High avidity WT1 T cell receptor and uses thereof

Abstract

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

Description

[Background technology]

[0001] [Background technology] Adoptive T cell immunotherapy with engineered T cells using antibody-based chimeric receptors to target tumor-associated antigens 1-3 or high affinity TCR 4-8 Several trials using high-affinity antigen receptors such as IgG1 and IgG2a have shown promise. Although the natural process of generating diversity in the thymus utilizes RAG-driven TCR gene rearrangements to generate highly diverse CDR3s that differ in length and amino acid composition, isolating effective high-affinity TCRs within the affinity limits imposed by central immune tolerance remains a challenge in implementing adoptive T cell immunotherapy for a variety of malignancies where candidate intracellular self / tumor antigens have been identified. 9,10 Additionally, TCR adoptive immunotherapy can detect intracellular antigens presented on the cell surface by MHC class I.

[0002] The WT1 protein is an attractive target for clinical development due to its immune signature (Cheever et al., Clin. Cancer Res. 15:5323, 2009) and its expression in many aggressive tumor types leading to poor prognosis. WT1 is involved in regulating gene expression that promotes proliferation and carcinogenesis (Oji et al., Jpn. J. Cancer Res. 90:194, 1999) and is overexpressed in most high-risk leukemias (Menssen et al., Leukemia 9:1060, 1995), up to 80% of NSCLCs (Oji et al., Int. J. Cancer 100:297, 2002), 100% of mesotheliomas (Tsuta et al., App. Immunohistochem. Mol. Morpho! 17:126, 2009), and ≥80% of gynecological malignancies (Coosemans and Van Gool, Expert Rev. Clin. Immunol. 10:705, 2014). Several peptides of the WT1 protein have been found to be tumor-associated antigen peptides, which are HLA-A*0201-restricted antigens. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Cheever et al.,Clin.Cancer Res.15:5323,2009 [Non-Patent Document 2] Oji et al.,Jpn.J.Cancer Res.90:194,1999 [Non-Patent Document 3] Menssen et al., Leukemia 9:1060,1995 [Non-Patent Document 4] Oji et al.,Int.J.Cancer 100:297,2002 [Non-Patent Document 5] Tsuta et al.,App.Immunohistochem.Mol.Morphol.17:126,2009 [Non-Patent Document 6] Coosemans and Van Gool,Expert Rev.Clin.Immunol.10:705,2014 Summary of the Invention [Problem to be solved by the invention]

[0004] There is a clear need for alternative, highly WT1 antigen-specific TCR immunotherapy methods for various cancers, including leukemia and tumors. [Means for solving the problem]

[0005] The embodiments disclosed herein address these needs and provide other related advantages. [Brief description of the drawings]

[0006] [Figure 1A]1A and 1B show how WT137-specific TCRs were identified by a high-throughput sequencing strategy. (A) Schematic of the initial sequencing strategy to identify TCR clonotypes with high binding to the WT137-45 peptide / MHC tetramer. [Figure 1B] 1A and 1B show how WT137-specific TCRs were identified by a high-throughput sequencing strategy. (B) The fold enrichment of the sorted population relative to the percentage of the total population is shown, highlighting selected TCRs. All TCRs, indicated by black circles, were synthesized and evaluated for antigen specificity (27 in total). [Diagram 2] Shown are the results of functional assessment of TCR binding with high levels of CD8-independent (CD8i) tetramers. TCR constructs were expressed in Jurkat cells lacking endogenous TCR α / β chains. Tetramer staining for CD3 expression of each TCR is shown (CD3 expression directly correlates with transgenic TCR surface expression). [Figure 3A] 3A-3C show how a modified high-throughput sequencing strategy using CD8-independent (CD8i) tetramers identified other WT137-specific TCRs. (A) Schematic of the modified sequencing strategy to identify TCR clonotypes with high binding to CD8-independent WT137 peptide / MHC tetramers. [Figure 3B] Figures 3A-3C show how other WT137-specific TCRs were identified by a modified high-throughput sequencing strategy using CD8-independent (CD8i) tetramers. (B) Fold enrichment of the initial sorted population as a percentage of the total population and (C) a similar analysis using CD8i tetramers are shown. An additional 14 TCRs were selected based on reduced surface CD3 levels and binding to CD8i tetramers. All TCRs, indicated by shaded circles, were synthesized and assessed for antigen specificity. [Figure 3C]Figures 3A-3C show how other WT137-specific TCRs were identified by a modified high-throughput sequencing strategy using CD8-independent (CD8i) tetramers. (B) Fold enrichment of the initial sorted population as a percentage of the total population and (C) a similar analysis using CD8i tetramers are shown. An additional 14 TCRs were selected based on reduced surface CD3 levels and binding to CD8i tetramers. All TCRs, indicated by shaded circles, were synthesized and assessed for antigen specificity. [Figure 4] CD8i tetramer binding of select WT137 TCRs is shown. TCR constructs were expressed in Jurkat cells lacking endogenous TCR α / β chains. Tetramer staining for CD3 expression of each TCR is shown (CD3 expression directly correlates with transgenic TCR surface expression). [Figure 5A] Figures 5A and 5B show the calculation of peptide EC50 in IFNγ assays when primary CD8+ PBMCs were transduced with selected TCRs. (A) CD8+ T cells isolated from donor PBMCs were transduced with selected TCRs. One week later, tetramer+ CD8+ T cells were sorted from the cells and expanded. Expanded antigen-specific cells were co-cultured with peptide-pulsed T2 target cells for 4-6 h, and IFNγ production was measured by flow cytometry. [Figure 5B] Figures 5A and 5B show the calculation of peptide EC50 in the IFNγ assay when selected TCRs were transduced into primary CD8+ PBMCs. (B) The percentage of IFNγ producing cells was fitted to a dose-response curve by non-linear regression to calculate the peptide EC50 for each TCR. [Figure 6]We show that primary CD8+ T cells expressing WT137-specific TCR efficiently kill the WT1+HLA-A2+ breast cancer cell line MDA-MB-468. Primary CD8+ T cells were transduced with the TCR, purified by selection for high tetramer binding, and mixed (in triplicate) at an 8:1 ratio with the breast cancer cell line MDA-MB-468 that had been pre-stained with CytoLight® Rapid Red dye. The total red object area (correlating to the total number of live target cells) was calculated over 72 hours for each TCR-transduced T cell population at the indicated time points. To assess the ongoing responsiveness of TCR-transduced T cells to persistent antigen, MDA-MB-468 cells were added after 48 hours. [Figure 7] We show that both CD4+ and CD8+ T cells expressing TCR10.1 can eliminate the WT1+A2+ pancreatic adenocarcinoma cell line PANC-1 after repeated in vitro challenge. Both CD4+ and CD8+ T cells were transduced to express WT137TCR10.1. CD4+ T cells were additionally transduced to express CD8α and CD8β genes. After 8 days, transduced cells were sorted and CD8+ tetramer+ T cells and CD4+ / CD8+ tetramer+ T cells were purified. Antigen-specific cells that were CD4+ / CD8+, CD8+ or a mixture of these two populations (CD4 and CD8) were mixed (in triplicates) at an 8:1 ratio with the pancreatic adenocarcinoma cell line PANC-1 that had previously been transduced to express NucLight® Red dye. The total red object area (correlated to the total number of live target cells) was calculated for each TCR-transduced T cell population at the indicated time points. PANC-1 cells were added after 48 hours to assess the ongoing responsiveness of TCR-transduced T cells to persistent antigen. [Figure 8]8A-8D show a comparison of tumor cell line killing by T cells transduced with the WT1p126 peptide-specific C4TCR of Schmitt et al. (Nat. Biotechnol. 35:1188, 2017) and killing by T cells transduced with the WT1p37 peptide-specific TCR (WT137-45TCR15.1) of the present disclosure. Note that the C4TCR has a lower affinity for the peptide:MHC complex compared to the WT1p37 peptide-specific TCR of the present disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] [Detailed Description] The present disclosure provides a WT1-derived antigen peptide (also known as WT1) consisting of amino acids 37-45 associated with the major histocompatibility complex (MHC) (e.g., human leukocyte antigen, HLA). 37-45 The present invention provides a T cell receptor (TCR) having high functional avidity for a peptide or p37 peptide antigen (e.g., VLDFAPPGA, SEQ ID NO: 59). Such p37 peptide antigen-specific TCRs are useful in adoptive immunotherapy to treat cancers, such as cancers that overexpress WT1.

[0008] By way of background, because tumors arise from previously normal tissues, the majority of tumor targets for T cell immunotherapy are self-antigens. For example, such tumor-associated antigens (TAAs) may be expressed at high levels on cancer cells but not, or only minimally, on other cells. During T cell development in the thymus, T cells that bind weakly to self-antigens are able to survive and further develop and mature within the thymus, whereas T cells that bind strongly to self-antigens are eliminated by the immune system as they generate undesirable autoimmune responses. Thus, T cells are selected for 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 natural T cells that can recognize tumor (self) antigens with high affinity, thereby eliminating T cells that can effectively eliminate tumor cells. As a result, it is difficult to isolate T cells with high-affinity TCRs specific for tumor antigens, as these cells are essentially eliminated by the immune system in large part.

[0009] In the present disclosure, a high-throughput sequencing approach was applied to immune cells from approximately 15 healthy donors to identify TCRs with high functional avidity for the p37:MHC complex. This strategy allows for the selection of TCRs even when the TCR expression level on the T cell surface is low. Enrichment of the sorting population relative to the percentage of the total population was used to select the TCRs of the present disclosure that have high affinity and functional avidity and are specific for p37 (i.e., those with the greatest anti-tumor effect) and their compositions. The T cells in which such TCRs with high functional avidity and specificity for p37 were identified were those that (a) bound p37 peptide / MHC tetramers in a CD8-independent manner, (b) underwent low in vitro expansion with peptide, and (c) in some cases expressed such TCRs at relatively low levels on the T cell surface compared to the expression of other TCRs on T cells without such characteristics. A total of 27 TCRs were synthesized and evaluated for p37 antigen specificity (see FIG. 1B).

[0010] In some embodiments, the T cell receptor (TCR) specific to the WT1 peptide includes a TCR α chain and a TCR β chain, and the TCR α chain includes a V α and an α chain constant domain having the amino acid sequence of SEQ ID NO: 47, and the TCR β chain comprises a V domain having an amino acid sequence set forth in any one of SEQ ID NOs: 253 to 263 and 23 to 33. β and a β chain constant domain having the amino acid sequence of SEQ ID NO: 45 or 46, such a TCR specifically binds to the VLDFAPPGA (SEQ ID NO: 59): human leukocyte antigen (HLA) complex on the surface of a T cell and binds to pEC 50 In some embodiments, the selected TCR promotes IFNγ production such that the IFNγ expression level is about 10 -8 K below M D or the high affinity TCR specifically binds to the VLDFAPPGA (SEQ ID NO:59): human leukocyte antigen (HLA) complex, or the high affinity TCR has a lower k compared to the TCR disclosed in Schmitt et al., Nat. Biotechnol. 35:1188, 2017. off It dissociates from the VLDFAPPGA (SEQ ID NO:59):HLA complex at high speed.

[0011] The compositions and methods described herein, in certain embodiments, will have therapeutic utility in treating diseases and conditions associated with WT1 expression or overexpression (e.g., detectable WT1 expression at levels statistically significantly higher than levels of WT1 expression detectable in normal or non-diseased cells). Such diseases include various hyperproliferative or proliferative disorders, such as hematopoietic malignancies and solid cancers. Non-limiting examples of these and related uses are described herein, including, but not limited to, WT1 peptides (e.g., VLDFAPPGA (SEQ ID NO: 59), also known as WT1 37-45 Examples of the method include in vitro, ex vivo, and in vivo stimulation of WT1 antigen-specific T cell responses, such as by using recombinant T cells expressing a high affinity TCR specific for the WT1 peptide or p37 peptide.

[0012] Before describing this disclosure in more detail, it is believed to be helpful to an understanding of the disclosure to provide definitions of certain terms used in the application. Additional definitions are set forth throughout this disclosure.

[0013] In the present description, all concentration ranges, percentage ranges, ratio ranges, or integer ranges are to be understood as including all integer values ​​within the stated range, and fractions thereof (such as tenths or hundredths of integers), where appropriate, unless otherwise specified. Additionally, all numerical ranges stated herein for any physical characteristic, such as polymer subunits, size, or thickness, are to be understood as including all integers within the stated range, unless otherwise specified. As used herein, the term "about" means ±10% of the specified range, number, or structure, unless otherwise specified. It should be understood that the indefinite article, as used herein, means "one or more" of the recited element. The use of alternatives (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "comprise", "have", and "comprise" are used interchangeably, and these terms and variations thereof should be construed as non-limiting.

[0014] Moreover, it will be understood that individual compounds or groups of compounds resulting from the various combinations of structures and substituents described herein are hereby disclosed to the same extent as if each compound or group of compounds was individually described, and thus, selection of a particular structure or particular substituents is within the scope of the disclosure.

[0015] The term "consisting essentially of" is not synonymous with "comprising" and means materials or steps specifically recited in a claim, or equivalent materials or steps to the extent that they do not materially affect the essential characteristics of the claimed subject matter. For example, a protein domain, region or module (e.g. a binding domain, hinge region, linker module) or a protein (which may have one or more domains, regions or modules) "consists essentially of" a particular amino acid sequence when the amino acid sequence of the domain, region, module or protein contains extensions (e.g. at the amino or carboxy termini or between domains), deletions, mutations or combinations thereof, which extensions etc. amount to up to 20% (e.g. up to 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of the domain, region, module or protein and do not substantially affect the activity of said domain, region, module or protein (e.g. the target binding affinity of a binding protein) (i.e. the activity is reduced by 50% or less, e.g. by 40% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less or 1% or less).

[0016] In some embodiments, the term "immune system cell" as used herein refers to any cell of the immune system derived from hematopoietic stem cells in the bone marrow that differentiate into two major lineages: myeloid progenitor cells (which differentiate into myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) and lymphoid progenitor cells (which differentiate into lymphoid cells such as T cells, B cells, and natural killer (NK) cells). Exemplary immune system cells include CD4+ T cells, CD8+ T cells, CD4-CD8- double negative T cells, γδ T cells, regulatory T cells, stem cell memory T cells, natural killer cells (e.g., NK cells or NK-T cells), B cells, and dendritic cells. Macrophages and dendritic cells are sometimes 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 APCs complexed with peptides interact with TCRs on the surface of T cells.

[0017] "Major histocompatibility complex (MHC)" in some embodiments can refer to glycoproteins that deliver peptide antigens to the cell surface. MHC class I molecules are heterodimers with a transmembrane α chain (with three α domains) and non-covalently associated β2-microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, α and β, both of which span the membrane. Each chain has two domains. MHC class I molecules deliver peptides from the cytosol to the cell surface, and the peptide:MHC complex is expressed by CD8 + MHC class II molecules deliver peptides from the vesicle system to the cell surface and are recognized by CD4 + Recognized by T cells. Human MHC is called human leukocyte antigen (HLA).

[0018] A "T cell" or "T lymphocyte" is a cell of the immune system that matures in the thymus and produces a T cell receptor (TCR). T cells are classified as naive T cells (e.g., T cells that have never been exposed to an antigen). CM These cells have higher expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and lower expression of CD45RO compared to normal T cells. M ) (e.g., have encountered antigen and are long-lived), and effector cells (have encountered antigen and are cytotoxic). M Furthermore, central memory T cells (T CM For example, compared to naive T cells, they have higher expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and lower expression of CD54RA) and effector memory T cells (T EM , e.g., naive T cells or T CM These subsets can be divided into effector T cell (T) subsets that display phenotypes or markers associated with T cells (lower expression of CD62L, CCR7, CD28, and CD45RA, and higher expression of CD127 compared to T cells). E ) are CD8 cells that have encountered an antigen + Cytotoxic T lymphocytes, T CMIt can refer to T lymphocytes that have lower expression of CD62L, CCR7, and CD28 compared to helper T cells, and are positive for granzymes and perforin. H ) is CD4, which releases cytokines that affect the activity of other immune cells. + Examples of cells include CD4 + T cells can activate or suppress adaptive immune responses, depending on the presence of other cells and signals. T cells can be harvested using known techniques and various subpopulations or combinations thereof can be enriched or depleted by known techniques such as affinity binding with antibodies, flow cytometry, or immunomagnetic selection. Other representative T cells include regulatory T cells such as Tr1, Th3, CD8+CD28-, and Qa-1 restricted T cells, as well as CD4+CD25+(Foxp3+) regulatory T cells and Treg17 cells.

[0019] By "T cell receptor (TCR)" in some embodiments is meant a member of the immunoglobulin superfamily capable of specifically binding to an antigenic peptide bound to an MHC receptor (having a variable binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail; see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3 rd Ed., Current Biology Publications, p. 4:33, 1997). In some embodiments, TCR refers to a binding protein comprising two TCR variable domains (Vα and Vβ) of the present disclosure. In some embodiments, the TCR comprises a single chain TCR (i.e., a single chain fusion protein comprising the TCR variable domains of the present disclosure), or a CAR comprising the TCR variable domains of the present disclosure (as described herein). In some embodiments, the TCR may be present on the surface of a cell or in a soluble form, and is generally composed of a heterodimer having an α chain and a β chain (also referred to as the TCR α chain and the TCR β chain, respectively) or a γ chain and a δ chain (also referred to as the TCR γ and TCR δ, respectively).

[0020] Similar to immunoglobulins, the extracellular portion of a TCR chain (e.g., α chain, β chain) consists of two immunoglobulin domains and an N-terminal variable domain (e.g., α chain variable domain or V α , β chain variable domain or V β generally amino acids 1 to 116 according to the Kabat numbering as described in Kabat et al., "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.) and one constant domain adjacent to the cell membrane (e.g., an α chain constant domain generally corresponding to amino acids 81 to 259 according to Kabat or a C chain constant domain generally corresponding to amino acids 81 to 259 according to Kabat α β chain constant domain, generally corresponding to amino acids 81 to 295 according to Kabat, or C β ). Further similar to immunoglobulins, variable domains contain complementarity determining regions (CDRs) flanked by framework regions (FRs) (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. USA 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In certain embodiments, TCRs are present on the surface of T cells (or T lymphocytes) and are associated with the CD3 complex. Sources of TCRs used in the present disclosure can be from various species, such as human, mouse, rat, rabbit, or other mammals.

[0021] The term "variable region" or "variable domain" refers to a domain of an immunoglobulin superfamily binding protein (e.g., a TCR α or β chain (or γ and δ chains for γδ TCRs)) involved in binding of the immunoglobulin superfamily binding protein (e.g., a TCR α or β chain (or γ and δ chains for γδ TCRs)). The variable domains of the α and β chains of native TCRs (Vα and Vβ, respectively) are generally similar in structure, with each domain containing four largely conserved framework regions (FR) and three CDRs. The Vα domain is encoded by two separate DNA segments, a variable gene segment and a joining gene segment (VJ), while the Vβ domain is encoded by three separate DNA segments, a variable gene segment, a diversity gene segment and a joining gene segment (VDJ). A single Vα or Vβ domain appears to be sufficient to confer antigen-binding specificity. Furthermore, a Vα or Vβ domain from a TCR that binds a particular antigen can be used to screen a library of complementary Vα or Vβ domains, respectively, to isolate TCRs that bind said antigen.

[0022] The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known in the art to refer to amino acid sequences within an immunoglobulin (e.g., TCR) variable region that confer antigen specificity and / or binding affinity and are separated from one another in the primary amino acid sequence by framework regions. Generally, there are three CDRs (αCDR1, αCDR2, αCDR3) in each TCR α chain variable region and three CDRs (βCDR1, βCDR2, βCDR3) in each TCR β chain variable region. In TCRs, CDR3 is believed to be the primary CDR involved in recognition of antigen after processing. Generally, CDR1 and CDR2 interact primarily or exclusively with MHC.

[0023] CDR1 and CDR2 are encoded within the variable gene segment of the sequence encoding the TCR variable region, and CDR3 is encoded by a region spanning the variable and joining segments in Vα, and by a region spanning the variable, diverse and joining segments in Vβ. Thus, if the types of variable gene segments of Vα or Vβ are known, their corresponding CDR1 and CDR2 sequences can be deduced, for example, according to the numbering scheme described herein. Compared to CDR1 and CDR2, CDR3 is generally significantly more diverse due to the addition and deletion of nucleotides during the recombination process.

[0024] TCR variable domain sequences can be aligned to a numbering scheme (e.g., Kabat, Chothia, EU, IMGT, Enhanced Chothia, and Aho), equivalent residue positions annotated, and different molecules compared using, for example, the ANARCI software tool (2016, Bioinformatics 15:298-300). The numbering scheme provides a standardized arrangement of framework regions and CDRs in the TCR variable domain. In certain embodiments, the CDRs of the present disclosure are identified according to the IMGT numbering scheme (Lefranc et al., Dev. Comp. Immunol. 27:55, 2003; imgt.org / IMGTindex / V-QUEST.php). In certain embodiments, the CDR3 amino acid sequence of the present disclosure includes one or more junction amino acids, such as may occur during rearrangement by RAG as described herein.

[0025] The term "CD8 coreceptor" or "CD8" as used herein refers to the cell surface glycoprotein CD8 as an α-α homodimer or an α-β heterodimer. The CD8 coreceptor assists the function of cytotoxic T cells (CD8+) and functions by signaling through their intracellular tyrosine phosphorylation pathway (Gao and Jakobsen, Immunol. Today 21:630-636, 2000; Cole and Gao, Cell. Mol. Immunol. 1:81-88, 2004). There are five known human CD8 β chain isoforms (see UniProtKB identifier P10966) and one known human CD8 α chain isoform (see UniProtKB identifier P01732).

[0026] "CD4" is an immunoglobulin coreceptor glycoprotein that helps the TCR communicate with antigen-presenting cells (see Campbell & Reece, Biology 909 (Benjamin Cummings, Sixth Ed., 2002); UniProtKB identifier P01730). CD4 is present on the surface of immune cells such as T helper cells, monocytes, macrophages, and dendritic cells, and contains four immunoglobulin domains (D1-D4) that are expressed on the cell surface. During antigen presentation, CD4 is recruited along with the TCR complex and binds to various regions of the MHCII molecule (CD4 binds to MHCIIβ2, and the TCR complex binds to MHCIIα1 / β1). Without intending to be bound by theory, it is believed that the proximity of the TCR complex allows CD4-associated kinase molecules to phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs) present in the intracellular domain of CD3. This activity is thought to amplify the signals generated by activated TCRs to generate or recruit various immune system cells, including T helper cells, to generate an immune response.

[0027] In some embodiments, the term "D / N / P region" as used herein refers to predicted nucleotides located within a diversity (D) gene segment or the amino acids encoded by said nucleotides, and can include non-template (N) and palindromic (P) nucleotides inserted (or deleted) during V(D)J recombination, which leads to T cell receptor diversity. Recombination activating genes (RAGs) rearrangement of variable (V), diversity (D) and joining (J) gene segments is an imprecise process that results in the variable addition or removal of nucleotides (termed palindromic or P nucleotides), which are then followed by the addition of further random non-template (N) nucleotides by terminal deoxynucleotidyl transferase (TdT) activity. Finally, unpaired nucleotides are removed by exonucleases, and gaps are filled by DNA synthesis and repair enzymes. Such trimming and repair mechanisms lead to joining diversity, facilitating efficient and specific recognition of different antigens by different TCRs. D gene segments can be identified using the annotation system provided by the international ImMunoGeneTics information system (IMGT; imgt.org).

[0028] In some embodiments, "CD3" is a multiprotein complex consisting of six protein chains (see Abbas and Lichtman, 2003; Janeway et al., p172 and 178, 1999). In mammals, the complex comprises a homodimer of CD3γCD3γ chain, CD3δCD3δ chain, two CD3ε chains, and CD3ζ chain. CD3γ, CD3δ and CD3ε chains are closely related cell surface proteins of the immunoglobulin superfamily that contain one immunoglobulin domain. The transmembrane regions of CD3γCD3γ chain, CD3δ chain and CD3ε chain are negatively charged, a feature that allows these chains to associate with positively charged regions of T cell receptor chains. The intracellular tails of CD3γ, CD3δ and CD3ε chains each contain one conserved motif called immunoreceptor tyrosine-based activation motif or ITAM, and each CD3ζ chain has three motifs. Without intending to be bound by theory, it is believed that ITAMs are important for the signaling ability of the TCR complex. The CD3 used in this disclosure can be derived from a variety of animal species, including human, mouse, rat, or other mammals.

[0029] In some embodiments, the term "TCR complex" as used herein refers to a complex formed by the association of CD3 and TCR. For example, the TCR complex can be composed of a CD3γCD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRα chain, and a TCRβ chain. Alternatively, the TCR complex can be composed of a CD3γCD3γ chain, a CD3δ chain, two CD3ε chains, a homodimer of a CD3ζ chain, a TCRγ chain, and a TCRδ chain.

[0030] In some embodiments, a "component of a TCR complex" as used herein refers to a TCR chain (i.e., TCR alpha, TCR beta, TCR gamma, or TCR delta), a CD3 chain (i.e., CD3 gamma, CD3 delta, CD3 epsilon, or CD3 zeta), or a complex formed from two or more TCR chains or CD3 chains (e.g., a complex of TCR alpha and TCR beta, a complex of TCR gamma and TCR delta, a complex of CD3 epsilon and CD3 delta, a complex of CD3 gamma and CD3 epsilon, or a sub-TCR complex of TCR alpha, TCR beta, CD3 gamma, CD3 delta, and two CD3 epsilon chains).

