Tgf-beta receptors and methods of use

Abstract

Provided herein are engineered receptors that engage cytokine signaling. Also provided herein are engineered receptors for modulating TGF-beta signaling, methods of modulating TGF-beta signaling, and methods of using chimeric antigen receptors to treat cancer.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 865,063, filed June 21, 2019, and U.S. Provisional Patent Application No. 62 / 951,217, filed December 20, 2019, both of which are incorporated herein by reference in their entirety.

[0003] STATED AS TO SEQUENCE LISTING

[0004] The sequence list associated with this application is provided in text format instead of a paper copy, and is incorporated herein by reference. The text file containing the sequence list is named "K1075_ST25". The text file is 109KB in size, created on June 17, 2020, and submitted electronically via EFS-Web at the time of filing this specification. Background Technology Technical Field

[0005] This disclosure relates to engineered receptors involved in TGF-β signaling. This disclosure also relates to engineered TGF-β receptors for regulating TGF-β signaling, and methods for regulating TGF-β signaling, cytokine signaling, and treating cancer.

[0006] TECHNICAL DESCRIPTION

[0007] Transforming growth factor β (TGF-β) is a pleiotropic immunosuppressive molecule secreted by many cell types that restricts the function and proliferation of effector T cells. Clinical and preclinical data have suggested that tumor cells, stromal cells, and suppressive immune subsets, including regulatory T cells, secrete TGF-β, and many groups have reported that TGF-β inhibits the function and proliferation of effector T cells in the tumor microenvironment (TME) (Thomas et al., Cancer Cell, 8(5):369-380 (2005); Yang et al., Trendsin Immunology, 31(6) (2010); and Pickup et al., Nat Rev Cancer, 13(11), 788-799 (2013)).

[0008] The TGF-b signaling pathway begins with the endogenous expression of two dimeric transmembrane transforming growth factor-b protein receptors: TGF-b receptor type I (TGF-bRI) and TGF-b receptor type II (TGF-bRII) (collectively referred to as “TGF-b receptors”). In T cells, TGF-bRII is constitutively phosphorylated. Upon TGF-b ligand binding, two molecules of each of TGF-bRI and TGF-bRII form a heterotetrameric receptor complex that induces TGF-bRII trans-phosphorylation of TGF-bRI at four key threonine sites between amino acids (aa) 185 to 205 of the TGF-bRI protein (Heldin et al., Cold Spring Harb Perspect Biol. Aug 1;8(8) (2016)). The phosphorylated TGF-bRI then initiates phosphorylation of a group of receptor-regulated signal transduction proteins, SMAD2 and SMAD3. Both pSMAD2 and pSMAD3 form a heterotrimeric oligomer with pSMAD4. The resulting activated SMAD complex then enters the nucleus and docks at target transcription factor binding sites to induce expression of target genes that support immune suppression (Inman et al., Mol Cell, 10(2):283-294 (2002)).

[0009] Some studies have attempted to limit the immunosuppressive effects of TGF-b on effector T cell function. These studies include the use of small molecule and antibody-based TGF-b binding and TGF-b signaling pathway inhibitors (Fabregat et al., Current Pharmaceutical Design, 20, 1-14 (2014)). Additionally, T cell receptor (TCR) constructs with modified TGF-b receptor molecules have been used. One example is a truncated form of TGF-bRII in which the intracellular portion of the type II receptor is removed, preventing downstream signaling events, and thereby acting as a dominant negative inhibitor of the TGF-b signaling pathway in engineered T cells (Wieser et al., Molecular and Cellular Biology 13(12):7239-7247 (1993) and Bollard et al., Blood. 99(9):3179-3187 (2002)). Nonetheless, no effective treatment based solely on TGF-b inhibition exists, and some systemic therapies have dose-limiting toxicities. Thus, there is a need to develop new ways to inhibit TGF-b. SUMMARY

[0010] One embodiment of the present disclosure is a recombinant polypeptide comprising an extracellular domain (ECD) and a transmembrane domain (TMD) from a TGF-β receptor, wherein the recombinant polypeptide lacks amino acid residues present in a wild-type TGF-β receptor that are responsible for signaling and phosphorylation.

