WT-1-specific T cell immunotherapy

Abstract

The present disclosure provides binding proteins specific for human Wilms tumor protein 1 (WT-1) epitopes, as well as host cells expressing the binding proteins. Polynucleotides encoding the binding proteins and vectors containing the polynucleotides are also provided. Methods and uses related to the compositions disclosed herein for treating diseases or disorders associated with WT-1 expression, such as various cancers, are also provided.

Description

[Technical Field]

[0001] The sequence listing associated with this application is provided in text format in lieu of print and is incorporated herein by reference. The name of the text file containing the sequence listing is 360056_473WO_SEQUENCE_LISTING.txt. The text file is 109 KB, was created on August 17, 2020, and has been submitted electronically via EFS-Web. [Background technology]

[0002] T cell receptor (TCR) gene therapy is an emerging treatment designed to overcome obstacles associated with traditional T cell adoptive immunotherapy, such as the extensive time and effort required to isolate, characterize, and expand tumor antigen-specific T cells (Schmitt et al., Hum. Gene Ther. 20:1240, 2009). Another challenge is that because the majority of identified tumor antigens targeted by T cell immunotherapy are overexpressed self-proteins, high-affinity T cells specific for these antigens are generally lost during thymic selection and are rare or absent in the peripheral repertoire.

[0003] Strategies have been considered to enhance the affinity of TCRs intended for use in TCR gene therapy (e.g., Udyavar et al., J. Immunol. 182:4439, 2009; Zhao et al., J. Immunol. 179:5845, 2007; Richman and Kranz, Biomol. Eng. 24:361, 2007).

[0004] A prerequisite for targeted T cell therapy that achieves maximum clinical efficacy while minimizing associated immunotoxicity involves identifying disease-associated antigens that are highly expressed and therefore present in malignant cell compartments but not significantly expressed in normal tissues. For example, several acute myeloid leukemia (AML)-associated antigens have been described, and Wilms' tumor protein 1 (WT-1) has been shown to be expressed at significantly higher levels in the leukemia stem cell (LSC) compartment of most AML patients than in physiological hematopoietic stem cells (HSC). WT-1 has also been targeted in clinical trials involving both adoptive T cell transfer and peptide vaccination (see, e.g., U.S. Patent Nos. 7,342,092; 7,608,685; and 7,622,119). In addition, WT-1 expression has been reported to be a marker of minimal residual disease, as elevated transcript levels in patients with AML in morphologic remission predicted overt clinical relapse (Inoue et al., Blood, 84:3071, 1994; Ogawa et al., Blood, 101:1698, 2003).

[0005] Because WT-1 is an intracellular (usually nuclear) protein, immunotherapy targeting WT-1 requires the use of WT-1-specific CD8 receptors that recognize peptides presented on the cell surface by MHC class I molecules. + Cellular methods have been used to generate cytotoxic T lymphocyte (CTL) responses. To induce CTL responses, intracellular proteins are typically degraded by proteasomes or endosomes / lysosomes, and the resulting peptide fragments bind to MHC class I or class II molecules. These peptide-MHC complexes are presented on the cell surface, where they are bound by T cells via peptide-MHC-TCR interactions. Peptides derived from the WT-1 protein could theoretically induce human leukocyte antigen (HLA)-restricted cytotoxic CD8 T cells, which could kill tumor cells. +WT-1 can be used in human vaccines to induce T cell responses. However, because WT-1 is a self-protein, immunization may only induce responses by T cells with low TCR affinity. In addition, antibodies against WT-1 can be detected in patients with hematopoietic malignancies and solid tumors, indicating that WT-1 may be a highly immunogenic antigen (Gaiger et al., Clin. Cancer Res. 7 (Suppl. 3):761, 2001). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 7,342,092 [Patent Document 2] U.S. Patent No. 7,608,685 [Patent Document 3] U.S. Patent No. 7,622,119 [Non-patent literature]

[0007] [Non-Patent Document 1] Schmitt et al., Hum.Gene Ther.20:1240, 2009 [Non-patent document 2] Udyavar et al., J.Immunol.182:4439, 2009 [Non-patent document 3] Zhao et al., J. Immunol. 179:5845, 2007 [Non-patent document 4] Richman and Kranz, Biomol.Eng.24:361, 2007 [Non-patent document 5] Inoue et al., Blood, 84:3071, 1994 [Non-patent document 6] Ogawa et al., Blood, 101:1698, 2003 [Non-Patent Document 7] Gaiger et al., Clin. Cancer Res. 7(Suppl. 3):761, 2001 Summary of the Invention [Problem to be solved by the invention]

[0008] There is clearly a need for alternative gene therapies for use as highly specific WT-1-targeted immunotherapies directed against a variety of cancers, including leukemias and tumors. The embodiments disclosed herein address this need and provide other related advantages. [Means for solving the problem]

[0009] (Abstract) According to certain embodiments, the present disclosure provides a binding protein capable of binding to the RMFPNAPYL (SEQ ID NO: 94): human leukocyte antigen (HLA) complex, comprising: (a) a T cell receptor (TCR) alpha chain variable (Vα) domain comprising a CDR3 amino acid sequence (CDR3α) according to any one of SEQ ID NOs: 19, 22, 25, or 28, or a variant thereof, and optionally comprising or consisting of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to the amino acid sequence set forth in any one of SEQ ID NOs: 5-8 or 13-16; The present invention provides a binding protein comprising a TCR Vα domain, or (c) the TCR Vα domain of (a) and the TCR Vβ domain of (b), optionally comprising or consisting of a Vβ domain or a mutant thereof and an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1 to 4 or 9 to 12.

[0010] In certain embodiments, the binding protein has an IFNγ-producing pEC of 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 or greater. 50 and RMFPNAPYL (SEQ ID NO: 94):human leukocyte antigen (HLA) complex (e.g., immune cells (e.g., T cells, NK-T cells, or NK cells) containing the binding protein have an IFNγ-producing pEC binding protein of 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, or greater for SEQ ID NO: 94:HLA). 50 ).

[0011] In certain embodiments, the HLA is HLA-A * Including 0201.

[0012] In certain embodiments, the binding protein is a human binding protein, a humanized binding protein, or a chimeric binding protein.

[0013] In any of the embodiments disclosed herein, the binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO: 94):human leukocyte antigen (HLA) complex, wherein the binding protein comprises a TCR Vβ domain and a TCR Vα domain, and (i) the Vβ domain comprises or consists of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to the amino acid sequence of any one of SEQ ID NOs: 5-8 or 13-16, and / or (ii) the Vα domain comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 1-4 or 9-12.

[0014] In certain embodiments, the encoded binding protein is a TCR, a CAR, or an scTCR.

[0015] Also provided herein are modified immune cells comprising a heterologous polynucleotide encoding a binding protein disclosed herein. In certain embodiments, the immune cells comprise T cells, NK cells, NK-T cells, or any combination thereof. In certain embodiments, immune cells expressing a binding protein disclosed herein are activated in the presence of a peptide according to SEQ ID NO:94 (e.g., contained within a peptide:HLA complex). In certain embodiments, immune cells expressing a binding protein disclosed herein are capable of killing target cells expressing a SEQ ID NO:94:HLA complex.

[0016] Also provided are isolated polynucleotides encoding binding proteins according to the present disclosure. In certain embodiments, the encoded binding proteins comprise, consist of, or are at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% identical) to (i) the amino acid sequence set forth in any one of SEQ ID NOs: 19, 22, 25, 28, 31, 34, 37, or 40; (ii) the amino acid sequence set forth in any one of SEQ ID NOs: 17, 20, 23, 26, 29, 32, 35, or 38; (iii) the amino acid sequence set forth in any one of SEQ ID NOs: 18, 21, 24, 27, 30, 33, 36, or 39; or (iv) the amino acid sequence set forth in any one of SEQ ID NOs: 1-4 or 9-12. , 98%, 99% or 100%) identity to the amino acid sequence set forth in any one of SEQ ID NOs: 5 to 8 or 13 to 16; (vi) an amino acid sequence comprising, consisting of or having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 46 to 53; (vii) an amino acid sequence comprising, consisting of or having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 54 to 61, or (viii) any combination of (i) to (vii).

[0017] In certain embodiments, the polynucleotide comprises or consists of a polynucleotide having at least 75% (e.g., 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the nucleotide sequence set forth in any one of SEQ ID NOs: 62-81. In certain embodiments, the polynucleotide is codon-optimized for expression in a host cell, such as an immune cell (e.g., a human T cell).

[0018] Also provided are vectors containing the polynucleotides described herein, as well as compositions comprising the modified cells, binding proteins, polynucleotides and / or vectors disclosed herein.

[0019] Also provided is a method for treating a subject having a disease or disorder associated with WT-1 expression or activity, the method comprising administering to the subject an effective amount of a modified immune cell, binding protein, polynucleotide, or vector disclosed herein.

[0020] In certain embodiments, the disease or disorder is a hematological malignancy or solid cancer. For example, the hematological malignancy to be treated can be acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia (AML, including refractory AML and relapsed AML, including acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelocytic leukemia, acute myelomonocytic leukemia (e.g., with or without eosinophilia), acute monocytic leukemia, acute erythroleukemia, and acute megakaryoblastic leukemia), chronic myelogenous leukemia (CML), chronic myelocytic leukemia, chronic eosinophilic leukemia (CEL), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or multiple myeloma (MM, including refractory MM and relapsed MM). Exemplary solid cancers to be treated include bile duct cancer, bladder cancer, bone and soft tissue cancer, brain tumor, breast cancer, breast carcinoma, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal carcinoma, desmoid tumor, embryonal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, glioblastoma, melanoma, diffuse peritoneal mesothelioma, malignant pleural mesothelioma, glioma, astrocytoma, gynecological tumors, head and neck squamous cell carcinoma, liver cancer , hepatocellular carcinoma, lung cancer, non-small cell lung cancer, malignant melanoma, osteosarcoma, ovarian cancer (e.g., epithelial ovarian cancer, ovarian carcinoma), fallopian tube cancer, endometrial cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytoma, primary thyroid cancer, prostate cancer, kidney cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, osteogenic sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, uterine carcinosarcoma, or uterine cancer.

[0021] These and other aspects and embodiments of the present disclosure will be further understood with reference to the following Detailed Description and the accompanying drawings. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the Application Data Sheet are hereby incorporated by reference in their entirety, as if each were individually incorporated. Aspects and embodiments of the present disclosure can be modified, if necessary, to utilize concepts from the various patents, applications, and publications to yield still further embodiments. [Brief explanation of the drawings]

[0022] [Figure 1] Figure 1 shows the identification of WT-1-specific T cell lines. Briefly, WT-1-specific polyclonal T cell lines (CD8+) obtained from four healthy donors were cultured with peptide-pulsed autologous dendritic cells, stained with CD8-independent (CD8i) WT-1 peptide / HLA-A tetramers, and sorted for high tetramer-staining cells. 24 WT-1-specific clonotypes were identified. TCR repertoire analysis was performed by sequencing. The fold enrichment of each clonotype relative to the unsorted population (y-axis) and surface expression of CD3 by each clonotype (x-axis) are shown. [Figure 2A]

[0023] Figure 1 shows the expression of exemplary WT-1-specific binding proteins in transduced host cells. Ten WT-1-specific TCRs derived from the 24 clonotypes shown in Figure 1 were used to transduce TCRα / TCRβ-deficient J76 Jurkat T cells. Binding to WT-1:HLA-A tetramers and CD3 expression by the transduced cells (shown in light gray) were compared with cells transduced with a WT-1-specific reference TCR (shown in dark gray) having an α chain according to SEQ ID NO: 82 and a β chain according to SEQ ID NO: 83, as described by Schmitt et al. (Nat. Biotechnol. 35:1188, 2017). [Figure 2B]

[0023] Figure 1 shows the expression of exemplary WT-1-specific binding proteins in transduced host cells. Ten WT-1-specific TCRs derived from the 24 clonotypes shown in Figure 1 were used to transduce TCRα / TCRβ-deficient J76 Jurkat T cells. Binding to WT-1:HLA-A tetramers and CD3 expression by the transduced cells (shown in light gray) were compared with cells transduced with a WT-1-specific reference TCR (shown in dark gray) having an α chain according to SEQ ID NO: 82 and a β chain according to SEQ ID NO: 83, as described by Schmitt et al. (Nat. Biotechnol. 35:1188, 2017). [Figure 2C]

[0023] Figure 1 shows the expression of exemplary WT-1-specific binding proteins in transduced host cells. Ten WT-1-specific TCRs derived from the 24 clonotypes shown in Figure 1 were used to transduce TCRα / TCRβ-deficient J76 Jurkat T cells. Binding to WT-1:HLA-A tetramers and CD3 expression by the transduced cells (shown in light gray) were compared with cells transduced with a WT-1-specific reference TCR (shown in dark gray) having an α chain according to SEQ ID NO: 82 and a β chain according to SEQ ID NO: 83, as described by Schmitt et al. (Nat. Biotechnol. 35:1188, 2017). [Figure 3A] Figure 1 shows that the binding proteins of the present disclosure are functional in transduced host cells. (A) Jurkat cells expressing the Nur77-dtTomato reporter (which reports antigen-specific signaling in human T cells; see Ahsouri and Weiss, J Immunol 198(2):657-668 (2017)) were transduced with a WT-1-specific TCR and incubated with the indicated concentrations of WT-1 peptide-loaded APCs for 24 hours. [Figure 3B] Figure 1 shows that binding proteins of the disclosure are functional in transduced host cells.(B) EC50 values ​​for the Nur77 activating peptide with the indicated TCRs. [Figure 4A]Figure 1 shows further functional characterization of exemplary binding proteins of the present disclosure: (A) Binding to peptide / MHC tetramers by CD8+ T cells transduced to express the indicated WT-1-specific TCRs. [Figure 4B] (B) IFNγ production (determined by flow cytometry) by antigen-specific CD8+ T cells expanded in 4-hour co-culture with T2 target cells pulsed with titrated concentrations of peptide. The percentage of IFN-γ+ T cells is shown. [Figure 4C] Figure 10 shows further functional characterization of exemplary binding proteins of the present disclosure. (C) The EC50 for IFNγ peptide was determined for each TCR by fitting the percentage of IFNγ-producing cells to a dose-response curve by linear regression. [Figure 5A] Figures 1A-1B show lysis of tumor cells by T cells expressing exemplary binding proteins of the present disclosure (TCR DL6 or DL10) compared to lysis by T cells expressing a reference TCR (alpha chain according to SEQ ID NO: 82; beta chain according to SEQ ID NO: 83). (A) Lysis of MDA-MB-468 cells, an HLA-A2-transduced tumor cell line. Both (A) and (B) show results from a 4-hour assay in which target cells were loaded with 51Cr and TCR-transduced T cell-mediated killing was calculated by measuring Cr release in response to decreasing doses of effector T cells (E:T; x-axis) against tumor cell targets. [Figure 5B] (A) and (B) show lysis of tumor cells by T cells expressing exemplary binding proteins of the present disclosure (TCR DL6 or DL10) compared to lysis by T cells expressing a reference TCR (alpha chain according to SEQ ID NO: 82; beta chain according to SEQ ID NO: 83). (B) Lysis of Panc-1 cells, an HLA-A2+ tumor cell line, over time. Both (A) and (B) show results from a 4-hour assay in which target cells were loaded with 51Cr and TCR-transduced T cell-mediated killing was calculated by measuring Cr release in response to decreasing doses of effector T cells (E:T; x-axis) against tumor cell targets. [Figure 6] FIG. 1 shows the production of IFN-γ by an exemplary TCR DL10 of the disclosure compared to a reference TCR (α chain according to SEQ ID NO: 82; β chain according to SEQ ID NO: 83) in the presence of peptide-pulsed T2 target cells. [Figure 7]

[0023] Figure 1 shows the reduction in proliferation of Panc-1 tumor cells in the presence of T cells expressing an exemplary TCR DL10 of the present disclosure compared to tumor cells alone or T cells expressing a reference TCR (alpha chain according to SEQ ID NO: 82; beta chain according to SEQ ID NO: 83). Cell proliferation / survival was monitored over 7 days in an IncuCyte assay. Error bars indicate the standard error of the mean. DETAILED DESCRIPTION OF THE INVENTION

[0023] In certain embodiments, the present disclosure provides binding proteins (e.g., TCRs, CARs, scTCRs) having specificity for WT-1 peptide antigens associated with major histocompatibility complex (MHC) (e.g., human leukocyte antigens (HLA)), polynucleotides encoding the binding proteins, vectors encoding and / or expressing the binding proteins, engineered immune cells, and related compositions. The WT-1-specific compositions disclosed herein are useful, for example, in adoptive immunotherapy to treat cancer. By way of background, the majority of tumor targets for T cell-based immunotherapy are autoantigens because tumors arise from previously normal tissues. For example, such tumor-associated antigens (TAAs) may be expressed at high levels in cancer cells but not or minimally expressed in other cells. During T cell development in the thymus, T cells that bind weakly to self-antigens are allowed to persist in the thymus and may undergo further development to increase their specificity for foreign invaders, whereas T cells that bind strongly to self-antigens are eliminated by the immune system because they trigger undesirable autoimmune responses. Thus, T cells are sorted according to their relative ability to bind antigens and prime the immune system to respond to foreign invaders (i.e., recognize non-self antigens) while simultaneously preventing autoimmune responses (i.e., recognize self-antigens). This tolerance mechanism limits naturally occurring T cells that can recognize tumor (self) antigens with high affinity, thereby eliminating T cells that effectively eliminate tumor cells. As a result, it is difficult to isolate T cells with high-affinity TCRs specific for tumor antigens because such cells are essentially eliminated by the immune system.

[0024] An advantage of the present disclosure is that it provides a binding protein specific to a WT-1 peptide, where the binding protein binds to WT-1 (SEQ ID NO: 94):HLA (e.g., HLA-A *0201) complex. In certain embodiments, the binding protein is capable of more efficiently associating with CD3 protein compared to endogenous TCRs capable of binding to WT-1 in a CD8-independent manner or in the absence of CD8, or is capable of inducing a signal to host cells to express the binding protein and produce cytokines, or any combination thereof. In certain embodiments, the binding protein has an IFNγ-producing pEC of 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 or greater. 50 and / or the binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO: 94):HLA complex with a higher level of IFNγ producing pEC compared to a TCR comprising an α chain amino acid sequence of SEQ ID NO: 82 and a β chain amino acid sequence of SEQ ID NO: 83. 50 It is capable of binding to the RMFPNAPYL (SEQ ID NO: 94):HLA complex with

[0025] In certain embodiments, the compositions and methods described herein have therapeutic utility for treating diseases and conditions associated with WT-1 expression (e.g., WT-1 expression detectable at a statistically significant level above that detectable in normal or disease-free cells). Such diseases include a variety of diseases and disorders, such as hematological malignancies and solid cancers. Non-limiting examples of these compositions and methods and related uses are described herein, including in vitro, ex vivo, and in vivo stimulation of WT-1 antigen-specific T cell responses, such as by using recombinant immune cells, such as T cells encoding and / or expressing a heterologous binding protein specific for a WT-1 peptide (e.g., RMFPNAPYL; SEQ ID NO: 94).

[0026] Before setting forth this disclosure in more detail, it may be helpful to an understanding thereof to set forth definitions of certain terms used herein. Additional definitions are set forth throughout this disclosure.

[0027] In this description, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range shall be understood to include any integer value within the recited range, and, where appropriate, fractions thereof (such as tenths and hundredths of integers). Also, unless otherwise indicated, any numerical range recited herein with respect to any physical characteristic, such as polymer subunits, size, or thickness, shall also be understood to include any integer within the recited range. Unless otherwise indicated, the term "about," as used herein, means ±20% relative to the stated range, value, or structure. As used herein, the terms "a" and "an" shall be understood to refer to "one or more" of the recited components. The use of disjunctions (e.g., "or") shall be understood to mean one, both, or any combination of the alternatives. As used herein, the terms "include," "having," and "comprise" are used interchangeably and it is intended that these terms and variations thereof be considered open-ended.

[0028] In addition, it is to be understood that individual compounds or groups of compounds resulting from the various combinations of structures and substituents described herein are disclosed by this application to the same extent as if each compound or group of compounds were individually indicated. Thus, selection of a particular structure or particular substituents is within the scope of this disclosure.

[0029] The term "consisting essentially of" is not synonymous with "comprising" and refers to specified materials or steps or materials or steps that do not materially affect the essential characteristics of the claimed invention. For example, a protein domain, region, or module (e.g., a binding domain, hinge region, linker module) or protein (which may have one or more domains, regions, or modules) "consists essentially of" a particular amino acid sequence if the amino acid sequence of the domain, region, module, or protein includes extensions, deletions, mutations, or combinations thereof (e.g., amino acids at the amino or carboxy termini, or between domains) that, in combination, contribute 20% or less (e.g., 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%) of the length of the domain, region, module, or protein and do not substantially affect (i.e., do not reduce activity by more than 50%, such as by 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., target binding affinity of a binding protein).

[0030] As used herein, the term "host" refers to a cell (e.g., a T cell) or microorganism that is targeted for genetic modification with a heterologous or exogenous nucleic acid molecule to produce a polypeptide of interest (e.g., an anti-WT-1 TCR with enhanced affinity). In certain embodiments, the host cell may already possess or be modified to contain other genetic modifications that confer desired properties (e.g., incorporation of a detectable marker; deletion, alteration, or truncation of an endogenous TCR; increased expression of a costimulatory factor), whether or not related to the biosynthesis of the heterologous or exogenous protein. In certain embodiments, the host cell is a human hematopoietic progenitor cell transduced with a heterologous or exogenous nucleic acid molecule encoding a TCR α chain specific for a WT-1 antigenic peptide.

[0031] As used herein, "immune system cell" refers to any cell of the immune system that is derived from hematopoietic stem cells in the bone marrow, which give rise to two major lineages: myeloid progenitor cells (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitor cells (which give rise to lymphoid cells such as T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4 + T cells, CD8 + T cells, CD4 - CD8 - These include double-negative T cells, γδ T cells, natural killer cells, and dendritic cells. Macrophages and dendritic cells may be referred to as "antigen-presenting cells" or "APCs," which are specialized cells that can activate T cells when major histocompatibility complex (MHC) receptors on the surface of the APC interact with TCRs on the surface of the T cell. In certain embodiments, the immune system cells are human immune system cells.

[0032] As used herein, a "hematopoietic progenitor cell" is a cell that can be derived from a hematopoietic stem cell or embryonic tissue and is capable of further differentiation into a mature cell type (e.g., an immune system cell). Exemplary hematopoietic progenitor cells are CD24 Lo Lin - CD117 + These include cells with the phenotype or cells found within the thymus (termed thymic progenitor cells).

[0033] "Major histocompatibility complex" (MHC) refers to glycoproteins that deliver peptide antigens to the cell surface. MHC class I molecules are heterodimers with a transmembrane α chain (with three α domains) that is non-covalently associated with β2-microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, α and β, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides from the cytosol to the cell surface, where the peptide:MHC complex binds to CD8 +MHC class II molecules deliver peptides from the vasculature to the cell surface, where the peptide:MHC complex is recognized by CD4 + Recognized by T cells. Human MHC is called human leukocyte antigen (HLA).

[0034] "T cells" are immune system cells that mature in the thymus and produce T cell receptors (TCRs). T cells include naive T cells (those that have not been exposed to antigen; T CM compared to normal T cells, they have increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO), and memory T cells (T M ) (antigen-experienced, long-lived) and effector cells (antigen-experienced, cytotoxic). M are central memory T cells (T CM : Compared to naive T cells, they have increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA), stem cell-like memory T cells, and effector memory T cells (T EM : Naive T cells or T CM They can be further divided into effector T cell (T) subsets (which have decreased expression of CD62L, CCR7, CD28, and CD45RA, and increased expression of CD127, compared to T cells). E ) is T CM Compared with antigen-experienced CD8 + Refers to cytotoxic T lymphocytes.

[0035] "T cell receptor" (TCR) 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," 3rd ed., Current Biology Publications, pp. 4-33, 1997) that is capable of specifically binding to an antigenic peptide bound to an MHC receptor. TCRs may be found on the surface of cells or in a soluble form and are generally composed of a heterodimer having an α chain and a β chain (also known as TCRα and TCRβ, respectively) or a γ chain and a δ chain (also known as TCRγ and TCRδ, respectively). Like other immunoglobulins (e.g., antibodies), the extracellular portions of TCR chains (e.g., α chain, β chain) contain two immunoglobulin domains: a variable domain (e.g., α chain variable domain or Vα, β chain variable domain or Vβ; typically, amino acids 1-116 according to Kabat numbering; Kabat et al., "Sequences of Proteins of Immunological Interest," U.S. Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.) at the N-terminus, and one constant domain adjacent to the cell membrane (e.g., α chain constant domain or Cα, typically, amino acids 117-259 according to Kabat; β chain constant domain or Cβ, typically, amino acids 117-295 according to Kabat). Again, like other immunoglobulins, the variable domains contain complementarity-determining regions (CDRs) separated by framework regions (FRs) (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. USA 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003).In certain embodiments, the TCR is found on the surface of a T cell (or T lymphocyte) and is associated with the CD3 complex. The source of the TCR used in this disclosure can be from a variety of animal species, such as human, mouse, rat, rabbit, or other mammals.

[0036] The term "variable region" or "variable domain" refers to a domain of an immunoglobulin superfamily binding protein (e.g., a TCR) (e.g., a TCRα chain or TCRβ chain (or the γ and δ chains for a γδ TCR)) that is involved in binding of the immunoglobulin superfamily binding protein to an antigen. In the case of a TCR, the variable domains of the α and β chains (Vα and Vβ, respectively) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs. The Vα domain is encoded by two separate DNA segments, a V (variable) gene segment and a J (joining) gene segment (VJ), and the Vβ domain is encoded by three separate DNA segments, a V (variable) gene segment, a D (diversity) gene segment, and a J (joining) gene segment (VDJ). A single Vα or Vβ domain, or a functional fragment or portion thereof, may be sufficient to confer antigen-binding specificity. Furthermore, Vα or Vβ domains derived from a TCR that binds to an antigen can be used to screen libraries of complementary Vα or Vβ domains, respectively, to isolate TCRs that bind to a specific antigen.

