. Based on the above data, cultures were sorted at various time points between day 20 and 30 for CD3⁺ TCRp⁺ CD27⁺ DN cells, which includes CD3⁺ TCRp⁺ CD8⁺ cells expressing high levels of CD27 as the populations most likely to contain agonist- selected cells, and TCRP libraries generated from the sorted populations.

Since the parental TCRp chain utilizes Vpl7 (TRBV19), Vpl7-restricted Cpl and Cp2 libraries were generated, as well as separate more diverse RACE PCR-derived libraries. The TCRP libraries were transduced into the CD8^" T cell line H9 previously transduced to express WTl₃₇a (H9.WTl₃₇a), and Vpi7 library -transduced cells were sorted for Vpi7 expression (Figure 7C). Library -transduced populations were enriched for WT1 tetramer-reactive cells by two rounds of WT1 tetramer-based sorting and subsequent expansion. Cells exhibiting the highest level of WT1 tetramer staining were then isolated, and library-derived TCRP chain genes recovered. WT1 tetramer⁺ cells could be detected in Vpi7 library-transduced cells at a frequency of -1% prior to enrichment by tetramer sorting (Figure 7C); and a range of tetramer reactivity was observed post-enrichment, suggestive of an enriched pool of TCRP chains with a range of affinities for HLA-A2AVT1 (Figure 7C). A total of 26 TCRp chains were detected multiple times, and these were co-expressed in the CD8^" TCRaP-deficient Jurkat76 cell line containing WTl₃₇a. Out of 26 total, 11 TCRP chains bound WT1 tetramer independent of CD8 when paired with WTl₃₇a (Vpl7#2-Vpl7#10, Vpl5#l and Vpl2#l) (Figure 8A). This group contained three Vp families (Vpl2, Vpl5, and Vpl7), four Jp families (Jpl-2, Jpl-5, Jp2-6, and Jp2-7), and CDR3 regions ranging from 13-16 amino acids in length (Figure 8B). Vpi7 was utilized by 9 of the 11 TCRP clones, and four of the Vpi7⁺ TCRP clones exhibiting the highest tetramer binding and one Vpi5+ TCRP clone were selected for further study (Figure 9A). Codon-optimized TCRaP constructs (TCRP-P2A-WTl₃₇a) contained in lentiviral constructs were synthesized for each TCRP chain, and expressed in Jurkat76 cells. WTl-specific tetramer staining relative to TCR surface expression for each the five candidate TCRP clones was compared to the wildtype WTl₃₇o (Figure 9B). The relative affinity of each TCR was determined by calculating the apparent K_D based on binding of decreasing concentrations of WTl tetramer to T cells expressing each of the selected TCRaP pairs (Figure 9C and 9E). The TCRs were transduced into primary human CD8⁺ T cells and sorted WTl tetramer⁺CD8⁺ T cells were expanded. Each Vpl7⁺ TCR expressed similar levels of the transgenic νβ17 chain, and all transduced populations expressed similar levels of CD8 (Figure 11 A). Four of the five selected TCRP chain clones (Vpl7#2, Vpl7#3, Vpl7#7, and Vpl5#l) exhibited enhanced tetramer binding compared to the wildtype TCRP chain when paired with WTl₃₇a. Of these four TCRP chain clones that showed enhanced tetramer binding, two (νβ17#2 and νβ17#3), as well as a third TCRP chain clone (νβ15#1), also induced enhanced IFNy production as compared to the parent TCR (νβ17 wt) when target cells were pulsed at lower levels of peptide (Figure 9D), and none of the TCRs produced IFNy in the absence of peptide (Figure 1 IB).

Two TCRβ chains with non-parental νβ chain usage (νβ12#1 and νβ15#1) were isolated from RACE libraries that bound tetramer with very high affinity when paired with WTl₃₇a. Notably, three of the five enhanced affinity human TCRβ chains isolated in this study have a longer CDR3 than the parental TCRβ chain (see Figures 8B and 9A).

Taken together, these data demonstrate enhanced affinity TCRs specific for human tumor/self-antigens were identified and isolated. Modifications directed to CDR3 are less likely to influence interactions with MHC independent of peptide⁴⁵ and minimizes the need for improving affinity by additional CDRl/2 mutations, which can change the docking geometry of the TCR on MHC. The TCRs identified by the methods described herein will be useful for treating WTl -associated disease.