[0011] In one aspect, the ECD is selected from TGF-βRI or TGF-βRII. In another aspect, the TMD is selected from TGF-βRI, TGF-βRII, PDGFR, CD4, CD8, CD28, CD127, CD132, CD3ζ, 4-IBB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, IL-5, IL-7, IL-7Rα, BTLA, or a mutant of any of the foregoing. In another aspect, the polypeptide further comprises a heterologous intracellular domain (ICD) that lacks amino acid residues present in a wild-type TGF-β receptor that are responsible for signaling and phosphorylation. In yet another aspect, the polypeptide further comprises a signal sequence.

[0012] In one aspect, the ECD comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 15 and the TMD comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 16. In another aspect, the ICD comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 6. In one aspect, the polypeptide comprises an amino acid sequence having at least 75% sequence identity to SEQ ID NO: 14.

[0013] In one aspect, the polypeptide binds to TGF-β1.

[0014] Another aspect of the present disclosure is an expression vector comprising a nucleic acid encoding a polypeptide described herein. In another aspect, the expression vector further comprises a nucleic acid sequence encoding a chimeric antigen receptor (CAR). In yet another aspect, the CAR binds to a tumor antigen comprising CD19, PSMA, or GPC3.

[0015] Another aspect of the present disclosure is a T cell transduced with an expression vector described herein.

[0016] Another aspect of the present disclosure is a method of treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of a T cell described herein.

[0017] In another embodiment of this disclosure, a recombinant nucleotide encodes a chimeric antigen receptor (CAR) comprising the nucleotide sequence SEQ ID NO:51. In one aspect, the recombinant nucleic acid further comprises the nucleotide sequence SEQ ID NO:48. In another aspect, the recombinant nucleic acid sequence encodes a polypeptide SEQ ID NO:47. In yet another aspect of this disclosure, an expression vector comprising a nucleic acid encoding the polypeptide described herein.

[0018] In another aspect, the CAR binds to tumor antigens including GPC3. Another aspect of this disclosure is T cells transduced using the expression vector described herein.

[0019] Another aspect of this disclosure is a method for treating liver cancer, comprising administering a therapeutically effective amount of the T cells described herein to a subject in need of treatment. Attached Figure Description

[0020] FIG. 1 Long-term efficacy and tumor growth delay were determined by progressing to a tumor volume endpoint set at four times the initial volume. Detailed Implementation

[0021] This disclosure relates to engineered receptors for regulating TGF-β signaling, and methods for regulating TGF-β signaling. In some aspects, this disclosure relates to an engineered receptor that can be used as a dominant-negative inhibitor to regulate TGF-β activity. In some aspects, this disclosure relates to dominant-negative TGF-β receptors, including TGF-β type I (TGF-βRI) or TGF-β type II (TGF-βRII), for inhibiting TGF-β activity. In one aspect, this disclosure relates to the use of dominant-negative TGF-β receptors for treating diseases or disorders, such as in T-cell immunotherapy. In another aspect, this disclosure relates to the use of dominant-negative TGF-β receptors to stimulate cytokine signaling that can enhance T-cell function in the TME. In yet another aspect, this disclosure relates to the use of dominant-negative TGF-β receptors in combination with other therapies.

[0022] DEFINITIONS

[0023] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of any discrepancy, this document (including the definitions) shall prevail. Methods and materials are described below, but similar or equivalent methods and materials may be used in the practice or testing of this disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods and examples disclosed herein are illustrative only and are not intended to be limiting.

[0024] The articles "a," "an," and "the" refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" can mean one element or more than one element.

[0025] As used herein, the term "about" or "approximately" means quantities, levels, values, numbers, frequencies, percentages, dimensions, sizes, amounts, weight, or lengths that vary by as much as 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In some embodiments, the term "about" or "approximately" when used before a numerical value indicates a range of that value plus or minus 15%, 10%, 5%, or 1%.

[0026] The terms "activated" and "activation" refer to the state of a T cell that has been sufficiently stimulated to induce detectable cell proliferation. In one embodiment, activation can also be associated with induced cytokine production and detectable effector function. The term "activated T cell" refers primarily to a T cell that is proliferating. A signal generated by the TCR alone can not be sufficient to fully activate a T cell, and can also require one or more secondary or costimulatory signals. Thus, T cell activation includes a primary stimulatory signal generated by the TCR / CD3 complex, as well as one or more secondary costimulatory signals. Costimulation can be evidenced by proliferation and / or cytokine production by T cells that have received a primary activation signal, such as stimulation by the TCR / CD3 complex.