[0031] As used herein, "antigen" or "Ag" refers to an immunogenic molecule that elicits an immune response. This immune response can include antibody production, activation of specific immunocompetent cells (e.g., T cells), or both. Antigens (immunogenic molecules) include, for example, peptides, glycopeptides, polypeptides, glycopolypeptides, polynucleotides, polysaccharides, lipids, and the like. Of course, antigens can be synthetic, recombinantly produced, or derived from biological samples. Exemplary biological samples that contain one or more antigens include tissue samples, tumor samples, cells, body fluids, or combinations thereof. Antigens can be produced by cells that have been modified or engineered to express the antigen or cells that endogenously (e.g., without modification or genetic engineering by human intervention) express an immunogenic mutation or polymorphism.

[0032] As used herein, a "neoantigen" refers to a host cell product that contains a structural change, mutation, or alteration that results in a novel antigen or antigenic epitope not previously found in the genome of a subject (i.e., in a sample of healthy tissue derived from a subject) or that is "encountered" or recognized by the host's immune system, and that (a) is processed by the cellular antigen processing and transport mechanisms and presented on the cell surface in association with an MHC (e.g., HLA) molecule; and (b) elicits an immune response (e.g., a cellular (T cell) response). Neoantigens can be derived, for example, from a coding polynucleotide that has a mutation (substitution, addition, deletion) that results in a mutated or mutant product, from cellular insertion of a foreign nucleic acid molecule or protein, or from exposure to an environmental agent (e.g., chemical, radiation) that results in a genetic change. Neoantigens can occur separately from, from, or in association with tumor antigens. "Tumor neoantigen" (or "tumor-specific neoantigen") refers to a protein that contains a neoantigenic determinant associated with, derived from, or generated within a tumor cell or multiple cells within a tumor. Tumor neo-antigenic determinants are present, for example, in antigenic tumor proteins or peptides that contain one or more somatic mutations or chromosomal rearrangements encoded by the DNA of tumor cells (e.g., pancreatic, lung, and colon cancers) and in proteins or peptides derived from viral open reading frames associated with virus-associated tumors.

[0033] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, T cell receptor (TCR), chimeric antigen receptor, or other binding molecule, domain, or protein. Epitopic determinants generally consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and can have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0034] In some embodiments, as used herein, "specifically binds" or "specific for" refers to an apparent affinity or K A(i.e., the equilibrium association constant for a particular binding interaction, expressed in units of 1 / M, and the association rate constant for this association reaction [k on ] and the dissociation rate constant [k off ] is equal to the ratio of 10 9 M -1 or more, or functional avidity or EC 50 10 -9 It means that a T cell receptor (TCR) or a binding domain thereof (e.g., an scTCR or a fusion protein thereof) associates or binds to a target molecule and does not significantly associate or bind to other molecules or components in a sample such that the K is greater than or equal to M. TCRs can be classified as "high affinity" binding proteins or binding domains (or fusion proteins thereof) or "low affinity" binding proteins or binding domains (or fusion proteins thereof). A "high affinity" TCR or binding domain is one that has a K A 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 A "low affinity" binding protein or binding domain refers to a TCR or binding domain thereof that is A 10 7 M -1 Up to 10 6 M -1 Up to 10 5 M -1 Alternatively, affinity is the equilibrium dissociation constant (K) of a particular binding interaction expressed in units of M. D ) (e.g., 10 -9 M~10 -13 M or less).

[0035] The term "functional avidity" refers to a biological measure or activation threshold of an in vitro T cell response to a given concentration of ligand, including cytokine production (e.g., IFNγ production, IL-2 production, etc.), cytotoxic activity, and proliferation. For example, T cells that respond biologically (immunologically) in vitro to a very low dose of antigen with cytokine production, cytotoxicity, or proliferation are considered to have high functional avidity, and T cells with low functional avidity require a higher amount of antigen to elicit an immune response equivalent to that of high avidity T cells. Of course, functional avidity is different from affinity and avidity. Affinity refers to the strength of the binding that occurs 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.

[0036] As used herein, "functional avidity" refers to a quantitative factor of the activation threshold of TCR expressed by T cells. In vivo, T cells are exposed to the same antigen dose regardless of TCR avidity, but there are many correlations between functional avidity and the effectiveness of immune responses. Some ex vivo studies have shown that different T cell functions (e.g., proliferation, cytokine production, etc.) can be induced at various thresholds (see, e.g., Betts et al., J.Immunol.172:6407,2004; Langenkamp et al., Eur.J.Immunol.32:2046,2002). Factors that influence functional avidity include (a) the affinity of the TCR for the pMHC complex, i.e., the strength of the interaction between the TCR and pMHC (Cawthon et al., J. Immunol. 167:2577, 2001), (b) the expression levels of the TCR and CD4 or CD8 co-receptors, and (c) the distribution and composition of signaling molecules (Viola and Lanzavecchia, Science 273:104, 1996), as well as the expression levels of molecules that reduce T cell function and TCR signaling.

[0037] The antigen concentration required to induce a response that is 50% between the baseline and maximum response after a specific exposure time is called the "50% effective concentration" or "EC 50 " EC 50 Values ​​are commonly expressed as molar (moles / liter) quantities, but can also be plotted as logarithmic values, such as log, which plots a sigmoid graph. 10 (EC 50 ) (see Figure 5A for an example). For example, EC 50 = 1 μM (10 -6 M), then log 10 (EC 50 ) value is -6. Another value is EC 50 The negative logarithm of (-log 10 (EC 50 pEC, defined as 50 In the above example, EC 50 = 1 μM, the pEC50 value is 6. In certain embodiments, the functional avidity of the TCRs of the disclosure is a measure of the ability of T cells to promote IFNγ production and can be measured using the assays described herein. A "high functional avidity" TCR or binding domain thereof is one that has a high EC 50 At least 10 -9 M, at least about 10 -10 M, at least about 10 -11 M, at least about 10 -12 M, or at least about 10 -13 In some embodiments, the response includes IFNγ production, e.g., the production of IFN-γ by an immune cell (such as a T cell, an NK cell, or an NK-T cell) that expresses a TCR in response to an antigen.

[0038] In some embodiments, "WT1 37-45 antigen" or "WT1 37-45 Peptide" or "WT1 37-45"Peptide antigen" or "p37 peptide" or "p37 antigen" or "p37 peptide antigen" each refer to a naturally or synthetically produced portion of the WT1 protein that is about 9 to about 15 amino acids long and comprises the amino acid sequence VLDFAPPGA (SEQ ID NO: 59), and that can form a complex with an MHC (e.g., HLA) molecule, and such a complex can bind to a TCR specific for the WT1 peptide:MHC (e.g., HLA) complex. Since WT1 is an internal host protein, the WT1 antigen peptide is presented by class I MHC. In a specific embodiment, the WT1 peptide VLDFAPPGA (SEQ ID NO: 59) can associate with the human class I HLA allele HLA-A*201.

[0039] In some embodiments, "WT1 37-45 Peptide-specific binding protein" or "WT1 37-45 Peptide-specific TCR" or "WT1 37-45 Antigen-specific TCR" or "WT1 37-45 The terms "peptide antigen-specific TCR" or "WT1p37 peptide-specific binding protein" or "WT1p37 peptide-specific TCR" or "WT1p37 antigen-specific TCR" or "WT1p37 peptide antigen-specific TCR" are used interchangeably herein and refer to a protein or polypeptide that specifically binds to a WT1p37 peptide complexed with an MHC or HLA molecule, e.g., on the cell surface, with an approximate or at least approximate specific affinity or functional avidity, preferably high functional avidity, as defined herein. Such binding proteins or polypeptides include a TCR variable domain as described herein. In some embodiments, a WT1-specific binding protein has a functional avidity log[EC 50 The EC 50 The range is approximately 3.16×10 as measured, for example, by the assay described in the following paragraph and in Example 1 of this application. -9M ~ approx. 1.58×10 -10 Equivalent to M.

[0040] Assays for assessing affinity, apparent affinity, relative affinity, or functional avidity are known. As described herein, the apparent affinity or functional avidity of the TCRs of the present disclosure is measured by assessing binding to various concentrations of tetramer associated with p37 peptide, for example by flow cytometry using labeled tetramer. In some examples, the apparent K of the TCR is measured using two-fold dilutions of various concentrations of labeled tetramer followed by non-linear regression to generate a binding curve. D or EC 50 For example, the apparent K D is determined as the concentration of ligand that produces 50% binding, and EC 50 is determined as the concentration of ligand that results in 50% production of, for example, a cytokine (eg, IFNγ, IL-2).

[0041] "MHC-peptide tetramer staining" in some aspects refers to an assay used to detect antigen-specific T cells utilizing a tetramer of MHC molecules containing identical peptides, each having a cognate (e.g., matching or related) amino acid sequence to at least one antigen (e.g., WT1), and the complex can bind to a T cell receptor specific for the cognate antigen. Each of the MHC molecules can be tagged with a biotin molecule. Fluorescently labelable streptavidin is added to tetramerize the biotinylated MHC / peptide. The tetramer can be detected by flow cytometry via the fluorescent label. In certain embodiments, the MHC-peptide tetramer assay is used to detect or select high affinity or high functional avidity TCRs of the present disclosure.

[0042] Cytokine concentrations can be measured by methods common in the art and described herein, such as ELISA, ELISPOT, intracellular cytokine staining, and flow cytometry, and combinations thereof (e.g., intracellular cytokine staining and flow cytometry). Immune cell proliferation and clonal expansion due to antigen-specific induction or stimulation of an immune response can be determined by isolating lymphocytes, such as circulating lymphocytes, in a sample of peripheral blood cells or lymph node-derived cells, stimulating the lymphocytes with an antigen, and measuring cytokine production, cell proliferation, and / or cell viability, such as by tritiated thymidine incorporation or non-radioactive assays such as MTT assays. For example, the effect of the immunogens described herein on the balance between Th1 and Th2 immune responses can be tested by measuring the concentrations 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.

[0043] In some embodiments, the “WT1p37-specific binding domain” or “WT1 37-45 "WT1p37 specific binding domain" or "WT1p37 specific binding fragment" or "WT1 37-45 The term "specific binding fragment" refers to a domain or portion of a WT1-specific TCR that is involved in specific binding to the WT1p37 antigen complexed with an MHC or HLA molecule. The WT1p37 antigen-specific binding domain derived from a TCR is soluble alone (i.e., without other portions of the WT1-specific TCR) and can be used in a variety of applications. -9 Less than M, about 10 -10 Less than M, about 10 -11 Less than M, about 10 -12 Less than M or about 10 -13 K under M D In another embodiment, the WT1p37 peptide-specific TCR has high functional avidity and specifically binds to the VLDFAPPGA (SEQ ID NO:59):Human Leukocyte Antigen (HLA) complex on the T cell surface and binds to the WT1p37 peptide:MHC complex at pEC 50is 8.5 or greater (e.g., up to about 9, up to about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, or about 13). Representative WT1p37 peptide-specific binding domains include WT1p37 peptide-specific scTCRs that are derived or can be derived from the anti-WT1p37 peptide TCRs of the present disclosure (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 TCR α and β variable domains, respectively, Cα and Cβ are TCR α and β constant domains, respectively, and L is a linker).

[0044] The principles of antigen presentation to T cells by antigen-presenting cells (APCs) (such as dendritic cells, macrophages, lymphocytes, or other cell types), including antigen processing by the APCs and major histocompatibility complex (MHC)-restricted presentation between immunocompatible APCs (e.g., sharing at least one allele of an MHC gene associated with antigen presentation) and T cells, are well known (see, e.g., Murphy, Janeway's Immunobiology (2006) 131:131-135). th (Ed.) 2011 Garland Science, NY; see Chapters 6, 9, and 16.) For example, processed antigenic peptides derived from the cytosol (e.g., tumor antigens, intracellular pathogens) will generally be about 7 to about 11 amino acids in length and will associate with class I MHC molecules, whereas peptides processed in the vesicular system (e.g., from bacteria, viruses) will be about 10 to about 25 amino acids in length and will associate with class II MHC molecules.

[0045] As used herein, the term "transmembrane domain" refers to any amino acid sequence that is thermodynamically stable in a cell membrane and has a three-dimensional structure generally about 15 to about 30 amino acids in length. The structure of the hydrophobic transmembrane domain may include an α-helix, a β-barrel, a β-sheet, a β-helix, or any combination thereof. Representative transmembrane domains are those derived from CD4, CD8, CD28, or CD27.

[0046] As used herein, an "immune effector domain" is an intracellular portion of a scTCR or CAR fusion protein that can stimulate an immune response directly or indirectly within a cell upon receipt of an appropriate signal. In certain embodiments, an immune effector domain is a portion of a protein or protein complex that receives a signal upon binding, or directly binds to a target molecule and elicits a signal from the immune effector domain. An immune effector domain may be a domain that contains an immune receptor tyrosine-based activation motif (i.e., an immunoreceptor tyrosine-based activation motif) or a target molecule that contains an immune receptor tyrosine-based activation motif (i.e., an immunoreceptor tyrosine-based activation motif). In some cases, the effector domain may directly stimulate an immune cell response, such as by comprising one or more signaling domains or motifs, such as tyrosine-based activation motifs (ITAMs). In other embodiments, the effector domain indirectly stimulates a cellular response by associating with one or more other proteins that directly stimulate the cellular response. Exemplary immune effector domains include intracellular signaling domains derived from 4-1BB, CD3ε, CD3δ, CD3ζ, CD27, CD28, CD79A, CD79B, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, Wnt, NKG2D, OX40, ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combination of two or three of such domains.

[0047] In some embodiments, a "linker" refers to an amino acid sequence that links two proteins, polypeptides, peptides, domains, regions, or motifs. One representative example of a linker is a "variable domain linker," specifically a linker between the T cell receptor V α / β Chain and C α / β Chain (e.g. V α -C α , V β -C β , V α -V β ) or V α -C α , V β -C β, V α -V β The term "variable domain linker" refers to a sequence of 5 to about 35 amino acids linking each pair of sub-binding domains with a hinge or transmembrane domain, which provides sufficient spacer function and flexibility for the interaction of the two sub-binding domains such that the resulting single-chain polypeptide maintains specific binding affinity or functional avidity for the same target molecule as the T cell receptor. In certain embodiments, the variable domain linker comprises about 10 to about 30 amino acids or about 15 to about 25 amino acids. In particular embodiments, the variable domain linker peptide comprises 1 to 10 Gly. x Ser y In the above formula, x and y are independently an integer of 0 to 10, provided that x and y are not both 0 (for example, Gly 4 Ser (SEQ ID NO: 171), Gly 3 Ser (SEQ ID NO: 172), Gly 2 Ser, or (Gly 3 Ser) n (Gly 4 Ser) 1 (SEQ ID NO: 173), (Gly 3 Ser) n (Gly 2 Ser) n (SEQ ID NO: 174), (Gly 3 Ser) n (Gly 4 Ser) n (SEQ ID NO: 175), or (Gly 4 Ser) n (SEQ ID NO: 171), where n is an integer of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and the linked variable domains form a functional binding domain (e.g., a scTCR).

[0048] In some embodiments, "junction amino acids" or "junction amino acid residues" refers to one or more (e.g., about 2-10) amino acid residues between two adjacent motifs, regions, or domains of a polypeptide, such as between a binding domain and an adjacent constant domain, or between a TCR chain and an adjacent self-cleaving peptide. Junction amino acids may result from the design of the fusion protein construct (e.g., amino acid residues resulting from restriction enzyme sites used during construction of a nucleic acid molecule encoding the fusion protein) or may be provided during genetic recombination or reassortment events (e.g., reassortment by RAG).

[0049] In some embodiments, a "mutated domain" or "mutated protein" refers to a motif, region, domain, peptide, polypeptide or protein that has at least 85% (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%) sequence identity to a wild-type motif, region, domain, peptide, polypeptide or protein (e.g., wild-type TCR alpha chain, TCR beta chain, TCR alpha constant domain, TCR beta constant domain), and wherein the CDR3 from each of the TCR alpha and beta variable domains is or is preferably not mutated.

[0050] In any of the embodiments disclosed herein, the TCR constant domain can be modified to enhance pairing of the TCR chain of interest. For example, the modification enhances pairing of the heterologous TCR α chain with the heterologous TCR β chain in the host T cell, favoring binding of the TCR comprising two heterologous chains over undesirable mispairing of the heterologous TCR chain with the endogenous TCR chain (see, e.g., Govers et al., Trends Mol. Med. 16(2):77 (2010), the TCR modifications of which are incorporated herein by reference). Exemplary modifications for enhancing pairing of the heterologous TCR chain include introducing complementary cysteine ​​residues into each of the heterologous TCR α chain and β chain. In some embodiments, the polynucleotide encoding the heterologous TCR α chain encodes a cysteine ​​at amino acid position 48 (corresponding to the full-length mature human TCR α chain sequence) and the polynucleotide encoding the heterologous TCR β chain encodes a cysteine ​​at amino acid position 57 (corresponding to the full-length mature human TCR β chain sequence).

[0051] "Chimeric Antigen Receptor (CAR)" refers to a fusion protein constructed to contain two or more naturally occurring amino acid sequences, domains, or motifs linked to each other in a manner that does not occur naturally or in a host cell, such a fusion protein being capable of functioning as a receptor when present on the surface of a cell. CARs can include an extracellular portion that includes an antigen-binding domain (e.g., obtained or obtained from an immunoglobulin or immunoglobulin-like molecule, such as a TCR binding domain obtained or obtained from a TCR specific for a cancer antigen, an scFv obtained or obtained from an antibody, or an antigen-binding domain obtained or obtained from a killer immune receptor derived from a NK cell) linked to a transmembrane domain and one or more intracellular signaling domains (optionally including a co-stimulatory domain) (see, e.g., Sadelain et al., Cancer Discov., 3(4):388 (2013); see also Harris and Kranz, Trends Pharmacol. Sci., 37(3):220 (2016); Stone et al., Cancer Immunol. Immunother., 63(11):1163 (2014); and Walseng et al., Scientific Reports 7:10713 (2017), the CAR constructs and methods of making them are incorporated herein by reference). A CAR of the present disclosure that specifically binds to a WT1 antigen (e.g., in the context of a peptide:HLA complex) comprises a TCR Vα domain and a Vβ domain.

[0052] In some aspects, the term "nucleic acid" or "nucleic acid molecule" or "polynucleotide" as used herein refers to any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotides, fragments generated, for example, by polymerase chain reaction (PCR) or in vitro translation, and fragments generated by ligation, cleavage, endonuclease action, or exonuclease action. In certain embodiments, the nucleic acids of the present disclosure are produced by PCR. Nucleic acids can be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (e.g., alpha enantiomers of naturally occurring nucleotides), or combinations of both. Modified nucleotides can have modifications or substitutions in the sugar moiety or in the pyrimidine or purine base moiety. Nucleic acid monomers can be linked by phosphodiester bonds or linkages similar to such bonds. Bonds similar to phosphodiester bonds include phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoroanilidate, phosphoroamidate, etc. Nucleic acid molecules may be single-stranded or double-stranded.

[0053] In some embodiments, the term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it occurs in nature). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide separated from some or all of the coexisting materials in the natural system is isolated. Such a nucleic acid can be part of a vector and / or such a nucleic acid or polypeptide can be part of a composition (e.g., a cell lysate), but the nucleic acid or polypeptide is still isolated because such a vector or composition is not part of the natural environment of the nucleic acid or polypeptide. The term "gene" refers to a DNA segment involved in producing a polypeptide chain, including "leader and trailer" regions before and after the coding region, and intervening sequences (introns) between individual coding segments (exons).

[0054] In some embodiments, the term "recombinant" as used herein refers to a cell, microorganism, nucleic acid molecule, or vector that has been genetically engineered by human intervention, i.e., modified by the introduction of a foreign or heterologous nucleic acid molecule, or to a cell or microorganism in which the expression of an endogenous nucleic acid molecule or gene has been modified to be regulated, deregulated, or constitutive. Genetic modifications made by humans include, for example, modifications that introduce nucleic acid molecules (which may include expression control elements such as promoters) that code for one or more proteins or enzymes, or the addition, deletion, substitution, or other functional disruption or addition of other nucleic acid molecules to the genetic material of a cell. Exemplary modifications include modifications of coding regions of heterologous or homologous polypeptides or functional fragments thereof from a reference or parent molecule.

[0055] As used herein in some aspects, "mutation" or "mutated" refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. Mutations can result in several different types of changes in the sequence, including nucleotide or amino acid substitutions, insertions, or deletions. In certain embodiments, the mutations are substitutions of 1-3 codons or amino acids, deletions of 1-5 codons or amino acids, or combinations thereof.

[0056] In some embodiments, a "conservative substitution" is recognized in the art as a substitution of one amino acid with another amino acid having similar properties. Representative conservative substitutions are well known in the art (e.g., WO 97 / 09433, p. 10; Lehninger, Biochemistry, 2 nd Edition; Worth Publishers, Inc. NY, NY, pp. 71-77, 1975; see Lewin, Genes IV, Oxford University Press, NY and Cell Press, Cambridge, MA, p. 8, 1990).

[0057] The term "construct" in some embodiments refers to any polynucleotide, including recombinant nucleic acid molecules. The construct may be inserted into a vector (e.g., bacterial vector, viral vector) or integrated into a genome. A "vector" is a nucleic acid molecule capable of transporting another nucleic acid molecule. A vector can be, for example, a plasmid, cosmid, virus, RNA vector, or a linear or circular DNA or RNA molecule, including chromosomal, non-chromosomal, semisynthetic, or synthetic nucleic acid molecules. Exemplary vectors are capable of autonomous replication (episomal vectors) or expression of linked nucleic acid molecules (expression vectors).

[0058] Representative viral vectors include retroviruses, adenoviruses, parvoviruses (e.g., adeno-associated viruses), negative-stranded RNA viruses such as coronaviruses, orthomyxoviruses (e.g., influenza viruses), rhabdoviruses (e.g., rabies and vesicular stomatitis viruses), paramyxoviruses (e.g., measles and Sendai), positive-stranded RNA viruses such as picornaviruses and alphaviruses, and double-stranded DNA viruses such as adenoviruses, herpesviruses (e.g., herpes simplex viruses types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxviruses (e.g., vaccinia, avipox, and canarypox). Other viruses include, for example, Norwalk virus, togaviruses, flaviviruses, reoviruses, papovaviruses, hepadnaviruses, and hepatitis viruses. Examples of retroviruses include avian leukosis sarcoma virus, mammalian type C, type B, and type D viruses, the HTLV-BLV group, lentiviruses, and spumaviruses (Coffin, JM, Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, BN Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

[0059] In some embodiments, the term "lentiviral vector" as used herein refers to an HIV-based lentiviral vector for gene delivery, which may be integrative or non-integrative, has a relatively high packaging capacity, and can transduce a variety of cell types. Lentiviral vectors are usually produced after transient transfection of three or more types of plasmids (packaging, envelope, and transfer) into producer cells. Similar to HIV, lentiviral vectors enter target cells through the interaction of viral surface glycoproteins with receptors on the cell surface. Once inside, viral RNA is reverse transcribed by the viral reverse transcription complex. The reverse transcription product is double-stranded linear viral DNA, which serves as the basis for viral integration into the DNA of infected cells.

[0060] The term "operably linked" in some embodiments refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment such that the function of one of the molecules is affected by the other. For example, a promoter is operably linked to a coding sequence when it is capable of affecting the expression of the coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Unlinked" means that the associated genetic elements are not in close association with each other and do not affect each other's functions.

[0061] In some embodiments, the term "expression vector" as used herein refers to a DNA construct in which a nucleic acid molecule is operably linked to suitable control sequences capable of effecting the expression of said nucleic acid molecule in a suitable host. Such control sequences include a promoter for transcription, an operator sequence that is optionally used to control such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and sequences that control the termination of transcription and translation. A vector can be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once introduced into a suitable host, the vector can replicate and function independently of the host genome, or in some cases can integrate into the genome itself. In this specification, "plasmid", "expression plasmid", "virus" and "vector" are often used interchangeably.

[0062] In some embodiments, the term "expression" as used herein refers to the process by which a polypeptide is produced based on a coding sequence of a nucleic acid molecule, such as a gene, including transcription, post-transcriptional regulation, post-transcriptional modification, translation, post-translational regulation, post-translational modification, or any combination thereof.

[0063] The term "introduction" in the context of inserting a nucleic acid molecule into a cell in some embodiments means "transfection" or "transformation" or "transduction" and includes the introduction of a nucleic acid molecule into a eukaryotic or prokaryotic cell, where the nucleic acid molecule may be incorporated into the genome of the cell (e.g., a chromosome, a plasmid, a plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., mRNA transfection).

[0064] As used herein in some aspects, a "heterologous" or "foreign" nucleic acid molecule, construct, or sequence refers to a nucleic acid molecule or portion of a nucleic acid molecule that does not naturally occur in a host cell, but may be homologous to the nucleic acid molecule or portion of a nucleic acid molecule originating from the host cell. The origin of a heterologous or foreign nucleic acid molecule, construct, or sequence may be from a different genus or species. In certain embodiments, a heterologous or foreign nucleic acid molecule is added to a host cell or host genome (i.e., is not endogenous or naturally occurring), e.g., by conjugation, transformation, transfection, electroporation, etc., and the added molecule may be integrated into the host genome or may exist as extrachromosomal genetic material (e.g., as a plasmid or other form of self-replicating vector), and may exist in multiple copies. "Heterologous" also refers to a non-native enzyme, protein, or other activity that is encoded by the foreign nucleic acid molecule when the foreign nucleic acid molecule is introduced into a host cell, even if the host cell encodes the homologous protein or activity. Furthermore, a cell that contains a "modified" or "heterologous" polynucleotide or binding protein includes the progeny of that cell, regardless of whether the progeny themselves have been transduced, transfected, or otherwise manipulated or altered.

[0065] As described herein, two or more heterologous or foreign nucleic acid molecules may be introduced into a host cell as separate nucleic acid molecules, as multiple individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof. For example, as disclosed herein, a host cell can be engineered to express two or more heterologous or foreign nucleic acid molecules encoding a TCR of interest (e.g., TCRα and TCRβ) specific to a WT1 antigen peptide. When two or more foreign nucleic acid molecules are introduced into a host cell, it is understood that the two or more foreign nucleic acid molecules may be introduced as a single nucleic acid molecule (e.g., in a single vector), introduced in separate vectors, integrated into one or more sites of a host chromosome, or any combination thereof. The number of heterologous nucleic acid molecules or protein activities referred to refers to the number of coding nucleic acid molecules or protein activities, and not the number of individual nucleic acid molecules introduced into the host cell.