[0037] The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to sequences of amino acids within an immunoglobulin (e.g., TCR) variable region that confer antigen specificity and / or binding affinity and are separated from each other by framework regions. Generally, within each TCR α chain variable region, there are three CDRs (αCDR1, αCDR2, αCDR3), and within each TCR β chain variable region, there are three CDRs (βCDR1, βCDR2, βCDR3). Within a TCR, CDR3 is considered to be the primary CDR responsible for recognition of processed antigen. Typically, CDR1 and CDR2 primarily interact with MHC.

[0038] While CDR1 and CDR2 are encoded within the V (variable) gene segment of the TCR variable region coding sequence, CDR3 is encoded by a region spanning the V (variable) segment and the J (joining) segment for Vα, or a region spanning the V (variable) segment, the D (diversity) segment, and the J (joining) segment for Vβ. Thus, if the identity of the V (variable) gene segment of Vα or Vβ is known, the sequences of their corresponding CDR1 and CDR2 can be deduced. Compared to CDR1 and CDR2, CDR3 is typically significantly more diverse due to the addition and loss of nucleotides during the recombination process.

[0039] TCR variable domain sequences can be aligned according to a numbering scheme (e.g., IMGT, Kabat, Chothia, Enhanced Chothia, Contact, and Aho) to annotate equivalent residue positions and allow for comparison of different molecules, e.g., using the ANARCI software tool (2016, Bioinformatics 15:298-300). The numbering scheme provides a standardized definition of framework regions and CDRs within a TCR variable domain. Thus, it is understood that a CDR derived from a TCR Vα or Vβ region or domain can have a particular sequence according to a particular numbering scheme, or a slightly shorter, extended, or shifted (e.g., partially overlapping) sequence according to a different numbering scheme. In certain embodiments disclosed herein, CDRs are determined using the IMGT numbering scheme, e.g., using IMGT V-Quest (imgt.org / IMGTindex / V-QUEST.php).

[0040] In the art, "CD3" is known as a six-chain multiprotein complex (see Abbas and Lichtman, 2003; Janeway et al., pp. 172 and 178, 1999). In mammals, the complex comprises a homodimer of the CD3γ chain, the CD3δ chain, two CD3ε chains, and the CD3ζ chain. The CD3γ, CD3δ, and CD3ε chains are closely related to cell surface proteins of the immunoglobulin superfamily, which contain a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a feature that is thought to enable these chains to associate with positively charged regions of T cell receptor chains. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, whereas each CD3ζ chain contains three conserved motifs. Without wishing to be bound by theory, ITAMs are believed to be important for the signaling ability of the TCR complex. The CD3 used in this disclosure may be derived from a variety of animal species, including human, mouse, rat, or other mammals.

[0041] As used herein, the term "TCR complex" refers to a complex formed by the association of CD3 with a TCR. For example, a TCR complex may be composed of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRα chain, and a TCRβ chain. Alternatively, a TCR complex may be composed of a CD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRγ chain, and a TCRδ chain.

[0042] As used herein, "a component of a TCR complex" refers to a TCR chain (i.e., TCRα, TCRβ, TCRγ, or TCRδ), a CD3 chain (i.e., CD3γ, CD3δ, CD3ε, or CD3ζ), or a complex formed by two or more TCR chains or CD3 chains (e.g., a complex of TCRα and TCRβ, a complex of TCRγ and TCRδ, a complex of CD3ε and CD3δ, a complex of CD3γ and CD3ε, or a TCR subcomplex of TCRα, TCRβ, CD3γ, CD3δ, and two CD3ε chains).

[0043] As used herein, the term "CD8 co-receptor" or "CD8" refers to CD8, a cell surface glycoprotein that can be expressed by T cells as a homodimer comprising two CD8 α chains or as a heterodimer comprising an α chain and a β chain. The CD8 co-receptor is a receptor for cytotoxic T cells (CD8 + ) and its function by signaling through cytoplasmic tyrosine phosphorylation pathways (Gao and Jakobsen, Immunol. Today 21:630-636, 2000; Cole and Gao, Cell. Mol. Immunol. 1:81-88, 2004). In particular, without wishing to be bound by theory, it is believed that the CD8 coreceptor binds to MHC-I protein complexes expressed on the surface of antigen-expressing cells, and that this binding in the context of TCR:antigen-MHC binding triggers or contributes to T cell signaling pathways that result in an immune response against the antigen-expressing cells (e.g., cytokine transcription and expression, calcium secretion, cytolytic activity, etc.). In humans, eight different CD8 beta chain isoforms are known ("M1" to "M8"; see UniProtKB Identification Numbers: P10966-1, 2, 3, 4, 6, 7, 8, and 9); of these, isoforms 1, 2, 4, and 5 are thought to be naturally associated with the cell membrane, whereas isoforms 3, 6, 7, and 8 are thought to be associated with the extracellular domain or secreted. Three CD8 alpha chain isoforms are also known in humans (see UniProtKB Identification Numbers P01732-1, 2, and 3).

[0044] "CD4" refers to the immunoglobulin coreceptor glycoprotein (see Campbell and Reece, Biology 909 (Benjamin Cummings, 6th ed., 2002); UniProtKB P01730) that aids in the interaction of the TCR with antigen-presenting cells. CD4 is found on the surface of immune cells, such as helper T cells, monocytes, macrophages, and dendritic cells, and typically contains four immunoglobulin domains expressed on the cell surface: D1 (containing an Ig-like V-type domain), D2, D3, and D4 (containing Ig-like C2-type domains 1, 2, and 3, respectively). During antigen presentation, CD4 is recruited along with the TCR complex to bind to distinct regions of the MHCII molecule (CD4 binds to MHCII β2, whereas the TCR complex binds to MHCII α1 / β1). Without wishing to be bound by theory, it is believed that proximity to the TCR complex allows CD4-associated kinase molecules to phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) present on the cytoplasmic domain of CD3. This activity is thought to amplify the signal generated by the activated TCR to result in multiple types of helper T cells.

[0045] As used herein, a "binding domain" (also referred to as a "binding region" or "binding moiety") refers to a peptide, oligopeptide, polypeptide, or protein that possesses the ability to specifically but noncovalently associate with, integrate with, or complex with a target (e.g., WT-1 or a WT-1 peptide:MHC complex). Binding domains include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biomolecule, molecular complex (i.e., a complex comprising two or more biomolecules), or other target of interest. Exemplary binding domains include single-chain immunoglobulin variable regions (e.g., scTCRs, scFvs), receptor extracellular domains, ligands (e.g., cytokines, chemokines), or synthetic polypeptides selected for their specific ability to bind to a biomolecule, molecular complex, or other target of interest.

[0046] As used herein, "specifically binds to" or "specific for" refers to the binding of a binding protein (e.g., a TCR receptor) or binding domain (or a fusion protein thereof) to a target molecule without significantly associating or associating with any other molecule or component in a sample. 5 M -1 (The on-rate [k on ], the off-speed [k off ] or higher affinity or K a (i.e., the equilibrium association constant of a particular binding interaction, in units of 1 / M). A binding protein or binding domain (or fusion protein thereof) can be classified as a "high affinity" binding protein or binding domain (or fusion protein thereof) or a "low affinity" binding protein or binding domain (or fusion protein thereof). A "high affinity" binding protein or binding domain is one that has at least 10 affinity. 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 Or at least 10 13 M -1 K a A "low affinity" binding protein or domain refers to a binding protein or domain having a binding affinity of 10 7 M -1 Below, 10 6 M -1 Below, 10 5 M -1 The following K a Alternatively, affinity refers to the equilibrium dissociation constant (K) for a particular binding interaction, in units of M.d ) (e.g., 10 -5 M~10 -13 M).

[0047] In certain embodiments, a receptor or binding domain may have "enhanced affinity," which refers to a selected or engineered receptor or binding domain that binds to a target antigen stronger than the wild-type (or parent) binding domain. For example, enhanced affinity may refer to a higher K for the target antigen than the wild-type binding domain. a (equilibrium association constant), which is the K d Less than K d (dissociation constant), which is the k off Off-rate (k off ), or a combination thereof. In certain embodiments, the affinity-enhanced TCR may be codon-optimized to enhance expression in certain host cells, such as T cells (Scholten et al., Clin. Immunol. 119:135, 2006).

[0048] The term "functional avidity" refers to the biological measure or activation threshold for the response of immune cells (e.g., T cells, NK cells, NK-T cells) to a given concentration of ligand in vitro, where biological measures can include cytokine production (e.g., IFNγ production, IL-2 production, etc.), cytotoxic activity, activation, and proliferation. For example, T cells that respond biologically (immunologically) to low antigen doses in vitro by producing cytokines, being cytotoxic, expressing activation markers, or proliferating are considered to have high functional avidity, whereas T cells with lower functional avidity require higher amounts of antigen to elicit the same immune response as T cells with higher avidity. It is understood that functional avidity is different from affinity and avidity. Affinity refers to the strength of any given binding between a binding protein and its antigen / ligand. Some binding proteins are multivalent and bind multiple antigens, in which case the overall strength of binding is the avidity.

[0049] There are numerous correlations between functional avidity and the efficacy of an immune response, and some ex vivo studies have shown that significantly different T cell functions (e.g., proliferation, cytokine production, etc.) can be elicited at different thresholds (see, e.g., Betts et al., J. Immunol. 172:6407, 2004; Langenkamp et al., Eur. J. Immunol. 32:2046, 2002). Factors that can influence functional avidity include (a) the affinity of the TCR for the pMHC complex, i.e., the strength of the interaction between the TCR and pMHC (Cawthon et al., J. Immunol. 167:2577, 2001), (b) the expression levels of the TCR and CD4 or CD8 co-receptors, and (c) the distribution and composition of signaling molecules (Viola and Lanzavecchia, Science 273:104, 1996), as well as the expression levels of molecules that attenuate T cell function and TCR signaling.

[0050] The concentration of antigen required to induce a half-maximal response between the baseline value and the maximum response after a specified exposure time is called the "50% effective concentration" or "EC 50 " EC 50 Values ​​are generally presented as molar quantities (moles per liter), but can also be expressed as: -log 10 (EC 50 ) is often converted to a logarithmic value (see, for example, Figure 4(C)). For example, EC 50 However, 1 μM (10 -6 M), log 10 (EC 50 ) value is -6. Another value that can be used is EC 50 The negative logarithm of (-log 10 (EC 50 )) defined as pEC 50 In the above example, EC equals 1 μM. 50 pEC 50 In certain embodiments, the functional avidity of a binding protein of the present disclosure is a measure of its ability to promote IFNγ production by immune cells (e.g., T cells, NK-T cells, NK cells), which may be measured using assays known in the art and / or described herein. A "high functional avidity" TCR or binding domain thereof has a functional avidity of at least 10 -4 M, at least about 10 -5 M or at least about 10 -6 M's EC 50 It refers to a TCR or a binding domain thereof having the following structure:

[0051] In some embodiments, pEC 50 is used to describe the amount of peptide antigen required for 50% of immune cells to express the activation marker Nur77.

[0052] A variety of assays are known for identifying binding domains of the present disclosure that specifically bind to a particular target, as well as for determining the affinity of a binding domain or fusion protein, including Western blot, ELISA, analytical ultracentrifugation, spectroscopy, and surface plasmon resonance (Biacore®) analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents), and peptide:MHC tetramer staining.

[0053] In certain embodiments, the term "immunoglobulin superfamily WT-1-specific binding protein" or "WT-1-specific binding protein" refers to a protein or polypeptide of the present disclosure that specifically binds to WT-1 or a peptide thereof, optionally complexed with an HLA molecule. The term "WT-1-binding domain" or "WT-1-binding fragment" refers to a domain or portion of a WT-1-specific binding protein that is responsible for specific binding to WT-1. A WT-1-specific binding domain alone (i.e., without any other portion of the WT-1-specific binding protein) may be soluble. Exemplary WT-1-specific binding domains include those derived from WT-1-specific TCRs that may be found within TCRs or scTCRs (e.g., single-chain αβTCR proteins such as Vα-L-Vβ, Vβ-L-Vα, Vα-Cα-L-Vα, or Vα-L-Vβ-Cβ (where Vα and Vβ are the variable domains of TCRα and TCRβ, respectively; Cα and Cβ are the constant domains of TCRα and TCRβ, respectively; and L is a linker)), and WT-1-specific binding domains derived from scFv fragments described herein, which may be derived from anti-WT-1 TCRs or anti-WT-1 antibodies.

[0054] The terms "WT-1 antigen" and "WT-1 peptide antigen" refer to a portion of the WT-1 protein, either natural or synthetic, ranging in length from about 7 to about 15 amino acids, that can form a complex with an MHC (e.g., HLA) molecule, and that can bind to a TCR specific for the WT-1 peptide:MHC (e.g., HLA) complex. The principles of antigen presentation, including antigen processing by antigen-presenting cells (APCs) (e.g., dendritic cells, macrophages, lymphocytes, or other cell types) and presentation by the APCs to T cells via major histocompatibility complex (MHC)-restricted presentation, between immunocompatible APCs (e.g., those that share at least one allelic form of an MHC gene involved in antigen presentation) and T cells, are well established (see, e.g., Murphy, Janeway's Immunobiology (8th ed.) 2011 Garland Science, NY, Chapters 6, 9, and 16). For example, processed antigenic peptides derived from the cytosol (e.g., tumor antigens, intracellular pathogens) are generally about 7 to about 11 amino acids in length and associate with class I MHC molecules, whereas peptides processed in the vasculature (e.g., bacterial, viral) vary in length from about 10 to about 25 amino acids and associate with class II MHC molecules. Because WT-1 is an internal host protein, WT-1 antigenic peptides are presented in the context of class I MHC. In a specific embodiment, WT-1 peptides are presented in the context of human class I HLA (and more specifically, the allele HLA-A). * 201) and RMFPNAPYL (SEQ ID NO: 94).

[0055] "Linker" refers to an amino acid sequence that can connect two proteins, polypeptides, peptides, domains, regions, or motifs and provide a spacer function compatible with the interaction of two binding subdomains such that the resulting polypeptide retains specific binding affinity for a target molecule (e.g., scTCR) or signaling activity (e.g., TCR complex). In certain embodiments, the linker is composed of about 2 to about 35 amino acids, for example, or about 4 to about 20 amino acids, or about 8 to about 15 amino acids, or about 15 to about 25 amino acids.

[0056] As used herein, a "fusion protein" refers to a protein having, within a single chain, at least two distinct domains or motifs that are not naturally found together in a protein. A polynucleotide encoding a fusion protein may be constructed using PCR, recombinantly engineered, etc., or such a fusion protein may be synthetic. A fusion protein may further contain other components, such as a tag, linker, or transduction marker. In certain embodiments, a fusion protein expressed or produced by a host cell (e.g., an immune cell, such as a T cell) is localized to the cell surface, where the fusion protein is anchored to the cell membrane (e.g., via a transmembrane component or domain) and comprises an extracellular component (e.g., capable of associating with an MHC molecule) and an intracellular component (e.g., containing a signaling domain or moiety, an effector domain or moiety, a costimulatory domain or moiety, or a combination thereof).

[0057] "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, for example, between a binding domain and an adjacent constant domain, or between a TCR chain and an adjacent self-cleaving peptide. Junction amino acids can arise from the design of a fusion protein (e.g., amino acid residues resulting from the use of restriction enzyme sites during construction of a nucleic acid molecule encoding the fusion protein).

[0058] A "modified domain" or "modified protein" refers to a motif, region, domain, peptide, polypeptide, or protein with at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%) sequence identity to a wild-type motif, region, domain, peptide, polypeptide, or protein (e.g., a wild-type TCR alpha chain, TCR beta chain, TCR alpha constant domain, TCR beta constant domain).

[0059] As used herein, "nucleic acid" or "nucleic acid molecule" or "polynucleotide" refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, such as fragments generated by polymerase chain reaction (PCR) or by in vitro translation, and fragments generated by any of ligation, cleavage, endonuclease action, or exonuclease action. In certain embodiments, polynucleotides of the present disclosure are generated by PCR. Polynucleotides can be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., α-enantiomer forms of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have modified or replaced 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 phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoroanilothioates, phosphoranilidates, phosphoramidates, etc. A polynucleotide may be single-stranded or double-stranded.

[0060] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if the material 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 coexisting materials in the natural system is isolated. Such a nucleic acid may be part of a vector and / or such a nucleic acid or polypeptide may be part of a composition (e.g., a cell lysate), but such a vector or composition may still be isolated, in the sense that it is not part of the natural environment for the nucleic acid or polypeptide. The term "gene" refers to the segment of DNA involved in producing a polypeptide chain, and includes the regions preceding and following the coding region, the "leader and trailer," as well as intervening sequences (introns) between individual coding segments (exons).

[0061] Any host cell, binding protein, polynucleotide, or vector of the present disclosure may be "separated / isolated."

[0062] As used herein, the terms "recombinant," "engineered," and "modified" refer to a cell, microorganism, nucleic acid molecule, or vector that has been altered by the introduction of an exogenous nucleic acid molecule, or to a cell or microorganism that has been altered so that expression of an endogenous nucleic acid molecule or endogenous gene is regulated, deregulated, or constitutive, where such an alteration or modification may be introduced by genetic engineering. Genetic alterations may include, for example, the addition, deletion, or substitution of one or more protein- or enzyme-encoding nucleic acid molecules (which may include expression control elements such as promoters) or other nucleic acid molecules, or modifications that introduce other functional disruptions or additions to the genetic material of a cell. Exemplary alterations include modifications within coding regions or functional fragments thereof of heterologous or homologous polypeptides derived from a reference or parent molecule.

[0063] As used herein, "mutation" refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule compared to a reference or wild-type nucleic acid molecule or a reference or wild-type polypeptide molecule, respectively. Mutations can result in several different types of sequence changes, including substitutions, insertions, or deletions of nucleotide(s) or amino acid(s). In certain embodiments, the mutation is a substitution of one or three codons or amino acids, a deletion of one to about five codons or amino acids, or a combination thereof.

[0064] In the art, a "conservative substitution" is recognized as a substitution of one amino acid for another amino acid with similar properties. Exemplary conservative substitutions are well known in the art (see, for example, WO97 / 09433, p. 10; Lehninger, "Biochemistry," 2nd ed., Worth Publishers, Inc. NY, NY, pp. 71-77, 1975; Lewin, "Genes IV," Oxford University Press, NY and Cell Press, Cambridge, MA, p. 8, 1990). Various criteria known to those skilled in the art indicate whether an amino acid substituted at a particular position in a peptide or polypeptide is conservative (or similar). For example, a similar amino acid substitution 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 classes: 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 difficult to classify, shares properties with amino acids with aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine). Under certain circumstances, substitution of glutamine for glutamic acid or asparagine for aspartic acid is considered to be similar substitutions 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 of a polypeptide and its alternative conserved amino acid sequences to the sequence of a second polypeptide (e.g., using the GENEWORKS, Align, BLAST algorithms or other algorithms described herein and practiced in the art).

[0065] In certain embodiments, proteins (e.g., binding proteins) according to the present disclosure comprise a variant sequence compared to a reference sequence (e.g., a variant TCR CDR compared to a reference TCR CDR disclosed herein). In certain embodiments, the variant proteins, peptides, polypeptides, and amino acid sequences of the present disclosure may comprise one or more conservative substitutions compared to the reference amino acid sequence. Variants of the polynucleotides and polypeptides of the present disclosure are also contemplated. A variant nucleic acid molecule or polypeptide is at least 70%, 75%, 80%, 85%, or 90% identical, and preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to a specified or reference polynucleotide or polypeptide described herein, respectively, or, for polynucleotides, hybridizes to a reference polynucleotide under stringent hybridization conditions of 0.015 M sodium chloride, 0.0015 M sodium citrate at about 65-68° C., or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at about 42° C. Variant nucleic acid molecules retain the ability to encode a binding protein or binding domain thereof having a functionality described herein, such as specific binding to a target molecule. For further details and explanations regarding stringency of hybridization reactions, see Ausubel, FM (1995), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.Furthermore, one skilled in the art can also follow the instructions given in the manuals Boehringer Mannheim GmbH (1993) "The DIG System Users Guide for Filter Hybridization", Boehringer Mannheim GmbH, Mannheim, Germany and in Liebl, W., Ehrmann, M., Ludwig, W. and Schleifer, KH (1991) International Journal of Systematic Bacteriology 41:255-260 on how to identify DNA sequences by hybridization.

[0066] A variant may also refer to a fragment of a defined or reference sequence (e.g., a portion resulting from truncation, cleavage, etc.), and a fragment may be of any length less than the length of the defined or reference sequence. As used herein, a "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide that comprises only a domain, portion, or fragment of a parent or reference compound, wherein the polypeptide or encoded polypeptide retains at least 50% of the activity associated with the domain, portion, or fragment of the parent or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% of the activity of the parent polypeptide, or provides a biological benefit (e.g., effector function). A "functional portion" or "functional fragment" of a polypeptide of the present disclosure or an encoded polypeptide has "similar binding" or "similar activity" if the functional portion or fragment exhibits no more than a 50% reduction in functionality (preferably no more than a 20% or 10% or less log difference in affinity compared to the parent or reference) compared to a parent or reference polypeptide in a selected assay, such as an assay for measuring binding affinity or measuring effector function (e.g., cytokine release). In certain embodiments, a functional portion refers to a "signaling portion" of an effector molecule, effector domain, costimulatory molecule, or costimulatory domain.

[0067] In certain embodiments, a variant binding protein or portion (e.g., binding domain) or fragment thereof may comprise one or more amino acid substitutions relative to a parent or reference binding protein or domain, where the one or more amino acid substitutions remove, alter, or attenuate a potentially undesirable trait or characteristic present in the parent or reference binding domain or domain, such as a potentially immunogenic amino acid sequence or an amino acid sequence that may result in undesired glycosylation sites, undesired deamidation sites, undesired oxidation sites, undesired isomerization sites, or reduced thermodynamic stability, or that may result in mispairing or misfolding within the binding protein (e.g., adjacent unpaired cysteine ​​residues). Amino acid sequences, patterns, and motifs that may result in undesirable traits or characteristics are known (see, e.g., Seeliger et al., mAbs, 7(3):505-515 (2015)).

[0068] In certain embodiments, amino acid substitutions include substitutions that eliminate somatic mutations, such as reversion to germline-expressed amino acids. For example, in certain embodiments, variants of a reference CDR amino acid sequence or TCR variable domain sequence or TCR constant region sequence include substitutions that eliminate or attenuate potential undesirable features or characteristics. It is understood that such variants are selected so as not to impair or substantially impair a desired function (e.g., binding specificity and / or binding affinity for the WT-1 antigen:HLA complex).

[0069] The term "construct" refers to any polynucleotide containing a recombinant nucleic acid. A construct may be present in a vector (e.g., bacterial vector, viral vector) or may be integrated into a genome. A "vector" is a nucleic acid molecule capable of carrying another nucleic acid. A vector may be, for example, a plasmid, cosmid, virus, RNA vector, or a linear or circular DNA or RNA molecule, which may contain chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acid. Exemplary vectors are vectors capable of autonomous replication (episomal vectors) or vectors capable of expression of nucleic acids to which they are linked (expression vectors).

[0070] Viral vectors include negative-strand RNA viruses such as retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles virus and Sendai virus), positive-strand RNA viruses such as picornaviruses and alphaviruses, as well as adenoviruses, double-stranded DNA viruses including herpesviruses (e.g., herpes simplex virus type 1 and herpes simplex virus type 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia virus, fowlpox virus, and canarypox virus). Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses include avian leukosis sarcoma viruses, mammalian type C viruses, mammalian type B viruses, mammalian type D viruses, the HTLV-BLV complex, lentiviruses, and spumaviruses (Coffin, JM, "Retroviridae: The viruses and their replication," in Fundamental Virology, 3rd ed., BN Fields et al., eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0071] As used herein, "lentiviral vector" refers to an HIV-based lentiviral vector for gene delivery, which may be integrative or non-integrative, may have a relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are typically generated after transient transfection of three or more plasmids (packaging plasmid, envelope plasmid, and transfer plasmid) into producer cells. Similar to HIV, lentiviral vectors also enter target cells through the interaction of viral surface glycoproteins with receptors on the cell surface. Upon entry, viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.

[0072] The term "operably linked" 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 to 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 in close association with each other so that the function of one does not affect the other.

[0073] As used herein, "expression vector" refers to a DNA construct containing a nucleic acid molecule operably linked to suitable control sequences capable of effecting 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 ribosomal binding sites, and sequences that control the termination of transcription and translation. A 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 in some cases, may be integrated into the genome itself. As used herein, "plasmid," "expression plasmid," "virus," and "vector" are often used interchangeably.

[0074] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene. The process includes both transcription and translation.

[0075] The term "introduced," in the context of inserting a nucleic acid sequence into a cell, means "transfection" or "transformation" or "transduction," and includes reference to the incorporation of a nucleic acid sequence into a eukaryotic or prokaryotic cell, where the nucleic acid molecule may be integrated into the genome of the cell (e.g., chromosomal, plasmid, plastid, or mitochondrial DNA), converted into a self-replicating replicon, or transiently expressed (e.g., transfected mRNA).

[0076] As used herein, a "heterologous" nucleic acid molecule, "heterologous" construct, or "heterologous" sequence, or an "exogenous" nucleic acid molecule, "exogenous" construct, or "exogenous" sequence, refers to a nucleic acid molecule or portion of a nucleic acid molecule that is not native to the host cell, but may be homologous to a nucleic acid molecule or portion of a nucleic acid molecule derived from the host cell. The source of a heterologous nucleic acid molecule, heterologous construct, or heterologous sequence, or an exogenous nucleic acid molecule, exogenous construct, or exogenous sequence, may be from a different genus or species. In certain embodiments, a heterologous nucleic acid molecule or exogenous nucleic acid molecule is added to a host cell or host genome (i.e., is not endogenous or native) by, for example, conjugation, transformation, transfection, electroporation, etc., where the added molecule may be integrated into the host genome, may exist as extrachromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), or may exist in multiple copies. Additionally, "heterologous" refers to a non-native enzyme, protein, or other activity encoded by an exogenous nucleic acid molecule introduced into a host cell, even if the host cell encodes a homologous protein or activity. Furthermore, a cell containing a "modified" polynucleotide or "modified" binding protein or a "heterologous" polynucleotide or "heterologous" binding protein includes the progeny of that cell, whether the progeny are themselves transduced, transfected, or otherwise manipulated or altered.