[0027] "Administering" refers to the physical introduction of an agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Exemplary routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration (e.g., by injection or infusion). The phrase "parenteral administration" means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection as well as other parenteral routes of injection and infusion, and in vivo electroporation. In some embodiments, administration is via a non-parenteral route (e.g., oral). Other non-parenteral routes include topical, epidermal, or mucosal routes of administration, e.g., intranasal, vaginal, rectal, sublingual, or topical. Administration can also be performed, e.g., once, multiple times, and / or over one or more extended periods.

[0028] The term "amount" refers to an "effective amount" or "therapeutically effective amount" of an agent (such as a genetically modified therapeutic cell, e.g., a T cell) for achieving a beneficial or desired prophylactic or therapeutic result, including a clinical result. A "therapeutically effective amount" of a genetically modified therapeutic cell can vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the T cell to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the virus or transduced therapeutic cell are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the composition of the disclosure to be administered can be determined by a physician with consideration of individual patient characteristics such as age, size, tumor size, extent of infection or metastasis, and individual tolerance to the therapy.

[0029] The term "antibody" (Ab) includes, but is not limited to, a glycoprotein immunoglobulin that specifically binds an antigen. Generally, an antibody can comprise at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen-binding molecule thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2 and CH3. Each L chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with an antigen. The constant regions of the Abs can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Generally, a human antibody is a tetrameric agent of approximately 150 kD, composed of two identical heavy (H) chain polypeptides (each about 50 kD) and two identical light (L) chain polypeptides (each about 25 kD), which associate with one another to create a structure commonly called a "Y shape." The heavy and light chains are linked or joined to one another by a single disulfide bond; the other two disulfide bonds connect the heavy chain hinge regions to one another, such that dimers are linked to one another and form a tetramer. Naturally occurring antibodies are also glycosylated, e.g., on the CH2 domain.

[0030] The term "human antibody" is intended to include antibodies having variable domain sequences and constant domain sequences generated, assembled, or derived from human immunoglobulin sequences, or sequences that are indistinguishable therefrom. In some embodiments, antibodies (or antibody components) can be considered "human" even if their amino acid sequences comprise residues or elements not encoded by human germline immunoglobulin sequences (e.g., variations introduced by in vitro random or site-specific mutagenesis or variations introduced by in vivo somatic mutation). The term "humanized" is intended to include antibodies having variable domains with sequences originating from a variable domain of a non-human species (e.g., mouse) that have been modified to more closely resemble sequences encoded by human germline sequences. In some embodiments, "humanized" antibodies comprise one or more framework regions having an amino acid sequence substantially identical to a framework region of a human antibody, and one or more complementarity determining regions having an amino acid sequence substantially identical to a complementarity determining region of a non-human antibody. In some embodiments, humanized antibodies comprise at least a portion of an immunoglobulin constant region (Fc), typically of a human immunoglobulin constant domain. In some embodiments, humanized antibodies can comprise a human heavy chain constant domain of C H 1, hinge, C H 2, C H 3, and optionally, C H 4 regions.

[0031] A "monoclonal antibody" is an antibody produced by a single clone of B lymphocytes or by cells in which a single antibody's light chain gene and heavy chain gene have been transfected. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by preparing hybrid antibody-forming cells from a fusion of myeloma cells and immune spleen cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0032] A "chimeric antibody" has framework residues from one species (such as human), and CDRs (typically conferring antigen binding) from another species (such as mouse). In some embodiments, the CARs contemplated herein comprise an antigen-specific binding domain that is a chimeric antibody or an antigen-binding fragment thereof.

[0033] Antibodies can include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chains and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), heteroconjugate antibodies, single domain antibodies, univalent antibodies, single chain antibodies or single chain Fv (scFv), camelized antibodies, affibodies, Fab fragments, F(ab’)2 fragments, disulfide linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), and antigen binding fragments of any of the above. In certain embodiments, the antibodies described herein refer to a polyclonal antibody population. Antibodies can also include, for example, Fab’ fragments, Fd’ fragments, Fd fragments, isolated CDRs, single chain Fv, polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof), camelid antibodies, single chain diabodies or tandem diabodies minibodies, ankyrin repeat proteins or DARTs, TCR-like antibodies, miniprotein antibodies, and

[0034] Immunoglobulins can be derived from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG, IgE, and IgM. IgG subclasses are also well known to those of skill in the art, including but not limited to human IgG1, IgG2, IgG3, and IgG4. “Isotype” refers to the Ab class or subclass (e.g., IgM or IgG1) that is encoded by the heavy chain constant region genes. For example, the term “antibody” includes naturally occurring antibodies and non-naturally occurring antibodies; monoclonal antibodies and polyclonal antibodies; chimeric antibodies and humanized antibodies; human antibodies or non-human antibodies; wholly synthetic antibodies; and single chain antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless specifically indicated otherwise, and unless otherwise indicated by context, the term “antibody” also includes antigen binding fragments or antigen binding portions of any of the foregoing immunoglobulins, and includes monovalent and bivalent fragments or portions, as well as single chain antibodies.