[0066] In some embodiments, the term "endogenous" or "native" as used herein 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 modified compared to a parent gene, protein, or activity is also considered to be endogenous or native to that particular host cell. For example, an endogenous control sequence (e.g., promoter, translation downregulation sequence) from a first gene can be used to modify or regulate the expression of a second native gene or nucleic acid molecule, where the expression or regulation of the second native gene or nucleic acid molecule differs from the normal expression or regulation in the parent cell.

[0067] In some embodiments, the term "homologous" or "homolog" refers to a molecule or activity that is present or derived from a host cell, species, or lineage. For example, a heterologous or foreign nucleic acid molecule can be homologous to a native host cell gene, and can optionally have altered expression levels, a different sequence, altered activity, or any combination thereof.

[0068] In some embodiments, "sequence identity" as used herein refers to the percentage of amino acid residues in a first sequence that match those in another reference polypeptide sequence after aligning the first sequence with another reference polypeptide sequence, introducing gaps as necessary to reach the maximum sequence identity percentage, and excluding conservative substitutions from the sequence identity. Percentage sequence identity values ​​can be determined using the NCBI BLAST 2.0 software as defined in Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25: 3389-3402, with parameters set to default values.

[0069] In some embodiments, a "hematopoietic progenitor cell" as used herein can refer to a hematopoietic stem cell or a cell derived from fetal tissue that can further differentiate into a mature cell type, such as an immune system cell. Exemplary hematopoietic progenitor cells include those that express CD24 Lo Lin - CD81 + These include those that have a specific phenotype and those that reside in the thymus (called precursor thymocytes).

[0070] In some aspects, the term "host" as used herein refers to a cell (e.g., a T cell) or microorganism that is targeted for genetic modification with a heterologous or foreign nucleic acid molecule to produce a polypeptide of interest (e.g., a high affinity anti-WT1 TCR). In some embodiments, the host cell may already have other genetic modifications that confer desired properties, optionally related or unrelated to the biosynthesis of a heterologous or foreign protein, or may be engineered to include such modifications (e.g., introduction of a detectable marker; deletion, mutation, or truncation of an endogenous TCR; enhanced expression of a costimulatory factor). In some embodiments, the host cell is genetically modified to express a protein or fusion protein that modulates immune signaling in the host cell, for example, to aid in the survival and / or expansion advantage of the modified cell (see, e.g., immune-modulating fusion proteins in WO2016 / 141357, incorporated herein by reference in their entirety). In other embodiments, the host cell is genetically modified to introduce a TCR as provided herein or to knock down or minimize an immunosuppressive signal in the cell (e.g., a checkpoint inhibitor), such modification can be performed, for example, using a CRISPR / Cas system (see, for example, US2014 / 0068797, U.S. Patent No. 8,697,359; WO2015 / 071474). In some embodiments, the host cell is a human hematopoietic progenitor cell transduced with a heterologous or foreign nucleic acid molecule encoding a TCR alpha chain specific for a WT1 antigenic peptide.

[0071] In some embodiments, the term "hyperproliferative disorder" as used herein refers to excessive growth or proliferation as compared to normal or non-diseased cells. Exemplary hyperproliferative disorders include tumors, cancers, neoplastic tissues, carcinomas, sarcomas, malignant cells, pre-malignant cells, and non-neoplastic or non-malignant hyperproliferative disorders (e.g., adenomas, fibromas, lipomas, leiomyomas, hemangiomas, fibrosis, restenosis, as well as autoimmune diseases such as rheumatoid arthritis, osteoarthritis, psoriasis, and inflammatory bowel disease). Certain disorders in which abnormal or excessive proliferation occurs more slowly than in hyperproliferative disorders can be referred to as "proliferative disorders," including certain tumors, cancers, neoplastic tissues, carcinomas, sarcomas, malignant cells, pre-malignant cells, and non-neoplastic or non-malignant disorders.

[0072] Furthermore, "cancer" can refer to any accelerated proliferation of cells, including solid tumors, ascites tumors, hematologic or lymphatic 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.

[0073] TCR specific to WT1p37 antigen peptide In certain embodiments, the present disclosure provides a method for the detection of T cell receptor (TCR) alpha chain variable (V α ) domain and a TCR β chain variable (V β ) domain; (b) a TCRV having a CDR3 amino acid sequence set forth in any one of SEQ ID NOs: 12 to 22, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, and 238. α Domain and TCRV β domain; or (c) a TCRV having a CDR3 amino acid sequence set forth in any one of SEQ ID NOs: 12 to 22, 178, 184, 190, 196, 202, 208, 214, 220, 226, 232, and 238. αA TCRV having a domain and a CDR3 amino acid sequence set forth in any one of SEQ ID NOs: 1 to 11, 181, 187, 193, 199, 205, 211, 217, 223, 229, 235, and 241. β For example, any of the disclosed TCRs or binding domains thereof can specifically bind to a WT1p37 peptide:HLA complex on the surface of a cell (e.g., a T cell) and / or can bind to a pEC 50 can promote IFNγ production to a value of 8.5 or greater (e.g., up to about 8.6, up to about 8.65, up to about 8.7, up to about 8.72, up to about 8.75, up to about 8.8, up to about 9, up to about 9.1, up to about 9.2, up to about 9.3, up to about 9.4, up to about 9.5, up to about 9.6, up to about 9.68, up to about 9.7, up to about 9.75, up to about 10, up to about 10.5, up to about 11, up to about 11.5, up to about 12, up to about 12.5, or up to about 13). In certain embodiments, the TCR of the present disclosure can specifically bind to the VLDFAPPGA (SEQ ID NO:59):human leukocyte antigen (HLA) complex and promote IFNγ-producing pEC 50 Value is 9.0 or higher, or IFNγ-producing pEC 50 In some embodiments, the TCR or binding domain thereof (e.g., scTCR or a fusion protein thereof) of the present disclosure can specifically bind to the WT1p37 peptide:HLA complex and has a value of 9.0 or greater. 50 IFNγ production can be promoted so that the EC 50 is approximately 1.1 x 10 -9 M ~ approx. 3.0×10 -10 M, or any number within this range. In another example, any of the TCRs disclosed herein can specifically bind to a WT1 peptide:HLA complex on the cell surface in a CD8-independent or absence of CD8. In another embodiment, the TCR can be about 10 -9 K below M Dand specifically binds to the VLDFAPPGA (SEQ ID NO:59):human leukocyte antigen (HLA) complex. In some embodiments, the HLA comprises HLA-A*201. The peptide antigen VLDFAPPGA (SEQ ID NO:59) is a WT1 peptide antigen and corresponds to amino acids 37-45 of the WT1 protein.

[0074] In any of the embodiments described herein, the present disclosure provides a T cell receptor (TCR) comprising an α chain and a β chain, the TCR binding to a WT1:HLA-A*201 complex on the surface of a T cell; and (a) a pEC 50 or (b) promotes IFNγ production such that an IFNγ expression level is 8.5 or greater (e.g., up to about 9, up to about 9.5, up to about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, or about 13); or (b) binds to the cell surface independently of or in the absence of CD8.

[0075] In certain embodiments, V β The domain comprises or is derived from TRBV7-6*01 / TRBJ2-7*01, TRBV20-1*02 / TRBJ2-7*01, TRBV15*02 / TRBJ1-5*01, TRBV13*01 / TRBJ2-5*01, TRAJ50*01 / TRBJ2-7*01, TRBV11-3*01 / TRBJ1-1*01, TRBV19*01 / TRBJ1-6*02, TRBV27*01 / TRBJ2-7*01, TRBV13*01 / TRBJ2-7*01, TRBV11-1*01 / TRBJ1-4*01, or TRBV4-3*01 / TRBJ1-3*01. αThe domains comprise or are derived from TRAV21*02 / TRAJ58*01, TRAV38-1*01 / TRAJ40*01, TRAV29 / DV5*01 / TRAJ6*01, TRAV29 / DV5*01 / TRAJ20*01, TRAV41*01 / TRAJ50*01, TRAV12-2*01 / TRAJ11*01, TRAV1-2*01 / TRAJ20*01, TRAV20*02 / TRAJ8*01, TRAV26-1*02 / TRAJ26*01, TRAV24*01 / TRAJ48*01, or TRAV20*02 / TRAJ37*02. In certain embodiments, the TCR comprises or is derived from (a) TRBV7-6*01 / TRBJ2-7*01 β domain and V containing or derived from TRAV21*02 / TRAJ58*01 α (b) a V domain comprising or derived from TRBV27*01 / TRBJ2-7*01; β domain and V containing or derived from TRAV20*02 / TRAJ8*01 α domain; or (c) a V domain comprising or derived from TRBV13*01 / TRBJ2-5*01 β domain and V containing or derived from TRAV29 / DV5*01 / TRAJ20*01 α Includes the domain.

[0076] In some embodiments, the TCR of the present disclosure further comprises: (i) a CDR1α amino acid sequence set forth in any one of SEQ ID NOs: 194, 176, 182, 188, 200, 206, 212, 218, 224, 230 and 236, or a variant thereof comprising one or two amino acid substitutions, optionally wherein the one or two amino acid substitutions are conservative amino acid substitutions; and / or (ii) a CDR2α amino acid sequence set forth in any one of SEQ ID NOs: 195, 177, 183, 189, 201, 207, 213, 219, 225, 231 and 237, or a variant thereof comprising one or two amino acid substitutions, optionally wherein the one or two amino acid substitutions are conservative amino acid substitutions.

[0077] In some embodiments, the TCR of the disclosure further comprises: (i) a CDR1β amino acid sequence set forth in any one of SEQ ID NOs: 197, 179, 185, 191, 197, 203, 209, 215, 221, 227, 233, and 239, or a variant thereof comprising one or two amino acid substitutions, optionally wherein the one or two amino acid substitutions comprise conservative amino acid substitutions; and / or (ii) a CDR2β amino acid sequence set forth in any one of SEQ ID NOs: 198, 180, 186, 192, 204, 210, 216, 222, 228, 234, and 240, or a variant thereof comprising one or two amino acid substitutions, optionally wherein the one or two amino acid substitutions comprise conservative amino acid substitutions.

[0078] In some embodiments, the TCRs of the disclosure are selected from the group consisting of: (i) SEQ ID NOs: 194, 195, 196 or 12, 197, 198, and 199 or 1, respectively; (ii) SEQ ID NOs: 176, 177, 178 or 18, 179, 180, and 181 or 7, respectively; (iii) SEQ ID NOs: 182, 183, 184 or 20, 185, 186, and 187 or 9, respectively; (iv) SEQ ID NOs: 188, 189, 190 or 21, 191, 192, and 193 or 10, respectively; (v) SEQ ID NOs: 200, 201, 202 or 13, 203, 204, and 205 or 2, respectively; (vi) SEQ ID NOs: 206, 207, 208 or 14, 209, 210, and 2, respectively. (vii) SEQ ID NOs: 212, 213, 214 or 15, 215, 216, and 217 or 4, respectively; (viii) SEQ ID NOs: 218, 219, 220 or 17, 221, 222, and 223 or 6, respectively; (ix) SEQ ID NOs: 224, 225, 226 or 19, 227, 228, and 229 or 8, respectively; (x) SEQ ID NOs: 230, 231, 232 or 22, 233, 234, and 235 or 11, respectively; or (xi) SEQ ID NOs: 236, 237, 238 or 16, 238, 240, and 241 or 5, respectively.

[0079] Any of the polypeptides of the present disclosure, as encoded by the polynucleotide sequence, can include a "signal peptide" (also known as a leader sequence, leader peptide, or transit peptide). Signal peptides direct newly synthesized polypeptides to their appropriate location inside or outside the cell. Signal peptides can be removed from polypeptides during or after localization or secretion is completed. 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.

[0080] In some embodiments, amino acid residues 1-19 of SEQ ID NO: 23 are or comprise a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain and comprises or consists of the amino acid sequence of SEQ ID NO: 23 with amino acid residues 1-19 of SEQ ID NO: 23 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 242).

[0081] In some embodiments, amino acid residues 1-15 of SEQ ID NO: 24 are or comprise a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain and comprises or consists of the amino acid sequence of SEQ ID NO: 23 minus amino acid residues 1-15 of SEQ ID NO: 24 (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 243).

[0082] In some embodiments, amino acid residues 1-19 of SEQ ID NO: 25 are or comprise a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain and comprises or consists of the amino acid sequence of SEQ ID NO: 25 with amino acid residues 1-15 of SEQ ID NO: 25 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 244).

[0083] In some embodiments, amino acid residues 1-29 of SEQ ID NO: 26 are or comprise a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain and comprises or consists of the amino acid sequence of SEQ ID NO: 26 with amino acid residues 1-29 of SEQ ID NO: 26 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 245).

[0084] In some embodiments, amino acid residues 1-19 of SEQ ID NO: 27 are or comprise a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain and comprises or consists of the amino acid sequence of SEQ ID NO: 27 with amino acid residues 1-19 of SEQ ID NO: 27 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 246).

[0085] In some embodiments, amino acid residues 1-19 of SEQ ID NO: 28 are or comprise a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain and comprises or consists of the amino acid sequence of SEQ ID NO: 28 with amino acid residues 1-19 of SEQ ID NO: 28 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 247).

[0086] In some embodiments, amino acid residues 1-19 of SEQ ID NO: 29 are or comprise a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain and comprises or consists of the amino acid sequence of SEQ ID NO: 29 with amino acid residues 1-19 of SEQ ID NO: 29 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 248).

[0087] In some embodiments, amino acid residues 1-19 of SEQ ID NO: 30 are or comprise a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain and comprises or consists of the amino acid sequence of SEQ ID NO: 30 with amino acid residues 1-19 of SEQ ID NO: 30 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 249).

[0088] In some embodiments, amino acid residues 1-29 of SEQ ID NO: 31 are or comprise a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain and comprises or consists of the amino acid sequence of SEQ ID NO: 31 with amino acid residues 1-29 of SEQ ID NO: 31 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 250).

[0089] In some embodiments, amino acid residues 1-19 of SEQ ID NO: 32 are or comprise a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain and comprises or consists of the amino acid sequence of SEQ ID NO: 32 with amino acid residues 1-19 of SEQ ID NO: 32 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 251).

[0090] In some embodiments, amino acid residues 1-19 of SEQ ID NO: 33 are or comprise a signal peptide. In some embodiments, the TCR Vβ domain is a mature TCR Vβ domain and comprises or consists of the amino acid sequence of SEQ ID NO: 33 with amino acid residues 1-19 of SEQ ID NO: 33 removed (i.e., the TCR Vβ domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 252).

[0091] In some embodiments, amino acid residues 1-19 of SEQ ID NO: 34 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain and comprises or consists of the amino acid sequence of SEQ ID NO: 34 with amino acid residues 1-19 of SEQ ID NO: 34 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 253).

[0092] In some embodiments, amino acid residues 1-20 of SEQ ID NO: 35 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain and comprises or consists of the amino acid sequence of SEQ ID NO: 35 with amino acid residues 1-20 of SEQ ID NO: 35 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 254).

[0093] In some embodiments, amino acid residues 1-26 of SEQ ID NO: 36 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain and comprises or consists of the amino acid sequence of SEQ ID NO: 36 with amino acid residues 1-26 of SEQ ID NO: 36 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 255).

[0094] In some embodiments, amino acid residues 1-26 of SEQ ID NO: 37 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain and comprises or consists of the amino acid sequence of SEQ ID NO: 37 with amino acid residues 1-26 of SEQ ID NO: 37 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 256).

[0095] In some embodiments, amino acid residues 1-22 of SEQ ID NO: 38 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain and comprises or consists of the amino acid sequence of SEQ ID NO: 38 with amino acid residues 1-22 of SEQ ID NO: 38 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 257).

[0096] In some embodiments, amino acid residues 1-21 of SEQ ID NO: 39 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain and comprises or consists of the amino acid sequence of SEQ ID NO: 39 with amino acid residues 1-21 of SEQ ID NO: 39 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 258).

[0097] In some embodiments, amino acid residues 1-17 of SEQ ID NO: 40 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain and comprises or consists of the amino acid sequence of SEQ ID NO: 40 with amino acid residues 1-17 of SEQ ID NO: 40 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 259).

[0098] In some embodiments, amino acid residues 1-21 of SEQ ID NO: 41 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain and comprises or consists of the amino acid sequence of SEQ ID NO: 41 with the exception of amino acid residues 1-21 of SEQ ID NO: 41 (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 260).

[0099] In some embodiments, amino acid residues 1-17 of SEQ ID NO: 42 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain and comprises or consists of the amino acid sequence of SEQ ID NO: 42 with amino acid residues 1-17 of SEQ ID NO: 42 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 261).

[0100] In some embodiments, amino acid residues 1-22 of SEQ ID NO: 43 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain and comprises or consists of the amino acid sequence of SEQ ID NO: 43 with amino acid residues 1-22 of SEQ ID NO: 43 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 262).

[0101] In some embodiments, amino acid residues 1-21 of SEQ ID NO: 44 are or comprise a signal peptide. In some embodiments, the TCR Vα domain is a mature TCR Vα domain and comprises or consists of the amino acid sequence of SEQ ID NO: 44 with amino acid residues 1-21 of SEQ ID NO: 44 removed (i.e., the TCR Vα domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 263).

[0102] In some embodiments, a T cell receptor (TCR) specific for a WT1 peptide:HLA complex comprises or is composed of an amino acid sequence set forth in any one of SEQ ID NOs: 253 to 263 and 34 to 33. α A V domain having an amino acid sequence set forth in any one of SEQ ID NOs: 242 to 252 and 23 to 33, or a ... β In certain embodiments, the V α The domain comprises or consists of the amino acid sequence of SEQ ID NO: 34, β In another particular embodiment, the domain comprises or consists of the amino acid sequence of SEQ ID NO: 23. α The domain comprises or consists of the amino acid sequence of SEQ ID NO: 41, β The domain comprises or consists of the amino acid sequence of SEQ ID NO: 30; (b) V α The domain comprises or consists of the amino acid sequence of SEQ ID NO: 37, β The domain comprises or consists of the amino acid sequence of SEQ ID NO: 26; or (c) V α The domain comprises or consists of the amino acid sequence of SEQ ID NO: 42, β In another particular embodiment, the V domain comprises or consists of the amino acid sequence of SEQ ID NO: 31. α The domain comprises or consists of the amino acid sequence of SEQ ID NO: 24, β The domain comprises or consists of the amino acid sequence of SEQ ID NO:35.

[0103] In some embodiments, the Vα domain and the Vβ domain are selected from the group consisting of SEQ ID NOs: (i) 253 and 242, respectively; (ii) 259 and 248, respectively; (iii) 261 and 250, respectively; (iv) 262 and 251, respectively; (v) 257 and 246, respectively; (vi) 254 and 243, respectively; (vii) 255 and 244, respectively; (viii) 256 and 245, respectively; (ix) 258 and 247, respectively; (x) 260 and 249, respectively; (xi) 26 (xii) 34 and 23, respectively; (xiii) 40 and 29, respectively; (xiv) 42 and 31, respectively; (xv) 43 and 32, respectively; (xvi) 35 and 24, respectively; (xvii) 36 and 25, respectively; (xviii) 37 and 26, respectively; (xix) 39 and 28, respectively; (xx) 41 and 30, respectively; (xxi) 44 and 33, respectively; or (xxii) 38 and 27, respectively.

[0104] In certain embodiments, a recombinant TCR with high functional avidity specific for the WT1p37 peptide described in the present application includes a mutant polypeptide species having one or more amino acid substitutions, insertions or deletions in its amino acid sequence with respect to any one or more of the amino acid sequences of SEQ ID NOs: 48 to 58 as described in the present application, provided that CDR3 is not mutated and the TCR maintains or substantially maintains its specific WT1p37-binding function.

[0105] Conservative substitutions of amino acids are well known and may occur naturally or may be introduced during recombinant production of TCRs. Amino acid substitutions, deletions and additions can be introduced into proteins using mutagenesis techniques known in the art (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, NY, 2001). Oligonucleotide-based site-directed (or segment-directed) mutagenesis can be used to provide mutant polynucleotides in which specific codons are mutated according to the desired substitution, deletion or insertion. Alternatively, random or saturation mutagenesis techniques, such as alanine scanning mutagenesis, error-prone polymerase chain reaction mutagenesis and oligonucleotide-based mutagenesis, can be used to generate immunogenic polypeptide variants (see, e.g., Sambrook et al., supra).

[0106] Whether an amino acid substituted at a particular position in a peptide or polypeptide is conservative (or similar) can be indicated by various criteria known to those skilled in the art. For example, a similar amino acid or conservative amino acid substitution is when an amino acid residue is replaced with an amino acid residue having a similar side chain. Similar amino acids can be divided into the following categories: amino acids with basic side chains (e.g., lysine, arginine, histidine); amino acids with acidic side chains (e.g., aspartic acid, glutamic acid); amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine); amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan). Proline is considered difficult to classify and also shares properties with amino acids having aliphatic side chains (e.g., leucine, valine, isoleucine, and alanine). Glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively, so in some circumstances, substitutions of glutamic acid for glutamine or aspartic acid for asparagine can also be considered analogous substitutions. As is understood in the art, "similarity" between two polypeptides is determined by comparing the amino acid sequence of a polypeptide and its conserved substituted amino acid residues to the sequence of a second polypeptide (e.g., using the GENEWORKS, Align, BLAST algorithms, or other algorithms described herein and common in the art).

[0107] As a wild-type TCR or a mutant of its binding domain specific to the WT1p37 antigen:MHC complex, β CDR3 and V of domain αExamples of such TCRs include those having at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% amino acid sequence identity to any of the representative amino acid sequences disclosed herein (e.g., SEQ ID NOs: 23 to 58), provided that neither CDR3 of the domain contains a mutation and that mutations in other portions do not reduce the functional avidity (or relative affinity) by more than 10%, 15% or 20% compared to the wild-type TCR. In some optionally selected embodiments, the mutant TCR may further include any one of SEQ ID NOs: 34 to 44 (parent V α domain) or any one of SEQ ID NOs: 23 to 33 (parent V β V of CDR1 listed in α Domain, V of CDR2 α Domain, V of CDR1 β Domain, V of CDR2 β In each of these embodiments, the TCR does not include a mutation in the amino acid sequence of the pEC domain or any combination thereof. 50 or the TCR maintains its ability to specifically induce IFNγ production such that the TCR expression level is 8.5, 8.6, 8.7, 8.8, 8.9 or higher, ... -9 K below M D and maintains its ability to specifically bind to a peptide antigen:HLA complex (e.g., VLDFAPPGA (SEQ ID NO: 59):HLA complex), specifically binding up to 1.5, 2, 2.5, 3, 3.3, 3.5, 5 times better than a wild-type TCR composed of any one of SEQ ID NOs: 48-58.

[0108] In another embodiment, the present disclosure provides a TCR α chain variable (V α ) domain and a TCR β chain variable (V β) domain; (b) a TCRV having at least 92% sequence identity to the amino acid sequence of SEQ ID NO: 36 or 37. α A domain and a TCRV 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: 23 to 25, 27, 28, 30, 32 and 33. β domain; or (c) a TCRV comprising or consisting of the amino acid sequence of SEQ ID NO: 34 to 44. α A domain and a TCRV having at least 90% sequence identity to any one of the amino acid sequences set forth in SEQ ID NOs: 23 to 25, 27, 28, 30, 32, and 33. β The present invention provides a p37-specific TCR or a binding domain thereof, comprising the p37-specific TCR or a binding domain thereof.

[0109] In yet another embodiment, the present disclosure provides a TCRV having (a) at least 90% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 34-35 and 38-44. α A domain having at least 92% (e.g., 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO:29. β domain; (b) a TCRV having at least 92% sequence identity to the amino acid sequence of SEQ ID NO: 36 or 37. α Domain and TCRV having at least 92% sequence identity to the amino acid sequence of SEQ ID NO: 29 β domain; or (c) a TCRV comprising or consisting of the amino acid sequence of SEQ ID NO: 34 to 44. α Domain and TCRV having at least 92% sequence identity to the amino acid sequence of SEQ ID NO: 29 β The present invention provides a p37-specific TCR or a binding domain thereof, comprising the p37-specific TCR or a binding domain thereof.

[0110] In yet another embodiment, the present disclosure provides a TCRV having (a) at least 90% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 34-35 and 38-44. α domain and a V domain having at least 93% sequence identity to the amino acid sequence of SEQ ID NO:31 β domain; (b) a TCRV having at least 92% sequence identity to the amino acid sequence of SEQ ID NO: 36 or 37. α Domain and TCRV having at least 93% sequence identity to the amino acid sequence of SEQ ID NO:31 β domain; or (c) a TCRV comprising or consisting of the amino acid sequence of SEQ ID NO: 34 to 44. α Domain and TCRV having at least 93% sequence identity to the amino acid sequence of SEQ ID NO:31 β The present invention provides a p37-specific TCR or a binding domain thereof, comprising the p37-specific TCR or a binding domain thereof.

[0111] In another embodiment, the present disclosure provides a TCRV having (a) at least 90% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 34-35 and 38-44. α domain and a V domain having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 26 β domain; (b) a TCRV having at least 92% sequence identity to the amino acid sequence of SEQ ID NO: 36 or 37. α Domain and TCRV having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 26 β domain; or (c) a TCRV comprising or consisting of the amino acid sequence of SEQ ID NO: 34 to 44. α Domain and TCRV having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 26 β The present invention provides a p37-specific TCR or a binding domain thereof, comprising the p37-specific TCR or a binding domain thereof.