[0077] As described herein, more than one heterologous or exogenous nucleic acid molecule may be introduced into a host cell as separate nucleic acid molecules, as individually regulated multiple genes, as a polycistronic nucleic acid molecule, as a nucleic acid molecule encoding a single fusion protein, or any combination thereof. For example, as disclosed herein, a host cell can be engineered to express two or more heterologous or exogenous nucleic acid molecules encoding desired TCRs (e.g., TCRα and TCRβ) specific for a WT-1 antigenic peptide. When two or more exogenous nucleic acid molecules are introduced into a host cell, the two or more exogenous nucleic acid molecules may be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, or integrated into a host chromosome at a single site or multiple sites. The number of heterologous nucleic acid molecules or heterologous protein activities referred to refers to the number of encoding nucleic acid molecules or protein activities, not the number of separate nucleic acid molecules introduced into the host cell. Exogenous nucleic acid molecules (e.g., encoding a binding protein or CD8 co-receptor of the present disclosure) may also be introduced into the genome of a host cell by gene editing methods, e.g., using CRISPR-Cas systems, meganucleases, etc.

[0078] As used herein, the term "endogenous" or "native" refers to a gene, protein, or activity that is normally present in a host cell. Furthermore, a gene, protein, or activity that is mutated, overexpressed, shuffled, duplicated, or otherwise altered compared to a parent gene, protein, or activity is also considered endogenous or native to that particular host cell. For example, an endogenous control sequence (e.g., promoter, translational repression sequence) from a first gene may be used to alter or regulate the expression of a second native gene or nucleic acid molecule, where the expression or regulation of the second native gene or nucleic acid molecule differs from its normal expression or regulation in the parent cell.

[0079] The terms "homologous" or "homolog" refer to a molecule or activity found in or derived from a host cell, host species, or host strain. For example, a heterologous or exogenous nucleic acid molecule can be homologous to a native host cell gene, and optionally, can have altered expression levels, differ in sequence, altered activity, or any combination thereof.

[0080] As used herein, "sequence identity" refers to the percentage of amino acid residues in one sequence that are identical to those in another reference polypeptide sequence, after aligning the sequences to achieve the maximum percent sequence identity, introducing gaps if necessary, and excluding conservative substitutions from the sequence identity.Percent sequence identity values ​​can be generated using the NCBI BLAST 2.0 software, with parameters set to the default values, 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.

[0081] As understood in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence of a polypeptide, and alternative conserved amino acid sequences thereof, to the sequence of a second polypeptide (e.g., using the GENEWORKS™, Align, Clustal™, BLAST algorithms, etc.).

[0082] As used herein, "hyperproliferative disorder" refers to excessive growth or proliferation compared to normal or non-diseased cells. Exemplary hyperproliferative disorders include tumors, cancers, neoplastic tissues, carcinomas, pre-malignant cells, as well as non-neoplastic or non-malignant hyperproliferative disorders (e.g., adenomas, fibroblastomas, lipomas, leiomyomas, hemangiomas, restenosis, as well as autoimmune diseases such as rheumatoid arthritis, osteoarthritis, psoriasis, and inflammatory bowel disease). Certain diseases involving abnormal or excessive growth that occurs more slowly than in the context of hyperproliferative disorders may be referred to as "proliferative disorders," and include certain tumors, cancers, neoplastic tissues, carcinomas, sarcomas, malignant cells, pre-malignant cells, as well as non-neoplastic or non-malignant disorders.

[0083] WT-1 antigen peptide-specific binding protein In certain embodiments, the present disclosure provides a T cell receptor (TCR) α chain variable (Vα) domain comprising a CDR3 amino acid sequence (CDR3α) according to any one of SEQ ID NOs: 19, 22, 25, or 28, or a variant thereof, and a TCR Vβ domain; (b) a TCR Vβ domain comprising a CDR3 amino acid sequence (CDR3β) according to any one of SEQ ID NOs: 31, 34, 37, or 40, or a variant thereof, and a TCR Vα domain; or (c) the TCR Vα domain of (a) and the TCR Vβ domain of (b), wherein the TCR Vα domain is capable of binding to the RMFPNAPYL (SEQ ID NO: 94):human leukocyte antigen (HLA) complex, and optionally wherein the HLA is HLA-A * Binding proteins containing 0201 are presented.

[0084] In certain embodiments, the binding protein binds to the RMFPNAPYL (SEQ ID NO: 94):HLA complex with an IFNγ-producing pEC of 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 or greater. 50 It is possible to combine with

[0085] In certain embodiments, the Vβ domain of (a), (b) or (c) comprises or consists of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) identity to the amino acid sequence set forth in any one of SEQ ID NOs: 5-8 or 13-16, and / or the Vα domain of (a), (b) or (c) comprises or consists of any amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4 or 9-12.

[0086] In certain embodiments, CDR3β comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31, and CDR3α comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, CDR3β comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34, and CDR3α comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, CDR3β comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37, and CDR3α comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, CDR3β comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40, and CDR3α comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28.

[0087] In certain embodiments, the binding protein comprises a Vα domain comprising CDR1α, CDR2α, and CDR3α, and a Vβ domain comprising CDR1β, CDR2β, and CDR3β.

[0088] In certain embodiments, the Vβ domain comprises a CDR1β amino acid sequence according to any one of SEQ ID NOs: 29, 32, 35, or 38, or a variant thereof, and / or a CDR2β amino acid sequence according to any one of SEQ ID NOs: 30, 33, 36, or 39, or a variant thereof. In certain embodiments, the Vα domain comprises a CDR1α amino acid sequence according to any one of SEQ ID NOs: 17, 20, 23, or 26, or a variant thereof, and / or a CDR2α amino acid sequence according to any one of SEQ ID NOs: 18, 21, 24, or 27, or a variant thereof.

[0089] In certain embodiments, the TCR Vα domain and the TCR Vβ domain comprise CDR1α, CDR2α, CDR3α and CDR1β, CDR2β, CDR3β, respectively, and the amino acid sequences of (i) SEQ ID NOs: 26-28 and 38-40, respectively; (ii) SEQ ID NOs: 23, 27, 28 and 38-40, respectively; (iii) SEQ ID NOs: 17-19 and 29-31, respectively; (iv) SEQ ID NOs: 20-22 and 32-34, respectively; or (v) SEQ ID NOs: 23-25 ​​and 35-37, respectively.

[0090] In any of the embodiments disclosed herein, the binding protein is a WT-1 peptide:HLA-A * In any of the embodiments disclosed herein, the binding protein is capable of binding to a WT-1 peptide:HLA complex on the cell surface in a CD8-independent manner or in the absence of CD8.

[0091] In certain embodiments, the binding proteins described herein include variant polypeptide species having one or more amino acid substitutions, insertions, or deletions within the amino acid sequence compared to the sequences of SEQ ID NOS: 1-61 presented herein. In certain embodiments, the substitutions are or include conservative substitutions. Conservative amino acid substitutions are well known and may occur naturally or may be introduced when the binding protein is produced recombinantly. Amino acid substitutions, deletions, and additions can be introduced into proteins using mutagenesis methods known in the art (see, e.g., Sambrook et al., "Molecular Cloning: A Laboratory Manual," 3rd ed., Cold Spring Harbor Laboratory Press, NY, 2001). Oligonucleotide-directed site-specific (or segment-specific) mutagenesis procedures can be utilized to generate modified polynucleotides with specific codons altered according to the desired substitution, deletion, or insertion. Alternatively, random or saturation mutagenesis methods, such as alanine scanning mutagenesis, error-prone polymerase chain reaction mutagenesis, and oligonucleotide-directed mutagenesis, can be used to prepare immunogenic polypeptide variants (see, e.g., Sambrook et al., supra).

[0092] In certain embodiments, a molecular species (or variant) of a particular binding protein specific for WT-1 may include a protein having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 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: 1-61).

[0093] In certain embodiments, binding proteins are provided that comprise a Vβ domain having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence set forth in any one of SEQ ID NOS:1-4 or 9-12 and / or a Vα domain having at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOS:5-8 or 13-16, and that are capable of binding to the RMFPNAPYL (SEQ ID NO:94):HLA complex. In further embodiments, any one or more of the βCDR or αCDR amino acid sequences presented herein may be present within the Vβ domain and / or Vα domain, respectively.

[0094] In certain embodiments, the binding protein has (a) at least three or four of the CDRs are unmutated; (b) the mutated CDRs have no more than two amino acid substitutions, no more than five consecutive amino acid deletions, or a combination thereof; and (c) the binding protein optionally has an IFNγ-producing pEC50 of 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 or greater. 50 and / or a Vα domain comprising or consisting of an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 1 to 4 or 9 to 12, provided that it retains its ability to bind to the RMFPNAPYL (SEQ ID NO: 94):HLA complex.

[0095] In certain embodiments, the binding protein comprises a TCR variable domain comprising an amino acid sequence encoded by a human TCR V allele, a human TCR D allele, and / or a human TCR J allele. By way of background, during lymphocyte development, Vα exons are assembled from different V (variable) and J (joining) gene segments (VJ), and Vβ exons are assembled from different V (variable), D (diversity), and J (joining) gene segments (VDJ). The TCRα chromosomal locus contains 70-80 V (variable) gene segments and 61 J (joining) gene segments. The TCRβ chromosomal locus contains 52 V (variable) gene segments and two distinct clusters, each containing a single D (diversity) gene segment along with six or seven J (joining) gene segments. Functional Vα and Vβ gene exons are generated by recombination of a V (variable) gene segment with a J (joining) gene segment for Vα, and a V (variable) gene segment with a D (diversity) gene segment and a J (joining) gene segment for Vβ. Nucleotide and amino acid sequences corresponding to the TCR gene segments of various alleles are known in the art and can be found on the ImMunoGeneTics website, for example, at imgt.org / IMGTrepertoire / LocusGenes / listIG_TR / TR / human / Hu_TRgroup.html.

[0096] The polynucleotide encoding the binding protein may comprise the same nucleotide sequence according to the TCR gene segments disclosed herein, although it is understood that within the context of this disclosure, any nucleotide sequence that encodes the amino acid sequence of the gene segments may be used.

[0097] In certain embodiments, the binding protein comprises a Vβ domain comprising (i) an amino acid sequence according to the TRBJ02-03 gene segment and / or (ii) an amino acid sequence according to the TRBV06-05 gene segment, the TRBV07-09 gene segment, or the TRBV20-01 gene segment. In certain embodiments, the binding protein comprises a Vα domain comprising (i) an amino acid sequence according to the TRAJ43 gene segment and / or (ii) an amino acid sequence according to the TRAV20-02 gene segment; an amino acid sequence encoded by the TRAV38DV08 gene segment, or the TRAV38-01 gene segment. In related embodiments, the Vβ domain comprises an amino acid sequence according to the TRBJ02-03 gene segment and the Vα domain comprises an amino acid sequence according to the TRAJ43 gene segment; the Vβ domain comprises an amino acid sequence according to the TRBV06-05 gene segment and the Vα domain comprises an amino acid sequence according to the TRAV20-02 gene segment; the Vβ domain comprises an amino acid sequence according to the TRBV07-09 gene segment and the Vα domain comprises an amino acid sequence according to the TRAV38DV08 gene segment; or the Vβ domain comprises an amino acid sequence according to the TRBV20-01 gene segment and the Vα domain comprises an amino acid sequence according to the TRAV38-01 gene segment.

[0098] In the above-described embodiments, the amino acid sequence according to the TRA or TRB gene segment is 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 amino acids in length, including any length between these exemplary lengths (e.g., 11, 12, 13, 14 amino acids, etc.).

[0099] In certain embodiments, a binding protein capable of binding to the RMFPNAPYL (SEQ ID NO:94):HLA complex is provided, the binding protein comprising a TCR Vα domain and a TCR Vβ domain. In certain embodiments, the Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:1 or 9, and the Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:5 or 13. In certain embodiments, the Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:2 or 10, and the Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:6 or 14. In certain embodiments, the Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:3 or 11, and the Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:7 or 15. In certain embodiments, the Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:4 or 12, and the Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO:8 or 16.

[0100] In any of the embodiments disclosed herein, the binding protein may comprise an alpha chain constant domain (Cα) or fragment thereof and / or a beta chain constant domain (Cβ) or fragment or portion thereof. In certain embodiments, Cα comprises or consists of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to any one of SEQ ID NOs: 41-44. In certain embodiments, Cβ comprises or consists of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 45.

[0101] In a further embodiment, the TCR Cβ comprises a cysteine ​​amino acid (e.g., GV(S→C)TD) in place of the native serine at amino acid position 57, and the TCR Cα comprises a cysteine ​​amino acid (e.g., DK(T→C)VL; see, e.g., Cohen et al., Cancer Res. 67(8):3898-3903 (2007)) in place of the native threonine at amino acid position 48.

[0102] In certain embodiments, the binding protein is a T cell receptor (TCR), a chimeric antigen receptor, or an antigen-binding fragment of a TCR, any of which may be a chimeric, humanized, or human binding protein. In further embodiments, the antigen-binding fragment of a TCR comprises a single-chain TCR (scTCR) or a chimeric antigen receptor (CAR).

[0103] "Chimeric antigen receptor" (CAR) refers to a fusion protein that has been engineered to contain two or more naturally occurring amino acid sequences linked together in a manner that does not occur in nature or within a natural host cell, and that can function as a receptor when present on the surface of a cell. CARs comprise an extracellular portion comprising an antigen-binding domain (e.g., an extracellular portion derived from or from an immunoglobulin or immunoglobulin-like molecule, such as a TCR-binding domain derived from or from a TCR specific for a cancer antigen, an scFv derived from or from an antibody, or an antigen-binding domain derived from a killer immune receptor derived from an NK cell), linked to a transmembrane domain and one or more intracellular signaling domains (optionally containing costimulatory domain(s)) (see, e.g., Sadelain et al., Cancer Discov., 3(4):388 (2013); also see Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2016), Stone et al., Cancer Immunol. Immunother., 63(11):1163 (2014), and Walseng et al., Scientific Reports, 2016), which are incorporated herein by reference. 7:10713 (2017). A CAR of the present disclosure that specifically binds to a WT-1 antigen (e.g., in the context of a peptide:HLA complex) comprises a TCR Vα domain and a TCR Vβ domain, or a functional fragment or portion thereof.

[0104] In certain embodiments, the WT-1-specific binding protein is a TCR. In related embodiments, the binding protein comprises (a) a TCR α chain having, comprising, or consisting of at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence set forth in SEQ ID NO: 50 or 46 and a TCR β chain having, comprising, or consisting of at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 58 or 54; (b) a TCR α chain having, comprising, or consisting of at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 51 or 47 and at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 59 or 55. (c) a TCR alpha chain having at least 90% identity to, comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 52 or 48 and a TCR beta chain having at least 90% identity to, comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 60 or 56; or (d) a TCR alpha chain having at least 90% identity to, comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 53 or 49 and a TCR beta chain having at least 90% identity to, comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 61 or 57.

[0105] In certain embodiments, the binding protein comprises a fusion protein comprising: (i) an extracellular component comprising a binding domain comprising a T cell receptor (TCR) alpha chain variable (Vα) domain and a T cell receptor (TCR) beta chain variable (Vβ) domain, wherein the Vα domain comprises a CDR3 amino acid sequence (CDR3α) according to any one of SEQ ID NOs: 19, 22, 25, or 28 or a variant thereof, and the TCR Vβ domain comprises a CDR3 amino acid sequence (CDR3β) according to any one of SEQ ID NOs: 31, 34, 37, or 40 or a variant thereof; (ii) an intracellular component; and (iii) a transmembrane component disposed between the extracellular component and the intracellular component. In certain embodiments, the fusion protein is a CAR.

[0106] In certain embodiments, the binding protein (i) specifically binds to the RMFPNAPYL (SEQ ID NO: 94) peptide:HLA complex, wherein the binding domain comprises a T cell receptor (TCR) alpha chain variable (Vα) domain and a T cell receptor (TCR) beta chain variable (Vβ) domain, and optionally, (a) at least three or four of the CDRs do not have mutations, (b) the CDRs that have mutations have no more than two amino acid substitutions, no more than five consecutive amino acid deletions, or a combination thereof; and (c) the fusion protein retains its ability to bind to the RMFPNAPYL (SEQ ID NO: 94):HLA complex. (ii) an extracellular component comprising a binding domain wherein the Vα domain comprises or consists of an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 9, 10, 11 or 12 or a variant thereof, and the Vβ domain comprises or consists of an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 5, 6, 7, 8, 13, 14, 15 or 16 or a variant thereof; (ii) an intracellular component; and (iii) a transmembrane component disposed between the extracellular component and the intracellular component, provided that:

[0107] It is understood that any of the binding domains disclosed herein, including the exemplary CDR and variable domain sequences and variants thereof, can be included in a fusion protein in accordance with the present disclosure.

[0108] In any of the embodiments described herein, the encoded polypeptide of the present disclosure can include a "signal peptide" (also known as a leader sequence, leader peptide, or transit peptide). Signal peptides target newly synthesized polypeptides to their appropriate location inside or outside a cell. Exemplary amino acid sequences of proteins or polypeptides of the present disclosure that include a signal peptide are provided, for example, in SEQ ID NOS: 1-8, 46-49, and 54-57. The signal peptide can be removed from the polypeptide during localization or secretion, or once localization or secretion is complete. Polypeptides having a signal peptide are referred to herein as "preproteins," and polypeptides from which their signal peptides have been removed are referred to herein as "mature" proteins or polypeptides. In any of the embodiments disclosed herein, the binding protein or fusion protein includes or is a mature protein, or is or is a preprotein. The amino acid sequences of exemplary mature proteins or polypeptides of the disclosure are provided in SEQ ID NOs: 9-16, 50-53, and 58-61.

[0109] In certain embodiments, the binding proteins of the present disclosure comprise one or more junction amino acids.

[0110] In certain embodiments, the binding protein comprises a linker. In certain embodiments, the linker is composed of about 2 to about 35 amino acids, for example, or about 4 to about 20 amino acids, or about 8 to about 15 amino acids, or about 15 to about 25 amino acids. Exemplary linkers include glycine-serine linkers (e.g., SEQ ID NOs: 95 and 96).

[0111] In certain embodiments, a composition is provided comprising a WT-specific binding protein according to any one of the above embodiments and a pharmaceutically acceptable carrier, diluent, or excipient.

[0112] In certain embodiments, WT-1-specific binding proteins are provided in a soluble form (see, e.g., Walseng et al., PLoS One doi:10.1371 / journal.pone.0119559 (2015)), which may optionally be conjugated to a cytotoxic agent and / or a detection agent. For example, a useful method for isolating and purifying recombinantly produced soluble TCRs may involve obtaining a supernatant from a suitable host cell / vector system that secretes the recombinant soluble TCR into the culture medium, followed by concentrating the medium using a commercially available filter. After concentration, the concentrate can be applied to a single suitable purification matrix or a series of suitable matrices, such as affinity matrices or ion exchange resins. One or more reverse-phase HPLC steps can be used to further purify the recombinant polypeptide. These purification methods can also be used to isolate immunogens from their natural environment. Methods for large-scale production of one or more of the isolated / recombinant soluble TCRs described herein include batch cell culture methods that are monitored and controlled to maintain appropriate culture conditions. Purification of soluble TCRs may be performed according to methods described herein, known in the art, and that comply with regulations and guidelines from domestic and international regulatory agencies.

[0113] In certain embodiments, nucleic acid molecules encoding WT-1-specific binding proteins are used to transfect / transduce host cells (e.g., T cells) for use in adoptive transfer therapy. The advantages of TCR sequencing have been described (e.g., Robins et al., 2009, Blood 114:4099; Robins et al., 2010, Sci. Translat. Med. 2:47-64, PMID:20811043; Robins et al., 2011 (Sep. 10), J. Imm. Meth. Epub, advance of print, PMID:21945395; Warren et al., 2011, Genome Res. 21:790) and can be utilized in the course of practicing embodiments in accordance with the present disclosure. Similarly, adoptive transfer procedures using T cells of desired antigen specificity (e.g., Schmitt et al., Hum. Gen. 20:1240, 2009; Dossett et al., Mol. Ther. 17:742, 2009; Till et al., Blood 112:2261, 2008; Wang et al., Hum. Gene Ther. 18:712, 2007; Kuball ...), as the adaptation of these methods to the embodiments disclosed herein is envisioned based on the teachings herein. 109:2331, 2007; US2011 / 0243972; US2011 / 0189141; Leen et al., Ann. Rev. Immunol. 25:243, 2007), as well as methods for transfecting / transducing T cells with a desired nucleic acid, including methods directed toward an affinity-enhanced TCR specific for the WT-1 peptide antigen RMFPNAPYL (SEQ ID NO: 94) complexed with HLA (e.g., US2004 / 0087025).

[0114] In certain embodiments, the WT-1-specific binding proteins or WT-1-specific binding domains described herein may be expressed by host T cells and functionally characterized according to any of a number of art-accepted methods for assaying for T cell activity, including determining T cell binding, activation, or induction, and also determining antigen-specific T cell responses. In certain embodiments, the binding proteins are capable of promoting an antigen-specific T cell response to human WT-1 in a class I HLA-restricted manner. In further embodiments, the class I HLA-restricted response is transporter-associated antigen processing (TAP)-independent. In certain embodiments, the antigen-specific T cell response is characterized by CD4 + Helper T lymphocyte (Th) response and CD8 + The assay includes at least one of a cytotoxic T lymphocyte (CTL) response. In a related embodiment, the CTL response is directed against WT-1-overexpressing cells. Further exemplary methods for assaying T cell activity include T cell proliferation, cytokine release by T cells, stimulation of antigen-specific T cells, MHC-restricted stimulation of T cells, CTL activity (e.g., from preloaded target cells), and the like. 51These assays include determining T cell phenotypic marker expression (by detecting the release of Cr), changes in T cell phenotypic marker expression, and other measures of T cell function. Procedures for performing these and similar assays can be found, for example, in Lefkovits (Immunology Methods Manual: The Comprehensive Sourcebook of Techniques, 1998). See also Current Protocols in Immunology; Weir, Handbook of Experimental Immunology, Blackwell Scientific, Boston, MA (1986); Mishell and Shigii (eds.), Selected Methods in Cellular Immunology, Freeman Publishing, San Francisco, CA (1979); Green and Reed, Science 281:1309 (1998) and references cited therein.

[0115] "MHC-peptide tetramer staining" refers to an assay used to detect antigen-specific T cells characterized by a tetramer of MHC molecules containing identical peptides, each having an amino acid sequence that is cognate (e.g., identical or related to) at least one antigen (e.g., WT-1), where the complex is capable of binding to a T cell receptor specific for the cognate antigen. Each MHC molecule may be tagged with a biotin molecule. The biotinylated MHC / peptide is tetramerized by the addition of streptavidin, which may 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 affinity-enhanced TCRs of the present disclosure.

[0116] Cytokine levels can be determined according to methods described herein and practiced in the art, including, for example, ELISA, ELISPOT, intracellular cytokine staining and flow cytometry, and combinations thereof (e.g., intracellular cytokine staining and flow cytometry). Proliferation and clonal expansion of immune cells resulting from antigen-specific induction of an immune response or antigen-specific stimulation can be determined by isolating lymphocytes, such as circulating lymphocytes, in a sample of peripheral blood cells or cells derived from lymph nodes, stimulating the cells with antigen, and measuring cytokine production, cell proliferation, and / or cell viability, such as by non-radioactive assays such as tritiated thymidine incorporation or MTT assays. The effect of the immunogens described herein on the balance between Th1 and Th2 immune responses can be examined by determining the levels of Th1 cytokines, such as IFN-γ, IL-12, IL-2, and TNF-β, and type 2 cytokines, such as IL-4, IL-5, IL-9, IL-10, and IL-13.

[0117] Polynucleotides and Vectors Also provided are polynucleotides (e.g., isolated polynucleotides) encoding the binding proteins according to the present disclosure, or fragments or portions thereof. The isolated or recombinant polynucleotides encoding the WT-1-specific binding proteins described herein can be generated and prepared according to a variety of methods and techniques in the fields of molecular biology or polypeptide purification.

[0118] In certain embodiments, polynucleotides are codon-optimized for efficient expression in target host cells. Codon optimization can be performed using known techniques and tools, such as the GenScript® OptimumGene™ tool; GeneArt® GeneOptimizer™ (Sigma), etc. Codon-optimized sequences include partially codon-optimized sequences (i.e., at least one codon is optimized for expression in a host cell) and fully codon-optimized sequences. Codon optimization for expression in certain host immune cells is disclosed, for example, in Scholten et al., Clin. Immunol. 119:135, 2006.

[0119] As one of skill in the art will recognize, polynucleotides can refer to single- or double-stranded DNA, cDNA, or RNA in any form, including complementary plus and minus strands of nucleic acids, including antisense DNA, cDNA, and RNA, as well as siRNA, microRNA, RNA-DNA hybrids, ribozymes, and various other naturally occurring or synthetic forms of DNA or RNA.

[0120] In some embodiments, a polynucleotide encoding a binding protein of the present disclosure comprises a polynucleotide having at least 75% (e.g., 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 62-81.

[0121] In certain embodiments, the polynucleotide encoding the binding protein comprises (i) a TCR β chain-encoding polynucleotide having at least 75% identity to the polynucleotide sequence set forth in SEQ ID NO: 74 and a TCR α chain-encoding polynucleotide having at least 75% identity to the polynucleotide sequence set forth in SEQ ID NO: 66; (ii) a TCR β chain-encoding polynucleotide having at least 75% identity to the polynucleotide sequence set forth in SEQ ID NO: 75 and a TCR α chain-encoding polynucleotide having at least 75% identity to the polynucleotide sequence set forth in SEQ ID NO: 67; (iii) a TCR β chain-encoding polynucleotide having at least 75% identity to the polynucleotide sequence set forth in SEQ ID NO: 76 and a TCR α chain-encoding polynucleotide having at least 75% identity to the polynucleotide sequence set forth in SEQ ID NO: 68; or (iv) a TCR β chain-encoding polynucleotide having at least 75% identity to the polynucleotide sequence set forth in SEQ ID NO: 77 and a TCR α chain-encoding polynucleotide having at least 75% identity to the polynucleotide sequence set forth in SEQ ID NO: 69.

[0122] In some embodiments, a single polynucleotide encodes a binding protein described herein, or alternatively, a binding protein may be encoded by more than one polynucleotide, in other words, components or portions of a binding protein may be encoded by two or more polynucleotides, which may be contained on a single nucleic acid molecule or on two or more nucleic acid molecules.