[0035] An "antigen binding molecule," "antigen binding moiety," or "antibody fragment" refers to any molecule comprising an antigen binding portion (e.g., CDRs) of an antibody from which the molecule is derived. An antigen binding molecule can comprise an antigenic complementarity determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen binding molecules. Peptibodies (i.e., Fc fusion molecules comprising a peptide binding domain) are another example of suitable antigen binding molecules. In some embodiments, an antigen binding molecule binds an antigen on a tumor cell. In some embodiments, an antigen binding molecule binds an antigen on a cell involved in a hyperproliferative disease or a viral or bacterial antigen. In further embodiments, an antigen binding molecule is an antibody fragment that specifically binds an antigen, including one or more complementarity determining regions (CDRs) thereof. In further embodiments, an antigen binding molecule is a single chain variable region fragment (scFv). In some embodiments, an antigen binding molecule comprises or consists of an avimer.

[0036] In some cases, a CDR is essentially identical to a CDR found in a reference antibody and / or CDR sequence provided in the present disclosure. In some embodiments, a CDR is essentially identical to a reference CDR (e.g., a CDR provided in the present disclosure) in that it is identical in sequence or contains 1, 2, 3, 4, or 5 (e.g., 1-5) amino acid substitutions compared to the reference CDR. In some embodiments, a CDR is essentially identical to a reference CDR in that it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) sequence identity to the reference CDR. In some embodiments, a CDR is essentially identical to a reference CDR in that it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference CDR. In some embodiments, a CDR is essentially identical to a reference CDR in that one amino acid is deleted, added, or substituted within the CDR, while the amino acid sequence of the CDR is otherwise identical to the amino acid sequence of the reference CDR. In some embodiments, a CDR is essentially identical to a reference CDR in that 2, 3, 4, or 5 (e.g., 2-5) amino acids are deleted, added, or substituted within the CDR, while the amino acid sequence of the CDR is otherwise identical to the amino acid sequence of the reference CDR. In various embodiments, an antigen binding fragment binds to the same antigen as a reference antibody.

[0037] Antigen binding fragments can be produced by any means. For example, in some embodiments, antigen binding fragments can be produced enzymatically or chemically by fragmentation of intact antibodies. In some embodiments, antigen binding fragments can be produced recombinantly (i.e., by expression of engineered nucleic acid sequences). In some embodiments, antigen binding fragments can be produced wholly or partially synthetically. In some embodiments, antigen binding fragments can have a length of at least about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or more amino acids; in some embodiments, a length of at least about 200 amino acids (e.g., 50 to 100, 50 to 150, 50 to 200, or 100 to 200 amino acids).

[0038] The terms“variable region” or“variable domain” are used interchangeably and have the ordinary meaning in the art. Variable region generally refers to the portion of an antibody, generally a portion of a light or heavy chain, typically about the first 110 to 120 amino acids in the amino terminus of a mature heavy chain and about 90 to 115 amino acids in a mature light chain, that varies in sequence between antibodies and is used for the binding and specificity of a particular antibody to its particular antigen. Variability in sequence is concentrated in those regions known as complementarity determining regions (CDRs), while the more highly conserved regions in the variable domain are known as framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction of an antibody with an antigen and specificity. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In particular embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0039] The terms“VL” and“VL domain” are used interchangeably to refer to the variable region of the light chain of an antibody or antigen binding molecule thereof.

[0040] Reference to“VH” or“V H ” refers to the variable region of an immunoglobulin heavy chain, including the variable region of an immunoglobulin heavy chain of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment as disclosed herein. Reference to“VL” or“V L ” refers to the variable region of an immunoglobulin light chain, including the variable region of an immunoglobulin light chain of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragment as disclosed herein.