The various embodiments described herein can be combined to provide further embodiments. All of the patents, patent application publications, patent applications, and non-patent publications referred to in this specification and/or listed in the

Application Data Sheet, including but not limited to U.S. Patent Application No. 62/558,550, are incorporated herein by reference in their entirety. In general, terms used in the following claims should not be construed as limited to 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. REFERENCES

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Claims

CLAIMS What is claimed is:

1. A binding protein, comprising:

(a) a T cell receptor (TCR) a-chain variable (V_a) domain and a TCR β-chain variable (V ) having a CDR3 amino acid sequence set forth in any one of SEQ ID NOS: l-l l;

(b) a TCR V_a domain having a CDR3 amino acid sequence of SEQ ID NO: 13, and a TCR νβ domain having a CDR3 amino acid sequence set forth in any one of SEQ ID NOS: 1-11; or

(c) a TCR V_a domain and a TCR ν_β domain comprising a CDR3 having a mutated SEQ ID NO: 12 amino acid sequence, wherein D/N/P region amino acids of SEQ ID NO: 12 comprise up to five amino acid substitutions, up to a contiguous three amino acid additions, or a combination thereof, provided that the CDR3 does not comprise the amino acid sequence of SEQ ID NO: 12;

wherein the binding protein is capable of specifically binding to a WT1 peptide:HLA complex on a cell surface independent of CD8 or in the absence of CD8.

2. The binding protein of claim 1, wherein the binding protein is capable of specifically binding to a VLDFAPPGA (SEQ ID NO:81):human leukocyte antigen (HLA) complex with a K_D of less than or equal to about 10^"8 M.

3. The binding protein of claim 2, wherein the HLA comprises

HLA-A*201.

4. The binding protein according to any one of claims 1-3, wherein the V_a domain comprises an amino acid sequence that is at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:26.

5. The binding protein according to any one of claims 1-4, wherein the

V_a domain comprises or consists of the amino acid sequence of SEQ ID NO:26.

6. The binding protein according to any one of claims 1-5, wherein the ν_β domain comprises an amino acid sequence that is at least:

(a) 98% identical to the amino acid sequence of SEQ ID NO: 14;

(b) 97%) identical to the amino acid sequence of SEQ ID NO: 15;

(c) 95% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 16-19;

(d) 93.5%) identical to the amino acid sequence of SEQ ID NO:20 or 21 ; or

(e) 91.5%) identical to the amino acid sequence of SEQ ID NO:22.

7. The binding protein according to any one of claims 1-5, wherein the ν_β domain comprises an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:23 or 24.

8. The binding protein of claim 6 or 7, wherein the ν_β domain comprises no change in the amino acid sequence of CDRl as compared to the CDRl present in any one of SEQ ID NOS: 14-25.

9. The binding protein of any one of claims 6-8, wherein the ν_β domain comprises no change in amino acid sequence of CDR2 as compared to the CDR2 present in any one of SEQ ID NOS: 14-25.

10. The binding protein according to any one of claims 1-9, wherein the νβ domain comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOS: 14-16, and 24.

1 1. The binding protein according to any one of claims 1-9, wherein the νβ domain comprises or consists of the amino acid sequence set forth in any one of SEQ ID NO: 17-19, and 23.

12. The binding protein according to any one of claims 1-9, wherein the νβ domain comprises or consists of the amino acid sequence as set forth in any one of SEQ ID NO:20-22.

13. The binding protein according to any one of claims 1-12, wherein the binding protein comprises a TCR a-chain constant domain having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:42.

14. The binding protein according to any one of claims 1-13, wherein the binding protein comprises a TCR β-chain constant domain having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO:40 or 41.

15. The binding protein according to any one of claims 1-14, wherein the binding protein comprises a TCR a-chain having an amino acid sequence that is at least about 90% identical to the amino acid sequence of SEQ ID NO:39.

16. The binding protein according to any one of claims 1-15, wherein the binding protein comprises a TCR a-chain comprising or consisting of the amino acid sequence of SEQ ID NO:39.

17. The binding protein according to any one of claims 1-16, wherein the binding protein comprises a TCR β-chain comprising or consisting of the amino acid sequence as set forth in any one of SEQ ID NO S: 27-37.

18. The binding protein according to any one of claims 1-17, wherein the binding protein comprises a T cell receptor (TCR).

19. The binding protein according to any one of claims 1-12, wherein the binding protein comprises an antigen-binding fragment of a TCR or a chimeric antigen receptor.

20. The binding protein according to claim 19, wherein the antigen-binding fragment of the TCR comprises a single chain TCR (scTCR).