[0112] In yet another embodiment, the present disclosure provides a TCRV having (a) at least 90% sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 34-35 and 38-44. α A domain and a V domain including or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 23 to 33. β domain; (b) a TCRV having at least 92% sequence identity to the amino acid sequence of SEQ ID NO: 36 or 37. α A TCRV comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 23 to 33. β domain; or (c) a TCRV comprising or consisting of the amino acid sequence of SEQ ID NO: 34 to 44. α A TCRV comprising or consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 23 to 33. β The present invention provides a p37-specific TCR or a binding domain thereof, comprising the p37-specific TCR or a binding domain thereof.

[0113] In any of the above embodiments, the TCR binds to a cell (e.g., T cell) surface WT1p37 peptide VLDFAPPGA (SEQ ID NO:59):HLA complex and binds to pEC 50 and / or the TCR is capable of specifically inducing IFNγ production such that the VLDFAPPGA (SEQ ID NO:59):HLA cell surface complex is CD8-independent or in the absence of CD8. β The domain does not contain any mutations in the amino acid sequence of CDR1 and / or CDR2 compared to CDR1 and / or CDR2, respectively, present in any one of SEQ ID NOs:23-33.

[0114] In some embodiments, any of the above WT1p37 peptide-specific T cell receptors (TCRs) can be an antigen-binding fragment of a TCR. In other embodiments, the antigen-binding fragment of a TCR can include a single-chain TCR (scTCR) and can be added to a chimeric antigen receptor (CAR). In some embodiments, the WT1p37 peptide-specific TCR can be, for example, a V α The TCR α chain, which contains the V domain and the α chain constant domain, β and a multi-chain binding protein comprising a TCR β chain comprising a TCR α chain constant domain and a β chain constant domain, wherein the TCR α chain constant domain has at least about 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 47, and the TCR β chain constant domain has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the amino acid sequence of SEQ ID NO: 45 or 46. In another embodiment, the disclosure provides a WT1p37 peptide-specific TCR comprising or consisting of an α chain constant domain having the amino acid sequence of SEQ ID NO: 47 and / or comprising or consisting of a β chain constant domain having the amino acid sequence of SEQ ID NO: 45 or 46.

[0115] In other embodiments, the present disclosure provides α providing a WT1p37 peptide-specific TCR comprising a TCR α chain and an α chain constant domain; α or (b) the V domain has 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: 34-35 and 38-44, and the α chain constant domain has at least about 98% sequence identity to the amino acid sequence of SEQ ID NO: 47; or αThe domain has at least 92% sequence identity to the amino acid sequence of SEQ ID NO:36 or 37, and the alpha chain constant domain has at least 98% sequence identity to the amino acid sequence of SEQ ID NO:47.

[0116] In some embodiments, the TCR is V α (a) a TCR α chain comprising a V domain and an α chain constant domain; α (b) the V domain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 242 to 252 and 34 to 44, and the α chain constant domain comprises the amino acid sequence of SEQ ID NO: 47; or α The domain is composed of an amino acid sequence set forth in any one of SEQ ID NOs: 242 to 252 and 34 to 44, and the alpha chain constant domain has at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence of SEQ ID NO: 47, or comprises such a sequence, or is composed of such a sequence.

[0117] In some embodiments, the α chain constant domain is present, and the Vα domain and the α chain constant domain together form a TCR α chain, in some embodiments, the β chain constant domain is present, and the Vβ domain and the β chain constant domain together form a TCR β chain.

[0118] In some embodiments, the TCR comprises a scTCR, or a scTCR derived from a TCR disclosed herein is provided. In some embodiments, the TCR comprises a CAR, or a CAR derived from a TCR disclosed herein (e.g., comprising one or more variable domains derived from a TCR disclosed herein) is provided.

[0119] In another embodiment, there is provided a composition comprising a highly functional avidity WT1-specific recombinant TCR or its binding domain according to any one of the above embodiments, and a pharma- ceutically acceptable carrier, diluent or excipient.

[0120] Useful methods for isolating and purifying recombinantly produced soluble TCRs include, for example, harvesting the supernatant from a suitable host cell / vector system that secretes recombinant soluble TCR into the medium, followed by concentrating the medium using a commercially available filter. After concentration, the concentrate may be applied to a suitable purification matrix or a series of suitable matrices, such as affinity matrices or ion exchange resins. One or more reverse phase HPLC steps may be used to further purify the recombinant polypeptide. These purification methods may also be used when isolating immunogens from their native environment. One or more large-scale production methods for isolated / recombinant soluble TCRs described herein include batch cell cultures that are monitored and controlled to maintain appropriate culture conditions. Purification of soluble TCRs may be performed according to methods known in the art and described herein, in accordance with the laws and guidelines of U.S. and foreign regulatory agencies.

[0121] In some embodiments, nucleic acid molecules encoding high affinity or functional avidity TCRs specific to WT1p37 peptide complexed with MHC are used to transfect / transduce host cells (e.g., T cells) for use in adoptive transfer therapy. Advances in TCR sequencing have been described in the literature (e.g., Robins et al., Blood 114:4099, 2009; Robins et al., Sci. Translat. Med. 2:47ra64, 2010; Robins et al., (Sept. 10) J. Imm. Meth. Epub ahead of print, 2011; Warren et al., Genome Res. 21:790, 2011) and can be used in the process of implementing embodiments of the present disclosure. Similarly, methods for transfecting / transducing T cells with a nucleic acid of interest (e.g., U.S. Patent Application Publication No. US2004 / 0087025) as well as adoptive transfer methods using T cells of desired antigen specificity have been described (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., J. Med. 1999:2010; J. Med. 1999:2011; J. Med. 1999:2012; J. Med. 1999:2013; ...1; J. Med. 1999:2012; J. Med. 1999:2011; J. Med. 1999:2011; J. Med. 1999:2012; J. Med. 1999:2011; J. Med. 1999:2011; J. Med. 1999:2011; J. Med. 1999:2011; J. Med. Based on the teachings of the present application, including the teachings regarding high affinity TCRs specific for WT1 peptide antigens complexed with HLA receptors (e.g., Ann. Rev. Immunol. 25:243, 2007), application of these methods to the embodiments disclosed herein is also contemplated.

[0122] The WT1-specific TCRs or binding domains thereof described herein (e.g., SEQ ID NOs: 23-58 and non-CDR3 variants thereof) can be functionally characterized according to any of a number of art-recognized methods for assaying T cell activity, including measuring T cell binding, activation or induction, and measuring antigen-specific T cell responses. Examples include T cell proliferation, T cell cytokine release, antigen-specific T cell stimulation, MHC-restricted T cell stimulation (e.g., target cell preloaded with T cells), and the like. 51 These assays include cytotoxic T lymphocyte (CTL) activity (by detecting Cr efflux), measuring changes in T cell phenotypic marker expression, and other measures of T cell function. For procedures for performing these and similar assays, see, e.g., 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 therein.

[0123] Polynucleotide encoding a TCR specific for the WT1p37 antigen peptide Heterologous, isolated or recombinant nucleic acid molecules encoding recombinant T cell receptors (TCRs) or binding domains thereof (e.g., scTCRs or fusion proteins thereof) with high affinity or functional avidity specific to the WT1p37 peptides described herein can be produced or prepared by various methods and techniques described herein (see Examples). The construction of expression vectors used to recombinantly produce artificial TCRs or binding domains thereof with high affinity or functional avidity specific to the WT1p37 peptides of interest can be carried out by using any suitable molecular bioengineering technique known in the art, for example, using restriction endonuclease digestion, ligation, transformation, plasmid purification and DNA sequencing as described in Sambrook et al. (1989 and 2001 editions; Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY) and Ausubel et al. (Current Protocols in Molecular Biology, 2003). To obtain efficient transcription and translation, the polynucleotide in each recombinant expression construct contains at least one appropriate expression control sequence (also referred to as a regulatory sequence), such as a leader sequence and / or promoter, specifically operably linked to the nucleotide sequence encoding the immunogen.

[0124] Certain embodiments relate to nucleic acids encoding polypeptides contemplated herein, such as artificial TCRs or binding domains thereof with high affinity or high functional avidity specific to the WT1p37 peptide:MHC complex. As will be appreciated by those skilled in the art, nucleic acid can refer to any form of single-stranded or double-stranded DNA, cDNA, or RNA, including complementary positive and negative strands of nucleic acid, including antisense DNA, cDNA, and RNA. It also includes siRNA, microRNA, RNA-DNA hybrids, ribozymes, and various other natural or synthetic forms of DNA or RNA.

[0125] In some embodiments, the present application provides an isolated polynucleotide encoding a functional high avidity artificial (e.g., codon-optimized) TCR or binding domain thereof specific for the WT1p37 peptide of the present disclosure, comprising a polynucleotide sequence that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 97, 98, and 101-107. α In a particular embodiment, the V domain can be encoded by a polynucleotide comprising or consisting of a nucleotide sequence set forth in any one of SEQ ID NOs: 97 to 107. α In another embodiment, the present application provides a polynucleotide encoding an artificial TCR or a binding domain thereof with high functional avidity specific to the WT1p37 peptide of the present disclosure, wherein the V domain is encoded by a polynucleotide that is at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100% identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 75 to 77, 79, 82, 84, and 85. β In a particular embodiment, the V domain is encoded by a polynucleotide comprising or consisting of a nucleotide sequence set forth in any one of SEQ ID NOs: 75 to 85. β Encode the domain.

[0126] In some embodiments, the TCR or binding domain thereof provided herein is a V that is encoded by a polynucleotide having at least 75% (75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or 100%) sequence identity to a polynucleotide sequence set forth in any one of SEQ ID NOs: 97, 98, and 101-107. α A domain or a V encoded by a polynucleotide having at least 94% sequence identity to the polynucleotide sequence of SEQ ID NO: 99 or 100. α A domain or a polynucleotide comprising or consisting of any one of the sequences set forth in SEQ ID NOs: 97 to 107. α A domain encoded by a polynucleotide having at least 75% sequence identity to any one of the polynucleotide sequences set forth in SEQ ID NOs: 75 to 77, 79, 82, 84, and 85. β A domain or a polynucleotide having at least 95% sequence identity to the polynucleotide sequence set forth in any one of SEQ ID NOs: 78, 80, 81 and 83. β A domain or a polynucleotide comprising or consisting of a nucleotide sequence set forth in any one of SEQ ID NOs: 75 to 85. β Includes the domain.

[0127] In any of the above embodiments, V α Domain, V βThe polynucleotides encoding the domains or both may further encode an alpha chain constant domain or a beta chain constant domain, respectively. In certain embodiments, the TCRs of the present disclosure comprise a TCR alpha chain constant domain, the alpha chain constant domain being encoded by a polynucleotide having at least 98%-100% sequence identity to SEQ ID NO: 110. In certain embodiments, the alpha chain constant domain is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 110. In other embodiments, the present application provides a beta chain constant domain encoded by a polynucleotide having at least 99.9%-100% sequence identity to SEQ ID NO: 108 or 109. In certain embodiments, the beta chain constant domain is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 108 or 109.

[0128] In any of the above embodiments, the polynucleotide encoding the TCR comprises a TCR alpha chain, a TCR beta chain, or both. In certain embodiments, the TCR of the present disclosure is encoded by a polynucleotide comprising a nucleotide sequence encoding a self-cleaving peptide disposed between the polynucleotide sequence encoding the TCR alpha chain and the polynucleotide sequence encoding the TCR beta chain. Exemplary self-cleaving peptides comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 60-63. Such self-cleaving peptides can be encoded by a polynucleotide comprising a polynucleotide sequence set forth in any one of SEQ ID NOs: 166-170, or can be encoded by a polynucleotide consisting of the polynucleotide sequence set forth in any one of SEQ ID NOs: 166-170.

[0129] In certain embodiments, the TCR α chain, the self-cleaving peptide, and the TCR β chain are encoded by a polynucleotide having at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) identity to any one of SEQ ID NOs: 155-165. In other embodiments, the TCR α chain, the self-cleaving peptide, and the TCR β chain are encoded by a polynucleotide comprising a polynucleotide sequence of any one of SEQ ID NOs: 155-165, or are encoded by a polynucleotide consisting of any one of the polynucleotide sequences of SEQ ID NOs: 155-165. In yet other embodiments, the encoded TCR α chain, the self-cleaving peptide, and the TCR β chain comprise an amino acid sequence having at least 95% (e.g., 95%, 96%, 97%, 98%, 99% or 100%) identity to any one of the polypeptides of SEQ ID NOs: 48-58, or the encoded TCR α chain, the self-cleaving peptide, and the TCR β chain comprise or consist of an amino acid sequence of any one of SEQ ID NOs: 48-58.

[0130] In any of the embodiments disclosed herein, the polynucleotide encoding the binding protein can further comprise: (i) a polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor alpha chain, optionally wherein the encoded polypeptide is or comprises the CD8 co-receptor alpha chain; (ii) a polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor beta chain, optionally wherein the encoded polypeptide is or comprises the CD8 co-receptor beta chain; or (iii) a polynucleotide of (i) and a polynucleotide of (ii). Without intending to be bound by theory, in certain embodiments, co-expression or parallel expression of the binding protein with the CD8 co-receptor protein or a portion thereof that functions to bind to an HLA molecule results in increased expression of the binding protein in a host cell (e.g., T cells, optionally CD4 +It is believed that one or more desired activities of a CD8 co-receptor polypeptide (such as an immune cell, such as a T cell) are improved. It will be appreciated that the polynucleotide encoding the binding protein and the polynucleotide encoding the CD8 co-receptor polypeptide can be present on a single nucleic acid molecule (e.g., in the same expression vector) or can be present on separate nucleic acid molecules within the host cell.

[0131] In certain other embodiments, the polynucleotide comprises: (a) a polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor alpha chain; (b) a polynucleotide encoding a polypeptide comprising an extracellular portion of a CD8 co-receptor beta chain; and (c) a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotides of (a) and (b). In other embodiments, the polynucleotide comprises a polynucleotide that encodes a self-cleaving peptide and is disposed (1) between the polynucleotide encoding a binding protein (e.g., a TCR of the disclosure) and the polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 co-receptor alpha chain; and / or (2) between the polynucleotide encoding the binding protein and the polynucleotide encoding a polypeptide comprising the extracellular portion of a CD8 co-receptor beta chain.

[0132] In yet other embodiments, the polynucleotides are operably linked in frame: (i) (pnCD8α)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnTCR; (ii) (pnCD8β)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnTCR; (iii) (pnTCR)-(pnSCP1)-(pnCD8α)-(pnSCP2)-(pnCD8β); (iv) (pnTCR)-(pnSCP1)-(pnCD8β)-(pnSCP2)-(pnCD8α; (v) (pnCD8α)-(pnSCP1)-(pnTCR)-(pnSCP2)-(pnCD8β); or (vi) (pnC pnCD8α)-(pnSCP1)-(pnTCR)-(pnSCP2)-(pnCD8α), where pnCD8α is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, pnCD8β is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, pnTCR is a polynucleotide encoding a TCR, and pnSCP1 and pnSCP2 are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different (e.g., P2A, T2A, F2A, E2A).

[0133] In some embodiments, the encoded TCR comprises a TCR α chain and a TCR β chain, and the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between a polynucleotide encoding a TCR α chain and a polynucleotide encoding a TCR β chain. In some embodiments, the polynucleotide comprises an in-frame operably linked (i) (pnCD8α)-(pnSCP 1 )-(pnCD8β)-(pnSCP 2 )-(pnTCRβ)-(pnSCP 3 )-(pnTCRα);(ii)(pnCD8β)-(pnSCP 1 )-(pnCD8α)-(pnSCP 2 )-(pnTCRβ)-(pnSCP 3)-(pnTCRα);(iii)(pnCD8α)-(pnSCP 1 )-(pnCD8β)-(pnSCP 2 )-(pnTCRα)-(pnSCP 3 )-(pnTCRβ);(iv)(pnCD8β)-(pnSCP 1 )-(pnCD8α)-(pnSCP 2 )-(pnTCRα)-(pnSCP 3 )-(pnTCRβ);(v)(pnTCRβ)-(pnSCP 1 )-(pnTCRα)-(pnSCP 2 )-(pnCD8α)-(pnSCP 3 )-(pnCD8β);(vi)(pnTCRβ)-(pnSCP 1 )-(pnTCRα)-(pnSCP 2 )-(pnCD8β)-(pnSCP 3 )-(pnCD8α);(vii)(pnTCRα)-(pnSCP 1 )-(pnTCRβ)-(pnSCP 2 )-(pnCD8α)-(pnSCP 3 )-(pnCD8β); or (viii) (pnTCRα)-(pnSCP 1 )-(pnTCRβ)-(pnSCP 2 )-(pnCD8β)-(pnSCP 3 )-(pnCD8α), where pnCD8α is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, pnCD8β is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain, pnTCRα is a polynucleotide encoding a TCR α chain, pnTCRβ is a polynucleotide encoding a TCR β chain, and pnSCP 1 , pnSCP 2 and pnSCP 3 are each independently a polynucleotide encoding a self-cleaving peptide, wherein said polynucleotides and / or said encoded self-cleaving peptides are optionally the same or different.

[0134] In other embodiments, the binding proteins are expressed as part of a transgene construct encoding one or more other 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, and / or the host cells of the present disclosure are capable of encoding such one or more other accessory proteins. Polynucleotides and transgene constructs useful for encoding and expressing the 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) are described in PCT application PCT / US2017 / 053112, and the polynucleotides, transgene constructs, and accessory components described therein, including the nucleotide and amino acid sequences, are incorporated herein by reference. It will be appreciated that any or all of the binding proteins, safety switch proteins, tags, selection markers, CD8 co-receptor beta chain, or CD8 co-receptor alpha chain of the present disclosure can be encoded by a single nucleic acid molecule or by polynucleotide sequences that are or are present on separate nucleic acid molecules.

[0135] Exemplary safety switch proteins include, for example, cetuximab (Erbitux) tEGF receptor (tEGFr; Wang et al., Blood 118:1255-1263, 2011), a truncated EGF receptor polypeptide (huEGFRt) that lacks an extracellular N-terminal ligand-binding domain and intracellular receptor tyrosine kinase activity but maintains its native amino acid sequence, exhibits cell surface localization of a type I transmembrane protein, and has a conformationally intact epitope bound by pharmaceutical grade anti-EGFR monoclonal antibodies, as well as caspase polypeptides (e.g., iCasp9; Straathof et al., Blood 105:4247-4254, 2005; Di Stasi et al., N. Engl. J. Med. 365:1673-1683, 2011; Zhou and Brenner, Exp. Hematol. pii:S0301-472X(16)30513-6. doi:10.1016 / j.exphem.2016.07.011), RQR8 (Philip et al., Blood 124:1277-1287, 2014); a 10 amino acid tag derived from the human c-myc protein (Myc) (Kieback et al., Proc. Natl. Acad. Sci. USA 105:623-628, 2008); and marker / safety switch polypeptides such as RQR (CD20+CD34; Philip et al., 2014).

[0136] Other accessory components useful for the modified host cells of the present disclosure include tags or selection markers that allow cells to be identified, sorted, isolated, enriched, or tracked. For example, labeled host cells with a desired characteristic (e.g., an antigen-specific TCR or a safety switch protein) can be selected from unlabeled cells in a sample and more efficiently activated and expanded for inclusion in a preparation of a desired purity.

[0137] As used herein, the term "selection marker" includes nucleic acid constructs (and encoded gene products) that confer a identifiable change to cells such that immune cells transduced with a polynucleotide containing the selection marker can be detected and positively selected. RQR is a selection marker that includes the large extracellular loop of CD20 and two minimal CD34 binding sites. In some embodiments, the polynucleotide encoding RQR includes a polynucleotide encoding a 16 amino acid CD34 minimal epitope. In some embodiments, the CD34 minimal epitope is incorporated into the amino-terminal position of the CD8 co-receptor stalk domain (Q8). In other embodiments, the CD34 minimal binding site sequence can be linked to a target epitope of CD20 to form a compact marker / suicide gene for T cells (RQR8) (Philip et al., 2014, incorporated herein by reference). This construct allows the selection of host cells expressing the construct, for example with CD34-specific antibodies coupled to magnetic beads (Miltenyi), and further allows the selective elimination of transgene-expressing artificial T cells using the clinically accepted pharmaceutical antibody rituximab (Philip et al., 2014).

[0138] Other representative selection markers include several truncated type I transmembrane proteins that are not normally expressed on T cells, namely 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 by reference in its entirety). A useful feature of CD19 and CD34 is that the off-the-shelf Miltenyi CliniMACs™ selection system can be used to apply these markers for clinical grade selection. However, CD19 and CD34 are relatively large surface proteins, which pose challenges to the vector packaging capacity and transcription efficiency of integrating vectors. Surface markers that include extracellular non-signaling domains or various proteins (e.g., CD19, CD34, LNGFR) may also be used. Any selection marker may be used and is deemed to conform to Good Manufacturing Practices. In certain embodiments, the selection marker is co-expressed with a polynucleotide encoding a gene product of interest (e.g., a binding protein of the present disclosure, such as a TCR or CAR). Other examples of selection markers include reporters, such as, for example, GFP, EGFP, β-gal, or chloramphenicol acetyltransferase (CAT). In certain embodiments, the cells express a selection marker, such as, for example, CD34, and CD34 can be used to select, enrich, or isolate transduced cells of interest (e.g., by immunomagnetic selection) for use in the methods described herein. The CD34 marker, as used herein, is distinct from anti-CD34 antibodies, or scFvs, TCRs, or other antigen recognition moieties that bind, for example, CD34.

[0139] 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.

[0140] Regarding RQR polypeptides, without intending to be bound by theory, it appears that the distance of the epitope or target sequence 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 incorporated into the beta chain, the alpha chain, or both, or a fragment or variant of either or both, of the encoded CD8 co-receptor. In certain embodiments, the modified host cell comprises a heterologous polynucleotide encoding iCasp9 and a heterologous polynucleotide encoding a recombinant CD8 co-receptor comprising a beta chain and a CD8 alpha chain incorporating an RQR polypeptide.

[0141] In any of the above embodiments, the polynucleotide encoding, for example, a TCR or binding domain thereof, or a CD8 co-receptor or extracellular portion thereof of the present disclosure, is codon-optimized for efficient expression in a target host cell. In some embodiments, the host cell comprises a human immune system cell, such as a T cell, a NK cell, or a NK-T cell (Scholten et al., Clin. Immunol. 119:135, 2006). Codon optimization can be performed using known techniques and tools, for example, the GenScript® OptimumGene™ tool or GeneArt (Life Technologies). Codon-optimized sequences include partially codon-optimized sequences (i.e., one or more, but fewer than all, codons are optimized for expression in a host cell) and fully codon-optimized sequences. It will be appreciated that in embodiments in which a polynucleotide encodes two or more polypeptides (e.g., a TCR alpha chain, a TCR beta chain, a CD8 co-receptor alpha chain, a CD8 co-receptor beta chain, and one or more self-cleaving peptides), each polypeptide may independently be fully codon optimized, partially codon optimized, or not codon optimized.

[0142] In certain embodiments, the disclosure provides a host cell comprising a heterologous polynucleotide encoding any one or more of the TCRs or binding domains thereof of the disclosure, such modified or recombinant host cell expressing the TCR or binding domain thereof encoded by the heterologous polynucleotide on its cell surface.

[0143] A variety of techniques may be used for recombinant (i.e., artificial) DNA, peptide and oligonucleotide synthesis, immunoassays, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's instructions, as commonly practiced in the art, or as described herein. These and related techniques and procedures may generally be performed according to conventional methods known in the art, or as described in the various general and specialized texts in microbiology, molecular biology, biochemistry, molecular genetics, cell biology, virology, and immunology, which are cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning:A Practical Approach,vol.I & II(IRL Press,Oxford Univ.Press USA,1985);Current Protocols in Immunology(Edited by:John E.Coligan,Ada M.Kruisbeek,David H.Margulies,Ethan M.Shevach,Warren Strober 2001 John Wiley & Sons,NY,NY);Real-Time PCR:Current Technology and Applications,Edited by Julie Logan,Kirstin Edwards and Nick Saunders,2009,Caister Academic Press,Norfolk,UK;Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992);Guthrie and Fink,Guide to Yeast Genetics and Molecular Biology(Academic Press,New York,1991);Oligonucleotide Synthesis(N.Gait,Ed.,1984);Nucleic Acid Hybridization(B.Hames & S.Higgins,Eds.,1985);Transcription and Translation(B.Hames & S.Higgins,Eds.,1984);Animal Cell Culture(R.Freshney,Ed.,1986);Perbal,A Practical Guide to Molecular Cloning(1984);Next-Generation Genome Sequencing(Janitz,2008 Wiley-VCH);PCR Protocols(Methods in Molecular Biology)(Park,Ed.,3. rdEdition, 2010 Humana Press); Immobilized Cells And Enzymes (IRL Press, 1986); Papers, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J.H. Miller and M.P. Calos eds., 1987, Cold Spring Harbor Laboratory); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D.M. Weir 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 Eds., 2008); Hematopoietic Stem Cell Protocols (Methods in Molecular Medicine) (Christopher A. Klug, and Craig T. Jordan Eds., 2001); Hematopoietic Stem Cell Protocols (Methods in Molecular Biology) (Kevin D. Bunting Ed., 2008) Neural Stem Cells: Methods and Protocols (Methods in Molecular Biology) (Leslie P. Weiner Ed., 2008) for reference.