[0041] A number of definitions of CDRs are commonly used: the Kabat numbering, the Chothia numbering, the AbM numbering, or the Contact numbering. The AbM definition is a compromise between the two definitions used by Oxford Molecular's AbM antibody modeling software. The Contact definition is based on an analysis of available complex crystal structures.

[0042]

[0043]

[0044] The terms "Kabat numbering" and like terms are art-recognized and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or antigen binding molecule thereof. In certain aspects, the CDRs of an antibody can be determined according to the Kabat numbering system (see, e.g., Kabat EA and Wu TT (1971) Ann NY Acad Sci 190:382-391, and Kabat EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are generally found at amino acid positions 31 to 35, which after 35 (referred to as 35A and 35B in the Kabat numbering scheme) optionally can include one or two additional amino acids (CDR1); amino acid positions 50 to 65 (CDR2); and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule are generally found at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3). In a particular embodiment, the CDRs of the antibodies described herein have been determined according to the Kabat numbering scheme.

[0045] In certain aspects, the CDRs of an antibody can be determined according to the Chothia numbering scheme, which refers to the location of immunoglobulin structural loops (see, e.g., Chothia C and Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Patent No. 7,709,226). Generally, when using the Kabat numbering convention, the Chothia CDR-H1 loop is present at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is present at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop is present at light chain amino acids 24 to 34, the Chothia CDR-L2 loop is present at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop is present at light chain amino acids 89 to 97. When numbered using the Kabat numbering convention, the end of the Chothia CDR-H1 loop varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is present, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).

[0046] The terms“constant region” and“constant domain” are used interchangeably and have their ordinary meaning in the art. A constant region is a portion of an antibody, e.g., a carboxy-terminal portion of a light chain and / or heavy chain, that is not directly involved in binding of an antibody to an antigen, but which can exhibit various effector functions, such as interaction with Fc receptors. The constant region of an immunoglobulin molecule generally has a more conserved amino acid sequence relative to an immunoglobulin variable domain.

[0047] The term“heavy chain” when used in reference to an antibody can refer to any of the different types, e.g., a, d, e, g, and m, which are respectively produced by g, IgD, IgE, IgG, and IgM class antibodies, including IgG subclasses, e.g., IgGl, IgG2, IgG3, and IgG4.

[0048] The term "light chain" when used in reference to a reference antibody can refer to any of the different types based on the amino acid sequence of the constant domain, e.g., kappa or lambda. Light chain amino acid sequences are well known in the art. In particular embodiments, the light chain is a human light chain.

[0049] The term "antigen (Ag)" refers to a compound, composition, or substance that can stimulate the production of an antibody or a T-cell response in a human or an animal, including compositions that are injected or absorbed into a human or an animal, such as compositions comprising tumor-specific proteins. Antigens react with the products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the antigens disclosed herein. A "target antigen" or "antigen of interest" is an antigen that is not substantially found on the surface of other normal (desirable) cells and for which the binding domain of a TCR or CAR contemplated herein is designed to bind. Those skilled in the art will readily understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. An antigen can be expressed endogenously, i.e., from genomic DNA, or can be expressed recombinantly. An antigen can be specific for a certain tissue, such as a cancer cell, or can be widely expressed. Furthermore, fragments of larger molecules can serve as antigens. In one embodiment, the antigen is a tumor antigen. A "target" is any molecule bound by a binding motif, antigen binding system, or binding agent (e.g., an antibody). In some embodiments, the target is an antigen or epitope of the present disclosure.

[0050] The term "cancer" relates generally to a class of diseases or disorders in which abnormal cells divide without control and can invade nearby tissues. Examples of cancers that can be treated by the methods of the present disclosure include, but are not limited to, cancers of the immune system, including lymphomas, leukemias, myelomas, and other white blood cell malignancies. In some embodiments, the methods of the present disclosure can be used to reduce the tumor size of a tumor derived from, for example, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, multiple myeloma, Hodgkin's Disease, non-Hodgkin's lymphoma (NHL), primary mediastinal large B-cell lymphoma (PMBC), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), transformed follicular lymphoma, splenic marginal zone lymphoma (SMZL), esophageal cancer, small bowel cancer, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia (ALL) (including non T-cell ALL), chronic lymphocytic leukemia (CLL), solid tumors of childhood, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary gland cancer, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers including those induced by asbestos, other B-cell malignancies, and combinations of said cancers. In a particular embodiment, the cancer is multiple myeloma. A particular cancer can be responsive to chemotherapy or radiation therapy, or the cancer can be refractory. Refractory cancer refers to a cancer that is inapt for surgical intervention and that is initially unresponsive or becomes unresponsive over time to chemotherapy or radiation therapy. Cancer also includes relapsed or refractory large B-cell lymphoma after two or more lines of systemic therapy, including diffuse large B-cell lymphoma (DLBCL) not otherwise specified, primary mediastinal large B-cell lymphoma after two or more lines of systemic therapy, high grade B-cell lymphoma, and DLBCL arising from follicular lymphoma.