[0123] In certain embodiments, a polynucleotide encoding two or more components or portions of a binding protein of the present disclosure comprises two or more coding sequences operably associated within a single open reading frame. Such an arrangement is advantageous because it may allow for coordinated expression of desired gene products, such as, for example, the synchronic expression of the alpha and beta chains of a TCR such that they are produced in an approximately 1:1 ratio. In certain embodiments, two or more alternative gene products of a binding protein of the present disclosure, such as a TCR (e.g., the alpha and beta chains) or a CAR, are expressed as separate molecules and post-translationally associate. In further embodiments, two or more alternative gene products of a binding protein of the present disclosure are expressed as a single polypeptide, with portions separated by a cleavable or removable segment. For example, self-cleaving peptides useful for expressing separate polypeptides encoded by a single polynucleotide or vector are known in the art, including, for example, porcine teschovirus type 1 2A (P2A-1) peptide, porcine teschovirus type 2A (P2A-2) peptide, Thosea asigna virus 2A (T2A) peptide, equine rhinitis virus type A (ERAV) 2A (E2A) peptide, and foot-and-mouth disease virus type 2A (F2A) peptide. Exemplary nucleotide and amino acid sequences of self-cleaving peptides are set forth in SEQ ID NOs: 84-93. In certain embodiments, a polynucleotide encoding a self-cleaving peptide comprises or consists of a polynucleotide having at least 75% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 89-93. In certain embodiments, the encoded self-cleaving peptide comprises or consists of an amino acid sequence having at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence set forth in any one of SEQ ID NOs: 84-88.

[0124] It is understood that various arrangements of polynucleotides encoding two polypeptides of interest separated by a self-cleaving peptide (e.g., β-chain-encoding polynucleotide-self-cleaving peptide-α-chain-encoding polynucleotide; α-chain-encoding polynucleotide-self-cleaving peptide-β-chain-encoding polynucleotide) are contemplated.

[0125] Thus, in some embodiments, the polynucleotide encoding the binding protein has a structure comprising or having, from the 5' end to the 3' end, (TCR β domain (e.g., chain)-encoding polynucleotide)-(self-cleaving peptide-encoding polynucleotide)-(TCR α domain (e.g., chain)-encoding polynucleotide).

[0126] In certain embodiments, the polynucleotide encoding the binding protein comprises or consists of a polynucleotide sequence having at least 75% (e.g., 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to the polynucleotide sequence set forth in any one of SEQ ID NOs: 78-81. In certain embodiments, the polynucleotide comprises a polynucleotide sequence that comprises or consists of the polynucleotide sequence set forth in any one of SEQ ID NOs: 78-81.

[0127] In a further embodiment, the encoded binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO: 94):HLA complex and comprises, consists of, or is at least 90% identical (e.g., 90%, 91%, 92%, 93% identical) to (i) the amino acid sequence set forth in any one of SEQ ID NOs: 19, 22, 25, 28, 31, 34, 37, or 40; (ii) the amino acid sequence set forth in any one of SEQ ID NOs: 17, 20, 23, 26, 29, 32, 35, or 38; (iii) the amino acid sequence set forth in any one of SEQ ID NOs: 18, 21, 24, 27, 30, 33, 36, or 39; or (iv) the amino acid sequence set forth in any one of SEQ ID NOs: 1-4 or 9-12. , 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence set forth in any one of SEQ ID NOs: 5 to 8 or 13 to 16; (vi) an amino acid sequence comprising, consisting of or having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 46 to 53; (vii) an amino acid sequence comprising, consisting of or having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 54 to 61; or (viii) an isolated polynucleotide encoding a binding protein comprising a TCR Vα domain and a TCR Vβ domain comprising any combination of (i) to (vii).

[0128] In further embodiments, the encoded binding protein may also be expressed as part of, and the engineered immune cells may express, a transgene construct encoding one or more additional accessory proteins, such as a safety switch protein; a tag, a selection marker, a CD8 co-receptor beta chain; a CD8 co-receptor alpha chain, or both or any combination thereof. Polynucleotides and transgene constructs useful for encoding and expressing binding proteins and accessory components (e.g., one or more of a safety switch protein, a selection marker, a CD8 co-receptor beta chain, or a CD8 co-receptor alpha chain), including their nucleotide and amino acid sequences, are described in PCT Publication No. WO2018 / 058002, which is incorporated by reference. It is understood that any or all of the binding proteins, safety switch proteins, tags, selection markers, CD8 co-receptor beta chains, or CD8 co-receptor alpha chains of the present disclosure may be encoded by a single nucleic acid molecule or may be encoded by polynucleotide sequences that are or are present on separate nucleic acid molecules.

[0129] Exemplary safety switch proteins include, for example, tEGF receptor (tEGFr; Wang et al., Blood 118:1255-1263, 2011) that lacks the extracellular N-terminal ligand-binding domain and intracellular receptor tyrosine kinase activity but retains its native amino acid sequence and exhibits cell surface localization of a type I transmembrane protein, as mediated by the pharmaceutical-grade anti-EGFR monoclonal antibody cetuximab (Erbitux); caspase polypeptides (e.g., iCasp9; Straathof et al., Blood 105:4247-4254, 2005); Stasi et al., N. Engl. J. Med. 365:1673–1683, 2011; Zhou and Brenner, Exp. Hematol. pii:S0301-472X(16)30513-6. doi:10.1016 / j.exphem.2016.07.011), RQR8 (Philip et al., Blood 124:1277-1287, 2014); a 10 amino acid tag derived from the human c-myc protein (Myc) (Kieback et al., Proc. Natl. Acad. Sci. USA, 105:623-628, 2008); and a truncated EGF receptor polypeptide (huEGFRt) with a conformationally intact binding epitope for a marker / safety switch polypeptide such as RQR (CD20+CD34; Philip et al., 2014).

[0130] Other accessory components useful for the engineered immune cells of the present disclosure include tags or selection markers that allow cells to be identified, sorted, isolated, enriched, or tracked. For example, marked immune cells with desired characteristics (e.g., antigen-specific TCR and safety switch protein) can be sorted from unmarked cells in a sample and more efficiently activated and expanded for incorporation into a product with the desired purity.

[0131] As used herein, the term "selection marker" includes nucleic acid constructs (and encoded gene products) that confer identifiable changes to cells that allow for the detection and positive selection of immune cells transduced with a polynucleotide containing the selection marker. RQR is a selection marker that contains a major CD20 extracellular loop and two minimal CD34-binding sites. In some embodiments, the RQR-encoding polynucleotide comprises a polynucleotide encoding a minimal CD34 epitope of 16 amino acids. In some embodiments, the minimal CD34 epitope is incorporated into the amino-terminal position of the CD8 co-receptor stalk domain (Q8). In further embodiments, the minimal CD34-binding site sequence can be combined with a target epitope for CD20 to form a compact marker / suicide gene for T cells (RQR8) (Philip et al., 2014, incorporated herein by reference). This construct allows for the selection of immune cells expressing the construct, for example, by CD34-specific antibodies coupled to magnetic beads (Miltenyi), allowing for the selective elimination of engineered T cells expressing the transgene (Philip et al., 2014), using the clinically accepted pharmaceutical antibody rituximab.

[0132] Further exemplary selection markers also include several truncated type I transmembrane proteins not normally expressed on T cells: truncated low-affinity nerve growth factor, truncated CD19, and truncated CD34 (see, e.g., Di Stasi et al., N. Engl. J. Med. 365:1673-1683, 2011; Mavilio et al., Blood 83:1988-1997, 1994; Fehse et al., Mol. Ther. 1:448-456, 2000, each of which is incorporated herein in its entirety). A useful feature of CD19 and CD34 is the commercial availability of Miltenyi's CliniMACs™ selection system, which can target these markers for clinical-grade sorting. However, CD19 and CD34 are relatively large surface proteins that impose a burden on vector packaging capacity and vector integration transcription efficiency. Surface markers containing extracellular non-signaling domains or various proteins (e.g., CD19, CD34, LNGFR) can also be used. Any selectable marker can be used, provided that it is acceptable to Good Manufacturing Practice. In certain embodiments, a selectable marker is expressed along with a polynucleotide encoding a gene product of interest (e.g., a binding protein of the present disclosure, such as a TCR or CAR). Further examples of selectable markers include reporters, such as GFP, EGFP, β-gal, or chloramphenicol acetyltransferase (CAT). In certain embodiments, a selectable marker, such as CD34, is expressed by the cells, and CD34 can be used to selectively enrich or isolate (e.g., by immunomagnetic selection) transduced cells of interest for use in the methods described herein. As used herein, the CD34 marker is distinct from anti-CD34 antibodies, or, for example, scFvs, TCRs, or other antigen recognition moieties that bind to CD34.

[0133] In certain embodiments, the selectable marker comprises an RQR polypeptide, a truncated low-affinity nerve growth factor (tNGFR), a truncated CD19 (tCD19), a truncated CD34 (tCD34), or any combination thereof.

[0134] By way of background, CD4 + Incorporation of T cells into immunotherapy cell products results in antigen-induced IL-2 secretion and transfer of cytotoxic CD8 + It can enhance T cell survival and function (see, e.g., Kennedy et al., Immunol. Rev. 222:129 (2008); Nakanishi et al., Nature 462(7272):510 (2009)). Under certain circumstances, CD4 + Class I-restricted TCRs in T cells may require the introduction of a CD8 coreceptor to enhance the sensitivity of the TCR to class I HLA-peptide complexes. The CD4 coreceptor is structurally distinct from the CD8 coreceptor and has been shown to be an effective alternative to the CD8 coreceptor (see, e.g., Stone and Kranz, Front. Immunol., 4:244 (2013); see also Cole et al., Immunology 137(2):139 (2012)). Thus, another accessory protein for use in the compositions and methods of the present disclosure includes the CD8 coreceptor or a component thereof.

[0135] In certain embodiments, engineered immune cells comprising a heterologous polynucleotide encoding a binding protein of the present disclosure may further comprise a heterologous polynucleotide encoding a CD8 co-receptor protein or its beta chain component or alpha chain component.

[0136] In some embodiments, the encoded CD8 co-receptor comprises an alpha chain or a fragment or variant thereof. The amino acid sequence of the human CD8 co-receptor alpha chain precursor is known and is provided, for example, in UniProtKB-P30433 (see also UniProtKB-P31783; -P10732; and -P10731).

[0137] In some embodiments, the encoded CD8 co-receptor comprises a β chain or a fragment or variant thereof. The amino acid sequence of the human CD8 co-receptor β chain precursor is known and is provided, for example, in UniProtKB-P10966 (see also UniProtKB-Q9UQ56; -E9PD41; Q8TD28; and -P30434; and -P05541).

[0138] Without wishing to be bound by theory, it is believed that distance from the host cell surface is important for the RQR polypeptide to function as a selection marker / safety switch (Philip et al., 2010, supra). In some embodiments, the encoded RQR polypeptide is contained within the beta chain, alpha chain, or both, or fragments or variants, of one or both of the encoded CD8 co-receptors. In specific embodiments, the engineered immune cells comprise a heterologous polynucleotide encoding iCasp9 and a heterologous polynucleotide encoding a recombinant CD8 co-receptor protein comprising a beta chain containing an RQR polypeptide, and further comprising a CD8 alpha chain.

[0139] The isolated polynucleotides of the present disclosure can further comprise polynucleotides encoding a safety switch protein disclosed herein, a selectable marker, a CD8 co-receptor beta chain, or a CD8 co-receptor alpha chain, or can comprise polynucleotides encoding any combination thereof.

[0140] In certain embodiments, the engineered immune cells of the disclosure further comprise: (i) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 co-receptor alpha chain, where optionally the encoded polypeptide is or comprises a CD8 co-receptor alpha chain; (ii) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 co-receptor beta chain, where optionally the encoded polypeptide is or comprises a CD8 co-receptor beta chain; or (iii) the polynucleotide of (i) and the polynucleotide of (ii), where optionally the host cell comprises a CD4+ T cell, a CD8+ T cell, or both.

[0141] In a further embodiment, the engineered immune cell comprises: (a) a heterologous polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor alpha chain, optionally wherein the encoded polypeptide is or comprises a CD8 co-receptor alpha chain; (b) a heterologous polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor beta chain, optionally wherein the encoded polypeptide is or comprises a CD8 co-receptor beta chain; and (c) a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide of (a) and the polynucleotide of (b).

[0142] In some embodiments, the polynucleotide encoding the binding protein further comprises a polynucleotide encoding a selectable marker.

[0143] Standard techniques for recombinant DNA, peptide and oligonucleotide synthesis, immunoassays, and tissue culture and transformation (e.g., electroporation, lipofection) may be used. Enzymatic reactions and purification methods may be performed according to manufacturer's specifications, as commonly accomplished in the art, or as described herein. These and related techniques and procedures are generally well known in the art and may be performed according to conventional methods, as described in the various general and more specific references in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology techniques, which are cited and discussed throughout this specification. For example, Sambrook et al., "Molecular Cloning: A Laboratory Manual," 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; "Current Protocols in Molecular Biology" (John Wiley and Sons, revised 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", Volumes I and II (IRL Press, Oxford Univ. Press USA, 1985); "Current Protocols in Immunology" (John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M."Real-Time PCR: Current Technology and Applications", Julie Logan, Kirstin Edwards and Nick Saunders (eds.), 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” (ed. N. Gait, 1984); “Nucleic Acid Hybridization” (ed. B. Hames and S. Higgins, 1985); “Transcription and Translation' (B. Hames and S. Higgins, eds., 1984); "Animal Cell Culture" (R. Freshney, eds., 1986); Perbal, "A Practical Guide to Molecular Cloning" (1984); "Next-Generation Genome Sequencing" (Janitz, 2008 Wiley-VCH); "PCR Protocols" (Methods in Molecular Biology) (Park, ed., 3rd ed., 2010 Humana Press); "Immobilized Cells and Enzymes" (IRL Press, 1986); articles in "Methods in Enzymology" (Academic Press, Inc., NY); "Gene Transfer Vectors For Mammalian Cells" (JH Miller and MPCalos, ed., 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 and C.C. 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, 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, eds., 2008); “Neural Stem Cells: Methods and See "Methods in Molecular Biology" (ed. Leslie P. Weiner, 2008). .

[0144] Certain embodiments include a polynucleotide contained within a vector. Those skilled in the art can readily identify vectors suitable for use with certain embodiments disclosed herein. A typical vector can contain a polynucleotide capable of transporting another nucleic acid to which it has been linked or capable of replication in a host organism. Some examples of vectors include plasmids, viral vectors, cosmids, etc. While some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and mammalian episomal vectors), other vectors can be integrated into the genome of the host cell upon introduction into the host cell, and thereby replicated along with the host genome. In addition, some vectors are capable of directing the expression of genes to which they are operably linked. It is further understood that, according to related embodiments, when one or more agents (e.g., polynucleotides encoding WT-1-specific binding proteins or variants thereof described herein) are co-administered to a subject, the agents may be present in separate vectors or in the same vector, or multiple vectors (each containing a different agent or the same agent) may be introduced into a cell or cell population and administered to a subject.

[0145] In certain embodiments, polynucleotides of the present disclosure may be operably linked to certain elements of a vector. For example, polynucleotide sequences required for the expression and processing of the coding sequence to which they are ligated may be operably linked. Expression control sequences may include appropriate transcription initiation sequences, transcription termination sequences, promoter and enhancer sequences; effective 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 operably linked when they are adjacent to a gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0146] In certain embodiments, the vector comprises a plasmid vector or a viral vector (e.g., a vector selected from a lentivirus vector or a gamma-retrovirus vector). Viral vectors include negative-strand RNA viruses such as retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus), paramyxoviruses (e.g., measles virus and Sendai virus), positive-strand RNA viruses such as picornaviruses and alphaviruses, and adenoviruses, double-stranded DNA viruses including herpesviruses (e.g., herpes simplex virus type 1 and herpes simplex virus type 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia virus, fowlpox virus, and canarypox virus). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reovirus, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis sarcoma viruses, mammalian type C retroviruses, mammalian type B retroviruses, mammalian type D retroviruses, HTLV-BLV group retroviruses, lentiviruses, and spumaviruses (Coffin, JM, "Retroviridae: The viruses and their replication," in Fundamental Virology, 3rd ed., BN Fields et al., eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0147] A "retrovirus" is a virus with an RNA genome, which is reverse transcribed into DNA using reverse transcriptase, and the reverse-transcribed DNA is then integrated into the host cell genome. "Gammaretrovirus" refers to a genus of the retroviridae family. Examples of gammaretroviruses include murine stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticulocytosis virus. As used herein, "lentiviral vector" refers to an HIV-based lentiviral vector for gene delivery, which can be integrative or non-integrative, have a relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are typically generated after transient transfection of three or more plasmids (packaging plasmid, envelope plasmid, and transfer plasmid) into producer cells. Like HIV, lentiviral vectors enter target cells through interaction of viral surface glycoproteins with receptors on the cell surface. Upon entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.

[0148] In certain embodiments, the viral vector may be a gammaretrovirus, such as a Moloney murine leukemia virus (MLV)-derived vector. In other embodiments, the viral vector may be a more complex retrovirus-derived vector, such as a lentivirus-derived vector. HIV-1-derived vectors belong to this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods using retroviral and lentiviral vectors and packaging cells to transduce viral particles containing a TCR or CAR transgene into mammalian host cells are known in the art and have been previously described, for example, in U.S. Pat. No. 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010, and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors may also be used for polynucleotide delivery, including, for example, DNA viral vectors, including adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; amplicon vectors, herpes simplex virus (HSV)-derived vectors, including replication-deficient HSV and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998).

[0149] Other vectors developed for use in gene therapy can also be used with the compositions and methods of the present disclosure, including baculovirus and alphavirus-derived vectors (Jolly, D J. 1999, "Emerging Viral Vectors," pp. 209-40; Friedmann T., ed., "The Development of Human Gene Therapy," New York: Cold Spring Harbor Lab) or plasmid vectors (such as Sleeping Beauty or other transposon vectors).

[0150] When the viral vector genome contains multiple polynucleotides that are expressed as separate transcripts in a host cell, the viral vector may also contain additional sequences between the two (or more) transcripts that allow for bicistronic or multicistronic expression. Examples of such sequences used in viral vectors include an internal ribosome entry site (IRES), a furin cleavage site, a viral 2A peptide, or any combination thereof.

[0151] In certain embodiments, the vector is capable of delivering a polynucleotide construct or transgene construct to a host cell (e.g., a hematopoietic progenitor cell or a cell of the human immune system). In specific embodiments, the vector is capable of delivering a polynucleotide construct or transgene construct to a cell of the human immune system, such as, for example, a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double negative T cell, a stem cell memory T cell, a γδ T cell, a natural killer cell, a dendritic cell, or any combination thereof. In further embodiments, the vector is capable of delivering a transgene construct to a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof. In some embodiments, a vector encoding a polynucleotide construct or transgene construct of the present disclosure may further comprise a polynucleotide encoding a nuclease (e.g., a CRISPR-Cas endonuclease or another endonuclease disclosed herein) that may be used to perform a chromosomal knockout in a host cell or that may be used to deliver a therapeutic polynucleotide or transgene, or portion thereof, to a host cell in gene replacement or gene repair therapy. Alternatively, the nuclease used for chromosomal knockout or gene replacement or gene repair therapy may be delivered to a host cell independently from a vector encoding a polynucleotide construct or transgene construct of the present disclosure.

[0152] host cell Also provided herein are host cells comprising a heterologous polynucleotide of the present disclosure (e.g., encoding any of the binding proteins or fusion proteins disclosed herein, and optionally encoding one or more additional proteins, such as a selectable marker self-cleaving peptide, a CD8 co-receptor polypeptide, or any combination thereof) and / or host cells expressing any of the binding proteins disclosed herein. Such host cells can be generated, for example, by transfection or transduction with a vector of the present disclosure and / or gene editing.

[0153] In some embodiments, the host cells comprise immune system cells, which can be any human immune system cell, such as the exemplary immune system cells described herein. In certain embodiments, the host cells comprise T cells, NK cells, NK-T cells, or any combination thereof. In further embodiments, the T cells are CD8 + T cells, CD4 + T cells or both.

[0154] Thus, in certain embodiments, the engineered immune cells comprise a heterologous polynucleotide encoding a binding protein according to the present disclosure, wherein the encoded binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO: 94):human leukocyte antigen (HLA) complex.

[0155] In certain embodiments, the encoded binding protein binds to SEQ ID NO:94:HLA complex with an IFNγ-producing pEC of 4.0, 4.5, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 or greater. 50 It is possible to combine with

[0156] In certain embodiments, the peptide:HLA complex is peptide:HLA-A * Contains 201 complexes.

[0157] In any of the embodiments disclosed herein, the population of modified immune cells is cultured at a peptide concentration in the co-culture of 10 -4 μM or 10 -5 μM or 10 -6 Antigen-presenting cells (e.g., HLA-A) pulsed with a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 94) at 1 μM * When co-cultured with T2 cells, Jurkat cells, dendritic cells expressing an HLA such as 0201 for 4 hours, 50% or more of the modified immune cells in the population produce interferon-gamma (IFN-γ). In certain embodiments, the population of modified immune cells produces interferon-gamma (IFN-γ) when the peptide concentration in the co-culture is increased to about 10 -2 When co-cultured with antigen-presenting cells (also referred to in some contexts as "target cells") pulsed with a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 94) at 1 μM, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more of the modified immune cells in the population express Nur77 + (i.e., express Nur77), in which the antigen-presenting cells optionally comprise T2 cells, Jurkat cells, or both, and optionally express HLA-A * It is 0201+.

[0158] In some embodiments, the population of engineered immune cells increases the peptide concentration in the co-culture to 10 -3 When co-cultured with antigen-presenting cells pulsed with a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 94) at 1 μM, 10%, 15%, 20% or more of the modified immune cells in the population express Nur77. + is.

[0159] In some embodiments, the binding protein has a binding affinity of 1.0, 1.1, 1.2, 1.3, 1.4, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6 or more of Nur77-expressing pEC50 (SEQ ID NO: 94: HLA (HLA-A *0201 etc.)) complex.

[0160] In any of the embodiments disclosed herein, the modified immune cells and HLA-A * 0201 + When the modified immune cells and MDA-MB-468 cells are present in the sample at a ratio of 30:1, the modified immune cells bind to HLA-A in the sample. * 0201 + In certain embodiments, the modified immune cells and HLA-A can be used to kill at least 11%, 12%, 13%, 14%, 15%, 20%, 25% or more of the MDA-MB-468 cells. * 0201 + When the modified immune cells and MDA-MB-468 cells are present in the sample at a ratio of 10:1, the modified immune cells bind to the HLA-A * 0201 + In certain embodiments, the modified immune cells and HLA-A * 0201 + When the modified immune cells and MDA-MB-468 cells are present in a sample at a 1:1 ratio, the modified immune cells bind to HLA-A in the sample. * 0201 + It is possible to kill approximately 5% or more of the MDA-MB-468 cells.

[0161] In any of the embodiments disclosed herein, killing can optionally be achieved by using labeled chromium (e.g., 51 Cr) can be measured using a 4 hour co-culture assay.

[0162] In certain embodiments, the modified immune cells are capable of killing 21%, 22%, 23%, 24%, 25% or more of the Pancl cells in a sample when the modified immune cells and Pancl cells are present at a ratio of 30:1. In further embodiments, the modified immune cells are capable of killing 10%, 15% or more of the Pancl cells in a sample when the modified immune cells and Pancl cells are present at a ratio of 10:1. In further embodiments, the modified immune cells are capable of killing 10%, 15% or more of the Pancl cells in a sample when the modified immune cells and Pancl cells are present at a ratio of 9:1, 8:1, 7:1, 6:1, or 5:1.

[0163] In certain embodiments, the engineered immune cells exhibit a higher affinity for HLA-A in co-culture for 4 hours compared to reference immune cells comprising a polynucleotide encoding a T cell receptor comprising a TCR α chain having the amino acid sequence set forth in SEQ ID NO: 82 and a TCR β chain having the amino acid sequence set forth in SEQ ID NO: 83. * 0201 + It increased killing activity against MDA-MB-468 cells and / or Panc1 cells, and / or improved IFN-γ production in co-culture with peptide-pulsed target cells for 4 hours, and / or further reduced proliferation of Panc-1 cancer cells (e.g., over 60, 75, 100, 125, 150 hours as measured using an IncuCyte assay at an effector:target cell ratio of 8:1).

[0164] In some embodiments, the modified immune cells of the present disclosure prevent or substantially prevent the growth and / or proliferation of Panc-1 cancer cells for 50, 100, 150 hours or more, optionally wherein the modified immune cells and Panc-1 cancer cells are present at an effector:target cell ratio of 8:1.

[0165] In any of the embodiments disclosed herein, killing can optionally be achieved by using labeled chromium (e.g., 51 Cr) can be measured using a 4 hour co-culture assay.

[0166] It is understood that a "reference" immune cell is of the same immune cell type and is phenotypically identical or substantially identical to the modified immune cell of interest, except for the encoded binding protein. For example, in modified CD8+ human T cells, the reference immune cell is a CD8+ human T cell, and can be derived, for example, from the same source or donor as the modified immune cell.

[0167] In any of the above-described embodiments, the encoded binding protein may be a TCR, a CAR, or an scTCR. In any of the above-described embodiments, the encoded binding protein is capable of binding to a WT-1 peptide (SEQ ID NO: 94):HLA complex on the cell surface in a CD8-independent manner or in the absence of CD8.

[0168] In any of the embodiments disclosed herein, the binding protein expressed by the engineered immune cell may comprise a TCR, a single-chain TCR (scTCR), a chimeric antigen receptor (CAR), or any combination thereof.

[0169] Any suitable immune cell, including, for example, a T cell, a NK cell, a NK-T cell, a macrophage, a monocyte, or a dendritic cell, can be modified to contain a polynucleotide encoding a heterologous binding protein of the present disclosure. In some embodiments, the modified immune cell is a CD4 + T cells, CD8 + In some embodiments, the modified immune cells include CD4 T cells, CD4 T cells, or both. - CD8 -In some embodiments, the T cells are naive T cells, central memory T cells, effector memory T cells, or any combination thereof. Adaptation of these methods to the embodiments disclosed herein is contemplated based on the teachings herein, as well as adoptive transfer procedures using T cells of the desired target specificity (e.g., Schmitt et al., Hum. Gen. 20:1240, 2009; Dossett et al., Mol. Ther. 17:742, 2009; Till et al., Blood 112:2261, 2008; Wang et al., Hum. Gene Ther. 18:712, 2007; Kuball et al., Blood 109:2331, 2007; US2011 / 0243972; US2011 / 0189141; Leen et al., Ann. Rev. Immunol. 25:243, 2007), as well as methods for transfecting / transducing T cells with a desired nucleic acid (e.g., U.S. Patent Application Publication No. 2004 / 0087025).