[0144] In any of the above embodiments, the polynucleotides of the present disclosure can be incorporated into a host cell, or in certain embodiments, into a vector, and the vector incorporating the polynucleotide can be introduced into a host cell. Thus, vectors are provided that include the polynucleotides provided herein. In some embodiments, the polynucleotides are operably linked to an expression control sequence. Vectors suitable for use in certain embodiments disclosed herein are known and can be selected for a particular purpose or cell. An exemplary vector can include a nucleic acid molecule capable of linking and transporting another nucleic acid molecule or capable of replicating in a host organism. Examples of vectors include plasmids, viral vectors, cosmids, and the like. Some vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and mammalian episomal vectors having a bacterial replication origin), while others can integrate into the genome of a host cell or, when introduced into a host cell, can promote integration of a polynucleotide insert and replicate in tandem with the host genome (e.g., lentiviral vectors). Additionally, some vectors can induce expression of a gene to which they are operably linked (these vectors are sometimes referred to as "expression vectors"). According to related embodiments, it will be further understood that when one or more agents (e.g., a polynucleotide encoding a recombinant TCR or binding domain thereof with high affinity or high functional avidity specific for WT1p37 as described herein) are co-administered to a subject, each agent may be carried on a separate vector or on the same vector, and multiple vectors (each carrying a different agent or the same agent) can be introduced into a cell or cell population or administered to a subject.

[0145] In certain embodiments, a polynucleotide encoding a recombinant TCR or its binding domain with high affinity or high functional avidity specific to the WT1p37 peptide:MHC of the present disclosure can be operably linked to a predetermined expression control element of a vector. For example, a polynucleotide sequence necessary for its expression and processing when ligated to a coding sequence can be operably linked. The expression control sequence can include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing signals and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion. An expression control sequence can be said to be operably linked when it is located adjacent to a gene of interest so as to act in trans, or when it is located at a distance so as to control the gene of interest. In certain embodiments, a polynucleotide encoding a TCR or its binding domain of the present disclosure is carried in an expression vector that is a viral vector such as a lentivirus vector, a gamma-retrovirus vector, or an adenovirus vector.

[0146] In certain embodiments, the recombinant expression vector is delivered to a suitable cell, e.g., a T cell or an antigen-presenting cell, i.e., a cell that presents peptide / MHC complexes on its cell surface (e.g., a dendritic cell) and lacks CD8. In certain embodiments, the host cell is a hematopoietic progenitor cell or a cell of the human immune system. For example, the immune system cell can be a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double negative T cell, a γδ T cell, a natural killer cell, a dendritic cell, or any combination thereof, including CD4+ T cells and CD8+ T cells, when combinations are present. In certain embodiments, the host is a T cell, the T cell can be a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof. Thus, the recombinant expression vector can further include a lymphoid tissue-specific transcriptional regulator (TRE), e.g., a B lymphocyte, a T lymphocyte, or the like, or a dendritic cell-specific TRE. Lymphoid tissue-specific TREs are known in the art (see, e.g., Thompson et al., Mol. Cell. Biol. 12:1043, 1992; Todd et al., J. Exp. Med. 177:1663, 1993; Penix et al., J. Exp. Med. 178:1483, 1993).

[0147] In addition to vectors, certain embodiments relate to host cells comprising a heterologous polynucleotide or vector disclosed herein. In certain embodiments, the host cell expresses the TCR encoded by the polynucleotide on its cell surface, the polynucleotide being heterologous to the host cell. As will be appreciated by those of skill in the art, many suitable host cells are available in the art. A host cell includes any individual cell or cell culture into which a vector can be introduced or into which a nucleic acid and / or protein can be incorporated, and any progeny cells. The term also refers to the progeny of a host cell, whether identical or different in genotype or phenotype. Suitable host cells vary depending on the vector and include mammalian cells, animal cells, human cells, monkey cells, insect cells, yeast cells, and bacterial cells. The vectors or other materials can be incorporated into these cells by the use of viral vectors, transformation by calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory, 1989).

[0148] In some embodiments, the V of the TCR expressed by the host cell α The domain is encoded by a polynucleotide having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100%) sequence identity to any one of the polynucleotides of SEQ ID NOs: 97, 98 and 101-107, or at least 94% sequence identity to SEQ ID NO: 99 or 100. α The domain is encoded by a polynucleotide that (a) comprises any one of the polynucleotide sequences set forth in SEQ ID NOs: 97 to 107, or (b) is composed of any one of the polynucleotide sequences set forth in SEQ ID NOs: 97 to 107.

[0149] In some embodiments, the V of the host cellβ The domain is encoded by a polynucleotide having at least 75% sequence identity to any one of the polynucleotides of SEQ ID NOs: 75-77, 79, 82, 84, and 85, or at least 95% sequence identity to any one of the polynucleotides of SEQ ID NOs: 78, 80, 81, and 83. β The domain is encoded by a polynucleotide that (a) comprises any one of the polynucleotide sequences set forth in SEQ ID NOs: 75 to 85, or (b) is composed of any one of the polynucleotide sequences set forth in SEQ ID NOs: 75 to 85.

[0150] In some embodiments, the TCR alpha chain comprises an alpha chain constant domain encoded by a polynucleotide having at least 98% identity to SEQ ID NO: 110. In some embodiments, the TCR alpha chain comprises an alpha chain constant domain encoded by a polynucleotide that (a) comprises the polynucleotide sequence of SEQ ID NO: 110, or (b) consists of the polynucleotide sequence of SEQ ID NO: 110. In some embodiments, the TCR beta chain comprises a beta chain constant domain encoded by a polynucleotide having at least 99.9% sequence identity to SEQ ID NO: 108 or 109. In some embodiments, the TCR beta chain comprises a beta chain constant domain encoded by a polynucleotide that (a) comprises the polynucleotide sequence of SEQ ID NO: 108 or 109, or (b) consists of the polynucleotide sequence of SEQ ID NO: 108 or 109.

[0151] In some embodiments, the polynucleotide comprises a nucleotide sequence encoding a self-cleaving peptide disposed between the polynucleotide sequence encoding the TCR α chain and the polynucleotide sequence encoding the TCR β chain.

[0152] In some embodiments, the encoded self-cleaving peptide (a) comprises the amino acid sequence of any one of the polypeptides set forth in SEQ ID NOs: 60-63; or (b) consists of the sequence of any one of the polypeptides set forth in SEQ ID NOs: 60-63.

[0153] In some embodiments, the polynucleotide encoding the self-cleaving peptide (a) comprises any one of the polynucleotide sequences set forth in SEQ ID NOs: 166 to 170; or (b) consists of any one of the polynucleotide sequences set forth in SEQ ID NOs: 166 to 170.

[0154] In some embodiments, the TCR α chain, the self-cleaving peptide, and the TCR β chain are encoded by a polynucleotide having at least 95% identity to any one of SEQ ID NOs: 155-165.

[0155] In some embodiments, the TCR α chain, self-cleaving peptide, and TCR β chain are encoded by a polynucleotide that (a) comprises any one of the sequences of the polynucleotides set forth in SEQ ID NOs: 155 to 165; or (b) is composed of any one of the sequences of the polynucleotides set forth in SEQ ID NOs: 155 to 165.

[0156] In some embodiments, the encoded TCR α chain, autocleaving peptide, and TCR β chain comprise an amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, 99.1%, 99.5%, 99.9% or 100% identity to any one of the polypeptides in SEQ ID NOs: 48 to 58. In some embodiments, the encoded TCR α chain, autocleaving peptide, and TCR β chain (a) comprise the amino acid sequence of any one of the polypeptides in SEQ ID NOs: 48 to 58; or (b) consist of the amino acid sequence of any one of the polypeptides in SEQ ID NOs: 48 to 58.

[0157] In some embodiments, the host cell is a hematopoietic progenitor cell or a human immune system cell, hi some embodiments, the immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double negative T cell, a γδ T cell, a natural killer cell, a natural killer T cell, a dendritic cell, or any combination thereof, optionally, the combination comprises a CD4+ T cell and a CD8+ T cell.

[0158] In some embodiments, the host immune system cells are T cells, hi some embodiments, the T cells are naive T cells, central memory T cells, effector memory T cells, or any combination thereof.

[0159] In some embodiments, the TCR has a higher surface expression on a T cell compared to an endogenous TCR (e.g., when the endogenous TCR has not been artificially silenced or inhibited in expression).

[0160] In some embodiments, the host cell further comprises: (i) a heterologous polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor alpha chain, optionally wherein the encoded polypeptide is or comprises the CD8 co-receptor alpha chain; (ii) a heterologous polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor beta chain, optionally wherein the encoded polypeptide is or comprises the 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.

[0161] In some embodiments, the host cell comprises: (a) a heterologous polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor alpha chain; (b) a heterologous polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor beta chain; and (c) a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotides of (a) and (b).

[0162] In any of the embodiments disclosed herein, the host cell (e.g., an immune cell such as a human T cell) is capable of killing the tumor cell when simultaneously present in a sample with: (i) a tumor cell of the breast cancer cell line MDA-MB-468; (ii) a tumor cell of the pancreatic adenocarcinoma cell line PANC-1; (iii) a tumor cell of the breast cancer cell line MDA-MB-231; (iv) a tumor cell of the myeloid leukemia cell line K562 that expresses HLA-A2, optionally wherein the HLA-A2 comprises HLA-A*201; (v) a tumor cell of the colon cancer cell line RKO that expresses HLA-A2, optionally wherein the HLA-A2 comprises HLA-A*201; or (vi) any combination of the tumor cells of (i)-(v).

[0163] In certain embodiments, the host cells are capable of killing the tumor cells when the host cells and the tumor cells are present in the sample at a host cell:tumor cell ratio of 32:1, 16:1, 8:1, 4:1, 2:1, or 1.5:1. Target cell killing can be determined, for example, with the Incucyte® bioimaging platform (Essen Bioscience). In certain embodiments, this platform uses caspase activation and tumor cell marker (e.g., RapidRed or NucRed) signals to measure overlap, and an increase in the overlap area is considered as tumor cell death by apoptosis. Target cells are then immunized with chromium ( 51 Cr) and in the supernatant after 4 hours of co-incubation with immune cells expressing the binding protein of the present disclosure. 51 Killing can also be measured using a 4 hour assay measuring Cr.

[0164] In any of the above embodiments, the host cell (e.g., immune cell) may be modified to reduce or silence expression of one or more endogenous genes encoding polypeptides involved in immune signaling or other related activities. Exemplary gene knockouts include those encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, HLA molecules, TCR molecules, etc. Without intending to be bound by theory, it is believed that certain endogenously expressed immune cell proteins may be recognized as foreign by the allogeneic host that has taken up the modified immune cell, thereby eliminating the modified immune cell (e.g., HLA alleles), downregulating the immune activity of the modified immune cell (e.g., PD-1, LAG-3, CTLA4, FasL, TIGIT, TIM3), or interfering with the binding activity of the heterologously expressed binding proteins of the present disclosure (e.g., the endogenous TCR of the modified T cell binds non-Ras antigens, preventing the modified immune cell from binding to cells expressing Ras antigens).

[0165] Thus, reducing or silencing the expression or activity of such endogenous genes or proteins can improve the activity, tolerance or persistence of the modified cells under autologous or allogeneic host conditions, allowing the cells to be administered universally (e.g., to any recipient regardless of HLA type). In certain embodiments, the modified cells are donor cells (e.g., allogeneic) or autologous cells. In certain embodiments, the host cells of the disclosure comprise one or more chromosomal gene knockouts of genes encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, FasL, 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 TCR component (e.g., a gene encoding a TCR variable region or a TCR constant region) (see, e.g., Torikai et al., Nature Sci. Rep. 6:21757 (2016); Torikai et al., Blood 119(24):5697 (2012); and Torikai et al., Blood 122(8):1341 (2013), all of which are incorporated by reference in their entirety for their gene editing techniques, compositions, and adoptive cell therapy techniques).

[0166] As used herein, the term "chromosomal gene knockout" refers to the introduction of a gene mutation or inhibitor in a host cell that prevents (e.g., reduces, slows, suppresses or stops) the production of a functionally active endogenous polypeptide product by the host cell. Mutations that result in a chromosomal gene knockout can include, for example, the introduction of nonsense mutations (including the formation of premature stop codons), missense mutations, gene deletions and strand breaks, and the heterologous expression of inhibitory nucleic acid molecules that suppress endogenous gene expression in the host cell.

[0167] In certain embodiments, chromosomal gene knock-out or knock-in is performed by chromosomal editing of the host cell. Chromosomal editing can be performed, for example, using endonucleases. As used herein, "endonuclease" refers to an enzyme capable of catalyzing the cleavage of phosphodiester bonds in a polynucleotide strand. In certain embodiments, the endonuclease can inactivate or "knock out" a target gene by cleaving the target gene. The endonuclease can be a natural, recombinant, genetically modified, or fusion endonuclease. Nucleic acid strand breaks caused by endonucleases are usually repaired by the respective mechanisms of 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, to "knock out" a target gene, and optionally to inactivate a target gene by a donor gene knock-in or target gene knock-out event. NHEJ is an error-prone repair process that often results in a mutation, e.g., a substitution, deletion, or addition of at least one base, in the DNA sequence at the cleavage site. 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.

[0168] As used herein, "zinc finger nuclease (ZFN)" refers to a fusion protein in which a zinc finger DNA binding domain is fused to a non-specific DNA cleavage domain such as Fokl endonuclease. Each zinc finger motif of about 30 amino acids binds to about 3 base pairs of DNA, and the specificity of the 3 base sequence can be changed by mutating amino acids at certain residues (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 generate binding specificity for a desired DNA sequence, such as a region of about 9 to about 18 base pairs in length. By way of background, ZFNs mediate genome editing by catalyzing the formation of site-specific DNA double-strand breaks (DSBs) in the genome, and homologous repair facilitates targeted integration of transgenes containing flanking sequences homologous to the genome at the DSB site. Alternatively, ZFN-induced DSBs can result in knockout of targeted genes by repair via non-homologous end joining (NHEJ), an error-prone cellular repair pathway that results in the insertion or deletion of nucleotides at the break site. In some embodiments, gene knockout includes insertion, deletion, mutation, or a combination thereof, performed using ZFN molecules.

[0169] As used herein, "Transcription Activator-Like Effector Nuclease (TALEN)" refers to a fusion protein that contains a TALE DNA-binding domain and a DNA cleavage domain, such as FokI endonuclease. A "TALE DNA-binding domain" or "TALE" is composed of one or more TALE repeat domains / units, each of which typically has a highly conserved 33-35 amino acid sequence, except for the 12th and 13th amino acids, which are variable. The TALE repeat domains are involved in the binding of the TALE to the target DNA sequence. The variable amino acid residues are called Repeat Variable Diresidues (RVDs) and correlate with specific nucleotide recognition. The natural (canonical) code for DNA recognition of these TALEs has been determined such that if positions 12 and 13 of the TALE are HD (histine-aspartic acid) sequences, the TALE binds to cytosine (C), if NG (asparagine-glycine), the TALE binds to T nucleotides, if NI (asparagine-isoleucine), the TALE binds to A, if NN (asparagine-asparagine), the TALE binds to G or A nucleotides, and if NG (asparagine-glycine), the TALE binds to T nucleotides. Non-canonical (atypical) RVDs are also known (see, for example, U.S. Patent Publication No. US2011 / 0301073, the entire contents of which are incorporated herein by reference). TALENs can be used to generate site-specific double-strand breaks (DSBs) in the genome of T cells. Non-homologous end joining (NHEJ) introduces errors and knocks out gene expression by joining DNA from both sides of a double-stranded break with little or no terminal sequence overlap to anneal. Alternatively, a transgene can be introduced at the site of a DSB by homologous repair if the transgene has homologous flanking sequences. In some embodiments, gene knockout includes insertion, deletion, mutation, or a combination thereof, and is performed using TALEN molecules.

[0170] As used herein, a "clustered regularly interspaced short palindromic repeats / Cas (CRISPR / Cas)" nuclease system refers to a system that utilizes a CRISPR RNA (crRNA)-guided Cas nuclease to recognize a target site (called a protospacer) in the genome by base-pairing complementarity and then cleaves DNA if a short conserved protospacer-associated motif (PAM) is present immediately 3' of 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. In types I and III, the crRNA-guided surveillance complex requires multiple Cas subunits. Type II systems are the most well-studied and contain at least three components: an RNA-guided Cas9 nuclease, a crRNA, and a trans-acting crRNA (tracrRNA). The tracrRNA contains a duplex-forming region. The crRNA and tracrRNA form a duplex and can interact with Cas9 nuclease to guide the Cas9 / crRNA:tracrRNA complex to a specific site on the target DNA by Watson-Crick base pairing between the spacer on the crRNA and the protospacer on the target DNA upstream of the PAM. Cas9 nuclease cleaves the double-stranded break within the region defined by the crRNA spacer. When repaired by NHEJ, insertions and / or deletions are generated and expression of the target locus is stopped. Alternatively, a transgene containing homologous flanking sequences can be introduced at the site of the DSB by homology repair. The crRNA and tracrRNA can be complexed to construct a single guide RNA (sgRNA or gRNA) (see, for example, Jinek et al., Science 337:816-21, 2012).Additionally, the region of the guide RNA complementary to the target site can be modified or programmed to target the desired sequence (Xie et al., PLOS One 9:e100448, 2014; U.S. Patent Application Publication No. US2014 / 0068797, U.S. Patent Application Publication No. US2014 / 0186843, U.S. Patent No. 8,697,359, and PCT Publication No. WO2015 / 071474, each of which is incorporated herein by reference). In certain embodiments, gene knockout comprises an insertion, deletion, mutation, or a combination thereof, and is performed using a CRISPR / Cas nuclease system. Exemplary gRNA sequences and methods for their use to knock out endogenous genes encoding immune cell proteins are described in Ren et al., Clin. Cancer Res. 23(9):2255-2266 (2017), all of which are incorporated herein by reference.

[0171] As used herein, "meganuclease" is also referred to as "homing endonuclease" and refers to an endo-deoxyribonuclease characterized by a large recognition site (a double-stranded DNA sequence of about 12 to about 40 base pairs). Based on their sequences and structural motifs, meganucleases can be classified into five families: LAGLIDADG, GIY-YIG, HNH, His-Cys box, and PD-(D / E)XK. Representative meganucleases include 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, and their 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).

[0172] In certain embodiments, natural meganucleases can be used to facilitate site-specific genome modification of targets selected from genes encoding PD-1, LAG3, TIM3, CTLA4, TIGIT, FasL, HLA, or genes encoding TCR components. In other embodiments, artificial meganucleases with novel binding specificities for target genes are used for 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. 10:895-905, 2002). Ther. 7:49-66, 2007; US Patent Publication Nos. US2007 / 0117128, US2006 / 0206949, US2006 / 0153826, US2006 / 0078552, and US2004 / 0002092). In another embodiment, homing endonucleases modified with modular DNA-binding domains of TALENs are used to generate fusion proteins called MegaTALs to generate chromosomal gene knockouts. MegaTALs can be used not only to knock out one or more target genes, but also to introduce (knock in) heterologous or foreign polynucleotides when used in conjunction with an exogenous donor template that encodes a polypeptide of interest.

[0173] In some 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, said inhibitory nucleic acid molecule encoding a target-specific inhibitor, and said encoded target-specific inhibitor inhibits endogenous gene expression (e.g., of PD-1, TIM3, LAG3, CTLA4, TIGIT, FasL, an HLA component, or a TCR component, or any combination thereof) in the host cell.

[0174] Chromosomal gene knockout can be confirmed directly by DNA sequencing of host immune cells following the use of knockout methods or agents. Chromosomal gene knockout can also be inferred from the absence of gene expression following knockout (e.g., the absence of mRNA or the polypeptide product encoded by this gene).

[0175] In some embodiments, the chromosomal gene knockout comprises 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.

[0176] In some embodiments, the chromosomal gene knockout comprises knocking out a TCR component gene selected from a TCR alpha variable region gene, a TCR beta variable region gene, a TCR constant region gene, or a combination thereof.

[0177] It will further be appreciated that any of the gene editing techniques and tools disclosed herein may be used to introduce a polynucleotide encoding a TCR and / or a polynucleotide encoding a CD8 co-receptor of the present disclosure into a host cell genome.

[0178] In another aspect, the present application provides compositions and unit dose formulations comprising the modified host cells of the present disclosure and a pharma- ceutically acceptable carrier, diluent or excipient.

[0179] In certain embodiments, a host cell composition or unit dose formulation contains about a 1:1 ratio of (i) a composition that contains at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% modified CD4+ T cells, and (ii) a composition that contains 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, wherein the unit dose formulation has a low or substantially no content of naive T cells (i.e., 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% of the naive T cell population present in a unit dose compared to a patient sample having a comparable number of PBMCs).

[0180] In some embodiments, the host cell composition or unit dose formulation comprises (i) a composition comprising at least about 50% modified CD4+ T cells and (ii) a composition comprising at least about 50% modified CD8+ T cells in a ratio of about 1:1, said host cell composition or unit dose formulation comprising a low or substantially zero content of naive T cells. In other embodiments, the host cell composition or unit dose formulation comprises (i) a composition comprising at least about 60% modified CD4+ T cells and (ii) a composition comprising at least about 60% modified CD8+ T cells in a ratio of about 1:1, said unit dose formulation comprising a low or substantially zero content of naive T cells. In yet other embodiments, the host cell composition or unit dose formulation comprises (i) a composition comprising at least about 70% artificial CD4+ T cells and (ii) a composition comprising at least about 70% artificial CD8+ T cells in a ratio of about 1:1, said unit dose formulation comprising a low or substantially zero content of naive T cells. In some embodiments, the host cell composition or unit dose formulation comprises (i) a composition comprising at least about 80% modified CD4+ T cells and (ii) a composition comprising at least about 80% modified CD8+ T cells in a ratio of about 1:1, said host cell composition or unit dose formulation comprising a low or substantially no naive T cells. In some embodiments, the host cell composition or unit dose formulation comprises (i) a composition comprising at least about 85% modified CD4+ T cells and (ii) a composition comprising at least about 85% modified CD8+ T cells in a ratio of about 1:1, said host cell composition or unit dose formulation comprising a low or substantially no naive T cells. In some embodiments, the host cell composition or unit dose formulation comprises (i) a composition containing at least about 90% modified CD4+ T cells and (ii) a composition containing at least about 90% modified CD8+ T cells in about a 1:1 ratio, wherein the host cell composition or unit dose formulation has low or substantially no content of naive T cells.

[0181] Of course, the host cell composition or unit dose formulation of the present disclosure can contain any host cell or any combination of host cells described herein. In some embodiments, for example, the host cell composition or unit dose formulation contains modified CD8+ T cells, modified CD4+ T cells, or both, modified to encode a binding protein specific for the Ras peptide:HLA-A*02:01 complex, and further contains modified CD8+ T cells, modified CD4+ T cells, or both, modified to encode a binding protein specific for the WT1 peptide:HLA-A*02:01 complex.Additionally or alternatively, the host cell compositions or unit dose formulations of the present disclosure can contain any host cell or combination of host cells described herein and can further contain another antigen (e.g., another WT1 antigen, or an antigen such as BCMA, CD3, CEACAM6, c-Met, EGFR, EGFRvIII, ErbB2, ErbB3, ErbB4, EphA2, IGF1R, GD2, O-acetyl GD2, O-acetyl GD3, O-acetyl GD4, O-acetyl GD5, O-acetyl GD6, O-acetyl GD7, O-acetyl GD8, O-acetyl GD9, O-acetyl GD10, O-acetyl GD11, O-acetyl GD12, O-acetyl GD13, O-acetyl GD14, O-acetyl GD15, O-acetyl GD16, O-acetyl GD17, O-acetyl GD18, O-acetyl GD19, O-acetyl GD20, O-acetyl GD19, O-acetyl GD11, O-acetyl GD12, O-acetyl GD15, O-acetyl GD16, O-acetyl GD17, O-acetyl GD18, O-acetyl GD19 ... 3, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CD151, CA125, CEA, CTLA-4, GITR, BTLA, TGFBR2, TGFBR1, IL6R, gp130, Lewis A, Le IS Y, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM, MAGE-A (e.g. MAGE-A1, MAGE-A3 and MAGE-A4), Mesothelin, NY-ESO-1, PSMA, RANK , ROR1, TNFRSF4, CD40, CD137, TWEAK-R, HLA, tumor- or pathogen-associated peptides bound to HLA, hTERT peptides bound to HLA, tyrosinase peptides bound to HLA, WT-1 peptides bound to HLA, LTβR, LIFRβ, LRP5, MUC1, OSMRβ, TCRα, TCRβ, CD19, CD20, CD22, CD25, CD28, CD30, CD33, CD52, CD5 The host cell may contain modified cells (e.g., immune cells such as T cells) expressing a binding protein specific for an antigen derived from another protein or target, such as CD123, CD171, CD276, B7H4, TLR7, TLR9, PTCH1, WT-1, HA1-H, Robo1, alpha-fetoprotein (AFP), frizzled, OX40, PRAME, and SSX-2. For example, a unit dose formulation may contain modified CD8+ T cells expressing a binding protein that specifically binds to the WT1-HLA complex and modified CD4+ T cells (and / or modified CD8+ T cells) expressing a binding protein that specifically binds to the HER2 antigen (e.g., CAR). It will also be appreciated that any of the host cells disclosed herein may be administered in a combination therapy.

[0182] In any of the embodiments described herein, the host cell composition or unit dose formulation contains equal or nearly equal amounts of engineered CD45RA-CD3+CD8+ cells and modified CD45RA-CD3+CD4+TM cells.

[0183] Methods of Use and Treatment In certain aspects, the disclosure relates to a method of treating a hyperproliferative or proliferative disorder or condition characterized by Wilms' tumor protein 1 (WT1) expression or overexpression by administering to a human subject in need of treatment a composition containing a recombinant high affinity or high functional avidity TCR specific for human WT1 or a binding domain thereof according to any of the above TCRs or any binding domain thereof described herein, or a host cell, such as a T cell, engineered to express the above TCR, or a composition containing any of the TCRs or binding domains thereof or host cells described herein. In some embodiments, the TCR is expressed by a host cell, such as a hematopoietic progenitor cell or a cell of the human immune system. In some embodiments, the cell of the immune system is a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double negative T cell, a gamma delta T cell, a natural killer cell, a natural killer T cell, a dendritic cell, or any combination thereof.