[0051] The terms “cancer cell,” “cancer cell,” “tumor cell,” or variants thereof refer to the individual cells of a cancerous growth or tissue. A tumor generally refers to a swelling or lesion formed by the abnormal growth of cells, which can be benign, pre-malignant, or malignant. Most cancers form tumors, but some (e.g., leukemia) do not necessarily form tumors. For those cancers that form tumors, the terms cancer (cells) and tumor (cells) are used interchangeably. The amount of tumor in an individual is the “tumor burden,” which can be measured as the number, volume, or weight of the tumor. Unless the context otherwise requires, the word “comprising” will be understood to imply the inclusion of the stated step or element, or a group of steps or elements, but does not exclude any other step or element, or a group of steps or elements. The phrase “consisting of” means including and limited to anything following the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed element is necessary or mandatory and that other elements may be absent. The phrase “substantially consisting of” means including any element listed following the phrase and is limited to other elements that do not interfere with or contribute to the activity or function of the listed elements specified in this disclosure.

[0052] "Conservative amino acid substitution" refers to the substitution of an amino acid residue by an amino acid residue having a similar side chain. Families of amino acid residues with side chains are defined in the art. These families include 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, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some embodiments, one or more amino acid residues within the CDR or within the framework region of an antibody or its antigen-binding molecule may be substituted with amino acid residues having similar side chains. Generally, two sequences are considered "substantially similar" if they contain conserved amino acid substitutions at corresponding positions. For example, some amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids and / or have "polar" or "nonpolar" side chains. A substitution of one amino acid with another of the same type can be considered a conserved substitution. Exemplary amino acid classifications are summarized in Table 2 below.

[0053] In Table 3:

[0054]

[0055]

[0056]

[0057] By "reducing" or "decreasing" or "diminishing", it is generally meant the ability of a composition contemplated herein to produce, elicit, or cause a lesser physiological response (i.e., downstream effect) as compared to the physiological response elicited by the vehicle alone (i.e., active moiety) or control molecule / composition. A "reduced" or "decreased" amount is generally a "statistically significant" amount, and can include a reduction that is 1 / 1.1, 1 / 1.2, 1 / 1.5, 1 / 2, 1 / 2.5, 1 / 3, 1 / 3.5, 1 / 4, 1 / 4.5, 1 / 5, 1 / 5.5, 1 / 6, 1 / 6.5, 1 / 7, 1 / 7.5, 1 / 8, 1 / 8.5, 1 / 9, 1 / 9.5, 1 / 10, 1 / 15, 1 / 20, 1 / 30, or less (e.g., 1 / 500, 1 / 1000) (including all integers and fractions greater than 1 between the foregoing numbers, e.g., 1.5, 1.6, 1.7, 1.8, etc., in the denominator) of the response produced by the vehicle, i.e., control composition.

[0058] The term "dominant negative" refers to a truncation or mutation of a polypeptide coding sequence that results in an altered gene product that inhibits downstream signaling functions normally transmitted by the unmodified gene product.

[0059] The terms "enhancing" or "promoting", or "increasing" or "augmenting" generally mean the ability of a composition contemplated herein to produce, elicit, or cause a greater physiological response (e.g., downstream effect) as compared to the physiological response elicited by the vehicle or control molecule / composition. Measurable physiological responses can include an increase in T cell expansion, activation, persistence, and / or cancer cell death killing capacity, as well as increases in other aspects that will be apparent in light of the understanding in the art and the descriptions herein. An "increased" or "enhanced" amount is generally a "statistically significant" amount, and can include an increase that is 1.1, 1.2, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 15, 20, 30, or more (e.g., 500, 1000) times (including all integers and fractions greater than 1 between the foregoing numbers, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response produced by the vehicle or control composition.