[0170] Any suitable method can be used to transfect or transduce cells, such as T cells, or administer the polynucleotide or composition of the present method. Known methods for delivering polynucleotides to host cells include, for example, the use of cationic polymers, lipid-like molecules, and certain commercially available products, such as IN-VIVO-JET PEI. Other methods include ex vivo transduction, injection, electroporation, DEAE-dextran, sonication loading, liposome-mediated transfection, receptor-mediated transduction, biolistics, transposon-mediated transfer, and the like. Still further methods for transfecting or transducing host cells utilize vectors, as described in more detail herein. In certain embodiments, the host cell is genetically edited to include in its genome a polynucleotide encoding the binding protein (or other protein or polypeptide) presented herein.

[0171] In any of the foregoing embodiments, the engineered immune cells may be engineered to reduce or eliminate expression of one or more endogenous genes (e.g., by chromosomal gene knockouts as described herein) that encode polypeptides involved in immune signaling or other related activities, and / or may be engineered to include a heterologous polynucleotide as provided herein. Exemplary gene knockouts include gene knockouts encoding PD-1, LAG-3, CTLA4, TIM3, HLA molecules, TCR molecules, etc. Without wishing to be bound by theory, certain endogenously expressed proteins of immune cells may be recognized as foreign proteins by the allogeneic host receiving the modified immune cells, which may result in the loss of the modified immune cells (e.g., HLA alleles), down-regulate the immune activity of the modified immune cells (e.g., PD-1, LAG-3, CTLA4), interfere with the binding activity of heterologously expressed binding proteins of the present disclosure (e.g., endogenous TCRs of modified T cells that bind non-WT-1 antigens and may interfere with binding of modified immune cells to cells expressing WT-1 antigen), or compete with heterologous binding proteins for expression.

[0172] Thus, reducing or eliminating the expression or activity of such endogenous genes or proteins can improve the activity, tolerability, expression of the binding protein, or persistence of the modified immune cells in autologous or allogeneic host settings, and may allow for universal administration of the cells (e.g., to any recipient, regardless of HLA type). In certain embodiments, the modified immune cells are donor cells (e.g., allogeneic cells) or autologous cells. In certain embodiments, the engineered immune cells of the present disclosure comprise a chromosomal gene knockout for one or more of the genes encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, an HLA component (e.g., a gene encoding alpha 1 macroglobulin, alpha 2 macroglobulin, alpha 3 macroglobulin, beta 1 microglobulin, or beta 2 microglobulin), or a component of a TCR (e.g., a gene encoding a TCR variable region or a TCR constant region) (see, e.g., Torikai et al., Nature Sci. Rep. 6:21757 (2016); Torikai et al., Blood 119(24):5697 (2012); and Torikai et al., Blood, 122(8):1341 (2013), the gene editing methods, compositions, and adoptive cell therapies of which are incorporated by reference in their entireties).

[0173] As used herein, the term "chromosomal gene knockout" refers to a genetic alteration or introduction of an inhibitory agent in a host cell that prevents (e.g., reduces, delays, suppresses, or prevents) the production of a functionally active endogenous polypeptide product by the host cell. Alterations that result in a chromosomal gene knockout can include, for example, nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletions, and strand breaks, as well as the introduction of heterologous expression of an inhibitory nucleic acid molecule that inhibits expression of an endogenous gene in the host cell.

[0174] In certain embodiments, chromosomal gene knockout or knock-in is achieved by chromosomal editing of a host cell. Chromosomal editing can be performed, for example, using an endonuclease. As used herein, "endonuclease" refers to an enzyme capable of catalyzing the cleavage of phosphodiester bonds within a polynucleotide chain. In certain embodiments, the endonuclease cleaves a targeted gene, thereby inactivating or "knocking out" the targeted gene. The endonuclease may be a naturally occurring endonuclease, a recombinant endonuclease, a genetically modified endonuclease, or a gene fusion endonuclease. Nucleic acid strand breaks caused by an endonuclease are generally repaired via distinctly different mechanisms: homologous recombination or non-homologous end joining (NHEJ). During homologous recombination, a donor nucleic acid molecule can be used to "knock in" a donor gene, "knock out" a target gene, or, optionally, inactivate a target gene via a donor gene knock-in event or a target gene knock-out event. NHEJ is an error-prone repair process that often results in a change to the DNA sequence at the site of the break, such as a substitution, deletion, or addition of at least one nucleotide. NHEJ can be used to "knock out" a target gene. Examples of endonucleases include zinc finger nucleases, TALE-nucleases, CRISPR-Cas nucleases, meganucleases, and megaTALs.

[0175] As used herein, "zinc finger nuclease" (ZFN) refers to a fusion protein containing a zinc finger DNA-binding domain fused to a nonspecific DNA cleavage domain, such as FokI endonuclease. Each zinc finger motif of about 30 amino acids binds to about three base pairs of DNA, and amino acids at certain residues can be varied to alter the sequence specificity of the triplet (see, e.g., Desjarlais et al., Proc. Natl. Acad. Sci. 90:2256-2260, 1993; Wolfe et al., J. Mol. Biol. 285:1917-1934, 1999). Multiple zinc finger motifs can be linked in tandem to create binding specificity to a desired DNA sequence, such as a region having a length ranging from about 9 to about 18 base pairs. By way of background, ZFNs mediate genome editing by catalyzing the formation of site-specific DNA double-strand breaks (DSBs) in the genome, and homology-directed repair facilitates targeted integration of transgenes containing flanking sequences homologous to the genome at the site of the DSB. Alternatively, ZFN-created DSBs can result in knockout of the target gene via repair by non-homologous end joining (NHEJ), an intracellular error-prone repair pathway that results in the insertion or deletion of nucleotides at the break site. In certain embodiments, the gene knockout comprises an insertion, deletion, mutation, or a combination thereof, achieved using ZFN molecules.

[0176] As used herein, "transcription activator-like effector nuclease" (TALEN) refers to a fusion protein comprising a TALE DNA-binding domain and a DNA-cleavage domain, such as a FokI endonuclease. A "TALE DNA-binding domain" or "TALE" generally consists of one or more TALE repeat domains / units, each of which has a highly conserved 33-35 amino acid sequence that differs at the 12th and 13th amino acids. The TALE repeat domain is responsible for binding of the TALE to the target DNA sequence. The different amino acid residues, termed repeat variable dimers (RVDs), correlate with specific nucleotide recognition. The natural (canonical) code for DNA recognition by these TALEs has been determined such that an HD (histidine-aspartic acid) sequence at positions 12 and 13 of the TALE results in the TALE binding to cytosine (C), NG (asparagine-glycine) binds to a T nucleotide, NI (asparagine-isoleucine) binds to an A, NN (asparagine-asparagine) binds to a G or A nucleotide, and NG (asparagine-glycine) binds to a T nucleotide. Non-canonical (atypical) RVDs are also known (see, for example, U.S. Patent Application Publication No. 2011 / 0301073, the atypical RVDs of which are incorporated herein by reference in their entirety). TALENs can be used to direct site-specific double-strand breaks (DSBs) in the genome of T cells. Non-homologous end joining (NHEJ) ligates DNA from both sides of a double-strand break, which has little or no overlapping sequence for annealing, thereby introducing an error that knocks out gene expression. Alternatively, homology-directed repair can introduce a transgene into the site of the DSB, provided that there is a homologous flanking sequence within the transgene. In certain embodiments, gene knockout includes insertion, deletion, mutation, or a combination thereof, and is achieved using TALEN molecules.

[0177] As used herein, the term "CRISPR / Cas" ("clustered regularly interspaced short palindromic repeats / Cas") nuclease system refers to a system that utilizes a CRISPR RNA (crRNA)-guided Cas nuclease to recognize a target site (known as a protospacer) within the genome through base-pairing complementarity, and then cleaves DNA when a short, conserved protospacer-associated motif (PAM) follows immediately 3' to the complementary target sequence. CRISPR / Cas systems are classified into three types (i.e., Type I, Type II, and Type III) based on the sequence and structure of the Cas nuclease. The crRNA-guided and surveillance complexes in Types I and III require multiple Cas subunits. The most well-studied Type II system contains at least three components: an RNA-guided Cas9 nuclease, a crRNA, and a trans-activating crRNA (tracrRNA). The tracrRNA contains a duplex-forming region. The crRNA and tracrRNA interact with the Cas9 nuclease, forming a duplex that can guide the Cas9 / crRNA:tracrRNA complex to a specific site on the target DNA through Watson-Crick base pairing between the spacer on the crRNA and the protospacer on the target DNA upstream from the PAM. The Cas9 nuclease creates a double-stranded break within the region defined by the crRNA spacer. Repair by NHEJ results in an insertion and / or deletion that disrupts expression of the targeted locus. Alternatively, a transgene with homologous flanking sequences can be introduced at the site of the DSB via homology-directed repair. The crRNA and tracrRNA can be engineered into single-stranded guide RNAs (sgRNAs or gRNAs) (see, e.g., Jinek et al., Science 337:816-21, 2012).Additionally, the region of the guide RNA that is complementary to the target site can be altered or programmed to target a desired sequence (Xie et al., PLOS One 9:e100448, 2014; U.S. Patent Application Publication No. 2014 / 0068797; U.S. Patent Application Publication No. 2014 / 0186843; U.S. Patent No. 8,697,359; and PCT Publication WO2015 / 071474, each of which is incorporated by reference). In certain embodiments, the gene knockout comprises an insertion, deletion, mutation, or a combination thereof, and is achieved using a CRISPR / Cas nuclease system.

[0178] Exemplary gRNA sequences and methods of using them to knock out endogenous genes encoding proteins in immune cells include the gRNA sequences and knockout methods described in Ren et al., Clin. Cancer Res. 23(9):2255-2266 (2017), which gRNAs, Cas9 DNA, vectors, and gene knockout methods are incorporated herein by reference in their entirety.

[0179] Alternative Cas nucleases may be used, including, but not limited to, Cas12, Cas13, and Cas14 nucleases and variants thereof, for example, the Cas nucleases disclosed in WO2019 / 178427, which is incorporated herein by reference in its entirety (including the Cas nucleases, CRISPR-Cas systems, and related methods disclosed therein).

[0180] As used herein, "meganuclease," also referred to as "homing endonuclease," refers to an endodeoxyribonuclease characterized by a large recognition site (a double-stranded DNA sequence of about 12 to about 40 base pairs). Meganucleases can be divided into five families based on sequence and structural motifs: LAGLIDADG (SEQ ID NO: 97), GIY-YIG (SEQ ID NO: 98), HNH, His-Cys box, and PD-(D / E)XK (SEQ ID NO: 99). Exemplary meganucleases are those whose recognition sequences are known (e.g., U.S. Pat. Nos. 5,420,032 and 6,833,252; Belfort et al., Nucleic Acids Res. 25:3379-3388, 1997; Dujon et al., Gene 82:115-118, 1989; Perler et al., Nucleic Acids Res. 22:1125-1127, 1994; Jasin, Trends Genet.12:224~228, 1996; Gimble et al., J.Mol.Biol.263:163~180, 1996; Argast et al., J.Mol.Biol.280:345~353, 1998), I-SceI , I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI, I-TevI, I-TevII and I-TevIII.

[0181] In certain embodiments, naturally occurring meganucleases can be used to facilitate site-specific genome modification of targets selected from genes encoding PD-1, LAG3, TIM3, CTLA4, TIGIT, HLA, or genes encoding TCR components. In other embodiments, engineered meganucleases with novel binding specificities for target genes can be used to facilitate site-specific genome modification (e.g., Porteus et al., Nat. Biotechnol. 23:967-73, 2005; Sussman et al., J. Mol. Biol. 342:31-41, 2004; Epinat et al., Nucleic Acids Res. 31:2952-62, 2003; Chevalier et al., Molec. Cell 10:895-905, 2002; Ashworth et al., Nature 441:656-659, 2006; Paques et al., Curr. Genet. 2004). Ther. 7:49-66, 2007; U.S. Patent Application Publication Nos. 2007 / 0117128; 2006 / 0206949; 2006 / 0153826; 2006 / 0078552 and 2004 / 0002092). In a further embodiment, homing endonucleases engineered with modular DNA-binding domains of TALENs are used to generate fusion proteins known as megaTALs, resulting in the knockout of chromosomal genes. megaTALs can be used not only to knock out one or more target genes when used in combination with an exogenous donor template encoding a polypeptide of interest, but also to introduce (knock in) heterologous or exogenous polynucleotides when used in combination with an exogenous donor template encoding a polypeptide of interest.

[0182] In certain embodiments, the chromosomal gene knockout comprises an inhibitory nucleic acid molecule introduced into a host cell (e.g., an immune cell) that comprises a heterologous polynucleotide encoding an antigen-specific receptor that specifically binds to a tumor-associated antigen, wherein the inhibitory nucleic acid molecule encodes a target-specific inhibitor that inhibits expression of an endogenous gene (i.e., PD-1, TIM3, LAG3, CTLA4, TIGIT, a component of an HLA, or a component of a TCR, or any combination thereof) in the host immune cell.

[0183] Chromosomal gene knockout can be directly confirmed by DNA sequencing of host immune cells after the knockout procedure or agent is used. Chromosomal gene knockout can also be inferred from the absence of gene expression (e.g., the absence of the mRNA product or polypeptide product encoded by the gene) after the knockout.

[0184] Any of the foregoing gene editing methods can be used to introduce a polynucleotide of the present disclosure (e.g., encoding a binding protein and / or a CD8 co-receptor polypeptide) into a host cell genome. In some embodiments, the heterologous polynucleotide is introduced into a locus encoding an endogenous TCR component, an HLA component, the PD-1 locus, the LAG-3 locus, the CTLA4 locus, the TIM3 locus, or the TIGIT locus, or a safe harbor locus such as Rosa26, AAVS1, or CCR5. In certain embodiments, the heterologous polynucleotide encoding a binding protein and / or a heterologous polynucleotide encoding a CD8 co-receptor polypeptide is introduced into the host cell TRAC locus. In further embodiments, a chromosomal knockout of the TRBC locus of the host cell is introduced.

[0185] Thus, in certain embodiments, a host cell (e.g., an engineered immune cell) is provided that comprises, within the endogenous TRAC locus, a heterologous polynucleotide encoding a binding protein of the present disclosure, a CD8 co-receptor of the present disclosure, or both. In further embodiments, the host cell comprises a chromosomal knockout of the endogenous TRAC locus.

[0186] In another aspect, provided herein are compositions comprising the modified immune cells (and / or polynucleotides, vectors, or binding proteins) of the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient.

[0187] Also provided herein are unit doses comprising an effective amount of the modified immune cells or compositions comprising the modified immune cells. In certain embodiments, a unit dose comprises (i) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4+ T cells in combination with (ii) a composition comprising at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD8+ T cells in about a 1:1 ratio, where the unit dose contains a reduced amount of or is substantially free of naive T cells (i.e., the population of naive T cells present in the unit dose is less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, or less than about 1% compared to a patient sample having an equivalent number of PBMCs).

[0188] In some embodiments, a unit dose comprises (i) a composition comprising at least about 50% modified CD4+ T cells in combination with (ii) a composition comprising at least about 50% modified CD8+ T cells in a ratio of about 1:1, where the unit dose contains a reduced amount of naive T cells or is substantially free of naive T cells. In further embodiments, a unit dose comprises (i) a composition comprising at least about 60% modified CD4+ T cells in combination with (ii) a composition comprising at least about 60% modified CD8+ T cells in a ratio of about 1:1, where the unit dose contains a reduced amount of naive T cells or is substantially free of naive T cells. In yet further embodiments, a unit dose comprises (i) a composition comprising at least about 70% modified CD4+ T cells in combination with (ii) a composition comprising at least about 70% modified CD8+ T cells in a ratio of about 1:1, where the unit dose contains a reduced amount of naive T cells or is substantially free of naive T cells. In some embodiments, a unit dose comprises (i) a composition comprising at least about 80% modified CD4+ T cells in combination with (ii) a composition comprising at least about 80% modified CD8+ T cells in a ratio of about 1:1, where the unit dose contains a reduced amount of naive T cells or substantially no naive T cells. In some embodiments, a unit dose comprises (i) a composition comprising at least about 85% modified CD4+ T cells in combination with (ii) a composition comprising at least about 85% modified CD8+ T cells in a ratio of about 1:1, where the unit dose contains a reduced amount of naive T cells or substantially no naive T cells. In some embodiments, a unit dose comprises (i) a composition comprising at least about 90% modified CD4+ T cells in combination with (ii) a composition comprising at least about 90% modified CD8+ T cells in a ratio of about 1:1, where the unit dose contains a reduced amount of naive T cells or substantially no naive T cells.

[0189] A unit dose of the present disclosure may comprise modified immune cells as described herein (i.e., expressing a binding protein specific for the WT-1 antigen according to SEQ ID NO: 94) and a different antigen (e.g., a different WT-1 antigen (e.g., comprising SEQ ID NO: 100) or a combination thereof, such as BCMA, CD3, CEACAM6, c-Met, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis A, Lewis B, Lewis C, Lewis D, Lewis E, Lewis F, Lewis H, Lewis I, Lewis J, Lewis H, Lewis J, Lewis H, Lewis I, Lewis H ... Y, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A (including, for example, MAGE-A1, MAGE-A3, and MAGE-A4), mesothelin, NY-ESO-1, PSMA, RANK, ROR1, TNFRSF4, CD40, CD137, TWEAK-R, HLA, HLA-bound tumor-associated peptide or pathogen-associated peptide, HLA-bound hTERT peptide, HLA-bound tyrosinase peptide, HLA-bound KRAS peptide, LTβR It is understood that the unit dose may include engineered immune cells expressing binding proteins specific for antigens derived from different proteins or targets, such as LIFRβ, LRP5, MUC1, OSMRβ, TCRα, TCRβ, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD52, CD56, CD79a, CD79b, CD80, CD81, CD86, CD123, CD171, CD276, B7H4, TLR7, TLR9, PTCH1, HA1-H, Robo1, alpha-fetoprotein (AFP), Frizzled, OX40, PRAME, BRAF, core binding factor, MR-1 (Crowther et al., Nature Immunol. 21:175-185 (2020)), and SSX-2. For example, a unit dose may include engineered CD8 cells expressing a binding protein that specifically binds to the WT-1:HLA complex. + T cells and modified CD4 expressing a binding protein (e.g., CAR) that specifically binds to the CD19 antigen. + It may comprise T cells (and / or modified CD8+ T cells).

[0190] In any of the embodiments described herein, the unit dose may comprise an equal or approximately equal number of engineered CD45RA - CD3 + CD8 + TM cells and modified CD45RA - CD3 + CD4 + Contains TM cells.

[0191] In some embodiments, the modified immune cells (i) comprise the modified immune cells disclosed herein; and (ii) comprise a TCR Vα and a TCR Vβ, and are capable of specifically binding to a VLDFAPPGA (SEQ ID NO: 100):HLA complex, and optionally, the HLA is HLA-A *

[0201] Optionally, the modified immune cells of (i) and the immune cells of (ii) are each independently selected from a T cell, an NK cell, and an NK-T cell, and further comprise an immune cell comprising a polynucleotide encoding the binding protein.

[0192] In certain further embodiments, the immune cell binding protein in (ii) comprises (a) the amino acid sequences of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of SEQ ID NOs: 101-103 and 105-107, respectively, and optionally, (1) Vα has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 104, and / or (2) Vβ has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 108; or (b) the amino acid sequences of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of SEQ ID NOs: 109-110 and 113-115, respectively, and optionally, (1) Vα has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 112. and / or (2) Vβ has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 116; or (c) comprises the amino acid sequences of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β set forth in SEQ ID NOs: 117-119 and 121-123, respectively, and optionally, (1) Vα has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 120; and / or (2) Vβ has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 124.

[0193] use In certain aspects, the present disclosure is directed to methods for treating a disease or disorder associated with expression of WT-1 by administering to a human subject in need thereof an effective amount of a binding protein, polynucleotide, vector, cell, or composition according to any embodiment disclosed herein.

[0194] "Treating" or "treatment" or "ameliorating" refers to the medical management of a disease, disorder, or condition in a subject (e.g., a human or non-human mammal such as a primate, horse, cat, dog, goat, mouse, or rat). Therapeutic or prophylactic / preventative benefit includes improved clinical outcome; relief or alleviation of symptoms associated with the disease; reduced incidence of symptoms; improved quality of life; prolonged disease-free state; reduced extent of disease, stabilization of the disease state; delayed disease progression; remission; survival; extended survival, or any combination thereof.

[0195] As used herein, a "therapeutically effective amount" or "effective amount" refers to an amount of modified immune cells sufficient to result in a therapeutic benefit, including, in a statistically significant manner, improved clinical outcome; reduction or alleviation of symptoms associated with the disease; reduced incidence of symptoms; improved quality of life; prolonged disease-free state; reduced extent of disease, stabilization of the disease state; slowed disease progression; remission; survival or prolonged survival.

[0196] As used herein, "statistically significant" refers to a p-value of 0.050 or less, as calculated using Student's t-test, indicating that the particular event or result measured is unlikely to have occurred by chance.

[0197] Subjects that can be treated according to the methods of the present disclosure are generally humans and other primate subjects, such as monkeys and apes for veterinary and / or research purposes. Mice or rats can also be used for research purposes. In any of the above-described embodiments, the subject can be a human subject. The subject can be male or female and of any appropriate age, including infant, pediatric, juvenile, adult, and elderly subjects. Cells according to the present disclosure can be administered in a manner appropriate to the disease, condition, or disorder being treated, as determined by one of ordinary skill in the medical arts. In any of the above embodiments, the engineered immune cells or unit doses described herein are administered intravenously, intraperitoneally, intratumorally, into the bone marrow, into a lymph node, or into the cerebrospinal fluid so as to encounter target cells (e.g., leukemia cells). The appropriate dose, duration, and frequency of administration of the composition are determined by factors such as the patient's condition; the size, type, and severity of the disease, condition, or disorder; the particular form of the active ingredient; and the method of administration.

[0198] When referring to an individual active ingredient or cells expressing a single active ingredient administered alone, a therapeutically effective amount refers to the effect of that ingredient or cell expressing that ingredient alone. When referring to a combination, a therapeutically effective amount refers to the combined amount of supplementary active ingredients, whether administered sequentially or simultaneously, combined with the active ingredient or cells expressing the active ingredient that results in a therapeutic effect. A combination can also be cells expressing more than one active ingredient.

[0199] As used herein, the terms "adoptive immune therapy" or "adoptive immunotherapy" refer to the administration of naturally occurring or genetically engineered disease- or antigen-specific immune cells (e.g., T cells). Adoptive cellular immunotherapy can be autologous (where the immune cells are derived from the recipient), allogeneic (where the immune cells are derived from a donor of the same species), or syngeneic (where the immune cells are derived from a donor who is genetically identical to the recipient).

[0200] Conditions associated with WT-1 expression include any disorder or condition in which there is a cellular or molecular event of under-, over-, or inappropriate WT-1 activity, typically resulting from high (statistically significant) levels of WT-1 expression in diseased cells (e.g., leukemic cells) compared to normal cells. Subjects with such disorders or conditions would benefit from treatment with the compositions or methods of the embodiments described herein. Thus, some conditions associated with WT-1 overexpression can include acute disorders and diseases, such as pathological conditions that predispose a subject to a particular disorder, as well as chronic disorders and diseases.

[0201] Diseases and disorders associated with WT-1 expression are known in the art, and criteria for diagnosis and classification have been established, including the presence in a subject of dysplastic, cancerous, and / or transformed cells (e.g., solid cancers; blood cancers, including lymphomas and leukemias, such as acute myeloid leukemia and chronic myeloid leukemia), including neoplastic cells, tumor cells, non-contact inhibitory cells, or tumorigenic transformed cells (e.g., Hanahan and Weinberg, 2011 Cell 144:646; Hanahan and Weinberg, 2000 Cell 100:57; Cavallo et al., 2011 Canc. Immunol. Immunother. 60:319; Kyrigideis et al., 2010 J. Carcinog. 9:3). In certain embodiments, such cancer cells may be cells of acute myeloid leukemia, B-cell lymphoblastic leukemia, T-cell lymphoblastic leukemia, or myeloma, including cancer stem cells capable of inducing and sequentially transplanting any of these types of cancer (see, e.g., Park et al., 2009 Molec. Therap. 17:219). According to certain embodiments, virtually any type of cancer characterized by overexpression of WT-1 may be treated through the use of the compositions and methods disclosed herein, including hematological cancers (e.g., leukemias including acute myeloid leukemia (AML), T-cell or B-cell lymphomas, myelomas, etc.). Furthermore, "cancer" may refer to any accelerated cell proliferation, including solid tumors, ascites tumors, hematologic or lymphoid malignancies or other malignancies; connective tissue malignancies; metastatic disease; minimal residual disease after organ or stem cell transplantation; multidrug-resistant cancers, primary or secondary malignancies, angiogenesis associated with malignancies, or other forms of cancer. Also contemplated within the embodiments disclosed herein are specific embodiments in which only one of the above types of diseases is included, or specific conditions may be excluded, whether or not characterized by overexpression of WT-1.

[0202] Some examples of conditions associated with overexpression of WT-1 include hyperproliferative and proliferative disorders in a subject, including tumors, neoplasms, cancers, malignancies, etc. In addition to activated or proliferative cells, hyperproliferative or proliferative disorders can also involve abnormalities or dysregulation of the cell death process, whether by necrosis or apoptosis. Such abnormalities in the cell death process can be associated with a variety of conditions, including cancer (including primary malignancies, secondary malignancies, as well as metastatic malignancies) or other conditions.

[0203] In certain embodiments, the methods of the present invention are useful for treating hematological malignancies or solid cancers. Exemplary hematological malignancies include acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia (AML, including refractory and relapsed AML, including acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelocytic leukemia, acute myelomonocytic leukemia (e.g., with or without eosinophilia), acute monocytic leukemia, acute erythroleukemia, and acute megakaryoblastic leukemia), chronic myeloid leukemia (CML), chronic myelocytic leukemia, chronic eosinophilic leukemia (CEL), myelodysplastic syndromes (MDS), non-Hodgkin's lymphoma (NHL), or multiple myeloma (MM, including refractory and relapsed MM).