21. The binding protein of claim 19 or 20, wherein the binding protein is a chimeric antigen receptor.

22. A composition comprising a binding protein according to any one of claims 1-21 and a pharmaceutically acceptable carrier, diluent, or excipient.

23. An isolated polynucleotide encoding the binding protein according to any one of claims 1-21.

24. The polynucleotide according to claim 23, wherein the polynucleotide encoding the binding protein is codon optimized for a host cell of interest.

25. An expression vector, comprising the polynucleotide of claim 23 or 24 operably linked to an expression control sequence.

26. The expression vector according to claim 25, wherein the vector is capable of delivering the polynucleotide to a host cell.

27. The expression vector according to claim 26, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.

28. The expression vector according to claim 27, wherein 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.

29. The expression vector according to claim 28, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

30. The expression vector according to any one of claims 25-29, wherein the vector is a viral vector.

31. The expression vector according to claim 30, wherein the viral vector is a lentiviral vector or a γ-retroviral vector.

32. A host cell, comprising the polynucleotide according to claim 23 or 24 or the expression vector according to any one of claims 25-31, wherein the recombinant host cell expresses on its cell surface the binding protein encoded by the polynucleotide.

33. The host cell according to claim 32, wherein the V_a domain is encoded by a polynucleotide comprising at least 75% identity to SEQ ID NO: 100.

34. The host cell according to claim 32 or 33, wherein V_a domain is encoded by a polynucleotide comprising at least 75% identity to SEQ ID NO: 113.

35. The host cell according to any one of claims 32-34, wherein the νβ domain is encoded by a polynucleotide comprising at least 75% identity to SEQ ID NO: 99, provided that the polynucleotide encodes a CDR3 that does not have the amino acid sequence of SEQ ID NO: 12.

36. The host cell according to any one of claims 32-35, wherein the νβ domain is encoded by a polynucleotide that comprises or consists of the

polynucleotide sequence of any one of SEQ ID NOS:88-98.

37. The host cell according to any one of claims 32-36, wherein the a-chain is encoded by a polynucleotide comprising at least 75% identity to SEQ ID NO: 132.

38. The host cell according to any one of claims 32-37, wherein the a-chain is encoded by a polynucleotide that comprises or consists of the polynucleotide sequence of SEQ ID NO: 145.

39. The host cell according to any one of claims 32-38, wherein the β-chain is encoded by a polynucleotide comprising at least 75% identity to any one of SEQ ID NOS: 120-130, provided that the polynucleotide encodes a CDR3 that does not have the amino acid sequence of SEQ ID NO: 12.

40. The host cell according to any one of claims 32-39, wherein the β-chain is encoded by a polynucleotide that comprises or consists of the polynucleotide sequence of any one of SEQ ID NOS: 133-143.

41. The host cell according to any one of claims 32-40, wherein the polynucleotide comprises a nucleotide sequence encoding a self-cleaving peptide disposed between the polynucleotide sequence encoding the a-chain and the polynucleotide sequence encoding the β-chain.

42. The host cell according to claim 41, wherein the encoded self-cleaving peptide comprises or consists of the amino acid sequence as set forth in any one of SEQ ID NOS:83-86.

43. The host cell according to claim 41 or 42, wherein the polynucleotide encoding the self-cleaving peptide comprises or consists of the polynucleotide sequence as set forth in any one of SEQ ID NOS:87 and 146-148.

44. The host cell according to any one of claims 32-43, wherein the a-chain, self-cleaving peptide, and β-chain are encoded by a polynucleotide comprising at least about 75% identity to any one of SEQ ID NOS:55-65, provided that the polynucleotide encoding the β-chain encodes a CDR3 that does not have the amino acid sequence of SEQ ID NO: 12.

45. The host cell of claim 44, wherein the encoded a-chain, self-cleaving peptide, and β-chain comprises or consists of the amino acid sequence as set forth in any one of SEQ ID NOS:43-53.

46. The host cell according to any one of claims 32-45, wherein the TCR a-chain, self-cleaving peptide, and TCR β-chain are encoded by a polynucleotide comprising or consisting of the polynucleotide sequence of any one of SEQ ID

NOS:67-77.

47. The host cell according to any one of claims 32-46, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.

48. The host cell according to claim 47, wherein 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.

49. The host cell according to claim 47, wherein the immune system cell is a

T cell.

50. The host cell according to claim 49, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

51. The host cell according to claim 49 or 50, wherein the binding protein expressed by the T cell is capable of more efficiently associating with a CD3 protein as compared to an endogenous TCR.

52. The host cell according to any one of claims 49-51, wherein the binding protein has higher surface expression on a T cell as compared to an endogenous TCR.