[0060] As used herein, the term "epitope" refers to a region of an antigen to which a binding agent binds. An epitope can be, for example, a contiguous amino acid of a polypeptide (linear epitope or contiguous epitope), or an epitope can be, for example, two or more noncontiguous regions from one or more polypeptides (conformational epitope, nonlinear epitope, discontinuous epitope, or noncontiguous epitope). In certain embodiments, an epitope to which an antibody binds can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange coupled to mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be achieved using any of the methods known in the art (e.g., Giegé R, et al. (1994) Acta Crystallogr D Biol Crystallogr 50 (Part 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques, and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) Vols. 114 and 115, Wyckoff HW, et al., eds.; U.S. 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Part 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, Carter CW, ed.; Roversi P, et al. (2000) Acta Crystallogr D Biol Crystallogr 56 (Part 10): 1316-1323). Mutagenesis mapping studies can be accomplished using any method known to one of skill in the art. See, e.g., Champe M, et al. (1995) J Biol Chem 270: 1388-1394 and Cunningham BC and Wells JA (1989) Science 244: 1081-1085 for descriptions of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0061] The term“genetically engineered” or“engineered” refers to a method of modifying the genome of a cell, including but not limited to deleting a coding or non-coding region or a portion thereof, or inserting a coding region or a portion thereof. In some embodiments, the modified cell is a lymphocyte, e.g., a T cell, which can be obtained from a patient or a donor. The cell can be modified to express an exogenous construct, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR), incorporated into the genome of the cell. Engineering typically involves manipulation by the hand of man. For example, a polynucleotide is considered to be“engineered” when two or more sequences that are not linked or joined together in that order in nature are manipulated by the hand of man to be linked or joined directly to one another in an engineered polynucleotide. In the case of cells manipulated by molecular biology techniques, a cell or organism is considered to be“engineered” if it has been manipulated so that its genetic information has been altered, e.g., new genetic material not previously present has been introduced, e.g., by transformation, somatic cell hybridization, transfection, transduction, or other mechanisms, or previously present genetic material has been altered or removed, e.g., by substitution or deletion mutation, or by other means. In some embodiments, the binding agent is a modified lymphocyte, e.g., a T cell, which can be obtained from a patient or a donor. The engineered cell can be modified to express an exogenous construct, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR), incorporated into the genome of the cell. Progeny of an engineered polynucleotide or binding agent are generally referred to as“engineered,” even if the actual manipulation was performed on a prior entity. In some embodiments,“engineered” refers to an entity that has been designed and produced. The term“designed” refers to an agent that (i) has a structure selected by the hand of man; (ii) is produced by a process requiring the hand of man; and / or (iii) is different from a natural substance and other known agents. A“T cell receptor” or“TCR” refers to an antigen recognition molecule present on the surface of T cells. During normal T cell development, each of the four TCR genes, alpha, beta, gamma, and delta, can rearrange, resulting in a highly diverse TCR protein. The terms“genetically modified cell,”“modified cell,” and“redirected cell” are used interchangeably.

[0062] An“immune effector cell” is any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and / or CDC). Illustrative immune effector cells contemplated herein are T lymphocytes, e.g., pan-CD3 + T cells, cytotoxic T cells (CTLs; CD8 + T cells), TILs, and helper T cells (HTLs; CD4 + T cells).

[0063] The terms“individual” and“subject” are often used interchangeably and refer to any animal that can be treated by the methods disclosed herein. Suitable subjects (e.g., patients) include laboratory animals such as mouse, rat, rabbit, or guinea pig, farm animals, and domestic animals or pets such as a cat or dog. Non-human primates and human patients are included. In one embodiment, a subject can include a human patient having cancer, who has been diagnosed with cancer, who is suspected of having cancer, or who is at risk of or is developing cancer. The term“patient” as used herein refers to a subject who can receive treatment for a disease or condition.

[0064] The terms“isolated peptide” or“isolated polypeptide” and the like refer to a peptide or polypeptide molecule that has been separated from the cellular environment and from association with other components of a cell in vitro, i.e., the peptide or polypeptide molecule is not significantly associated with in vivo material. Similarly, an“isolated cell” refers to a cell that has been obtained from in vivo tissue or organ and is substantially free of extracellular matrix.

[0065] The term“isolated polynucleotide” refers to a polynucleotide that has been purified away from the sequences that flank it in its naturally occurring state, e.g., a DNA fragment that has been removed from the sequences that normally flank it. An“isolated polynucleotide” also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not occur in nature and have been made by the hand of man.