[0204] In certain embodiments, the methods disclosed herein are for the treatment of bile duct cancer, bladder cancer, bone and soft tissue cancer, brain tumor, breast cancer, breast carcinoma, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal carcinoma, desmoid tumor, embryonal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, glioblastoma, melanoma, diffuse peritoneal mesothelioma, malignant pleural mesothelioma, glioma, astrocytoma, gynecological tumors, head and neck squamous cell carcinoma, liver cancer, liver cirrhosis ... The compounds are useful for treating solid cancers such as hepatocellular carcinoma, lung cancer, non-small cell lung cancer, malignant melanoma, osteosarcoma, ovarian cancer (e.g., epithelial ovarian cancer, ovarian carcinoma), fallopian tube cancer, endometrial cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, kidney cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, osteogenic sarcoma, testicular germ cell tumor, urothelial cancer, uterine sarcoma, uterine carcinosarcoma, or uterine cancer.

[0205] Also provided are pharmaceutical compositions comprising the binding proteins, polynucleotides, vectors, host cells, and / or engineered immune cells (optionally in combination with immune cells expressing a binding protein capable of binding to SEQ ID NO:100:HLA complex) for use according to any of the methods described herein. Pharmaceutical compositions can be administered in a manner appropriate to the disease or condition being treated (or prevented), as determined by one skilled in the medical arts. The appropriate dose of administration of the composition and the appropriate duration and frequency of administration will be determined by factors such as the patient's health, the patient's size (i.e., weight, body size, or body surface area), the type and severity of the patient's condition, the particular form of the active ingredient, and the method of administration. Generally, an appropriate dose and treatment regimen will administer the composition(s) in an amount sufficient to provide a therapeutic and / or prophylactic benefit (such as the therapeutic and / or prophylactic benefit described herein, including improved clinical outcomes such as an increased frequency of complete or partial remission, or an increase in disease-free and / or overall survival, or a reduction in the severity of symptoms).

[0206] An effective amount of a pharmaceutical composition refers to a sufficient amount, at a dosage required for a period of time necessary to achieve the desired clinical results or beneficial treatments described herein. An effective amount may be delivered in one or more administrations. When administration is to a subject known or confirmed to have a disease or disease state, the term "therapeutic amount" may be used to refer to treatment, whereas a "prophylactically effective amount" may be used to describe administration of an effective amount as a preventative course to a subject susceptible to or at risk of developing a disease or disease state (e.g., recurrence thereof).

[0207] In certain embodiments, the method comprises a TCR Vα and a TCR Vβ, capable of specifically binding to a VLDFAPPGA (SEQ ID NO: 100):HLA complex, and optionally, the HLA is HLA-A *

[0201] The method includes administering the engineered immune cells provided herein to a subject who has received, is receiving, or will receive immune cells comprising a polynucleotide encoding a binding protein, wherein the engineered immune cells and the immune cells are each independently selected from T cells, NK cells, and NK-T cells. In certain embodiments, the binding protein capable of specifically binding to a VLDFAPPGA (SEQ ID NO: 100):HLA complex comprises (a) the amino acid sequences of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of SEQ ID NOs: 101-103 and 105-107, respectively, and optionally, (1) Vα has, comprises, or consists of at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 104, and / or (2) Vβ has, comprises, or consists of at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 108; (b) comprising the amino acid sequences of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of SEQ ID NOs: 109 to 110 and 113 to 115, respectively, and optionally, (1) Vα has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 112, and / or (2) Vβ has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 116; (c) comprises the amino acid sequences of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of SEQ ID NOs: 117-119 and 121-123, respectively, and optionally, (1) Vα has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 120, and / or (2) Vβ has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 124.

[0208] Generally, appropriate dosages and treatment regimens administer active molecules or cells in amounts sufficient to produce benefit. Such responses can be monitored by establishing improved clinical outcomes in treated subjects compared to untreated subjects (increased frequency of complete or partial remission or prolonged disease-free survival). An increase in a pre-existing immune response to tumor proteins generally correlates with improved clinical outcomes. Such immune responses can generally be assessed using standard proliferation, cytotoxicity, or cytokine assays that are conventional in the art and can be performed using samples obtained from subjects before and after treatment.

[0209] For prophylactic use, the dose shall be sufficient to prevent or delay the onset of, or reduce the severity of, the disease or disorder or its associated symptoms. The prophylactic benefit of the compositions administered in accordance with the methods described herein may be determined by conducting preclinical (including in vitro and in vivo animal studies) and clinical studies and analyzing the data obtained therefrom via appropriate statistical, biological, and clinical methods and techniques, all of which may be readily performed by one of ordinary skill in the art.

[0210] Certain treatment or prevention methods contemplated herein involve administering host cells (which may be autologous, allogeneic, or syngeneic) containing a desired nucleic acid molecule described herein, stably integrated into the cellular chromosome or present as an extrachromosomal nucleic acid molecule. For example, such cellular compositions can be generated ex vivo using autologous, allogeneic, or syngeneic immune system cells (e.g., T cells, antigen-presenting cells, natural killer cells) to administer a desired WT-1-targeted T cell composition to a subject as adoptive immunotherapy. In certain embodiments, the immune cells are CD4 + T cells, CD8 + T cells, CD4 - CD8 -In a further embodiment, the immune cells are naive T cells, central memory T cells, effector memory T cells, or any combination thereof.

[0211] The amount of cells in a composition or unit dose may be at least one cell (e.g., one modified CD8 + T cell subpopulations (e.g., optionally, memory CD8 + T cells and / or naive CD8 + T cells); one modified CD4 + T cell subpopulations (e.g., optionally, memory CD4 + T cells and / or naive CD4 + T cells) or cells 10 2 pieces, e.g., 10 cells 4 Less than or equal to 10 5 Less than or equal to 10 6 Less than or equal to 10 7 Less than or equal to 10 8 Less than or equal to 10 9 Less than or equal to 10 10 10 or less 11 In certain embodiments, the cells are present in an amount of about 10 or less, more typically in amounts greater than these numbers. 4 ~about 10 11 cells / 1m 2 in the range of about 10 5 ~about 10 9 cells / 1m 2 In some embodiments, the administered dose is about 3.3 x 10 5 In some embodiments, the administered dose comprises about 1 x 10 cells / kg or less. 6 In some embodiments, the administered dose comprises about 3.3 x 10 cells / kg or less. 6 In some embodiments, the administered dose comprises about 1 x 10 cells / kg or less. 7 In certain embodiments, the engineered immune cells comprise about 5 x 10 cells / kg or less. 4 ≤ 5 x 10 cells / kg5 5 x 10 cells / kg 6 cells / kg or approximately 5 x 10 7 In certain embodiments, the modified immune cells are administered to a subject at a dose comprising at least about 5 x 10 cells / kg or less. 4 5 x 10 cells / kg 5 5 x 10 cells / kg 6 cells / kg or approximately 5 x 10 7 The composition is administered to a subject at a dose containing 10 or fewer cells / kg. The number of cells depends on the intended end use of the composition and the type of cells contained therein. For example, cells modified to contain a binding protein include a cell population containing at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of such cells. For the uses provided herein, the cells are generally in a volume of 1 liter or less, 500 ml or less, 250 ml or less, or 100 ml or less. In some embodiments, the desired cell density is typically 10 cells / ml or less. 4 Generally, more than 10 cells per ml 7 Generally, more than 10 cells per ml 8 The cells may be administered as a single infusion or as multiple infusions over a range of time periods. A clinically relevant number of immune cells is cumulatively greater than 10 cells. 6 , 10 7 , 10 8 , 10 9 , 10 10 or 10 11 The modified immune cells may be divided into multiple infusions equal to or greater than 1000 cells. In certain embodiments, the unit dose of modified immune cells may be co-administered with (e.g., simultaneously or contemporaneously with) hematopoietic stem cells derived from an allogeneic donor. In some embodiments, one or more of the modified immune cells included in the unit dose are autologous to the subject.

[0212] 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 use. In certain embodiments, a unit dose contains about 10 recombinant host cells described herein. 4 cells / 1m 2 ~about 10 11 cells / 1m 2 Suitable administration regimens and treatment regimen developments are provided for use with certain compositions described herein in a variety of treatment regimens, including, for example, parenteral or intravenous administration or formulations for parenteral or intravenous administration.

[0213] In certain embodiments, pharmaceutical compositions comprising the engineered immune cells disclosed herein further comprise a pharmaceutically acceptable carrier, diluent, or excipient. The term "pharmaceutically acceptable excipient or carrier" or "physiologically acceptable excipient or carrier" refers to a biologically compatible medium, such as physiological saline, which is described in more detail herein and is suitable for administration to a human or other non-human mammalian subject and is generally recognized to be safe or not cause serious adverse events.

[0214] Suitable excipients include water, saline, dextrose, glycerol, etc., and combinations thereof. In some embodiments, the compositions comprising the fusion proteins or host cells disclosed herein further comprise a suitable infusion medium. The suitable infusion medium can be any isotonic medium formulation, typically normal saline, Normosol R (Abbott), or Plasma-Lyte A (Baxter), 5% dextrose in water, or Ringer's lactate. The infusion medium may be supplemented with human serum albumin or other human serum components.

[0215] When the subject compositions are administered parenterally, they may also comprise sterile, aqueous or oily solutions or suspensions. Suitable, non-toxic, parenterally acceptable diluents or solvents include water, Ringer's solution, isotonic saline, 1,3-butanediol, ethanol, propylene glycol, or polyethylene glycol in a mixture with water. Aqueous solutions or suspensions may further contain one or more buffering agents, such as sodium acetate, sodium citrate, borax, or sodium tartrate. Of course, any material used in preparing any dosage unit formulation shall be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, active compounds may be incorporated into sustained-release preparations and formulations. As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages for the subject to be treated, each unit containing a predetermined quantity of recombinant cells or active compound calculated to produce the desired therapeutic effect in association with a suitable pharmaceutical carrier.

[0216] As used herein, administration of a composition or therapy refers to delivering the composition or therapy to a subject, regardless of the route or mode of delivery. Administration can be continuous, intermittent, or parenteral. Administration can be to treat a subject already identified as having a recognized condition, disease, or disease state, or to treat a subject susceptible to or at risk of developing such a condition, disease, or disease state. Co-administration with adjunctive therapy can include simultaneous and / or sequential delivery of multiple agents in any order and on any administration schedule (e.g., modified immune cells with one or more cytokines; immunosuppressive therapy such as a calcineurin inhibitor, a corticosteroid, a microtubule inhibitor, a low-dose mycophenolate prodrug, or any combination thereof).

[0217] In certain embodiments, the subject receives multiple doses of the modified immune cells or binding proteins described herein, with the multiple doses administered at intervals of about 2 to about 4 weeks. In further embodiments, the cytokines are administered sequentially, provided that the subject receives at least three or four doses of the modified immune cells prior to cytokine administration. In certain embodiments, the cytokines are administered subcutaneously (e.g., IL-2, IL-15, IL-21). In yet further embodiments, the subject being treated is receiving immunosuppressive therapy, such as a calcineurin inhibitor, a corticosteroid, a microtubule inhibitor, a low-dose mycophenolic acid prodrug, or any combination thereof.

[0218] The treatment or prevention methods of the present disclosure can be administered to a subject as part of a treatment course or regimen, which can include additional treatments before or after administration of a unit dose, cells, or compositions disclosed herein. For example, in certain embodiments, the subject receiving the unit dose of modified immune cells is undergoing or has previously undergone hematopoietic cell transplantation (HCT; including myeloablative and non-myeloablative HCT). In some embodiments, the modified immune cells can be administered at least two to at least three months after a non-myeloablative hematopoietic cell transplant. Techniques and regimens for performing HCT are known in the art and can include the transplantation of any suitable donor cells, such as cells derived from umbilical cord blood, bone marrow or peripheral blood, hematopoietic stem cells, mobilized stem cells, or amniotic fluid. Thus, in certain embodiments, the modified immune cells of the present disclosure can be administered together with or immediately after hematopoietic stem cells in a modified HCT regimen. In some embodiments, the HCT comprises donor hematopoietic cells that contain a chromosomal knockout of a gene encoding an HLA component, a chromosomal knockout of a gene encoding a TCR component, or both.

[0219] In some embodiments, the subject receiving the modified immune cells or binding proteins has previously undergone lymphodepleting chemotherapy, hi further embodiments, the lymphodepleting chemotherapy comprises cyclophosphamide, fludarabine, antithymocyte globulin, or a combination thereof.

[0220] In certain embodiments, the subject has previously undergone treatment for AML or is at risk of developing or progressing to AML.

[0221] Methods according to the present disclosure may further comprise administering one or more additional agents to treat the disease or disorder via combination therapy. In some embodiments, the method further comprises administering the modified immune cells or binding proteins together (concurrently, simultaneously, or sequentially) with an immune checkpoint inhibitor. In some embodiments, the method comprises administering the modified immune cells together with an agonist of a stimulatory immune checkpoint agent. In further embodiments, the combination therapy comprises administering the modified immune cells together with a secondary therapy, such as a chemotherapeutic agent, radiation therapy, surgery, an antibody, or any combination thereof.

[0222] As used herein, the term "immune suppression agent" or "immunosuppressant" refers to one or more cells, proteins, molecules, compounds, or complexes that provide inhibitory signals that help control or suppress an immune response. For example, immunosuppressants include molecules that partially or completely block immune stimulation; reduce, prevent, or delay immune activation; or increase, activate, or upregulate immune suppression. Exemplary immunosuppressive agents to target (e.g., with immune checkpoint inhibitors) include PD-1, PD-L1, PD-L2, LAG3, CTLA4, B7-H3, B7-H4, CD244 / 2B4, HVEM, BTLA, CD160, TIM3, GAL9, KIR, PVR1G (CD112R), PVRL2, adenosine, A2aR, immunosuppressive cytokines (e.g., IL-10, IL-4, IL-1RA, IL-35), IDO, arginase, VISTA, TIGIT, LAIR1, CEACAM-1, CEACAM-3, CEACAM-5, Treg cells, or any combination thereof.

[0223] The immunosuppressant inhibitor (also referred to as an immune checkpoint inhibitor) can be a chemical compound, an antibody, an antibody fragment, or a fusion polypeptide (e.g., an Fc fusion such as CTLA4-Fc or LAG3-Fc), an antisense molecule, a ribozyme, or an RNAi molecule, or a small organic molecule. In any of the embodiments disclosed herein, the method can include the modified immune cell or binding protein together with one or more inhibitors of any one of the following immunosuppressive components, alone or in any combination:

[0224] In certain embodiments, the engineered immune cells or binding proteins are used in combination with a PD-1 inhibitor, e.g., a PD-1-specific antibody or binding fragment thereof, such as pidilizumab, nivolumab, pembrolizumab, MEDI0680 (formerly known as AMP-514), AMP-224, BMS-936558, or any combination thereof. In further embodiments, the engineered immune cells or binding proteins of the disclosure are used in combination with a PD-L1-specific antibody or binding fragment thereof, such as BMS-936559, durvalumab (MEDI4736), atezolizumab (RG7446), avelumab (MSB0010718C), MPDL3280A, or any combination thereof.

[0225] In certain embodiments, the modified immune cells or binding proteins of the disclosure are used in combination with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof.

[0226] In certain embodiments, the modified immune cells or binding proteins are used in combination with an inhibitor of CTLA4, hi certain embodiments, the modified immune cells or binding proteins are used in combination with a CTLA4-specific antibody or binding fragment thereof, such as ipilimumab, tremelimumab, a CTLA4-Ig fusion protein (e.g., abatacept, belatacept), or any combination thereof.

[0227] In certain embodiments, the engineered immune cells or binding proteins are used in combination with a B7-H3-specific antibody or binding fragment thereof, such as enoblituzumab (MGA271), 376.96, or both. The B7-H4 antibody binding fragment can be, for example, an scFv or fusion protein thereof, as described in Dangaj et al., Cancer Res. 73:4820, 2013, as well as binding fragments described in U.S. Patent No. 9,574,000 and PCT Publication Nos. WO2016 / 40724A1 and WO2013 / 025779A1.

[0228] In certain embodiments, the modified immune cells or binding proteins are used in combination with an inhibitor of CD244.

[0229] In certain embodiments, the engineered immune cells or binding proteins are used in combination with inhibitors of BLTA, HVEM, CD160, or any combination thereof. Anti-CD160 antibodies are described, for example, in PCT Publication WO2010 / 084158.

[0230] In certain embodiments, the modified immune cells or binding proteins are used in combination with an inhibitor of TIM3.

[0231] In certain embodiments, the modified immune cells or binding proteins are used in combination with an inhibitor of Gal9.

[0232] In certain embodiments, the modified immune cells or binding proteins are used in combination with inhibitors of adenosine signaling, such as decoy adenosine receptors.

[0233] In certain embodiments, the modified immune cells or binding proteins are used in combination with an inhibitor of A2aR.

[0234] In certain embodiments, the modified immune cells or binding proteins are used in combination with an inhibitor of KIR, such as ililumab (BMS-986015).

[0235] In certain embodiments, the engineered immune cells are used in combination with an inhibitory cytokine (typically a cytokine other than TGFβ) or an inhibitor of Treg development or activity.

[0236] In certain embodiments, the engineered immune cells or binding proteins are used in combination with an IDO inhibitor, such as levo-1-methyltryptophan, epacadostat (INCB024360; Liu et al., Blood 115:3520-30, 2010), ebselen (Terentis et al., Biochem. 49:591-600, 2010), indoximod, NLG919 (Mautino et al., American Association for Cancer Research 2013 104th Annual Meeting, April 6-10, 2013), 1-methyl-tryptophan (1-MT)-tirapazamine, or any combination thereof.

[0237] In certain embodiments, the engineered immune cells or binding proteins are used in combination with an arginase inhibitor, such as N(omega)-nitro-L-arginine methyl ester (L-NAME), N-omega-hydroxy-nor-l-arginine (nor-NOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine ​​(BEC), or any combination thereof.

[0238] In certain embodiments, the modified immune cells or binding proteins are used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.).

[0239] In certain embodiments, the modified immune cells are used in combination with an inhibitor of TIGIT, such as, for example, COM902 (Compugen, Toronto, Ontario Canada), an inhibitor of CD155, such as, for example, COM701 (Compugen), or both.

[0240] In certain embodiments, the engineered immune cells or binding proteins are used in combination with inhibitors of PVRIG, PVRL2, or both. Anti-PVRIG antibodies are described, for example, in PCT Publication WO2016 / 134333. Anti-PVRL2 antibodies are described, for example, in PCT Publication WO2017 / 021526.

[0241] In certain embodiments, the modified immune cells or binding proteins are used in combination with a LAIR1 inhibitor.

[0242] In certain embodiments, the modified immune cells or binding proteins are used in combination with inhibitors of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.

[0243] In certain embodiments, the modified immune cells or binding proteins are used in combination with agents that increase the activity of (i.e., are agonists of) stimulatory immune checkpoint molecules. For example, the modified immune cells or binding proteins may be used in combination with agents that increase the activity of (i.e., are agonists of) stimulatory immune checkpoint molecules, such as CD137 (4-1BB) agonists (e.g., urelumab), CD134 (OX-40) agonists (e.g., MEDI6469, MEDI6383, or MEDI0562), lenalidomide, pomalidomide, CD27 agonists (e.g., CDX-1127), CD28 agonists (e.g., TGN1412, CD80 or and CD86 agonists), CD40 agonists (e.g., CP-870,893, rhuCD40L, or SGN-40), CD122 agonists (e.g., IL-2), agonists of GITR (e.g., the humanized monoclonal antibodies described in PCT Publication WO2016 / 054638), agonists of ICOS (CD278) (e.g., GSK3359609, mAb 88.2, JTX-2011, Icos 145-1, Icos 314-8, or any combination thereof). In any of the embodiments disclosed herein, the method may include administering the engineered immune cells or binding proteins with one or more agonists of stimulatory immune checkpoint molecules, including any of the foregoing, alone or in any combination.

[0244] In certain embodiments, the combination therapy includes a second-line therapy comprising one or more of an engineered immune cell or binding protein and an antibody or antigen-binding fragment thereof specific for a cancer antigen expressed by a non-inflammatory solid tumor, radiation treatment, surgery, a chemotherapeutic agent, a cytokine, RNAi, or any combination thereof.

[0245] In certain embodiments, the combination therapy comprises administering modified immune cells or binding proteins and further performing radiation treatment or surgery. Radiation therapy is well known in the art and includes X-ray therapy, such as gamma irradiation, and radiopharmaceutical therapy. Appropriate surgeries and procedures for treating a given cancer in a subject are well known to those skilled in the art.

[0246] In certain embodiments, the combination therapy comprises administering the modified immune cells or binding proteins and further administering a chemotherapeutic agent, including, but not limited to, chromatin function inhibitors, topoisomerase inhibitors, microtubule inhibitors, DNA damaging agents, antimetabolites (such as folate antagonists, pyrimidine analogs, purine analogs, and sugar modification analogs), DNA synthesis inhibitors, DNA interacting agents (such as intercalating agents), and DNA repair inhibitors. Exemplary chemotherapeutic agents include, but are not limited to, the following groups: antimetabolites / anticancer drugs such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine); microtubule disruptors such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and navelbine; epidipofen cyclotoxins (etoposide, teniposide), DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine, oxaliplatin, ifosfamide, melphalan, merchlorthamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, temozolomide, teniposide, triethylenethiophosphoramide, and etoposide (VP Antiproliferative / antimitotic agents, including 16); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin), and mitomycin; enzymes (L-asparaginase, which metabolizes L-asparagine systemically and depletes cells that do not have the ability to synthesize their own asparagine); antiplatelet agents;Antiproliferative / antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates (busulfan), nitrosoureas (carmustine (BCNU) and analogs, streptozocin), triazenes (dacarbazine (DTIC)); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogues (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; anti-migratory drugs Chemotactic agents; antisecretory agents (brefeldin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (TNP470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors; fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab, rituximab); chimeric antigen receptors; cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone and prednisolone); growth factor signaling kinase inhibitors;Mitochondrial dysfunction inducers, toxins such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin or diphtheria toxin and caspase activators; and chromatin disruptors.

[0247] Cytokines can be used to manipulate host immune responses toward anti-cancer activity. See, e.g., Floros and Tarhinis, Semin. Oncol. 42(4):539-548, 2015. Cytokines useful for promoting immune anti-cancer or anti-tumor responses include, for example, IFN-α, IL-2, IL-3, IL-4, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-24, and GM-CSF, alone or in any combination with the modified immune cells or binding proteins of the disclosure.

[0248] In yet a further aspect, a method for producing the composition of the present disclosure is provided. In certain embodiments, the method comprises (i) combining an aliquot of host cells transduced with a vector of the present disclosure with (ii) a pharmaceutically acceptable carrier. In certain embodiments, the vector of the present disclosure is used to transfect / transduce host cells (e.g., T cells) for use in adoptive transfer therapy (e.g., targeting cancer antigens).

[0249] In some embodiments, the method further comprises culturing the transduced host cells and selecting the transduced cells as cells that have integrated the vector (i.e., express the vector) prior to aliquoting. In further embodiments, the method comprises expanding the transduced host cells after culturing and selection prior to aliquoting. In any of the embodiments of the method, the produced composition or unit dose can be frozen for later use. Any suitable host cells can be used to produce compositions or unit doses according to the method, including, for example, hematopoietic stem cells, T cells, primary T cells, T cell lines, NK cells, or NK-T cells. In a specific embodiment, the method comprises host cells that are CD8+ T cells, CD4+ T cells, or both.

[0250] The present disclosure also includes the following exemplary embodiments.

[0251] Embodiment 1. An engineered immune cell comprising a heterologous polynucleotide encoding a binding protein, wherein the encoded binding protein comprises: (a) a T cell receptor (TCR) α chain variable (Vα) domain or a variant thereof comprising a CDR3 amino acid sequence (CDR3α) according to any one of SEQ ID NOs: 28, 19, 22, or 25, and a TCR β chain variable (Vβ) domain; or (b) a TCR Vβ domain or a variant thereof comprising a CDR3 amino acid sequence (CDR3β) according to any one of SEQ ID NOs: 40, 31, 34, or 37, and a TCR Vα domain; or (c) the TCR Vα domain of (a) and the TCR Vβ domain of (b), wherein the encoded binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO: 94):human leukocyte antigen (HLA) complex, and optionally, the HLA is HLA-A. * 0201, comprising an engineered immune cell.

[0252] Embodiment 2. The encoded binding protein is an IFNγ-producing pEC 502. The modified immune cell of embodiment 1, wherein the modified immune cell is capable of binding to the RMFPNAPYL (SEQ ID NO: 94):HLA complex with

[0253] Embodiment 3. The encoded binding protein is a pEC IFNγ-producing ... 50 3. The modified immune cell of embodiment 1 or 2, wherein the modified immune cell is capable of binding to the RMFPNAPYL (SEQ ID NO: 94):HLA complex with

[0254] Embodiment 4. The encoded binding protein is a IFNγ-producing pEC 50 4. The modified immune cell of any one of embodiments 1-3, wherein the modified immune cell is capable of binding to the RMFPNAPYL (SEQ ID NO: 94):HLA complex with

[0255] Embodiment 5. The encoded binding protein is a pEC IFNγ-producing ... 50 5. The modified immune cell of any one of embodiments 1-4, wherein the modified immune cell is capable of binding to the RMFPNAPYL (SEQ ID NO: 94):HLA complex with

[0256] Embodiment 6. The encoded binding protein is an IFNγ-producing pEC 50 6. The modified immune cell of any one of embodiments 1-5, wherein the modified immune cell is capable of binding to the RMFPNAPYL (SEQ ID NO: 94):HLA complex with

[0257] Embodiment 7. The HLA is HLA-A * 201. The modified immune cell of any one of embodiments 1-6, comprising:

[0258] Embodiment 8. The population of modified immune cells is a peptide in a co-culture of 10 -4 μM, 10 -5 μM or 10 -6 Antigen-presenting cells (e.g., HLA-A) pulsed with a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 94) at 1 μM *8. The modified immune cells of any one of embodiments 1-7, wherein when co-cultured with a T cell-expressing antibody (T cell-expressing antibody) or a T cell-expressing antibody (T cell-expressing antibody) for 4 hours, 50% or more of the modified immune cells in the population produce IFN-γ, and the antigen-presenting cells optionally comprise T2 cells.

[0259] Embodiment 9. The population of modified immune cells is a peptide in a co-culture of 10 -2 When co-cultured (e.g., for about 24 hours) with antigen-presenting cells pulsed with a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 94) at 100 μM, 10% or more of the modified immune cells in the population express Nur77 + and the antigen-presenting cells optionally comprise T2 cells, Jurkat cells, or both.