[0184] The presence of a hyperproliferative or proliferative disorder or malignant condition in a subject refers to the presence in said subject of dysplastic, cancerous and / or transformed cells, such as neoplastic cells, tumor cells, non-contact inhibitory cells or oncogenically transformed cells (e.g., solid cancers; hematopoietic cancers, including lymphomas and leukemias, such as acute myeloid leukemia, chronic myeloid leukemia, etc.), which are known in the art and have established criteria for their diagnosis and classification (e.g., Hanahan and Weinberg, Cell 144:646, 2011; Hanahan and Weinberg, Cell 100:57, 2000; Cavallo et al., Canc. Immunol. Immunother. 60:319, 2011; Kyrigideis et al., J. Carcinog. 9:3, 2010). In certain embodiments, such cancer cells can be acute myeloid leukemia, B-cell lymphoblastic leukemia, T-cell lymphoblastic leukemia, or myeloma cells, including cancer stem cells that can initiate and sequentially engraft any of these types of cancer (see, e.g., Park et al., Molec. Therap. 17:219, 2009).

[0185] In certain embodiments, methods are provided for treating hyperproliferative or proliferative disorders, such as hematopoietic malignancies and solid cancers (see, e.g., Nakatsuka et al., Modern Pathology 19:804-714 (2006)). Exemplary hematopoietic malignancies include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic eosinophilic leukemia (CEL), myelodysplastic syndromes (MDS), non-Hodgkin's lymphoma (NHL), or multiple myeloma (MM).

[0186] In another embodiment, a method is provided for treating a hyperproliferative or proliferative disorder, such as a solid cancer selected from biliary tract cancer, bladder cancer, bone and soft tissue carcinoma, brain tumor, breast cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colon cancer, desmoid tumor, embryonal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumors, head and neck squamous cell carcinoma, liver cancer, lung cancer, mesothelioma, malignant melanoma, osteosarcoma, ovarian cancer (see, e.g., Hylander et al., Gynecologic Oncology 101:12-17 (2006)), pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, kidney cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, or uterine carcinoma.

[0187] In some embodiments, the TCR is capable of promoting an antigen-specific T cell response against human WT1 in a class I HLA-restricted manner. In some embodiments, the class I HLA-restricted response is independent of transporter associated with antigen processing (TAP). In some embodiments, the antigen-specific T cell response comprises at least one of a CD4+ helper T lymphocyte (Th) response and a CD8+ cytotoxic T lymphocyte (CTL) response. In some embodiments, the CTL response is specific to cells overexpressing WT1.

[0188] Also provided herein are any of the above TCRs, polynucleotides, compositions, vectors and host cells (including any combination) for use in a method for treating a proliferative or hyperproliferative disorder associated with Wilms Tumor Protein 1 (WT1) expression or overexpression.

[0189] Also provided herein are any of the above TCRs, polynucleotides, compositions, vectors and host cells (including any combination) for use in a method for manufacturing a medicament for the treatment of a proliferative or hyperproliferative disorder associated with Wilms Tumor Protein 1 (WT1) expression or overexpression.

[0190] As will be appreciated by those skilled in the medical field, the terms "treat" and "treatment" refer to the medical management of a disease, disorder, or condition in a subject (i.e., a patient, host, which can be a human or non-human animal) (see, for example, Stedman's Medical Dictionary). In general, an appropriate dosage and treatment regimen provides one or more recombinant TCRs with high functional avidity specific to human WT1 or binding domains thereof (e.g., SEQ ID NOs: 23 to 58 and variants thereof provided herein) or host cells expressing said TCRs, etc., in an amount sufficient to provide a therapeutic or prophylactic effect, and optionally an adjuvant therapy (e.g., cytokines such as IL-2, IL-15, IL-21, or any combination thereof). Therapeutic or prophylactic effects obtained by therapeutic treatment or prophylactic or protective methods include, for example, improved clinical outcomes, the purpose of which is to prevent, delay, or otherwise inhibit undesirable physiological changes or disorders (e.g., to reduce statistically significant amounts compared to untreated controls), or to prevent, delay, or otherwise inhibit the spread or severity of such diseases or disorders. Beneficial or desirable clinical outcomes obtained by treating a subject include suppression, reduction, or alleviation of symptoms caused by or associated with the disease or disorder being treated; reduced incidence of symptoms; improved quality of life; extended disease-free state (i.e., reducing the probability or tendency of a subject to develop symptoms that are diagnostic of a disease); reduced extent of disease; stabilization (i.e., non-worsening) of the disease state; slowing or deceleration of disease progression; improvement or palliative disease state; and remission, whether detectable or undetectable (whether partial or complete), or extended overall survival.

[0191] "Treatment" may also mean extending the survival of a subject as compared to the expected survival if the subject had not received the treatment. Subjects in need of the methods and compositions described herein include those already suffering from a disease or disorder as well as those prone to or at risk of developing a disease or disorder. Subjects in need of prophylactic treatment include those in whom a disease, condition or disorder is to be prevented (i.e., reducing the probability of the occurrence or recurrence of a disease or disorder). The clinical benefits provided by the compositions (and formulations containing the compositions) and methods described herein can be evaluated by designing and conducting in vitro assays, preclinical and clinical studies in subjects who would benefit from administration of the compositions, as described in the Examples.

[0192] In another aspect, the disclosure relates to a method of treating a hyperproliferative disorder or a condition characterized by Wilms' tumor protein 1 (WT1) overexpression or expression by administering to a human subject in need of treatment a composition containing an isolated polynucleotide encoding a recombinant high affinity or high functional avidity TCR or binding domain thereof specific for human WT1 associated with any of the encoded TCRs or binding domains thereof, or a host cell, such as a T cell, comprising said polynucleotide, or a composition containing any of the TCRs or binding domains thereof or host cells described herein. In certain embodiments, the polynucleotide encoding the TCR or binding domain thereof specific for human WT1p37 peptide:MHC is codon-optimized for the host cell of interest. In other embodiments, any of the polynucleotides described above are operably linked to an expression control sequence and are optionally carried in an expression vector, such as a viral vector. Exemplary viral vectors include lentiviral vectors and gamma-retroviral vectors. In related embodiments, the vector is capable of delivering the polynucleotide to a host cell, such as a hematopoietic progenitor cell or a cell of the immune system (e.g., a human hematopoietic progenitor cell or a cell of the immune system). Exemplary immune system cells include CD4+ T cells, CD8+ T cells, CD4-CD8- double negative T cells, γδ T cells, natural killer cells, dendritic cells, or any combination thereof (e.g., of human origin). In some embodiments, the immune system cells are T cells, such as naive T cells, central memory T cells, effector memory T cells, or any combination thereof, optionally all of which are of human origin.

[0193] In yet another aspect, the disclosure relates to a method of treating a hyperproliferative or proliferative disorder or a condition characterized by Wilms tumor protein 1 (WT1) expression or overexpression by administering to a human subject in need of treatment an effective amount of a host cell comprising a heterologous polynucleotide or expression vector according to any of the above embodiments or any of the above described herein, wherein the artificial or recombinant host cell expresses a TCR encoded by a heterologous polynucleotide specific for human WT1p37:MHC on its cell surface. In some embodiments, the disclosure relates to a method of treating a hyperproliferative or proliferative disorder or a condition characterized by Wilms tumor protein 1 (WT1) p37 peptide production or the presence of a WT1p37 peptide:MHC complex by administering to a human subject in need of treatment an effective amount of a host cell comprising a heterologous polynucleotide or expression vector according to any of the above embodiments or any of the above described herein, wherein the artificial or recombinant host cell expresses a TCR encoded by a heterologous polynucleotide specific for human WT1p37:MHC on its cell surface.

[0194] Also provided is an adoptive immunotherapy method for treating a condition characterized by WT1 overexpression in cells of a subject having a hyperproliferative or proliferative disorder, comprising administering to the subject an effective amount of a host cell or composition of the present disclosure.

[0195] In some embodiments, the host cells are modified ex vivo. In some embodiments, the host cells are allogeneic, syngeneic or autologous to the subject. In some embodiments, the host cells are hematopoietic progenitor cells or human immune system cells. In some embodiments, the immune system cells are CD4+ T cells, CD8+ T cells, CD4-CD8- double negative T cells, γδ T cells, natural killer cells, dendritic cells, or any combination thereof.

[0196] In some embodiments, the T cells are naive T cells, central memory T cells, effector memory T cells, or any combination thereof.

[0197] In some embodiments, the hyperproliferative or proliferative disorder is a hematopoietic malignancy or solid cancer.

[0198] In some embodiments, the hematopoietic malignancy is selected from acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic eosinophilic leukemia (CEL), myelodysplastic syndromes (MDS), non-Hodgkin's lymphoma (NHL), or multiple myeloma (MM).

[0199] In some embodiments, the solid cancer is selected from breast cancer, ovarian cancer, lung cancer, biliary tract cancer, bladder cancer, bone and soft tissue carcinoma, brain cancer, cervical cancer, colon cancer, colorectal adenocarcinoma, colon cancer, desmoid tumor, embryonal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumors, head and neck squamous cell carcinoma, liver cancer, mesothelioma, malignant melanoma, osteosarcoma, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, kidney cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, or uterine cancer.

[0200] In some embodiments, the host cells are administered parenterally.

[0201] In some embodiments, the method comprises administering multiple doses of the host cells to the subject, hi some embodiments, the multiple doses are administered at intervals of about 2 to about 4 weeks.

[0202] A cell expressing a recombinant TCR or its binding domain specific (e.g., high affinity or high functional avidity) to the human WT1p37 peptide described in the present application can be administered to a subject by blending it with a pharma- ceutically or physiologically acceptable or appropriate excipient or base. A pharma- ceutically acceptable excipient is a biocompatible solvent suitable for administration to a human or other non-human mammalian subject, such as physiological saline, and is described in detail in the present application.

[0203] A therapeutically effective amount is the amount of host cells (expressing recombinant TCR or its binding domain with high affinity or high functional avidity specific to human WT1p37 peptide:MHC) used in adoptive transfer that is capable of producing a clinically desirable result in a human or non-human mammal following administration (i.e., an amount sufficient to statistically significantly induce or enhance a specific T cell immune response (e.g., a cytotoxic T cell response) against cells overexpressing WT1 or cells producing WT1p37 peptide). As is well known in the medical field, the dosage administered to any one patient will vary depending on a number of factors, including the patient's size, weight, body surface area, age, the particular therapy administered, sex, time and route of administration, general health, and other drugs administered in parallel. Although dosages vary, a preferred dosage for administration of host cells containing a recombinant expression vector described herein is about 10 cells / mL. 4 pieces / m 2 , about 5×10 4 pieces / m 2 , about 10 5 pieces / m 2 , about 5×10 5 pieces / m 2 , about 10 6 pieces / m 2 , about 5×10 6 pieces / m 2 , about 10 7 pieces / m 2 , about 5×10 7 pieces / m 2 , about 10 8 pieces / m 2 , about 5×10 8 pieces / m 2 , about 10 9 pieces / m 2 , about 5×10 9 pieces / m 2 , about 10 10 pieces / m 2 , about 5×10 10 pieces / m 2 Or about 10 11 pieces / m 2 In some embodiments, the dose is about 10 cells. 7 pieces / m 2 , about 5×10 7 pieces / m 2 , about 10 8 pieces / m 2, about 5×10 8 pieces / m 2 , about 10 9 pieces / m 2 , about 5×10 9 pieces / m 2 , about 10 10 pieces / m 2 , about 5×10 10 pieces / m 2 Or about 10 11 pieces / m 2 Includes.

[0204] The pharmaceutical composition may be administered as appropriate for the disease or condition to be treated (or prevented) based on the judgment of one of ordinary skill in the medical arts. The appropriate dose and the appropriate duration and frequency of administration of the composition will be determined by factors such as the health of the patient, the size of the patient (i.e., body weight, body mass index or body surface area), the type and severity of the patient's disease, the particular form of the active ingredient, and the method of administration. In general, an appropriate dosage and treatment regimen provides a sufficient amount of the composition to produce a therapeutic and / or prophylactic effect (as described herein, including an improved clinical outcome, such as an increased frequency of complete or partial remission, or an increased disease-free and / or overall survival, and a reduced severity of symptoms). For prophylactic applications, the dosage should be sufficient to prevent, delay the onset of, or reduce the severity of the associated disease or disorder. The prophylactic effect of the immunogenic composition administered according to the methods described herein can be determined by conducting preclinical and clinical studies (including in vitro and in vivo animal studies) and analyzing the resulting data using appropriate statistical, biological and clinical methods and techniques, all of which are readily accessible to one of ordinary skill in the art.

[0205] Pathologies associated with WT1 overexpression (or expression, in some embodiments) include any disorder or condition in which there is underactivity, overactivity, or inappropriate activity of WT1 cellular or molecular events, and generally result from (statistically significant) abnormally high levels of WT1 expression in diseased cells (e.g., leukemia cells) compared to normal cells. Subjects with such disorders or diseases would benefit from treatment with the compositions or methods of the embodiments described herein. Thus, pathologies associated with WT1 overexpression include acute and chronic disorders and diseases, including conditions that predispose the subject to a particular disorder.

[0206] Examples of pathologies associated with WT1 overexpression include hyperproliferative disorders, which in some embodiments refer to a state in which cells are activated and / or proliferated (or may be in a state of excessive transcriptional activity) in a subject, and include tumors, neoplasms, cancers, malignant tumors, and the like. In addition to cell activation or proliferation, hyperproliferative disorders also include abnormalities or dysregulation of the cell death process, whether by necrosis or apoptosis. Such abnormalities in the cell death process appear to be associated with a variety of pathologies, including cancer (including primary and secondary malignancies and metastases) and other pathologies.

[0207] According to certain embodiments, the compositions and methods disclosed herein can be used to treat almost any type of cancer characterized by WT1 overexpression, including hematopoietic cancers (e.g., leukemias, including acute myeloid leukemia (AML), T- or B-cell lymphomas, myelomas, etc.). Additionally, "cancer" may refer to any accelerated proliferation of cells, including solid tumors, ascites tumors, hematologic or lymphatic 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. The embodiments disclosed herein include certain embodiments that include only one of the above types of diseases, and also include certain embodiments that may exclude a particular pathology, whether or not characterized by WT1 overexpression.

[0208] Certain therapeutic or prophylactic methods contemplated herein include stably incorporating a nucleic acid molecule of interest as described herein into the chromosomes of a host cell (which may be autologous, allogeneic, or syngeneic) and administering the host cell. For example, a desired WT1-targeting T cell composition can be produced ex vivo using autologous, allogeneic, or syngeneic immune system cells (e.g., T cells, antigen-presenting cells, natural killer cells) to administer such a cell composition to a subject as adoptive immunotherapy.

[0209] In some embodiments, administering a composition or therapy as used herein means delivering said composition or therapy to a subject, regardless of the route or mode of delivery. Administration can be continuous or intermittent, parenterally. Administration can be performed to treat a subject already identified as having a given pathology, disease or disease state, or to treat a subject who may or may be at risk of developing such a pathology, disease or disease state. Co-administration with adjuvant therapy includes simultaneous and / or sequential delivery of multiple agents in any order according to any administration schedule (e.g., one or more cytokines; immunosuppressive therapy such as calcineurin inhibitors, corticosteroids, microtubule inhibitors, low-dose mycophenolic acid prodrugs, or any combination thereof, in combination with WT1-specific modified (i.e., recombinant or artificial) host cells). For example, the therapies of the present disclosure can be combined with specific inhibitors or modulators of immune suppressive components, such as inhibitors or modulators of immune checkpoint molecules (e.g., anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibodies; see, e.g., Pardol, Nature Rev. Cancer 12:252, 2012; Chen and Mellman, Immunity 39:1, 2013).

[0210] In some embodiments, the host cells are cultured at about 10 7 pieces / m 2 ~about 10 11 pieces / m 2In some embodiments, the method further comprises administering a cytokine. In some embodiments, the cytokine is IL-2, IL-15, IL-21, or any combination thereof. In some embodiments, the cytokine is IL-2 and is administered simultaneously or sequentially with the host cells. In some embodiments, the cytokine is administered sequentially, provided that the subject is administered at least three or four times with the host cells prior to administration of the cytokine.

[0211] In some embodiments, the cytokine is IL-2 and is administered subcutaneously.

[0212] In some embodiments, the subject is further undergoing immunosuppressive therapy.

[0213] In some embodiments, the immunosuppressive therapy is selected from a calcineurin inhibitor, a corticosteroid, a microtubule inhibitor, a low-dose mycophenolic acid prodrug, or any combination thereof.

[0214] In some embodiments, the subject has undergone a non-myeloablative or myeloablative hematopoietic cell transplant.

[0215] In some embodiments, the host cells are administered to the subject at least 3 months following non-myeloablative hematopoietic cell transplantation.

[0216] In some embodiments, the host cells are administered to the subject at least two months after myeloablative hematopoietic cell transplantation. Techniques and regimens for performing HCT are known in the art and can include 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 cells derived from amniotic fluid. Thus, in certain embodiments, modified immune cells of the present disclosure can be administered simultaneously or immediately after hematopoietic stem cells in a modified HCT regimen. In some embodiments, the HCT includes donor hematopoietic cells that include a chromosomal knockout of genes encoding HLA components, a chromosomal knockout of genes encoding TCR components, or both.

[0217] In other embodiments, the subject has undergone lymphodepleting chemotherapy prior to receiving the composition or HCT. In some embodiments, the lymphodepleting chemotherapy comprises a conditioning regimen comprising cyclophosphamide, fludarabine, antithymocyte globulin, or combinations thereof.

[0218] In some embodiments, the recombinant host cells described herein can be administered to the subject multiple times, with the administration interval being about 2 to about 4 weeks. In other embodiments, the cytokines are administered sequentially, provided that the subject is administered at least 3 or 4 doses of the host cells prior to cytokine administration. In some embodiments, the cytokine is administered subcutaneously (e.g., IL-2, IL-15, IL-21).

[0219] In yet other embodiments, the subject is further undergoing immunosuppressive therapy, such as an antibody specific for PD-1 (e.g., pidilizumab, nivolumab, or pembrolizumab), an antibody specific for PD-L1 (e.g., MDX-1105, BMS-936559, MEDI4736, MPDL3280A, or MSB0010718C), an antibody specific for CTLA4 (e.g., tremelimumab or ipilimumab), a calcineurin inhibitor, a corticosteroid, a microtubule inhibitor, a low-dose mycophenolic acid prodrug, or any combination thereof. In yet other embodiments, the subject has undergone a non-myeloablative or myeloablative hematopoietic cell transplant, and the host cells can be administered at least 2 months to at least 3 months after the non-myeloablative hematopoietic cell transplant.

[0220] An effective amount of a therapeutic or pharmaceutical composition in some embodiments refers to an amount sufficient to achieve a desired clinical outcome or beneficial treatment at the dosage and duration required, as described herein. An effective amount can be delivered in one or more administrations. The term "therapeutic amount" can be used in the context of treatment when administered to a subject already known or identified to have a disease or disease state, and a "prophylactically effective amount" can be used to refer to administration of an effective amount as a prophylactic course to a subject likely or at risk of developing (e.g., recurring) a disease or disease state.

[0221] The level of a cytotoxic T lymphocyte (CTL) immune response can be measured by any one of a number of immunological methods routinely practiced in the art and described herein. The level of a CTL immune response can be measured, for example, before and after administration of any one of the WT1-specific TCRs described herein expressed by T cells. A cytotoxicity assay for measuring CTL activity can be performed using any one of several techniques and methods routinely practiced in the art (see, for example, Henkart et al., "Cytotoxic T-Lymphocytes" in Fundamental Immunology, Paul (ed.) (2003 Lippincott Williams & Wilkins, Philadelphia, PA), pp. 1127-50 and references therein).

[0222] Antigen-specific T cell responses are generally measured by comparing the observed T cell responses according to any of the T cell functional parameters described herein (e.g., proliferation, cytokine release, CTL activity, changes in cell surface marker phenotype, etc.), and such comparisons can be made between T cells exposed to a cognate antigen in an appropriate context (e.g., an antigen used to prime or activate T cells when presented by an immunocompatible antigen-presenting cell) and T cells from the same cognate population exposed to a structurally different or unrelated control antigen. A response to the cognate antigen is deemed antigen-specific if it is statistically significantly greater than the response to the control antigen.

[0223] A biological sample can be obtained from a subject to measure the presence and level of an immune response to the WT1-derived antigenic peptide described herein. As used herein, a "biological sample" can be a blood sample (from which serum or plasma can be prepared), a biopsy specimen, a body fluid (e.g., lung lavage, ascites, mucosal lavage, synovial fluid), bone marrow, lymph node, tissue explant, organ culture, or any other tissue or cell preparation derived from the subject or biological source. A biological sample can also be obtained from a subject before administration of an immunogenic composition, and such a biological sample is useful as a control to establish baseline (i.e., pre-immunization) data.

[0224] The pharmaceutical compositions described herein may be in the form of unit-dose or multi-dose containers, such as sealed ampoules or vials. Such containers may be frozen to maintain the stability of the formulation until use. In certain embodiments, a unit-dose formulation contains about 10 recombinant host cells described herein. 7 pieces / m 2 ~about 10 11 pieces / m 2 The development of suitable administration and treatment regimens for use with the particular compositions described herein may include a variety of treatment regimens, including, for example, parenteral or intravenous administration or formulations.

[0225] When the composition of the present application is administered parenterally, the composition may further comprise a sterile aqueous or oily solution or suspension. Suitable non-toxic diluents or solvents acceptable for parenteral administration include water, Ringer's solution, isotonic saline, 1,3-butanediol, ethanol, propylene glycol, or a mixture of polyethylene glycol and water. The aqueous solution or suspension may further comprise one or more buffering agents, such as sodium acetate, sodium citrate, sodium borate, or sodium tartrate. Of course, all materials used in the preparation of any dosage unit formulation must be pharma- ceutically pure and substantially non-toxic in the amounts employed. Additionally, the active compound may be incorporated into sustained release preparations and formulations. As used herein, dosage unit form refers to physically discrete units suitable as a unitary dose for the subject to be treated, each unit containing a predetermined amount of recombinant cells or active compound calculated to produce the desired therapeutic effect, together with a suitable pharmaceutical carrier.

[0226] In general, an appropriate dosing and treatment regimen provides a sufficient amount of active molecules or cells to provide a therapeutic or prophylactic effect. Such a response can be monitored by determining an improved clinical outcome (e.g., a higher frequency of complete or partial remission, or a longer disease-free survival) in treated subjects compared to untreated subjects. An increase in a pre-existing immune response to a tumor protein generally correlates with an improved clinical outcome. Such an immune response can generally be assessed using standard proliferation, cytotoxicity, or cytokine assays, which are routinely performed in the art and can be performed using samples obtained from the subject before and after treatment.

[0227] The method of the present disclosure can further include administering one or more other agents to treat the disease or disorder with a combination therapy. For example, in certain embodiments, the combination therapy includes administering (concurrently, simultaneously, or sequentially) a composition of the present disclosure with an immune checkpoint inhibitor. In some embodiments, the combination therapy includes administering a composition of the present disclosure (e.g., a TCR, a polynucleotide, a vector, or a host cell, or a combination thereof) with an agonist of a stimulatory immune checkpoint agent. In other embodiments, the combination therapy includes administering a composition of the present disclosure with a secondary therapy, such as a chemotherapeutic agent, radiation therapy, surgery, an antibody, or any combination thereof.

[0228] The term "immunosuppressant" as used herein refers to one or more cells, proteins, molecules, compounds, or complexes that provide an inhibitory signal to help control or suppress an immune response. For example, immunosuppressants include molecules that partially or completely block immune stimulation; molecules that reduce, prevent, or delay immune activation; or molecules that enhance, activate, or upregulate immune suppression. Exemplary immunosuppressive targets (e.g., of 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.

[0229] Techniques and regimens for performing HCT are known in the art and can include 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 cells derived from amniotic fluid. Thus, in certain embodiments, modified immune cells of the present disclosure can be administered simultaneously or immediately after hematopoietic stem cells in a modified HCT regimen. In some embodiments, the HCT comprises donor hematopoietic cells that include a chromosomal knockout of genes encoding HLA components, a chromosomal knockout of genes encoding TCR components, or both.

[0230] In other embodiments, the subject has undergone lymphodepleting chemotherapy prior to receiving the composition or HCT. In some embodiments, the lymphodepleting chemotherapy comprises a conditioning regimen comprising cyclophosphamide, fludarabine, antithymocyte globulin, or combinations thereof.

[0231] The method of the present disclosure can further include administering one or more other agents to treat the disease or disorder with a combination therapy. For example, in certain embodiments, the combination therapy includes administering the composition of the present disclosure in combination with an immune checkpoint inhibitor (concurrently, simultaneously or sequentially). In some embodiments, the combination therapy includes administering the composition of the present disclosure in combination with an agonist of a stimulatory immune checkpoint agent. In other embodiments, the combination therapy includes administering the composition of the present disclosure in combination with a secondary therapy, such as a chemotherapeutic agent, radiation therapy, surgery, an antibody, or any combination thereof.