53. A composition, comprising the host cell of any one of claims 32-52 and a pharmaceutically acceptable carrier, diluent, or excipient.

54. A method for treating a hyperproliferative disorder, comprising administering to human subject in need thereof a composition comprising the binding protein specific for human Wilms tumor protein 1 (WT1) according to any one of claims 1-21, or the composition of claim 22.

55. The method according to claim 54, wherein the hyperproliferative disorder is a hematological malignancy or a solid cancer.

56. The method according to claim 55, wherein the hematological malignancy is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or multiple myeloma (MM).

57. The method according to claim 55, wherein the solid cancer is selected from biliary cancer, bladder cancer, bone and soft tissue carcinoma, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonal cancer, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumor, head and neck squamous cell carcinoma, hepatic cancer, lung cancer, mesothelioma, malignant melanoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, renal cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ-cell tumor, urothelial cancer, uterine sarcoma, or uterine cancer.

58. The method according to any one of claims 54-57, wherein the binding protein is capable of promoting an antigen-specific T cell response against a human WT1 in a class I HLA-restricted manner.

59. The method according to claim 58, wherein the class I HLA-restricted response is transporter-associated with antigen processing (TAP)-independent.

60. The method according to claim 58 or 59, 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.

61. The method according to claim 60, wherein the CTL response is directed against a WTl-overexpressing cell.

62. An adoptive immunotherapy method for treating a condition

characterized by WTl overexpression in cells of a subject having a hyperproliferative disorder, comprising administering to the subject an effective amount of the host cell according to any one of claims 32-52, or the composition of claim 53.

63. The method according to claim 62, wherein the host cell is modified ex vivo.

64. The method according to claim 62 or 63, wherein the host cell is an allogeneic cell, a syngeneic cell, or an autologous cell to the subject.

65. The method according to any one of claims 62-64, wherein the host cell is a hematopoietic progenitor cell or a human immune system cell.

66. The method according to claim 65, wherein 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.

67. The method according to claim 66, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

68. The method according to any one of claims 62-67, wherein the hyperproliferative disorder is a hematological malignancy or a solid cancer.

69. The method according to claim 68, wherein the hematological malignancy is selected from acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or multiple myeloma (MM).

70. The method according to claim 68, wherein the solid cancer is selected from biliary cancer, bladder cancer, bone and soft tissue carcinoma, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonal cancer, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumor, head and neck squamous cell carcinoma, hepatic cancer, lung cancer, mesothelioma, malignant melanoma, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, renal cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ-cell tumor, urothelial cancer, uterine sarcoma, or uterine cancer.

71. The method according to any one of claims 62-70, wherein the host cell is administered parenterally.

72. The method according to any one of claims 62-71, wherein the method comprises administering a plurality of doses of the host cell to the subject.

73. The method according to claim 72, wherein the plurality of doses are administered at intervals between administrations of about two to about four weeks.

74. The method according to any one of claims 62-73, wherein the host cell is administered to the subject at a dose of about 10⁷ cells/m² to about 10¹¹ cells/m².

75. The method according to any one of claims 62-74, wherein the method further comprises administering a cytokine.

76. The method according to claim 75, wherein the cytokine is IL-2, IL-15, IL-21 or any combination thereof.

77. The method according to claim 76, wherein the cytokine is IL-2 and is administered concurrently or sequentially with the host cell.

78. The method according to claim 77, wherein the cytokine is administered sequentially, provided that the subject was administered the host cell at least three or four times before cytokine administration.

79. The method according to any one of claims 76-78, wherein the cytokine is IL-2 and is administered subcutaneously.

80. The method according to any one of claims 62-79, wherein the subject is further receiving immunosuppressive therapy.

81. The method according to claim 80, wherein the immunosuppressive therapy is selected from calcineurin inhibitors, corticosteroids, microtubule inhibitors, low dose of a mycophenolic acid prodrug, or any combination thereof.

82. The method according to any one of claims 62-81, wherein the subject has received a non-myeloablative or a myeloablative hematopoietic cell transplant.

83. The method according to claim 82, wherein the subject is administered the host cell at least three months after the non-myeloablative hematopoietic cell transplant.

84. The method according to claim 82, wherein the subject is administered the host cell at least two months after the myeloablative hematopoietic cell transplant.

85. A unit dose form comprising the host cell according to any one of claims 32-52 or the composition of claim 53.

86. The unit dose form according to claim 85, wherein the host cell is at a dose of about 10⁷ cells/m² to about 10¹¹ cells/m².