[0260] Embodiment 10. The population of modified immune cells is a peptide in a co-culture of 10 -2 When co-cultured (e.g., for about 24 hours) with antigen-presenting cells pulsed with a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 94) at 1 μM, 15% or more of the modified immune cells in the population express Nur77 + 10. The modified immune cell of embodiment 9, wherein the modified immune cell is

[0261] Embodiment 11. The population of modified immune cells is a peptide in a co-culture of 10 -2 When co-cultured (e.g., for about 24 hours) with antigen-presenting cells pulsed with a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 94) at 100 μM, 20% or more of the engineered immune cells in the population express Nur77 + 11. The modified immune cell of embodiment 9 or 10, wherein

[0262] Embodiment 12. The population of modified immune cells is a peptide in a co-culture of 10 -2When co-cultured (e.g., for about 24 hours) with antigen-presenting cells pulsed with a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 94) at 100 μM, 40% or more of the engineered immune cells in the population express Nur77 + The modified immune cell of any one of embodiments 9 to 11, wherein

[0263] Embodiment 13. The population of modified immune cells is a peptide in a co-culture of 10 -2 When co-cultured (e.g., for about 24 hours) with antigen-presenting cells pulsed with a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 94) at 1 μM, 50% or more of the modified immune cells in the population express Nur77 + The modified immune cell of any one of embodiments 9 to 12, wherein

[0264] Embodiment 14. The population of modified immune cells is a peptide in a co-culture of 10 -3 When co-cultured (e.g., for about 24 hours) with antigen-presenting cells pulsed with a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 94) at 100 μM, 10% or more of the modified immune cells in the population express Nur77 + 14. The modified immune cell of any one of embodiments 1 to 13, wherein

[0265] Embodiment 15. The population of modified immune cells is a peptide in a co-culture of 10 -3 When co-cultured (e.g., for about 24 hours) with antigen-presenting cells pulsed with a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 94) at 1 μM, 15% or more of the modified immune cells in the population express Nur77 + 15. The modified immune cell of embodiment 14, wherein the modified immune cell is

[0266] Embodiment 16. The population of modified immune cells is a co-culture of the peptide concentration of about 10 -3When co-cultured (e.g., for about 24 hours) with antigen-presenting cells pulsed with a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 94) at 100 μM, 20% or more of the engineered immune cells in the population express Nur77 + 16. The modified immune cell of embodiment 14 or 15, wherein

[0267] Embodiment 17. Modified immune cells and HLA-A * 0201 + When MDA-MB-468 cells and HLA-A in the sample were present at a ratio of 30:1, * 0201 + 17. The modified immune cell of any one of embodiments 1-16, which is capable of killing 11% or more of MDA-MB-468 cells.

[0268] Embodiment 18. Modified immune cells and HLA-A * 0201 + When MDA-MB-468 cells and HLA-A in the sample were present at a ratio of 30:1, * 0201 + 18. The modified immune cell of embodiment 17, wherein the modified immune cell is capable of killing 12% or more of MDA-MB-468 cells.

[0269] Embodiment 19. Modified immune cells and HLA-A * 0201 + When MDA-MB-468 cells and HLA-A in the sample were present at a ratio of 30:1, * 0201 + 19. The modified immune cell of embodiment 17 or 18, wherein the modified immune cell is capable of killing 14% or more of MDA-MB-468 cells.

[0270] Embodiment 20. Modified immune cells and HLA-A * 0201 + When MDA-MB-468 cells and HLA-A in the sample were present at a ratio of 30:1, * 0201 +20. The modified immune cell of any one of embodiments 17-19, which is capable of killing 15% or more of MDA-MB-468 cells.

[0271] Embodiment 21. Modified immune cells and HLA-A * 0201 + When MDA-MB-468 cells and HLA-A in the sample were present at a ratio of 30:1, * 0201 + 21. The modified immune cell of any one of embodiments 17-20, which is capable of killing 20% ​​or more of MDA-MB-468 cells.

[0272] Embodiment 22. Modified immune cells and HLA-A * 0201 + When MDA-MB-468 cells and HLA-A in the sample were present at a ratio of 30:1, * 0201 + 22. The modified immune cell of any one of embodiments 17-21, which is capable of killing 25% or more of MDA-MB-468 cells.

[0273] Embodiment 23. Modified immune cells and HLA-A * 0201 + When MDA-MB-468 cells and HLA-A in the sample were present at a ratio of 10:1, * 0201 + 23. The modified immune cell of any one of embodiments 1-22, which is capable of killing 10% or more of MDA-MB-468 cells.

[0274] Embodiment 24. Modified immune cells and HLA-A * 0201 + When MDA-MB-468 cells and HLA-A in the sample are present at a 1:1 ratio in the sample, * 0201 + 24. The modified immune cell of any one of embodiments 1-23, which is capable of killing about 5% or more of MDA-MB-468 cells.

[0275] Embodiment 25. The modified immune cell of any one of embodiments 1 to 24, wherein the modified immune cell is capable of killing 21% or more of Panc1 cells in a sample when the modified immune cell and Panc1 cells are present at a ratio of 30:1.

[0276] Embodiment 26. The modified immune cell of embodiment 25, which is capable of killing 22% or more of Panc1 cells in a sample when the modified immune cell and Panc1 cells are present in a ratio of 30:1.

[0277] Embodiment 27. The modified immune cell of embodiment 25 or 26, which is capable of killing 23% or more of Panc1 cells in a sample when the modified immune cell and Panc1 cells are present in a ratio of 30:1.

[0278] Embodiment 28. The modified immune cells of any one of embodiments 25 to 27, which are capable of killing 24% or more of Panc1 cells in a sample when the modified immune cells and Panc1 cells are present at a ratio of 30:1.

[0279] Embodiment 29. The modified immune cells of any one of embodiments 25 to 28, which are capable of killing 25% or more of Panc1 cells in a sample when the modified immune cells and Panc1 cells are present in a ratio of 30:1.

[0280] Embodiment 30. A method for treating a T cell receptor comprising administering to a subject a subject a subject in need thereof, comprising administering to a subject a subject a subject in need thereof, a method for treating a T cell receptor ... * 0201 + 30. The modified immune cell of any one of embodiments 1-29, having increased killing activity against MDA-MB-468 cells and / or Panc1 cells.

[0281] Embodiment 31. The modified immune cell of any one of Embodiments 1 to 30, wherein the encoded binding protein is capable of binding to a WT-1 peptide:HLA complex on the cell surface in a CD8-independent manner or in the absence of CD8.

[0282] Embodiment 32. The modified immune cell of any one of embodiments 1 to 31, wherein (i) the encoded Vβ domain comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 16, 5, 6, 7, 8, 13, 14, or 15, and / or (ii) the encoded Vα domain comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence of any one of SEQ ID NOs: 12, 1, 2, 3, 4, 9, 10, or 11.

[0283] Embodiment 33. (i) at least three or four of the CDRs are free of mutations; (ii) the mutated CDRs have no more than two amino acid substitutions, no more than five consecutive amino acid deletions, or a combination thereof; and (iii) the encoded binding protein optionally has an interferon-gamma (IFNγ)-producing pEC 50 33. The modified immune cell of any one of embodiments 1-32, wherein the encoded Vβ domain comprises or consists of an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 16, 5, 6, 7, 8, 13, 14 or 15, and / or the encoded Vα domain comprises or consists of an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 12, 1, 2, 3, 4, 9, 10 or 11, provided that the modified immune cell retains the ability to bind to the RMFPNAPYL (SEQ ID NO: 94):HLA complex, with

[0284] Embodiment 34. The modified immune cell of any one of embodiments 1 to 33, wherein (i) the encoded CDR3β comprises or consists of the amino acid sequence set forth in SEQ ID NO: 40, and the encoded CDR3α comprises or consists of the amino acid sequence set forth in SEQ ID NO: 28; (ii) the encoded CDR3β comprises or consists of the amino acid sequence set forth in SEQ ID NO: 31, and the encoded CDR3α comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19; (iii) the encoded CDR3β comprises or consists of the amino acid sequence set forth in SEQ ID NO: 34, and the encoded CDR3α comprises or consists of the amino acid sequence set forth in SEQ ID NO: 22, or (iv) the encoded CDR3β comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37, and the encoded CDR3α comprises or consists of the amino acid sequence set forth in SEQ ID NO: 25.

[0285] Embodiment 35. The modified immune cell of any one of embodiments 1 to 34, wherein (i) the encoded Vβ domain comprises a CDR1β amino acid sequence of any one of SEQ ID NOs: 38, 29, 32, or 35 and / or a CDR2β amino acid sequence of any one of SEQ ID NOs: 39, 30, 33, or 36; and / or (ii) the encoded Vα domain comprises a CDR1α amino acid sequence of any one of SEQ ID NOs: 26, 17, 20, or 23 and / or a CDR2α amino acid sequence of any one of SEQ ID NOs: 27, 18, 21, or 24.

[0286] Embodiment 36. The modified immune cell of any one of embodiments 1 to 35, wherein the encoded TCR Vα domain and TCR Vβ domain comprise the amino acid sequences of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of (i) SEQ ID NOs: 26 to 28 and 38 to 40, respectively; (ii) SEQ ID NOs: 23, 27, 28, and 38 to 40, respectively; (iii) SEQ ID NOs: 17 to 19 and 29 to 31, respectively; (iv) SEQ ID NOs: 20 to 22 and 32 to 34, respectively; or (v) SEQ ID NOs: 23 to 25 and 35 to 37, respectively.

[0287] Embodiment 37. The modified immune cell of any one of embodiments 1 to 36, wherein the encoded TCR Vβ domain comprises (i) an amino acid sequence according to the TRBJ02-03 gene segment and / or (ii) an amino acid sequence according to the TRBV06-05 gene segment, an amino acid sequence according to the TRBV07-09 gene segment, or an amino acid sequence according to the TRBV20-01 gene segment.

[0288] Embodiment 38. The modified immune cell of any one of embodiments 1 to 37, wherein the encoded TCR Vα domain comprises (i) an amino acid sequence according to the TRAJ43 gene segment and / or (ii) an amino acid sequence according to the TRAV20-02 gene segment; an amino acid sequence according to the TRAV38DV08 gene segment, or an amino acid sequence according to the TRAV38-01 gene segment.

[0289] Embodiment 39. 39. The modified immune cell of embodiment 38, wherein (i) the encoded TCR Vβ domain comprises an amino acid sequence according to the TRBJ02-03 gene segment; and (ii) the encoded TCR Vα domain comprises an amino acid sequence according to the TRAJ43 gene segment.

[0290] Embodiment 40. 39. The modified immune cell of embodiment 38, wherein (i) the encoded TCR Vβ domain comprises an amino acid sequence according to the TRBV06-05 gene segment; and (ii) the encoded TCR Vα domain comprises an amino acid sequence according to the TRAV20 gene segment.

[0291] Embodiment 41. 39. The modified immune cell of embodiment 38, wherein (i) the encoded TCR Vβ domain comprises an amino acid sequence according to the TRBV07-09 gene segment; and (ii) the encoded TCR Vα domain comprises an amino acid sequence according to the TRAV38DV08 gene segment.

[0292] Embodiment 42. The modified immune cell of embodiment 38, wherein (i) the encoded TCR Vβ domain comprises an amino acid sequence according to the TRBV20-01 gene segment; and (ii) the encoded TCR Vα domain comprises an amino acid sequence according to the TRAV38-01 gene segment.

[0293] Embodiment 43. The modified immune cell of any one of embodiments 1 to 42, wherein (i) the encoded Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 or 8, and the encoded Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12 or 4; (ii) the encoded Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13 or 5, and the encoded Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9 or 1; (iii) the encoded Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 14 or 6, and the encoded Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10 or 2, or (iv) the encoded Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15 or 7, and the encoded Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11 or 3.

[0294] Embodiment 44. The modified immune cell of any one of embodiments 1 to 43, wherein the encoded binding protein further comprises (i) a TCR alpha chain constant domain (Cα) or a fragment thereof and / or (ii) a TCR beta chain constant domain (Cβ) or a fragment thereof.

[0295] Embodiment 45. The modified immune cell of embodiment 44, wherein the encoded Cα comprises or consists of an amino acid sequence having at least 90% identity to any one of SEQ ID NOs: 41-44.

[0296] Embodiment 46 The modified immune cell of embodiment 44 or 45, wherein the encoded Cβ comprises or consists of an amino acid sequence having at least 90% identity to SEQ ID NO: 45.

[0297] Embodiment 47. The encoded binding protein is (i) a TCR β chain having at least 90% identity to, comprising, or consisting of the amino acid sequence set forth in SEQ ID NO: 61 or 57, and a TCR α chain having at least 90% identity to, comprising, or consisting of the amino acid sequence set forth in SEQ ID NO: 53 or 49; (ii) a TCR β chain having at least 90% identity to, comprising, or consisting of the amino acid sequence set forth in SEQ ID NO: 58 or 54, and a TCR α chain having at least 90% identity to, comprising, or consisting of the amino acid sequence set forth in SEQ ID NO: 50 or 46; (iii) 47. The modified immune cell of any one of embodiments 44 to 46, comprising a TCR beta chain having at least 90% identity to, comprising, or consisting of the amino acid sequence set forth in SEQ ID NO: 59 or 55, and a TCR alpha chain having at least 90% identity to, comprising, or consisting of the amino acid sequence set forth in SEQ ID NO: 51 or 52; or (iv) a TCR beta chain having at least 90% identity to, comprising, or consisting of the amino acid sequence set forth in SEQ ID NO: 60 or 56, and a TCR alpha chain having at least 90% identity to, comprising, or consisting of the amino acid sequence set forth in SEQ ID NO: 52 or 48.

[0298] Embodiment 48. The modified immune cell of any one of embodiments 1 to 47, wherein the encoded binding protein is a TCR, a chimeric antigen receptor (CAR), or a single-chain TCR (scTCR).

[0299] Embodiment 49. The modified immune cell of any one of embodiments 1 to 48, wherein the immune cell is a human immune system cell.

[0300] Embodiment 50. The immune cells are T cells, optionally CD4 + T cells, CD8 + T cells, CD4 - CD8 - 50. The modified immune cell of any one of embodiments 1-49, which is a double-negative T cell or γδ T cell, a natural killer cell, an NK-T cell, a dendritic cell, or any combination thereof.

[0301] Embodiment 51. The modified immune cell of embodiment 50, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, a stem cell memory T cell, or any combination thereof.

[0302] Embodiment 52 The modified immune cell of any one of embodiments 1 to 51, wherein the polynucleotide encoding the binding protein is codon-optimized for expression in the immune cell.

[0303] Embodiment 53. The modified immune cell of any one of embodiments 1 to 52, wherein the polynucleotide encoding the binding protein comprises a polynucleotide having at least 75% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 62 to 77.

[0304] Embodiment 54. The modified immune cell of embodiment 53, wherein the polynucleotide encoding the binding protein comprises: (i) a polynucleotide having at least 75% identity to the polynucleotide sequence set forth in SEQ ID NO: 77 and a polynucleotide having at least 75% identity to the polynucleotide sequence set forth in SEQ ID NO: 69; (ii) a polynucleotide having at least 75% identity to the polynucleotide sequence set forth in SEQ ID NO: 74 and a polynucleotide having at least 75% identity to the polynucleotide sequence set forth in SEQ ID NO: 66; (iii) a polynucleotide having at least 75% identity to the polynucleotide sequence set forth in SEQ ID NO: 75 and a polynucleotide having at least 75% identity to the polynucleotide sequence set forth in SEQ ID NO: 67; or (iv) a polynucleotide having at least 75% identity to the polynucleotide sequence set forth in SEQ ID NO: 76 and a polynucleotide having at least 75% identity to the polynucleotide sequence set forth in SEQ ID NO: 68.

[0305] Embodiment 55. The modified immune cell of any one of embodiments 1 to 54, wherein the polynucleotide encoding the binding protein further comprises a polynucleotide encoding a self-cleaving peptide disposed between the Vβ-encoding polynucleotide and the Vα-encoding polynucleotide.

[0306] Embodiment 56. The modified immune cell of embodiment 55, wherein the encoded self-cleaving peptide comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 84-88.

[0307] Embodiment 57. The modified immune cell of embodiment 55 or 56, wherein the polynucleotide encoding the self-cleaving peptide comprises or consists of a polynucleotide sequence having at least 75% identity to the polynucleotide sequence set forth in any one of SEQ ID NOs: 89-93.

[0308] Embodiment 58. The modified immune cell of any one of embodiments 55 to 57, wherein the polynucleotide encoding the binding protein has a structure from the 5' end to the 3' end of (TCR β chain-encoding polynucleotide)-(self-cleaving peptide-encoding polynucleotide)-(TCR α chain-encoding polynucleotide).

[0309] Embodiment 59. The modified immune cell of any one of embodiments 55-58, wherein the polynucleotide encoding the binding protein comprises a polynucleotide having at least 75% identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 78-81.

[0310] Embodiment 60. The modified immune cell of embodiment 53, wherein the polynucleotide encoding the binding protein comprises a polynucleotide comprising or consisting of a polynucleotide sequence set forth in any one of SEQ ID NOs: 78-81.

[0311] Embodiment 61. The modified immune cell of any one of embodiments 1 to 60, further comprising: (i) a heterologous polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor alpha chain, optionally wherein the encoded polypeptide is or comprises a CD8 co-receptor alpha chain; (ii) a heterologous polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor beta chain, optionally wherein the encoded polypeptide is or comprises a CD8 co-receptor beta chain; or (iii) the polynucleotide of (i) and the polynucleotide of (ii), optionally wherein the host cell comprises a CD4+ T cell.

[0312] Embodiment 62. (a) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, optionally wherein the encoded polypeptide is or comprises the CD8 co-receptor α chain; (b) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor β chain, optionally wherein the encoded polypeptide is or comprises the CD8 co-receptor β chain; and (c) a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide of (a) and the polynucleotide of (b). 62. The host cell of embodiment 61, comprising:

[0313] Embodiment 63. The modified immune cell of any one of embodiments 1-62, wherein the immune cell comprises a chromosomal gene knockout of a PD-1 gene, a LAG3 gene, a TIM3 gene, a CTLA4 gene, an HLA component gene, a TCR component gene, or any combination thereof.

[0314] Embodiment 64. The modified immune cell of embodiment 63, wherein the chromosomal gene knockout comprises a knockout of an HLA component gene selected from the alpha 1 macroglobulin gene, the alpha 2 macroglobulin gene, the alpha 3 macroglobulin gene, the beta 1 microglobulin gene, or the beta 2 microglobulin gene, or any combination thereof.

[0315] Embodiment 65. The modified immune cell of embodiment 63 or 64, wherein the chromosomal gene knockout comprises a knockout of a TCR component gene selected from a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, or any combination thereof.

[0316] Embodiment 66. An isolated polynucleotide encoding a binding protein, wherein the encoded binding protein comprises a TCR Vα domain and a TCR Vβ domain and is capable of binding to the RMFPNAPYL (SEQ ID NO: 94):HLA complex, wherein (i) the encoded Vα domain comprises a CDR3 amino acid sequence set forth in any one of SEQ ID NOs: 28, 19, 22, or 25; (ii) the encoded Vβ domain comprises a CDR3 amino acid sequence set forth in any one of SEQ ID NOs: 40, 31, 34, or 37; (iii) the encoded Vα domain comprises a CDR1 amino acid sequence set forth in any one of SEQ ID NOs: 26, 17, 20, or 23; (iv) the encoded Vβ domain comprises a CDR1 amino acid sequence set forth in any one of SEQ ID NOs: 38, 32, or 35; (v) the encoded Vα domain comprises a CDR1 amino acid sequence set forth in any one of SEQ ID NOs: (vi) the encoded Vβ domain comprises a CDR2 amino acid sequence set forth in any one of SEQ ID NOs: 39, 30, 33, or 36; (vii) the encoded Vα domain comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 4, 1-3, 12, or 9-11; (viii) the encoded Vβ domain comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 8, 5-7, 16, or 13-15; (ix) the encoded TCR (x) an isolated polynucleotide in which the encoded TCR Vβ is contained within a TCR β chain and the TCR β chain comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 53 and 46 to 52; or (xi) any combination of (i) to (x).

[0317] Embodiment 67. The isolated polynucleotide of embodiment 66, wherein the polynucleotide is codon-optimized for expression in a host cell, and optionally the host cell is a human T cell, a human NK cell, or a human NK-T cell.

[0318] Embodiment 68. The isolated polynucleotide of embodiment 66 or 67, wherein the polynucleotide comprises a polynucleotide having at least 75% identity to the polynucleotide sequence set forth in any one of SEQ ID NOs: 62 to 81.

[0319] Embodiment 69. A vector comprising the polynucleotide according to any one of embodiments 66 to 68.

[0320] Embodiment 70. The vector of embodiment 69, which is a lentiviral vector or a retroviral vector.

[0321] Embodiment 71. A host cell comprising a polynucleotide according to any one of embodiments 66 to 68 and capable of expressing the encoded binding protein.

[0322] Embodiment 72. A composition comprising: (i) a modified immune cell according to any one of embodiments 1 to 65; (ii) a polynucleotide according to any one of embodiments 66 to 68; (iii) a vector according to embodiment 69 or 70 and / or (iv) a host cell according to embodiment 71; and a pharmaceutically acceptable carrier, excipient or diluent.

[0323] Embodiment 73. (i) the modified immune cell of any one of embodiments 1 to 65; and (ii) an immune cell comprising a polynucleotide encoding a binding protein comprising a TCR Vα and a TCR Vβ, and capable of specifically binding to a VLDFAPPGA (SEQ ID NO: 100):HLA complex, wherein, optionally, the HLA is HLA-A *0201, and optionally wherein the modified immune cells of (i) and the immune cells of (ii) each independently comprise immune cells selected from T cells, NK cells, and NK-T cells.

[0324] The immune cell binding protein in embodiment 74 (ii) (a) comprises the amino acid sequences of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β of SEQ ID NOs: 101-103 and 105-107, respectively, and optionally (1) Vα has at least 90% identity to, comprises or consists of the amino acid sequence shown in SEQ ID NO: 104, and / or (2) Vβ has at least 90% identity to, comprises or consists of the amino acid sequence shown in SEQ ID NO: 108; (b) comprises the amino acid sequences of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β and CDR3β of SEQ ID NOs: 109-110 and 113-115, respectively, and optionally (1) Vα has at least 90% identity to, comprises or consists of the amino acid sequence shown in SEQ ID NO: 112. and / or (2) Vβ has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 116; or (c) comprises the amino acid sequences of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of SEQ ID NOs: 117-119 and 121-123, respectively, and optionally, (1) Vα has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 120; and / or (2) Vβ has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 124.

[0325] Embodiment 75. A method for treating a subject having a disease or disorder associated with expression of WT-1, comprising administering to the subject an effective amount of: (i) the modified immune cell of any one of embodiments 1 to 65; (ii) the polynucleotide of any one of embodiments 66 to 68; (iii) the vector of embodiment 69 or 70; (iv) the host cell of embodiment 71, and / or (v) the composition of any one of embodiments 72 to 74.

[0326] Embodiment 76. A method for the treatment of a patient comprising administering to a subject who has received, is receiving, or will receive immune cells comprising a polynucleotide encoding a binding protein comprising TCR Vα and TCR Vβ, and capable of specifically binding to the VLDFAPPGA (SEQ ID NO: 100):HLA complex, the modified immune cells of any one of embodiments 1 to 65, wherein the HLA is HLA-A. * 76. The method of embodiment 75, comprising: 0201, and optionally, the modified immune cell and the immune cell are each independently selected from a T cell, an NK cell, and an NK-T cell.

[0327] Embodiment 77. VLDFAPPGA (SEQ ID NO: 100): A binding protein capable of specifically binding to an HLA complex, comprising (a) the amino acid sequences of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of SEQ ID NOs: 101-103 and 105-107, respectively, and optionally, (1) Vα has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 104, and / or (2) Vβ has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 108; (b) the amino acid sequences of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of SEQ ID NOs: 109-110 and 113-115, respectively, and optionally, (1) Vα has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 109 , and / or (2) Vβ has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 112, and / or (3) Vβ has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 116, or (c) comprises the amino acid sequences of CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β of SEQ ID NOs: 117 to 119 and 121 to 123, respectively, and optionally, (1) Vα has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 120, and / or (2) Vβ has at least 90% identity to, comprises, or consists of the amino acid sequence set forth in SEQ ID NO: 124.

[0328] Embodiment 78. The method of any one of embodiments 75 to 77, wherein the disease or disorder is a hematological malignancy or solid cancer.

[0329] Embodiment 79. The method of embodiment 78, wherein the hematological malignancy is selected from acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia (AML, including refractory AML and relapsed AML, including acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelocytic leukemia, acute myelomonocytic leukemia (e.g., with or without eosinophilia), acute monocytic leukemia, acute erythroleukemia, and acute megakaryoblastic leukemia), chronic myeloid leukemia (CML), chronic myelocytic leukemia, chronic eosinophilic leukemia (CEL), myelodysplastic syndromes (MDS), non-Hodgkin's lymphoma (NHL), or multiple myeloma (MM, including refractory MM and relapsed MM).

[0330] Embodiment 80. The solid cancer is selected from the group consisting of bile duct cancer, bladder cancer, bone and soft tissue cancer, brain tumor, breast cancer, breast carcinoma, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, glioblastoma, melanoma, diffuse peritoneal mesothelioma, malignant pleural mesothelioma, glioma, astrocytoma, gynecological tumor, head and neck squamous cell carcinoma, liver cancer, hepatocellular carcinoma, and lung cancer. 79. The method of embodiment 78, wherein the cancer is selected from non-small cell lung cancer, malignant melanoma, osteosarcoma, ovarian cancer (e.g., epithelial ovarian cancer, ovarian carcinoma), fallopian tube cancer, endometrial cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, renal cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, osteogenic sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, uterine carcinosarcoma, or uterine cancer.

[0331] Embodiment 81 The method of any one of embodiments 75 to 80, wherein the modified immune cells are modified ex vivo to contain the heterologous polynucleotide.

[0332] Embodiment 82. The method of any one of embodiments 75-81, wherein the modified immune cells are allogeneic, syngeneic, or autologous to the subject.

[0333] Embodiment 83. The method of any one of embodiments 75 to 82, wherein the modified immune cells are hematopoietic progenitor cells or human immune system cells.