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

[0233] The immunosuppressant inhibitor (also known 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 RNAi molecule, or a small organic molecule. In any of the embodiments disclosed herein, the method can include the compositions of the present disclosure in combination with one or more inhibitors including any one of the following immunosuppressive components, alone or in any combination:

[0234] In certain embodiments, the compositions of the disclosure are used in combination with a PD-1 inhibitor, for example, a PD-1 specific antibody or binding fragment thereof, such as pidilizumab, nivolumab, pembrolizumab, MEDI0680 (formerly known as AMP-514), AMP-224, BMS-936558, or any combination thereof. In other embodiments, the compositions 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. Other possible candidates include cemiplimab; IBI-308; nivolumab + lelatolimab; BCD-100; camrelizumab; JS-001; spartalizumab; tislelizumab; AGEN-2034; BGBA-333 + tislelizumab; CBT-501; dostallimab; durvalumab + MEDI-0680; JNJ-3283; pazopanib hydrochloride + pembrolizumab; pidilizumab; REGN- 1979+cemiplimab;ABBV-181;ADUS-100+spartalizumab;AK-104;AK-105;AMP-224;BAT-1306;BI-754091;CC-90006;cemiplimab+REGN-3767;CS-1003;GLS-010;LZM-009;MEDI-5752;MGD-013;PF-06801591;Sym-021;tislelizumab+pamiparib;Xm Ab-20717;AK-112;ALPN-202;AM-0001;Antibodies for antagonizing PD-1 in Alzheimer's disease;BH-2922;BH-2941;BH-2950;BH-2954;Biopharmaceuticals for antagonizing CTLA-4 and PD-1 in solid tumors;Bispecific monoclonal antibodies for targeting PD-1 and LAG-3 in tumors;BLSM-101;CB-201;CB-213;CBT- 103;CBT-107;Cellular immunotherapy + PD-1 inhibitor;CX-188;HAB-21;HEISCOIII-003;IKT-202;JTX-4014;MCLA-134;MD-402;mDX-400;MGD-019;Monoclonal antibodies to antagonize PDCD1 in tumors;Monoclonal antibodies to antagonize PD-1 in tumors;Oncolytic viruses to inhibit PD-1 in tumors;OT-2;PD-1 antagonist + PEG interferon alpha-2b;PEGMP-7;PRS-332;RXI-762;STIA-1110;TSR-075;vaccines for targeting HER2 and PD-1 in tumors;vaccines for targeting PD-1 in tumors and autoimmune diseases;XmAb-23104;antisense oligonucleotides for inhibiting PD-1 in tumors;AT-16201;bispecific monoclonal antibody for inhibiting PD-1 in tumors;IMM-1802;PD-1 in solid tumors and hematopoietic malignancies Monoclonal antibodies for antagonizing CTLA-1 and CTLA-4;Nivolumab biosimilars;Recombinant proteins for agonizing CD278 and CD28 and antagonizing PD-1 in tumors;Recombinant proteins for agonizing PD-1 in autoimmune and inflammatory diseases;SNA-01;SSI-361;YBL-006;AK-103;JY-034;AUR-012;BGB-108;Agents for inhibiting PD-1, Gal-9 and TIM-3 in solid tumors;ENUM-244C8;ENUM -388D4;MEDI-0680;Monoclonal antibodies for antagonizing PD-1 in metastatic melanoma and metastatic lung cancer;Monoclonal antibodies for inhibiting PD-1 in tumors;Monoclonal antibodies for targeting CTLA-4 and PD-1 in tumors;Monoclonal antibodies for antagonizing PD-1 in NSCLC;Monoclonal antibodies for inhibiting PD-1 and TIM-3 in tumors;Monoclonal antibodies for inhibiting PD-1 in tumors;Recombinant antibodies for inhibiting PD-1 and VEGF-A in hematopoietic malignancies and solid tumors These include: proteins; small molecules to antagonize PD-1 in tumors; Sym-016; inebilizumab + MEDI-0680; vaccines to target PDL-1 and IDO in metastatic melanoma; anti-PD-1 monoclonal antibodies + cellular immunotherapy for the treatment of glioblastoma; antibodies to antagonize PD-1 in tumors; monoclonal antibodies to inhibit PD-1 / PD-L1 in hematopoietic malignancies and bacterial infections; monoclonal antibodies to inhibit PD-1 in HIV; or small molecules to inhibit PD-1 in solid tumors;

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

[0236] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of CTLA4. In particular embodiments, the compositions of the present disclosure are used in combination with CTLA4-specific antibodies or binding fragments thereof, such as ipilimumab, tremelimumab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), or any combination thereof.

[0237] In some embodiments, compositions of the disclosure are used in combination with B7-H3 specific antibodies or binding fragments 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, or as described in U.S. Patent No. 9,574,000 and PCT Patent Publication Nos. WO / 201640724A1 and WO2013 / 025779A1.

[0238] In certain embodiments, an inhibitor of CD244 is used in combination with a composition of the present disclosure.

[0239] In some embodiments, the compositions of the present disclosure are used in combination with inhibitors of BLTA, HVEM, CD160, or any combination thereof. Anti-CD160 antibodies are described, for example, in PCT Publication No. WO2010 / 084158.

[0240] In certain embodiments, an inhibitor of TIM3 is used in combination with the cell compositions of the disclosure.

[0241] In certain embodiments, an inhibitor of Gal9 is used in combination with a composition of the disclosure.

[0242] In certain embodiments, the compositions of the present disclosure are used in combination with an adenosine signaling inhibitor, such as a decoy adenosine receptor.

[0243] In certain embodiments, an inhibitor of A2aR is used in combination with a composition of the present disclosure.

[0244] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of KIR, such as lirilumab (BMS-986015).

[0245] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitory cytokine (generally a cytokine other than TGFβ) or an inhibitor of Treg development or activity.

[0246] In certain embodiments, the compositions of the present disclosure 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 104th Annual Meeting 2013; Apr 6-10, 2013), 1-methyltryptophan (1-MT)-tirapazamine, or any combination thereof.

[0247] In certain embodiments, the compositions of the present disclosure are used in combination with an arginase inhibitor, such as Nω-nitro-L-arginine methyl ester (L-NAME), Nω-hydroxynor-l-arginine (norNOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine ​​(BEC), or any combination thereof.

[0248] In certain embodiments, a composition of the present disclosure is used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.).

[0249] In certain embodiments, the compositions of the present disclosure 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.

[0250] In some embodiments, the compositions of the present disclosure are used in combination with inhibitors of PVRIG, PVRL2, or both. Anti-PVRIG antibodies are described, for example, in PCT Publication No. WO2016 / 134333. Anti-PVRL2 antibodies are described, for example, in PCT Publication No. WO2017 / 021526.

[0251] In certain embodiments, a LAIR1 inhibitor is used in combination with a composition of the present disclosure.

[0252] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.

[0253] In certain embodiments, the compositions of the present disclosure are used in combination with agents that enhance the activity of stimulatory immune checkpoint molecules (i.e., agonists), 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 CD86), CD137 (4-1BB) agonists (e.g., urelumab), CD134 (OX-40) agonists (e.g., MEDI6469, MEDI6383, or MEDI0562 ...137 (4-1BB) agonists (e.g., urelumab), CD134 (OX-40) agonists (e.g., MEDI6469, MEDI6383, or MEDI0562), CD137 (OX-40) agonists (e.g., MEDI6469, MEDI6383, or MEDI0562), CD137 (OX-40) agonists (e.g., MEDI6469, MEDI6383, or MEDI0562), CD137 (OX-40) agonists (e.g., MEDI6469, MEDI6383, or MEDI056 The compositions of the present disclosure can be administered in combination with D40 agonists, CD122 agonists (e.g., IL-2), agonists of GITR (e.g., the humanized monoclonal antibodies described in PCT Patent Publication No. WO2016 / 054638), agonists of ICOS (CD278) (e.g., GSK3359609, mAb88.2, JTX-2011, Icos145-1, Icos314-8, or any combination thereof). In any of the embodiments disclosed herein, the method can include administering the compositions of the present disclosure in combination with one or more agonists of stimulatory immune checkpoint molecules, including any of the above, alone or in any combination.

[0254] In some embodiments, the combination therapy includes a second therapy that includes, in addition to a composition of the present disclosure, one or more of an antibody or antigen-binding fragment thereof specific for a cancer antigen expressed by a non-inflammatory solid tumor, radiation therapy, surgery, a chemotherapeutic agent, a cytokine, RNAi, or any combination thereof.

[0255] In some embodiments, the combination therapy comprises administering a composition of the present disclosure and further performing radiation therapy or surgery. Radiation therapy is well known in the art and includes X-ray therapy, such as gamma radiation, and radiopharmaceutical therapy. Surgical therapies and techniques suitable for treating a given cancer in a subject are well known to those of ordinary skill in the art.

[0256] In some embodiments, the combination therapy comprises administering a composition of the present disclosure and further administering a chemotherapeutic agent, including but not limited to, inhibitors of chromatin function, topoisomerase inhibitors, microtubule inhibitors, DNA damaging agents, antimetabolites (such as antifolates, pyrimidine analogs, purine analogs, and glycosylation analogs), DNA synthesis inhibitors, DNA interactors (such as intercalating agents), and DNA repair inhibitors. Specific examples of 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, epothilone, and navelbine; epidipodophyllotoxins (etoposide, teniposide); DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin antiproliferative / antimitotic agents including cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, ifosfamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, temozolamide, teniposide, triethylenethiophosphoramide and etoposide (VP16); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase, which metabolizes L-asparagine throughout the body and kills 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), alkylsulfonic acids (busulfan), nitrosoureas (carmustine (BCNU) and analogs, streptozocin), triazenes (dacarbazine (DTIC)); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum complexes (cisplatin); , carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogues (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytics (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab ;Migration inhibitors;Secretion inhibitors (brefeldin);Immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil);Antiangiogenetic compounds (TNP470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors);Angiotensin receptor antagonists;Nitric oxide donors;Antisense oligonucleotides;Antibodies (trastuzumab, rituximab);Chimeric antigen receptors;Peripheral Phase inhibitors / 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;Toxins such as mitochondrial dysfunction inducers, cholera toxin, ricin, Pseudomonas aeruginosa exotoxin, Bordetella pertussis adenylate cyclase toxin or diphtheria toxin, and caspase activators; and chromatin disruptors.

[0257] Cytokines can be used to manipulate the host immune response to anti-cancer activity. See, e.g., Floros & Tarhini, Semin. Oncol. 42(4):539-548, 2015. Cytokines useful for promoting immune anti-cancer or anti-tumor responses include, for example, IFN-α, IL-2, IL-3, IL-4, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-24, and GM-CSF, alone or in any combination, in combination with the compositions of the present disclosure.

[0258] Also provided herein is a method of modulating adoptive immunotherapy, the method comprising administering to a subject that has previously been administered an engineered host cell of the present disclosure comprising a heterologous polynucleotide encoding a safety switch protein, an effective amount of a cognate compound of the safety switch protein to extinguish the previously administered engineered host cell in the subject.

[0259] In some embodiments, the safety switch protein comprises tEGFR and the cognate compound is cetuximab, or the safety switch protein comprises iCasp9 and the cognate compound is AP1903 (e.g., dimerized AP1903), or the safety switch protein comprises an RQR polypeptide and the cognate compound is rituximab, or the safety switch protein comprises a myc binding domain and the cognate compound is an antibody specific for the myc binding domain.

[0260] In yet another aspect, a method for producing a composition or unit dose formulation of the present disclosure is provided. In some embodiments, the method comprises adding (i) an aliquot of host cells into which a vector of the present disclosure has been introduced, and (ii) a pharma- ceutically acceptable carrier. In some 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 a cancer antigen).

[0261] In some embodiments, the method further comprises culturing the transduced host cells and selecting for those transduced cells that have taken up (i.e., express) the vector prior to splitting into aliquots. In other embodiments, the method comprises expanding the transduced host cells after said culturing and selection prior to splitting into aliquots. In any of the embodiments of the method, the composition or unit dose formulation after production can be frozen for later use. Any suitable host cell can be used to produce a composition or unit dose formulation 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 certain embodiments, the method comprises the step of: + T cells, CD4 + The host cell may be a T cell, a cellular or biological cell.

[0262] In some embodiments, the compositions of the present disclosure are used in combination with a LAG3 inhibitor, such as LAG525, IMP321, IMP701, 9H12, BMS-986016, or any combination thereof.

[0263] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of CTLA4. In particular embodiments, the compositions of the present disclosure are used in combination with CTLA4-specific antibodies or binding fragments thereof, such as ipilimumab, tremelimumab, CTLA4-Ig fusion proteins (e.g., abatacept, belatacept), or any combination thereof.

[0264] In some embodiments, compositions of the disclosure are used in combination with B7-H3 specific antibodies or binding fragments 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, or as described in U.S. Patent No. 9,574,000 and PCT Patent Publication Nos. WO / 201640724A1 and WO2013 / 025779A1.

[0265] In certain embodiments, an inhibitor of CD244 is used in combination with a composition of the present disclosure.

[0266] In some embodiments, the compositions of the present disclosure are used in combination with inhibitors of BLTA, HVEM, CD160, or any combination thereof. Anti-CD160 antibodies are described, for example, in PCT Publication No. WO2010 / 084158.

[0267] In certain embodiments, an inhibitor of TIM3 is used in combination with the cell compositions of the disclosure.

[0268] In certain embodiments, an inhibitor of Gal9 is used in combination with a composition of the disclosure.

[0269] In certain embodiments, the compositions of the present disclosure are used in combination with an adenosine signaling inhibitor, such as a decoy adenosine receptor.

[0270] In certain embodiments, an inhibitor of A2aR is used in combination with a composition of the present disclosure.

[0271] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of KIR, such as lirilumab (BMS-986015).

[0272] In certain embodiments, the compositions of the present disclosure are used in combination with an inhibitory cytokine (generally a cytokine other than TGFβ) or an inhibitor of Treg development or activity.

[0273] In certain embodiments, the compositions of the present disclosure 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 104th Annual Meeting 2013; Apr 6-10, 2013), 1-methyltryptophan (1-MT)-tirapazamine, or any combination thereof.

[0274] In certain embodiments, the compositions of the present disclosure are used in combination with an arginase inhibitor, such as Nω-nitro-L-arginine methyl ester (L-NAME), Nω-hydroxynor-l-arginine (norNOHA), L-NOHA, 2(S)-amino-6-boronohexanoic acid (ABH), S-(2-boronoethyl)-L-cysteine ​​(BEC), or any combination thereof.

[0275] In certain embodiments, a composition of the present disclosure is used in combination with an inhibitor of VISTA, such as CA-170 (Curis, Lexington, Mass.).

[0276] In certain embodiments, the compositions of the present disclosure 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.

[0277] In some embodiments, the compositions of the present disclosure are used in combination with inhibitors of PVRIG, PVRL2, or both. Anti-PVRIG antibodies are described, for example, in PCT Publication No. WO2016 / 134333. Anti-PVRL2 antibodies are described, for example, in PCT Publication No. WO2017 / 021526.

[0278] In certain embodiments, a LAIR1 inhibitor is used in combination with a composition of the present disclosure.

[0279] In certain embodiments, the compositions of the present disclosure are used in combination with inhibitors of CEACAM-1, CEACAM-3, CEACAM-5, or any combination thereof.

[0280] In certain embodiments, the compositions of the present disclosure are used in combination with agents that enhance the activity of stimulatory immune checkpoint molecules (i.e., agonists), 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 CD86), CD29 agonists (e.g., CP-870,893, rhuCD40L, or SGN-40), and the like. The compositions of the present disclosure can be administered in combination with CD40 agonists, CD122 agonists (e.g., IL-2), agonists of GITR (e.g., the humanized monoclonal antibodies described in PCT Patent Publication No. WO2016 / 054638), agonists of ICOS (CD278) (e.g., GSK3359609, mAb88.2, JTX-2011, Icos145-1, Icos314-8, or any combination thereof). In any of the embodiments disclosed herein, the method can include administering the compositions of the present disclosure in combination with one or more agonists of stimulatory immune checkpoint molecules, including any of the above, alone or in any combination.

[0281] In some embodiments, the combination therapy includes a second therapy that includes, in addition to a composition of the present disclosure, one or more of an antibody or antigen-binding fragment thereof specific for a cancer antigen expressed by a non-inflammatory solid tumor, radiation therapy, surgery, a chemotherapeutic agent, a cytokine, RNAi, or any combination thereof.

[0282] In some embodiments, the combination therapy comprises administering a composition of the present disclosure and further performing radiation therapy or surgery. Radiation therapy is well known in the art and includes X-ray therapy, such as gamma radiation, and radiopharmaceutical therapy. Surgical therapies and techniques suitable for treating a given cancer in a subject are well known to those of ordinary skill in the art.

[0283] In some embodiments, the combination therapy comprises administering a composition of the present disclosure and further administering a chemotherapeutic agent, including but not limited to, inhibitors of chromatin function, topoisomerase inhibitors, microtubule inhibitors, DNA damaging agents, antimetabolites (such as antifolates, pyrimidine analogs, purine analogs, and glycosylation analogs), DNA synthesis inhibitors, DNA interactors (such as intercalating agents), and DNA repair inhibitors. Specific examples of 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, epothilone, and navelbine; epidipodophyllotoxins (etoposide, teniposide); DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin antiproliferative / antimitotic agents including cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine oxaliplatin, ifosfamide, melphalan, mechlorethamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, temozolamide, teniposide, triethylenethiophosphoramide and etoposide (VP16); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase, which metabolizes L-asparagine throughout the body and kills 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), alkylsulfonic acids (busulfan), nitrosoureas (carmustine (BCNU) and analogs, streptozocin), triazenes (dacarbazine (DTIC)); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum complexes (cisplatin); , carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogues (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytics (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab ;Migration inhibitors;Secretion inhibitors (brefeldin);Immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil);Antiangiogenetic compounds (TNP470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors);Angiotensin receptor antagonists;Nitric oxide donors;Antisense oligonucleotides;Antibodies (trastuzumab, rituximab);Chimeric antigen receptors;Peripheral Phase inhibitors / 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;Toxins such as mitochondrial dysfunction inducers, cholera toxin, ricin, Pseudomonas aeruginosa exotoxin, Bordetella pertussis adenylate cyclase toxin or diphtheria toxin, and caspase activators; and chromatin disruptors.

[0284] Cytokines can be used to manipulate the host immune response to anti-cancer activity. See, e.g., Floros & Tarhini, Semin. Oncol. 42(4):539-548, 2015. Cytokines useful for promoting immune anti-cancer or anti-tumor responses include, for example, IFN-α, IL-2, IL-3, IL-4, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-21, IL-24, and GM-CSF, alone or in any combination, in combination with the compositions of the present disclosure.

[0285] Also provided herein is a method of modulating adoptive immunotherapy, the method comprising administering to a subject that has previously been administered an engineered host cell of the present disclosure comprising a heterologous polynucleotide encoding a safety switch protein, an effective amount of a cognate compound of the safety switch protein to extinguish the previously administered engineered host cell in the subject.

[0286] In some embodiments, the safety switch protein comprises tEGFR and the cognate compound is cetuximab, or the safety switch protein comprises iCasp9 and the cognate compound is AP1903 (e.g., dimerized AP1903), or the safety switch protein comprises an RQR polypeptide and the cognate compound is rituximab, or the safety switch protein comprises a myc binding domain and the cognate compound is an antibody specific for the myc binding domain.

[0287] In yet another aspect, a method for producing a composition or unit dose formulation of the present disclosure is provided. In some embodiments, the method comprises adding (i) an aliquot of host cells into which a vector of the present disclosure has been introduced, and (ii) a pharma- ceutically acceptable carrier. In some 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 a cancer antigen).

[0288] In some embodiments, the method further comprises culturing the transduced host cells and selecting for those transduced cells that have taken up (i.e., express) the vector prior to splitting into aliquots. In other embodiments, the method comprises expanding the transduced host cells after said culturing and selection prior to splitting into aliquots. In any of the embodiments of the method, the composition or unit dose formulation after production can be frozen for later use. Any suitable host cell can be used to produce a composition or unit dose formulation 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 certain embodiments, the method comprises the step of: + T cells, CD4 + The host cell may be a T cell, a cellular or biological cell. EXAMPLES

[0289] [Example 1] Method cell line T2 is a TAP-deficient T-cell leukemia / B-LCL hybrid cell line that expresses only HLA-A*02:01 11 and 293T / 17, a highly efficiently transfectable cell line, were purchased from ATCC. Jurkat76 cells are a TCRα / TCRβ-deficient derivative of the parent Jurkat cell line and do not naturally express CD8. 12Jurkat76 cells were previously transduced to express CD8αβ (Jurkat-CD8). The cell lines were maintained in HEPES-containing RPMI1640 medium (Invitrogen, GIBCO) supplemented with 10% heat-inactivated FBS (Hyclone, GE Healthcare Life Sciences), 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0290] Human T cell culture: Leukapheresis samples were collected from healthy donors at the Seattle Cancer Care Alliance after obtaining written informed consent and Institutional Review Board approval as protocol 868.01 in accordance with the Declaration of Helsinki. PBMCs were isolated from HLA-typed donors and analyzed as previously described. 13,14 , peptide WT1 37-45 Ten HLA-A*02:01-restricted T cell lines specific for VLDFAPPGA were generated per donor (total of 10 donors). + CD8 T cells were isolated using a T cell isolation kit (StemCell Technologies). + DCs were generated from autologous PBMCs by purifying T cells, adhering them to a plastic substrate, and culturing them with 1000U / ml IL-4 and 800U / ml GMCSF for 2 days, with the maturation cytokine cocktail added on the final day before harvesting. DCs were loaded with 1μg / ml peptide for 90 minutes, washed to remove excess peptide, and irradiated with 4000 rads. Peptide-pulsed DCs were supplemented with 30ng / ml IL-21, which was then counter-cultured against CD8 + T cells approximately 5 x 10 6T cells were maintained in HEPES-containing RPMI 1640 medium (Invitrogen, GIBCO) supplemented with 5% heat-inactivated pooled human serum (Bloodworks Northwest), 100 U / mL penicillin, 100 μg / mL streptomycin, and 55 μM 2-β-mercaptoethanol. Half of the medium was replaced every 2–3 days, and the cultures were supplemented with 12.5 U / mL IL-2 and 2250 U / mL IL-7 and IL-15. T cells were restimulated every 10 days by co-culturing them at a 1:2 ratio with peptide-pulsed, irradiated autologous PBMCs.

[0291] Cell sorting by flow cytometry T cell lines from all donors were pooled on ice after antigen-specific expansion. Pooled samples were aliquoted and stained with peptide / HLA-A2 tetramers under three conditions: (1) the wild-type tetramer concentration that gave the greatest separation of positive and negative populations was experimentally determined as described in the "Tetramer Binding and Affinity Measurements" section; (2) the optimal tetramer dose was diluted 100-fold; and (3) the HLA-A2 molecule was mutated to D227K and T228A at the CD8-interacting α3 domain positions. 15 This generates another engineered tetramer that has been shown to selectively bind high affinity, CD8-independent TCRs. 16,17 For each tetramer-stained sample, cells showing the highest levels of tetramer binding (top ~2% of labeled cells) were selected by flow cytometry. The selected populations were analyzed using the Adaptive Biotechnologies immunoseq assay to quantify the relative abundance of each clonotype. A separate sample containing a total tetramer-positive population was also selected from the optimal tetramer-stained sample and analyzed using the Adaptive Biotechnologies pairSeq assay. 18 TCRαβ pairing information was obtained by:

[0292] 4. Data Analysis Concentration factor calculation (Selected tetramers +An enrichment score for each clonotype was calculated as frequency in the population / frequency in the unsorted pooled sample. Clonotypes that were not detected in the pooled sample were assigned a frequency corresponding to one cell in the pooled sample for fold enrichment calculations.

[0293] TCR sequencing and α / β pairing: TCR repertoire analysis was performed using ImmunoSeq assays from Adaptive Biotechnologies. Single-cell V(D)J analysis (TCRα / β pairing) was performed using Chromium single-cell immune profiling from 10x genomics.

[0294] TCR transduction The codon-optimized TCR construct, arranged in the order TCRβ-p2a-TCRα, was synthesized on a BioXp™ 3200 (SGI-DNA) and cloned into the pRRLSIN.cPPT.MSCV.WPRE lentiviral expression plasmid (a gift from Dr. Richard Morgan, NCI) by Gibson assembly. The expression vector was then packaged into 293T cells using a third-generation lentiviral packaging system. After 48 hours, the lentiviral supernatant was harvested and filtered to remove cell debris. 2 ml of lentiviral supernatant was supplemented with 5 μg / ml polybrene and transfected into approximately 5 × 10 Jurkat76 cells. 5 The cells were centrifuged at 1000g for 90 min at 30°C to facilitate transduction. + EasySep(TM) human CD8 for TCR transduction into T cells + HLA-A2 was identified using a T cell isolation kit (StemCell Technologies). + CD8 from PBMCs + T cells were enriched and activated with Dynabeads™ Human T-Expander CD3 / CD28 (Gibco) for 4 hours. 2 ml of lentivirus supernatant was supplemented with 5 μg / ml protamine sulfate and 50 U / ml IL-2 to induce CD8 + T cells approximately 2 x 10 6The peptide / HLA-A*02:01 tetramer was used to express transgenic TCR + Cells were FACS sorted to obtain pure antigen-specific cell populations for downstream assays.

[0295] TCR binding data Evaluating the correct TCR pairing Jurkat76 cells were transfected with each TCR construct and tetramer binding was analyzed relative to CD3 surface expression, which reflects total transgenic TCR surface expression in these cells that lack endogenous TCR.

[0296] Tetramer binding and affinity measurements The optimal tetramer dose was determined by performing a tetramer titration on the positive T cell population and selecting the concentration that best separated the positive and negative populations without increasing background staining in the negative population.

[0297] TCR Functional Data IFN-γ production Primary CD8 + T cells were transfected with each TCR expression construct, selected, and then cultured at decreasing doses (1–10 -5 T2 target cells were mixed at a 1:1 ratio with peptide-pulsed T2 target cells (1 μM). Alternatively, where indicated, autologous PBMCs were used as APCs. After 4 h of incubation with Golgi inhibitors (BD GolgiPlug and GolgiStop), cells were surface stained with anti-CD8, fixed (BD Cytofix / Cytoperm), and then intracellularly labeled with anti-IFN-γ in BD Perm / Wash buffer. Cells were analyzed by flow cytometry and IFN-γ was detected in each sample. + The percentage of cells was determined. These data were fitted to dose-response curves by non-linear regression using Graphpad Prism (4 parameters - variable slope was selected and the bottom and top of the curve were constrained to 0 and 100, respectively).