[0334] Embodiment 84. The immune system cells are CD4 + T cells, CD8 + T cells, CD4 - CD8 - 84. The method of embodiment 83, wherein the cells are double-negative T cells, γδ T cells, natural killer cells, dendritic cells, or any combination thereof.

[0335] Embodiment 85. The method of embodiment 84, wherein the T cells are naive T cells, central memory T cells, effector memory T cells, or any combination thereof.

[0336] Embodiment 86 The method of any one of embodiments 75-85, comprising administering multiple doses of the engineered immune cells to the subject.

[0337] Embodiment 87. The method of embodiment 86, wherein the multiple doses are administered with an interval of about 2 to about 4 weeks between doses.

[0338] Embodiment 88. The modified immune cells are 4 cells / kg ~ approx. 10 11 The method of any one of embodiments 75 to 87, wherein the cell line is administered to the subject at a dose of cells / kg.

[0339] Embodiment 89. The method of any one of embodiments 75 to 88, further comprising administering a cytokine to the subject.

[0340] Embodiment 90. The method of embodiment 89, wherein the cytokine is IL-2, IL-15, IL-21, or any combination thereof.

[0341] Embodiment 91 The method of embodiment 90, wherein the cytokine is IL-2 and is administered simultaneously or sequentially with the modified immune cells.

[0342] Embodiment 92. The method of embodiment 91, wherein the cytokines are administered sequentially, provided that the subject has received at least three or four administrations of the modified immune cells prior to administration of the cytokines.

[0343] Embodiment 93. The method of any one of embodiments 89 to 92, wherein the cytokine is IL-2 and is administered subcutaneously.

[0344] Embodiment 94. The method of any one of embodiments 75 to 93, wherein the subject is undergoing immunosuppressive therapy.

[0345] Embodiment 95. The method of embodiment 94, wherein the immunosuppressive treatment is selected from a calcineurin inhibitor, a corticosteroid, a microtubule inhibitor, a low-dose mycophenolic acid prodrug, or any combination thereof.

[0346] Embodiment 96. The method of any one of embodiments 75 to 95, wherein the subject has undergone a non-myeloablative hematopoietic cell transplant or a myeloablative hematopoietic cell transplant.

[0347] Embodiment 97. The method of embodiment 96, wherein the subject is administered the modified immune cells at least 3 months after non-myeloablative hematopoietic cell transplantation.

[0348] Embodiment 98. The method of embodiment 97, wherein the subject is administered the modified immune cells at least 2 months after myeloablative hematopoietic cell transplantation.

[0349] Embodiment 99. The method of any one of embodiments 75 to 98, wherein the subject has previously received treatment for AML.

[0350] Embodiment 100. A composition comprising (i) the modified immune cell of any one of embodiments 1 to 65; (ii) the polynucleotide of any one of embodiments 66 to 68; (iii) the vector of embodiment 69 or 70, and / or (iv) the host cell of embodiment 71, for use in treating a disease or disorder associated with expression of WT-1.

[0351] Embodiment 101. A composition comprising: (i) the modified immune cell of any one of embodiments 1 to 65; (ii) the polynucleotide of any one of embodiments 66 to 68; (iii) the vector of embodiment 69 or 70; and / or (iv) the host cell of embodiment 71, for use in the manufacture of a medicament for the treatment of a disease or disorder associated with expression of WT-1.

[0352] Embodiment 102. A composition for use according to embodiment 100 or 101, wherein the disease or disorder is a hematological malignancy or solid cancer.

[0353] Embodiment 103. The composition for use of embodiment 102, wherein the hematological malignancy is selected from acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia (AML, including refractory AML and relapsed AML, including acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelocytic leukemia, acute myelomonocytic leukemia (e.g., with or without eosinophilia), acute monocytic leukemia, acute erythroleukemia, and acute megakaryoblastic leukemia), chronic myeloid leukemia (CML), chronic myelocytic leukemia, chronic eosinophilic leukemia (CEL), myelodysplastic syndromes (MDS), non-Hodgkin's lymphoma (NHL), or multiple myeloma (MM, including refractory MM and relapsed MM).

[0354] Embodiment 104. The solid cancer is selected from the group consisting of bile duct cancer, bladder cancer, bone and soft tissue cancer, brain tumor, breast cancer, breast carcinoma, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, glioblastoma, melanoma, diffuse peritoneal mesothelioma, malignant pleural mesothelioma, glioma, astrocytoma, gynecological tumor, head and neck squamous cell carcinoma, liver cancer, hepatocellular carcinoma, lung cancer, non-small cell lung cancer, 103. The composition for use of embodiment 102, wherein the cancer is selected from: cell lung cancer, malignant melanoma, osteosarcoma, ovarian cancer (e.g., epithelial ovarian cancer, ovarian carcinoma), fallopian tube cancer, endometrial cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, renal cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, osteogenic sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, uterine carcinosarcoma, or uterine cancer.

[0355] Embodiment 105. A binding protein comprising a T-cell receptor (TCR) alpha chain variable (Vα) domain and a TCR beta chain variable (Vβ) domain, wherein the Vα domain comprises: (i) a CDR3 amino acid sequence (CDR3α) according to any one of SEQ ID NOs: 28, 19, 22, or 25 or 28, or a variant thereof; (ii) a CDR1 amino acid sequence (CDR1α) according to any one of SEQ ID NOs: 26, 17, 20, or 23; (iii) a CDR2 amino acid sequence (CDR2α) according to any one of SEQ ID NOs: 27, 18, 21, or 24; (iv) an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1-4 or 9-12, or (v) any combination of (i) to (iv); Vβ 105. The binding protein of embodiment 105, wherein the domain comprises: (vi) a CDR3 amino acid sequence (CDR3β) according to any one of SEQ ID NOs: 40, 31, 34, or 37, or a variant thereof; (vii) a CDR1 amino acid sequence (CDR1β) according to any one of SEQ ID NOs: 38, 29, 32, or 35; (viii) a CDR2 amino acid sequence (CDR2β) according to any one of SEQ ID NOs: 39, 30, 33, or 36; (ix) an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 5-8 or 13-16, or (x) any combination of (vi) to (x), wherein the binding protein of embodiment 105 is capable of binding to the RMFPNAPYL (SEQ ID NO: 94):HLA complex, and optionally the HLA is HLA-A. * Binding proteins, including 0201.

[0356] Embodiment 106. The binding protein of embodiment 105, further comprising (1) a TCR alpha chain constant domain (Cα) or a fragment thereof and / or (2) a TCR beta chain constant domain (Cβ) or a fragment thereof.

[0357] Embodiment 107. A binding protein according to embodiment 106, wherein Cα comprises or consists of the amino acid sequence of any one of SEQ ID NOs: 41-44.

[0358] Embodiment 108. A binding protein according to embodiment 106 or 107, wherein Cβ comprises or consists of the amino acid sequence of SEQ ID NO: 45.

[0359] Embodiment 109. An isolated polynucleotide encoding a binding protein according to any one of embodiments 105 to 108.

[0360] Embodiment 110. The polynucleotide of embodiment 109, which is codon-optimized for expression in a host cell.

[0361] Embodiment 111. A modified immune cell comprising a polynucleotide according to any one of embodiments 109-110, wherein the polynucleotide is heterologous to the immune cell.

[0362] array

[0363] [ka] TIFF0007737978000002.tif239159TIFF0007737978000003.tif236158TIFF0007737 978000004.tif240159TIFF0007737978000005.tif244162TIFF0007737978000006.t if240162TIFF0007737978000007.tif246163TIFF0007737978000008.tif246160TIF F0007737978000009.tif241161TIFF0007737978000010.tif242163TIFF0007737978 000011.tif243164TIFF0007737978000012.tif243169TIFF0007737978000013.tif2 38163TIFF0007737978000014.tif242161TIFF0007737978000015.tif241157TIFF00 07737978000016.tif232158TIFF0007737978000017.tif238159TIFF0007737978000 018.tif245163TIFF0007737978000019.tif241157TIFF0007737978000020.tif38157 [Example]

[0364] [Example 1] Identification and characterization of TCR specific for WT-1 peptide CD8 from four donors +T cells were cultured with autologous dendritic cells (DCs) pulsed with the WT-1 peptide RMFPNAPYL (SEQ ID NO: 94) (Ho, Greenberg et al., 2006). T cell lines were combined with CD8-independent (CD8i) WT-1 peptide:HLA tetramers, stained, and sorted for high tetramer-staining cells. A total of 40 polyclonal T cell lines with high tetramer staining were identified. To quantify each TCR clonotype, both the sorted T cell samples and a fraction of the total unsorted sample were analyzed by TCR repertoire analysis (Adaptive Biotechnologies). The fraction of high tetramer-staining cells was also analyzed by single-cell RNA sequencing and TCR pairing (10X genomics). By comparing the fold enrichment of each clonotype within the sorted population with that of the unsorted population and by measuring CD3 surface expression by each clonotype, 24 TCR clonotypes were identified as WT-1-specific (Figure 1). Of these, 10 TCR clonotypes were selected for synthesis. TCRα / TCRβ-deficient J76 Jurkat T cells were transduced to express the candidate TCRs. The affinity of each candidate TCR for WT-1 was assessed by measuring relative WT-1 peptide:HLA tetramer staining relative to CD3 expression, a marker for total surface expression of the transgenic TCR. Results were compared with those from cells expressing a WT-1 peptide:HLA-specific TCR with an α chain according to SEQ ID NO: 82 and a β chain according to SEQ ID NO: 83. Data are shown in Figures 2A-2C.

[0365] TCR-transduced Jurkat cells expressing the Nur77-tomato activation marker were assessed for activation in the presence of peptide, and the data are shown in Figure 3A.

[0366] CD8+ T cells isolated from donor PMBCs were transduced to express one of eight TCRs, which were selected for further study. One week later, transduced cells were sorted for WT-1 peptide:HLA tetramer staining and CD8 expression (Figure 4A). + CD8 +T cells were expanded and assayed for IFNγ production. Expanded antigen-specific cells were cultured with T2 target cells pulsed with titrated concentrations of WT-1 peptide for 4 hours, and IFNγ production was determined by flow cytometry (Figure 4B). For each TCR, the EC200 / EC2000 response to the peptide was 0.01. 50 To calculate the ρ, the percentage of IFNγ-producing cells was fitted to a dose-response curve by nonlinear regression (Fig. 4C).

[0367] EC 50 T cells expressing the two lowest TCRs were compared with T cells expressing a WT-1-specific reference TCR for their ability to induce tumor cell lysis. + In the tumor cell line Panc-1, 51 TCR-transduced T cell-mediated killing was calculated by measuring the release of Cr in response to decreasing doses of effector T cells against tumor cell targets (Figures 5A and 5B).

[0368] T cells expressing the exemplary TCR DL10 were further explored for IFN-γ production ( FIG. 6 ) and their ability to reduce tumor cell line proliferation ( FIG. 7 ). For IFN-γ production, primary CD8+ T cells were transduced with a reference TCR or TCR DL10 and cultured with T2 target cells pulsed with the peptides indicated in FIG. 6 . After 4 hours, IFN-γ production was assessed by intracellular flow cytometry, and percent IFN-γ positivity was fitted to a dose-response curve by nonlinear regression in Graphpad Prism. To assess the ability to control tumor growth, a reference TCR (comprising a TCR α chain having the amino acid sequence set forth in SEQ ID NO: 82 and a TCR β chain having the amino acid sequence set forth in SEQ ID NO: 83) or TCRDL10 was transduced into primary CD8+ T cells and cultured in triplicate at an 8:1 ratio with the HLA-A2+ WT1+ pancreatic tumor cell line, Panc-1 (transduced to express red fluorescent protein) in an IncuCyte assay, and cell proliferation / survival was monitored over a 7-day period. Error bars indicate the standard error of the mean.

[0369] The various embodiments described above can be combined to produce further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the Application Data Sheet, including U.S. Provisional Patent Application No. 62 / 889,519, filed August 20, 2019, are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to utilize concepts from the various patents, applications, and publications to produce still further embodiments.

[0370] These and other changes can be made to the embodiments in light of the above Detailed Description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but rather to include all possible embodiments, along with the full range of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

1. A binding protein comprising a T cell receptor (TCR) alpha chain variable (Vα) domain and a TCR beta chain variable (Vβ) domain, wherein the Vα and Vβ domains comprise the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NOs:26-28 and 38-40, respectively, and wherein the binding protein is capable of binding to the RMFPNAPYL (SEQ ID NO:94):HLA complex. The binding protein.

2. The HLA is HLA-A ＊ 0201. The binding protein of claim 1 .

3. (i) the Vβ domain comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 16 or 8; and / or (ii) the Vα domain comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 12 or 4; The binding protein according to claim 1 or 2.

4. (1) (i) the Vβ domain comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 16; and (1)(ii) the Vα domain comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 12; or (2) (i) the Vβ domain comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 8; and (2) (ii) the Vα domain comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 4; The binding protein of claim 3.

5. 5. The binding protein of any one of claims 1 to 4, wherein the Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 16 or 8, and the Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 4 or 12.

6. 6. The binding protein of claim 5, wherein the Vβ domain comprises the amino acid sequence set forth in SEQ ID NO: 16 and the Vα domain comprises the amino acid sequence set forth in SEQ ID NO:

12.

7. (i) the TCR alpha chain constant domain (Cα) or a fragment thereof, and / or (ii) TCR β chain constant domain (Cβ) or a fragment thereof The binding protein of any one of claims 1 to 6, further comprising:

8. 8. The binding protein of claim 7, wherein the Cα comprises or consists of an amino acid sequence having at least 90% identity to SEQ ID NO: 44 and / or the Cβ comprises or consists of an amino acid sequence having at least 90% identity to SEQ ID NO:

45.

9. 9. The binding protein of claim 8, wherein the Cα comprises or consists of the amino acid sequence set forth in SEQ ID NO: 44 and the Cβ comprises or consists of the amino acid sequence set forth in SEQ ID NO:

45.

10. 10. The binding protein of any one of claims 7 to 9, wherein the binding protein is a TCR comprising a TCR beta chain having at least 90% identity to, comprising, or consisting of the amino acid sequence set forth in SEQ ID NO: 61 or 57, and a TCR alpha chain having at least 90% identity to, comprising, or consisting of the amino acid sequence set forth in SEQ ID NO: 53 or 49.

11. The binding protein is (1) (i) a TCR β chain comprising the amino acid sequence set forth in SEQ ID NO: 61; and (1) (ii) a TCRα chain comprising the amino acid sequence set forth in SEQ ID NO: 53; or (2) (i) a TCR β chain consisting of the amino acid sequence set forth in SEQ ID NO: 61; and (2) (ii) a TCRα chain consisting of the amino acid sequence shown in SEQ ID NO: 53 The binding protein of any one of claims 7 to 10, which is a TCR comprising:

12. An isolated polynucleotide encoding the binding protein of any one of claims 1 to 11.

13. 13. The polynucleotide of claim 12, wherein the polynucleotide is codon-optimized for expression in a host cell.

14. The polynucleotide of claim 12 or 13, wherein the polynucleotide further comprises a polynucleotide encoding a self-cleaving peptide positioned between the polynucleotide encoding the Vβ and the polynucleotide encoding the Vα or between the polynucleotide encoding the TCRβ chain and the polynucleotide encoding the TCRα chain.

15. 15. The polynucleotide of claim 14, wherein the encoded self-cleaving peptide comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 84-88.

16. The polynucleotide of any one of claims 12 to 15, comprising a polynucleotide having at least 90% identity to the polynucleotide sequence set forth in any one of SEQ ID NOs: 65, 69, 73, 77, and 81.

17. 17. The polynucleotide of claim 16, wherein the polynucleotide comprises or consists of the polynucleotide sequence set forth in SEQ ID NO:

81.

18. A vector comprising the polynucleotide according to any one of claims 12 to 17.

19. 19. The vector of claim 18, wherein the vector is a lentiviral vector or a retroviral vector.

20. A host cell comprising a polynucleotide according to any one of claims 12 to 17 and capable of expressing the encoded binding protein.

21. 21. The host cell of claim 20, wherein the host cell is an immune cell and the polynucleotide is heterologous to the immune cell.

22. The host cell of claim 20 or 21, wherein the host cell is a hematopoietic stem cell, a hematopoietic progenitor cell, or a T cell.

23. (i) the host cell is (1) a T cell, (2) a natural killer cell, (3) an NK-T cell, (4) a dendritic cell, or (5) any combination thereof; and / or (ii) the host cell is a human T cell; 22. The host cell of claim 21.

24. The host cell is a T cell, and the T cell is a CD4 ＋ T cells, CD8 ＋ T cells, CD4 － CD8 － 24. The host cell of claim 23, wherein the T cell is a double-negative T cell or a γδ T cell, and / or the T cell is a naive T cell, a central memory T cell, an effector memory T cell, a stem cell memory T cell, or any combination thereof.

25. 25. The host cell of any one of claims 20-24, wherein the immune cell comprises a chromosomal gene knockout of a PD-1 gene, a LAG3 gene, a TIM3 gene, a CTLA4 gene, an HLA component gene, a TCR component gene, or any combination thereof.

26. (i) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α-chain; (ii) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta chain; or (iii) the polynucleotide of (i) and the polynucleotide of (ii) The host cell of any one of claims 20 to 25, further comprising:

27. (i) a heterologous polynucleotide encoding a polypeptide that is or comprises a CD8 co-receptor alpha chain, and / or (ii) a heterologous polynucleotide encoding a polypeptide that is or comprises a CD8 co-receptor beta chain. The host cell of any one of claims 20 to 25, further comprising:

28. The host cells are CD4 ＋ 28. The host cell of claim 26 or 27, comprising a T cell.

29. the host cell is an immune cell modified to contain the polynucleotide; (i) the encoded binding protein has an IFNγ-producing pEC of 4.0 or greater, 4.5 or greater, 5.0 or greater, 5.5 or greater, 6.0 or greater, or 6.5 or greater. ５０ and / or (ii) the host cells are immune cells modified to contain the polynucleotide, and the population of modified immune cells is cultured at a peptide concentration of 10 －４ μM, 10 －５ μM, or 10 －６ When co-cultured with antigen-presenting cells pulsed with a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 94) at 2 μM for 4 hours, 50% or more of the modified immune cells in the population produce IFN-γ, and the antigen-presenting cells comprise T2 cells. A host cell according to any one of claims 20 to 28.

30. the host cell is an immune cell modified to contain the polynucleotide; (i) the population of modified immune cells is cultured at a peptide concentration of 10 －２ When co-cultured with antigen-presenting cells pulsed with a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 94) at 1 μM, 10% or more, 15% or more, 20% or more, 40% or more, or 50% or more of the modified immune cells in the population express Nur77. ＋ wherein the antigen-presenting cells comprise T2 cells, Jurkat cells, or both; and / or (ii) the population of modified immune cells is cultured at a peptide concentration of 10 －３ When co-cultured with antigen-presenting cells pulsed with a peptide comprising or consisting of the amino acid sequence RMFPNAPYL (SEQ ID NO: 94) at 1 μM, 10% or more, 15% or more, 20% or more of the modified immune cells in the population express Nur77. ＋ That is, A host cell according to any one of claims 20 to 29.

31. the host cell is an immune cell modified to contain the polynucleotide; (i) When the modified immune cells and MDA-MB-468 cells are present in a sample at a ratio of 30:1, the modified immune cells ＊ 0201 ＋ capable of killing 11% or more, 12% or more, 14% or more, 15% or more, 20% or more, or 25% or more of MDA-MB-468 cells; (ii) When the modified immune cells and MDA-MB-468 cells are present in a sample at a ratio of 10:1, the modified immune cells ＊ 0201 ＋ is capable of killing 10% or more of MDA-MB-468 cells; and / or (iii) When the modified immune cells and MDA-MB-468 cells are present in a sample at a ratio of 1:1, the modified immune cells are ＊ 0201 ＋ It is capable of killing 5% or more of MDA-MB-468 cells. A host cell according to any one of claims 20 to 30.

32. The host cell of any one of claims 20 to 31, wherein the host cell is an immune cell modified to contain the polynucleotide, and when the modified immune cell and Panc1 cells are present in a sample at a ratio of 30:1, the modified immune cell is capable of killing 21% or more, 22% or more, 23% or more, 24% or more, or 25% or more of Panc1 cells.

33. the host cell is an immune cell modified to contain the polynucleotide, and the modified immune cell exhibits a T cell receptor comprising a TCR α chain having the amino acid sequence set forth in SEQ ID NO: 82 and a TCR β chain having the amino acid sequence set forth in SEQ ID NO: 83 in a co-culture for 4 hours, compared to a reference immune cell comprising a polynucleotide encoding a T cell receptor comprising a TCR α chain having the amino acid sequence set forth in SEQ ID NO: 82 and a TCR β chain having the amino acid sequence set forth in SEQ ID NO:

83. ＊ 0201 ＋ The host cell of any one of claims 20 to 32, which has increased killing activity against MDA-MB-468 cells and / or Panc1 cells.

34. 34. The host cell of any one of claims 20 to 33, wherein the host cell is an immune cell modified to contain the polynucleotide, and the encoded binding protein is capable of binding to a WT-1 peptide:HLA complex on the cell surface in a CD8-independent manner or in the absence of CD8.

35. (i) a binding protein according to any one of claims 1 to 11; (ii) a polynucleotide according to any one of claims 12 to 17; (iii) a vector according to claim 18 or 19, or (iv) a host cell according to any one of claims 20 to 34, and a pharmaceutically acceptable carrier, excipient, or diluent. A composition comprising:

36. A pharmaceutical composition for use in a method for treating a subject having a disease or disorder associated with WT-1 expression, comprising the binding protein of any one of claims 1 to 11, the polynucleotide of any one of claims 12 to 17, the vector of claim 18 or 19, the host cell of any one of claims 20 to 34, or the composition of claim 35.

37. 37. The pharmaceutical composition of claim 36, wherein the disease or disorder is a hematological malignancy or solid cancer.

38. 38. The pharmaceutical composition of claim 37, wherein the hematological malignancy is selected from acute lymphoblastic leukemia (ALL), acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloid leukemia (AML, including refractory AML and relapsed AML, and including acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelocytic leukemia, acute myelomonocytic leukemia (e.g., with or without eosinophilia), acute monocytic leukemia, acute erythroleukemia, and acute megakaryoblastic leukemia), chronic myeloid leukemia (CML), chronic myelocytic leukemia, chronic eosinophilic leukemia (CEL), myelodysplastic syndromes (MDS), non-Hodgkin's lymphoma (NHL), or multiple myeloma (MM, including refractory MM and relapsed MM).

39. The solid cancer is selected from the group consisting of bile duct cancer, bladder cancer, bone and soft tissue cancer, brain tumor, breast cancer, breast carcinoma, cervical cancer, colon cancer, colorectal adenocarcinoma, colorectal cancer, desmoid tumor, embryonal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, glioblastoma, melanoma, diffuse peritoneal mesothelioma, malignant pleural mesothelioma, glioma, astrocytoma, gynecological tumor, head and neck squamous cell carcinoma, liver cancer, hepatocellular carcinoma, lung cancer, non-small cell lung cancer, and ovarian cancer.

38. The pharmaceutical composition of claim 37, wherein the cancer is selected from: cell lung cancer, malignant melanoma, osteosarcoma, ovarian cancer (e.g., epithelial ovarian cancer, ovarian carcinoma), fallopian tube cancer, endometrial cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, kidney cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, osteogenic sarcoma, testicular germ cell tumor, urothelial cancer, uterine sarcoma, uterine carcinosarcoma, or uterine cancer.

40. 40. The pharmaceutical composition of any one of claims 36-39, wherein the subject has been administered, is being administered, or will be administered immune cells comprising a polynucleotide encoding a binding protein that comprises TCRVα and TCRVβ and is capable of specifically binding to the VLDFAPPGA (SEQ ID NO: 100):HLA complex.

41. the binding protein capable of specifically binding to the VLDFAPPGA (SEQ ID NO: 100):HLA complex is (a) the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NOs: 101-103 and 105-107, respectively; (b) the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NOs: 109-110 and 113-115, respectively; or (c) the CDR1α, CDR2α, CDR3α, CDR1β, CDR2β, and CDR3β amino acid sequences set forth in SEQ ID NOs: 117-119 and 121-123, respectively.

41. The pharmaceutical composition of claim 40, comprising:

42. 42. The pharmaceutical composition of any one of claims 36-41, wherein the method comprises administering the modified immune cells to the subject in multiple doses.

43. 43. The pharmaceutical composition of claim 42, wherein the multiple doses are administered at intervals of about 2 to about 4 weeks.

44. The host cells are ４ cells / kg to about 10 １１ The pharmaceutical composition of any one of claims 36 to 43, wherein the composition is administered to the subject at a dose of cells / kg.

45. The pharmaceutical composition of any one of claims 36 to 44, further comprising administering to the subject a cytokine.

46. 46. ​​The pharmaceutical composition of claim 45, wherein the cytokine is IL-2, IL-15, IL-21, or any combination thereof.

47. The pharmaceutical composition of any one of claims 36 to 46, wherein the subject is further receiving immunosuppressive treatment.

48. 48. The pharmaceutical composition of claim 47, wherein the immunosuppressive treatment is selected from a calcineurin inhibitor, a corticosteroid, a microtubule inhibitor, a low dose of a mycophenolic acid prodrug, or any combination thereof.

49. The pharmaceutical composition according to any one of claims 36 to 48, wherein the subject has undergone non-myeloablative hematopoietic cell transplantation or myeloablative hematopoietic cell transplantation.

50. The method further comprises administering to the subject an immune checkpoint inhibitor. The pharmaceutical composition according to any one of claims 36 to 49.

51. (i) the immune checkpoint inhibitor is an inhibitor of CTLA4; (ii) the immune checkpoint inhibitor is selected from ipilimumab, tremelimumab, CTLA4-Ig fusion protein, or any combination thereof; (iii) the immune checkpoint inhibitor is a PD-1 inhibitor, and / or (iv) The pharmaceutical composition of claim 50, wherein the immune checkpoint inhibitor is selected from pidilizumab, nivolumab, pembrolizumab, MEDI0680 (formerly known as AMP-514), AMP-224, BMS-936558, or any combination thereof.

52. 35. A method of making a host cell according to any one of claims 20 to 34, comprising ex vivo transfecting or transducing said host cell with a polynucleotide according to any one of claims 12 to 17.

53. 53. The method of claim 52, further comprising introducing a chromosomal gene knockout into said host cell ex vivo, wherein said chromosomal gene knockout comprises a knockout of a TCR component gene selected from a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, or any combination thereof.

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