[0298] Figures 1(A) and 1(B) show the high-throughput sequencing strategy used to identify WT1 37-45 How peptide-specific TCRs were identified. TCR clonotypes enriched by selection for high tetramer binding compared to the total tetramer-positive population were identified as likely to have high affinity or high functional avidity for peptide / HLA-A2 ligands. (A)WT1 37-45 Schematic of the initial sequencing strategy to identify TCR clonotypes with high binding to peptide / MHC tetramers. (B) Fold enrichment of the sorted population relative to the percentage of the total population is shown, highlighting selected TCRs. All TCRs, indicated by black circles, were synthesized and assessed for antigen specificity (27 in total).

[0299] Figure 2 shows the results of tetramer binding studies assessing the specificity and relative tetramer binding affinity of selected TCRs. TCR constructs were expressed in Jurkat cells lacking endogenous TCR α / β chains. Tetramer staining for CD3 expression of each TCR is shown (CD3 expression directly correlates with transgenic TCR surface expression).

[0300] [Example 2] Identification of TCRs with high functional avidity Given that some high affinity TCRs were found to bind tetramers in a CD8-independent manner, a second experiment was performed to further identify TCRs that were specifically enriched as a select population with high tetramer binding when CD8-independent (CD8i) tetramers were used. Figures 3A-3C show that a modified high-throughput sequencing strategy using CD8-independent (CD8i) tetramers was used to identify other WT1 37-45 This shows how peptide-specific TCR was identified. CD8-independent WT1 37A schematic of the modified sequencing strategy to identify TCR clonotypes with high binding to peptide / MHC tetramers is shown in Figure 3A. The fold enrichment of the initial sorted population as a percentage of the total population and a similar analysis using CD8i tetramers are shown in Figures 3B and 3C. Fourteen additional TCRs were selected based on surface CD3 levels and binding to CD8i tetramers. All TCR clonotypes named in Figures 3B and 3C were synthesized and evaluated for antigen specificity. All TCRs shown in Figure 3C as shaded circles represent TCRs further identified using CD8i tetramers.

[0301] [Example 3] Tetramer staining for CD3 expression Other WT1 selected with CD8i tetramer 37-45 CD8i tetramer binding of peptide-specific TCRs is shown in Figure 4. TCR constructs were expressed in Jurkat cells lacking endogenous TCR α / β chains (and therefore lacking CD8 expression). Tetramer staining for each TCR relative to CD3 expression is shown in Figure 4 (CD3 expression directly correlates with transgenic TCR surface expression). TCRs that bound most avidly to the tetramer and gave high levels of tetramer staining compared to anti-CD3 staining were selected for further analysis.

[0302] [Example 4] Functional avidity (EC 50 IFNγ assay to measure Peptide EC is the amount of peptide that needs to be pulsed onto target cells to elicit a T cell response (e.g., IFNγ production) from 50% of T cells transduced with the TCR of the present application. 50 This measured the ability of a TCR to induce a signal for T cell activation at a limiting antigen concentration, which directly correlates with the ability of T cells expressing a given TCR to kill target cells expressing the antigen. 50 To determine the CD8 + T cells were transduced with each TCR (Figure 5A). After one week, tetramers were extracted from the cells. + CD8 +T cells were sorted and expanded. Expanded antigen-specific cells were co-cultured with peptide-pulsed T2 target cells for 4–6 h, and IFNγ production was measured by flow cytometry (Figure 5A). The percentage of IFNγ-producing cells was fitted to a dose-response curve by nonlinear regression to determine the peptide EC for each TCR. 50 was calculated (Figure 5B).

[0303] [Example 5] WT1 37-45 HLA-A2 by primary CD8+ T cells expressing peptide-specific TCRs + WT1 + In vitro killing of MDA-MB-468 cells Tumor cells that naturally express the WT1p37 antigen and present it on HLA-A2 are expressed by TCR-transduced CD8 + To directly assess whether T cells can lyse each of the selected TCRs, we cloned donor-derived CD8 + T cells were transduced and purified by selection for high tetramer binding. TCR-transduced T cells were then mixed (in triplicates) at an 8:1 ratio with breast cancer cell line MDA-MB-468, which had been pre-stained with CytoLight® Rapid Red dye. Total red object area (correlating to total number of live target cells) was calculated for each TCR-transduced T cell population over 72 hours at the indicated time points. The most potent tumor-reactive T cells appear to be responsive to tumor antigens for an extended period of time after in vivo transfer into patients. Therefore, MDA-MB-468 cells were added after 48 hours to assess the ongoing responsiveness of TCR-transduced T cells to persistent antigens. See Figure 6.

[0304] [Example 6] WT1 37-45 Primary CD4 expressing peptide-specific TCR + T cells and CD8 + HLA-A2 by T cells + WT1 + In vitro killing of PANC-1 cells CD4 + T cells and CD8 +Both CD8 T cells can play a role in tumor clearance in vivo. + CD4 rather than TCR, which can only activate T cells + MHC class I-restricted TCRs are preferred because they can induce antigen-specific responses in T cells. + The ability of T cells to function appears to depend, in part, on the affinity of the TCR for peptide MHC. In many cases, transfection of CD4+ T cells with genes encoding CD8α and CD8β helps them to elicit antigen-specific responses more efficiently. Thus, we show that CD4 T cells (transfected with CD8α / CD8β) express HLA-A2, compared with CD8 T cells expressing TCR10.1. + WT1 + To evaluate the ability to target tumor cells, WT1 37-45 CD4 to express TCR10.1 + T cells and CD8 + Both CD4 and T cells were transduced. + T cells were further transduced to express the CD8α and CD8β genes. After 8 days, the transduced cells were selected and CD8 + tetramer + T cells and CD4 + / CD8 + tetramer + T cells were purified and co-transduced 8:1 with the pancreatic adenocarcinoma cell line PANC-1, which had been previously transduced to express NucLight® Red dye. + , CD8 + or a mixture of these two populations (CD4 and CD8), antigen-specific cells were mixed (in triplicates). The total red object area (correlating to the total number of live target cells) was calculated for each TCR-transduced T cell population at the indicated time points. To assess the continued responsiveness of TCR-transduced T cells to persistent antigen, PANC-1 cells were added after 48 hours. Figure 7 shows the WT1 37-45 CD4 expressing TCR10.1 + T cells and CD8 + All T cells were positive for WT1 after repeated in vitro challenge. + A2+ We show that the pancreatic adenocarcinoma cell line PANC-1 can be eliminated.

[0305] The WT1p126 epitope is not necessarily efficiently processed / presented by cells expressing WT1 and HLA-A2 (Jaigirdar et al., J. Immunother. 39:105, 2017). In particular, several solid tumor-derived cell lines expressing WT1 and HLA-A2 are not efficiently targeted by WT1-p126-specific TCRs, regardless of whether they are pre-cultured with IFNγ to upregulate immunoproteasome expression. In some embodiments, the present disclosure relates in one aspect to the finding that the WT1-p37 epitope is more extensively processed and presented by a variety of tumor types compared to the WT1-p126 epitope. Figures 8A-8D show a comparison of lysis of various WT1+A2+ tumor cell lines by WT1-p126 peptide-specific TCRs and WT1p37 peptide-specific TCRs. These data support the fact that WT1p-37 peptide-specific TCRs appear to be able to more reliably target a broad range of WT1+A2+ tumors in general.

[0306] Various embodiments described herein can be combined to provide other embodiments. All patents, patent application publications, patent applications, and non-patent literature referenced herein and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety, including, but not limited to, U.S. Patent Application No. 62 / 816,746, filed March 11, 2019. In general, the terms used in the following claims should not be construed to be limited to the specific embodiments disclosed herein, but rather to include all possible embodiments along with the full scope of equivalents to which such claims are entitled.

[0307] References 1.Kalos,M.et al.T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced Leukemia.Sci.Transl.Med.3,95ra73-95ra73(2011). 2.Porter,D.L.,Levine,B.L.,Kalos,M.,Bagg,A.& June,C.H.Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.N Engl J Med 365,725-733(2011). 3.Kochenderfer,J.N.et al.Eradication of B-lineage cells and regression of lymphoma in a patient treated with autologous T cells genetically engineered to recognize CD19.Blood 116,4099-4102(2010). 4.Chapuis,A.G.et al.Transferred WT1-reactive CD8+ T cells can mediate antileukemic activity and persist in post-transplant patients.Sci Transl Med 5,174ra27(2013). 5.Chapuis,A.G.et al.Transferred melanoma-specific CD8+ T cells persist,mediate tumor regression,and acquire central memory phenotype.Proc Natl Acad Sci U S A(2012).doi:10.1073 / pnas.1113748109 6.Morgan,R.et al.Cancer Regression in Patients After Transfer of Genetically Engineered Lymphocytes.Science 314,126-129(2006). 7.Dudley,M.et al.Adoptive cell therapy for patients with metastatic melanoma:evaluation of intensive myeloablative chemoradiation preparative regimens.J Clin Oncol 26,5233-5239(2008). 8.Robbins,P.F.et al.Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1.Journal of Clinical Oncology 29,917-924(2011). 9.Stromnes,I.M.et al.Abrogation of SRC homology region 2 domain-containing phosphatase 1 in tumor-specific T cells improves efficacy of adoptive immunotherapy by enhancing the effector function and accumulation of short-lived effector T cells in vivo.The Journal of Immunology 189,1812-1825(2012). 10.Schmitt,T.M.,Ragnarsson,G.B.& Greenberg,P.D.T Cell Receptor Gene Therapy for Cancer.Hum Gene Ther 20,1240-1248(2009). 11.Salter,R.D.& Cresswell,P.Impaired assembly and transport of HLA-A and -B antigens in a mutant TxB cell hybrid.EMBO.J.5,943-949(1986). 12.Heemskerk,M.H.et al.Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-specific T-cell receptor complexes expressing a conserved alpha joining region.Blood 102,3530-3540(2003). 13.Ho,W.Y.,Nguyen,H.N.,Wolfl,M.,Kuball,J.& Greenberg,P.D.In vitro methods for generating CD8+ T-cell clones for immunotherapy from the naive repertoire.J Immunol Methods 310,40-52(2006). 14.Chapuis,A.G.et al.Transferred WT1-reactive CD8+ T cells can mediate antileukemic activity and persist in post-transplant patients.Sci Transl Med 5,174ra127(2013). 15.Luescher,I.F.et al.CD8 modulation of T-cell antigen receptor-ligand interactions on living cytotoxic T lymphocytes.Nature 373,353-356(1995). 16.Stone,J.D.& Kranz,D.M.Role of T cell receptor affinity in the efficacy and specificity of adoptive T cell therapies.Front Immunol 4,244(2013). 17.Pittet,M.J.et al.α3 Domain Mutants of Peptide / MHC Class I Multimers Allow the Selective Isolation of High Avidity Tumor-Reactive CD8 T Cells.The Journal of Immunology 171,1844-1849(2003). 18.Howie,B.et al.High-throughput pairing of T cell receptor alpha and beta sequences.Sci Transl Med 7,301ra131(2015).

Claims

1. A T cell receptor (TCR) comprising a TCR alpha chain variable (Vα) domain and a TCR beta chain variable (Vβ) domain, wherein the Vα domain comprises the CDR1α, CDR2α, and CDR3α amino acid sequences set forth in SEQ ID NO: 194, SEQ ID NO: 195, and SEQ ID NO: 196 or 12, respectively, and the Vβ domain comprises the CDR1β, CDR2β, and CDR3β sequences set forth in SEQ ID NO: 197, SEQ ID NO: 198, and SEQ ID NO: 199 or 1, respectively; A T cell receptor, wherein the TCR binds to the VLDFAPPGA (SEQ ID NO:59): human leukocyte antigen (HLA) complex.

2. The TCR is 8.5 or more, and IFNγ-producing pEC ５０ 2. The TCR of claim 1, which specifically binds to the VLDFAPPGA (sequence number 59): human leukocyte antigen (HLA) complex at a value of

3. The TCR of claim 1 or 2, further comprising a TCR that specifically binds to the VLDFAPPGA (sequence number 59): human leukocyte antigen (HLA) complex on the cell surface independently of CD8 or in the absence of CD8.

4. The TCR of any one of claims 1 to 3, wherein the HLA includes HLA-A*201.

5. (a) the Vα domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:34; and / or (b) the Vβ domain comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:23; A TCR according to any one of claims 1 to 4.

6. (a) the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO: 253 or 34; (b) the Vβ domain comprises or consists of the amino acid sequence of SEQ ID NO: 242 or 23; (c) the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO: 253, and the Vβ domain comprises or consists of the amino acid sequence of SEQ ID NO: 242; or (d) the Vα domain comprises or consists of the amino acid sequence of SEQ ID NO: 34, and the Vβ domain comprises or consists of the amino acid sequence of SEQ ID NO: 23; A TCR according to any one of claims 1 to 5.

7. the TCR comprises a TCR alpha chain constant domain having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 47; and / or The TCR comprises a TCR β chain constant domain having at least about 90% sequence identity to the amino acid sequence of SEQ ID NO: 45 or 46. A TCR according to any one of claims 1 to 6.

8. The Vα domain and the Vβ domain are (i) SEQ ID NO: 253 and SEQ ID NO: 242, respectively; or (ii) SEQ ID NO: 34 and SEQ ID NO: 23, respectively The TCR according to any one of claims 1 to 7, comprising an amino acid sequence as set forth in:

9. The Vα domain and the Vβ domain (i) SEQ ID NO: 253 and SEQ ID NO: 242, respectively; or (ii) SEQ ID NO: 34 and SEQ ID NO: 23, respectively. The TCR according to any one of claims 1 to 7, which consists of an amino acid sequence as set forth in:

10. The TCR of claim 8 or 9, further comprising an alpha chain constant domain and / or a beta chain constant domain, wherein the alpha chain constant domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 47, and the beta chain constant domain comprises an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO: 45 or 46.

11. A TCR according to any one of claims 1 to 10, comprising: (i) a TCR alpha chain comprising a Valpha domain and an alpha chain constant domain, (a) The V α the alpha chain constant domain has at least about 90% sequence identity to the amino acid sequence represented by SEQ ID NO:34, and the alpha chain constant domain has at least about 98% sequence identity to the amino acid sequence represented by SEQ ID NO:47; (b) the Vα domain comprises the amino acid sequence represented by SEQ ID NO: 253 and the α chain constant domain comprises the amino acid sequence represented by SEQ ID NO: 47; or (c) the Vα domain consists of the amino acid sequence represented by SEQ ID NO: 253 and the α chain constant domain consists of the amino acid sequence represented by SEQ ID NO: 47, and / or (ii) a TCR β chain comprising a Vβ domain and a β chain constant domain, (a) the Vβ domain has at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:23, and the β chain constant domain comprises the amino acid sequence of SEQ ID NO:45 or has at least about 95% sequence identity to the amino acid sequence of SEQ ID NO:46; (b) the Vβ domain comprises an amino acid sequence represented by SEQ ID NO: 242, and the β chain constant domain comprises an amino acid sequence represented by SEQ ID NO: 45 or 46; (c) the Vβ domain consists of the amino acid sequence represented by SEQ ID NO: 242, and the β chain constant domain consists of the amino acid sequence represented by SEQ ID NO: 45 or 46; or (d) the Vβ domain comprises or consists of the amino acid sequence represented by SEQ ID NO:23, and the β chain constant domain comprises or consists of the amino acid sequence represented by SEQ ID NO:

46.

12. (i) a TCR alpha chain comprising a Valpha domain comprising the amino acid sequence represented by SEQ ID NO: 253, and an alpha chain constant domain comprising the amino acid sequence represented by SEQ ID NO: 47; and (ii) a TCR β chain comprising a Vβ domain comprising an amino acid sequence represented by SEQ ID NO: 242, and a β chain constant domain comprising an amino acid sequence represented by SEQ ID NO: 46; The TCR of any one of claims 1 to 11, comprising:

13. The TCR of any one of claims 1 to 12, wherein the TCR comprises a scTCR.

14. The TCR of any one of claims 1 to 12, wherein the TCR comprises a CAR.

15. An isolated polynucleotide encoding the TCR of any one of claims 1 to 14.

16. 16. The polynucleotide of claim 15, wherein the polynucleotide encodes an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:48, or an amino acid sequence comprising the amino acid sequence set forth in SEQ ID NO:48, or an amino acid sequence consisting of the amino acid sequence set forth in SEQ ID NO:

48.

17. 17. The polynucleotide of any one of claims 15 or 16, comprising a polynucleotide sequence set forth in any one of SEQ ID NOs: 64, 75, 86, 97, 108, 109, 111, 122, 133, 144 and 155.

18. (i) a polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor alpha chain, optionally wherein the encoded polypeptide is or comprises the CD8 co-receptor alpha chain; (ii) a 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) A polynucleotide of (i) and a polynucleotide of (ii) The polynucleotide according to any one of claims 15 to 17, further comprising:

19. (a) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α-chain; (b) a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor beta chain; (c) a polynucleotide encoding a self-cleaving peptide, the polynucleotide being located between the polynucleotide of (a) and the polynucleotide of (b). The polynucleotide of claim 18, comprising:

20. Encoding a self-cleaving peptide, (1) between a polynucleotide encoding a binding protein and a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α-chain; and / or (2) Between a polynucleotide encoding a binding protein and a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 coreceptor β chain.

20. The polynucleotide of claim 18 or 19, further comprising a polynucleotide arranged in

21. In-frame functionally connected (i)(pnCD8α)-(pnSCP １ )-(pnCD8β)-(pnSCP ２ )-(pnTCR); (ii)(pnCD8β)-(pnSCP １ )-(pnCD8α)-(pnSCP ２ )-(pnTCR); (iii)(pnTCR)-(pnSCP １ )-(pnCD8α)-(pnSCP ２ )-(pnCD8β); (iv)(pnTCR)-(pnSCP １ )-(pnCD8β)-(pnSCP ２ )-(pnCD8α); (v) (pnCD8α)-(pnSCP １ )-(pnTCR)-(pnSCP ２ )-(pnCD8β); or (vi)(pntx8β)-(pniCP １ )-(pnTCR)-(pnSCP ２ )-(pntx8α) wherein pnCD8α is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain; pnCD8β is a polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α chain; pnTCR is a polynucleotide encoding a TCR, pnSCP １ and pnSCP ２ are each independently a polynucleotide encoding a self-cleaving peptide, wherein the polynucleotides and / or the encoded self-cleaving peptides are optionally the same or different; A polynucleotide according to any one of claims 18 to 20.

22. The polynucleotide of any one of claims 15 to 21, wherein the encoded TCR comprises a TCR alpha chain and a TCR beta chain, and the polynucleotide comprises a polynucleotide encoding a self-cleaving peptide disposed between the polynucleotide encoding the TCR alpha chain and the polynucleotide encoding the TCR beta chain.

23. 23. The polynucleotide of claim 22, wherein the encoded self-cleaving peptide comprises the amino acid sequence of any one of the polypeptides of SEQ ID NOs:60-63.

24. 24. The polynucleotide of claim 22 or 23, wherein the TCR alpha chain, the self-cleaving peptide and the TCR beta chain are encoded by a polynucleotide having at least 95% identity to SEQ ID NO:

155.

25. The polynucleotide of any one of claims 22 to 24, wherein the encoded TCR alpha chain, self-cleaving peptide and TCR beta chain comprise an amino acid sequence having at least 95% identity to the polypeptide represented by SEQ ID NO:

48.

26. An expression vector comprising the polynucleotide of any one of claims 15 to 25 operably linked to an expression control sequence.

27. The vector is capable of delivering the polynucleotide to a host cell, and optionally the host cell is a hematopoietic stem cell, a hematopoietic progenitor cell, or a human immune system cell; Further optionally, the immune system cells are CD4+ T cells, CD8+ T cells, CD4-CD8- double negative T cells, γδ T cells, natural killer cells, dendritic cells, or any combination thereof; Further optionally, the T cells are naive T cells, central memory T cells, effector memory T cells, or any combination thereof.

27. The expression vector of claim 26.

28. 28. The expression vector of claim 26 or 27, wherein the vector is a viral vector.

29. 29. The expression vector of claim 28, wherein the viral vector is an adenoviral vector, a lentiviral vector, a gamma-retroviral vector, or an alpha-viral vector.

30. 28. The expression vector of claim 26 or 27, which is a plasmid vector, a cosmid vector, an RNA vector, or a linear or circular DNA or RNA molecule.

31. A method for producing a recombinant host cell in vitro or ex vivo, comprising introducing a polynucleotide according to any one of claims 15 to 25 and / or an expression vector according to any one of claims 26 to 30 into a host cell, optionally wherein the host cell is an immune cell.

32. A host cell comprising a polynucleotide according to any one of claims 15 to 25 or an expression vector according to any one of claims 26 to 30.

33. 33. The host cell of claim 32, wherein the host cell expresses the TCR encoded by the polynucleotide on its cell surface, and the polynucleotide is heterologous to the host cell.

34. 34. The host cell of claim 32 or 33, wherein the host cell is a hematopoietic stem cell, a hematopoietic progenitor cell or a human immune system cell.

35. 35. The host cell of claim 34, wherein the immune system cell is a CD4+ T cell, a CD8+ T cell, a CD4-CD8- double negative T cell, a γδ T cell, a natural killer cell, a natural killer T cell, a dendritic cell or any combination thereof, optionally, when present, the combination comprises a CD4+ T cell and a CD8+ T cell.

36. 35. The host cell of claim 34, wherein the immune system cell is a T cell.

37. 37. The host cell of claim 36, wherein the T cell is a naive T cell, a central memory T cell, an effector memory T cell, or any combination thereof.

38. (i) a heterologous polynucleotide encoding a polypeptide comprising an extracellular portion of the CD8 co-receptor alpha chain, optionally wherein said encoded polypeptide is or comprises the 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) A polynucleotide of (i) and a polynucleotide of (ii) 38. The host cell of any one of claims 32 to 37, further comprising:

39. (a) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor α-chain; (b) a heterologous polynucleotide encoding a polypeptide comprising the extracellular portion of the CD8 co-receptor β chain; (c) a polynucleotide encoding a self-cleaving peptide, the polynucleotide being disposed between the polynucleotide of (a) and the polynucleotide of (b); 39. The host cell of claim 38, comprising:

40. The host cell comprises (i) Tumor cells of the breast cancer cell line MDA-MB-468; (ii) tumor cells of the pancreatic adenocarcinoma cell line PANC-1; (iii) tumor cells of the breast cancer cell line MDA-MB-231; (iv) tumor cells of the myeloid leukemia cell line K562 expressing HLA-A2, optionally wherein said HLA-A2 comprises HLA-A*201; (v) tumor cells of the colon cancer cell line RKO expressing HLA-A2, optionally wherein said HLA-A2 comprises HLA-A*201; or (vi) any combination of the tumor cells of (i) to (v). The host cell according to any one of claims 32 to 39, which is capable of killing said tumor cells when present in a sample at the same time as

41. 41. The host cell of claim 40, wherein the host cell is capable of killing the tumor cells when the host cells and the tumor cells are present in the sample at a host cell:tumor cell ratio of 32:1, 16:1, 8:1, 4:1, 2:1 or 1.5:

1.

42. A composition comprising a host cell according to any one of claims 32 to 41 and a pharma- ceutically acceptable carrier, diluent or excipient.

43. The composition of claim 42, comprising host CD4+ T cells and host CD8+ T cells.

44. A TCR according to any one of claims 1 to 14, and a pharma- ceutically acceptable base, excipient or diluent; A polynucleotide according to any one of claims 15 to 25, and a pharma- ceutically acceptable carrier, excipient or diluent; The expression vector according to any one of claims 26 to 30, and a pharma- ceutically acceptable base, excipient or diluent; A host cell according to any one of claims 32 to 41 and a pharma- ceutically acceptable carrier, excipient or diluent; or The composition according to claim 42 or 43.

13. A pharmaceutical composition for treating a hyperproliferative or proliferative disorder in a patient comprising:

45. 45. The pharmaceutical composition of claim 44, wherein the hyperproliferative or proliferative disorder is a hematopoietic malignancy or solid cancer.

46. 46. ​​The pharmaceutical composition of claim 45, wherein the hematopoietic malignancy is selected from acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic eosinophilic leukemia (CEL), myelodysplastic syndromes (MDS), non-Hodgkin's lymphoma (NHL) or multiple myeloma (MM).

47. 46. ​​The pharmaceutical composition of claim 45, wherein the solid cancer is selected from breast cancer, ovarian cancer, lung cancer, biliary tract cancer, bladder cancer, bone and soft tissue carcinoma, brain tumor, cervical cancer, colon cancer, colorectal adenocarcinoma, colon cancer, desmoid tumor, embryonal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, gastric adenocarcinoma, glioblastoma multiforme, gynecological tumors, head and neck squamous cell carcinoma, liver cancer, mesothelioma, malignant melanoma, osteosarcoma, pancreatic cancer, pancreatic ductal adenocarcinoma, primary astrocytic tumor, primary thyroid cancer, prostate cancer, kidney cancer, renal cell carcinoma, rhabdomyosarcoma, skin cancer, soft tissue sarcoma, testicular germ cell tumor, urothelial carcinoma, uterine sarcoma, or uterine cancer.

48. 48. The pharmaceutical composition of any one of claims 44 to 47, wherein the patient has undergone lymphodepleting chemotherapy prior to administration of the pharmaceutical composition, and optionally, said lymphodepleting chemotherapy comprises a conditioning regimen comprising cyclophosphamide, fludarabine, antithymocyte globulin, or a combination thereof.

49. The host cell is ７ pieces / m ２ ~about 10 １１ pieces / m ２ The pharmaceutical composition according to any one of claims 44 to 48, wherein the dosage is

50. 13. A TCR according to any one of claims 1 to 14, a polynucleotide according to any one of claims 15 to 25, a vector according to any one of claims 26 to 30, a host cell according to any one of claims 32 to 41, or a composition according to claim 42 or 43, or any combination thereof, for use in a method for the manufacture of a medicament for the treatment of a proliferative or hyperproliferative disorder associated with Wilms Tumor Protein 1 (WT1) expression or overexpression.

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