[0066] The term“lymphocyte” includes a natural killer (NK) cell, a T cell, or a B cell. NK cells are a type of cytotoxic (cell-poisoning) lymphocyte that represents a major component of the innate immune system. NK cells reject tumors and cells infected by viruses. It acts through the process of apoptosis or programmed cell death. They are called“natural killers” because they do not need to be activated to kill cells. T cells play a major role in cell-mediated immunity (without antibody involvement). Their T cell receptors (TCRs) distinguish them from other lymphocyte types. The thymus is a specialized organ of the immune system, primarily responsible for the maturation of T cells. There are six types of T cells, namely: Helper T cells (e.g., CD4 + cells), cytotoxic T cells (also known as TC, cytotoxic T lymphocytes, CTLs, T killer cells, cytolytic T cells, CD8 + T cells, or killer T cells), memory T cells ((i) stem memory T cells (TSCM), (ii) central memory T cells (TCM), (iii) effector memory T cells (TEM), (iv) terminally differentiated effector memory T cells (TEMRA), (v) mucosal-associated invariant T cells (MAIT), and (vi) gamma-delta T cells (Tgd)), regulatory T cells (Tregs), and gamma-delta T cells (Tgd). SCM Cells such as naive cells are CD45RO - , CCR7 + , CD45RA + , CD62L + (L-selectin), CD27 + , CD28 +and IL-7Ra + but they also express large amounts of CD95, IL-2R, CXCR3, and LFA-1, and show many functional attributes characteristic of memory cells); (ii) central memory T CM cells express L-selectin and CCR7, they secrete IL-2 but not IFNy or IL-4, whereas (iii) effector memory T EM cells do not express L-selectin or CCR7, but produce effector cytokines such as IFNy and IL-4), regulatory T cells (Tregs, suppressor T cells, or CD4 + CD25 + regulatory T cells), natural killer T cells (NKT), and gd T cells. T cells are intended to include gd T cells, pan-CD3 + T cells, including thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells can be T helper (Th) cells, such as T helper 1 (Thl) cells or T helper 2 (Th2) cells. T cells can be helper T cells (HTL; CD4 + T cells) CD4 + T cells, cytotoxic T cells (CTL; CD8 + T cells), tumor infiltrating cytotoxic T cells (TIL; CD8 + T cells), CD4 + CD8 + T cells, CD4-CD8- T cells, or any other subset of T cells. Other illustrative T cell populations suitable for use in some embodiments include naive T cells and memory T cells. B cells, on the other hand, play a major role in humoral immunity (involving antibodies). They manufacture antibodies and act as antigen presenting cells (APCs) and, upon activation by antigen interaction, transform into memory B cells. In mammals, immature B cells are formed in the bone marrow, from which they derive their name.

[0067] The term“modified T cell” refers to a T cell that has been modified by the introduction of a polynucleotide encoding an engineered polypeptide as described herein. Modified T cells include both genetically modified and non-genetically modified (e.g., episomally or extrachromosomally modified) T cells. In one embodiment, a modified T cell can include a TGF-b receptor or CAR-DN TGF-b receptor as described herein.

[0068] By "maintain" or "preserve" or "retain" or "unchanged" or "no significant change" or "no significant decrease" is generally meant the ability of a composition contemplated herein to produce, elicit, or cause a lesser physiological response (i.e., downstream effects) in a cell as compared to the response elicited by a vehicle, control molecule / composition. A comparable response is one that is not significantly different or measurably different from the reference response.

[0069] The term "malignant" refers to a cancer in which a group of tumor cells displays one or more of uncontrolled growth (i.e., division beyond the normal limits), invasion (i.e., intrusion into and destruction of adjacent tissues), and metastasis (i.e., spread to other locations in the body via lymph or blood). As used herein, the term "metastasis" refers to the spread of a cancer from one part of the body to another. A tumor formed from cells that have spread is called a "metastatic tumor" or "metastasis." A metastatic tumor contains cells similar to those in the original (primary) tumor. As used herein, the term "benign" or "non-malignant" refers to a tumor that can grow larger but does not spread to other parts of the body. Benign tumors are self-limited and generally do not invade or metastasize.

[0070] The term "proliferation" refers to an increase in cell division (symmetric or asymmetric cell division). In some embodiments, "proliferation" refers to symmetric or asymmetric division of a T cell. "Increased proliferation" occurs when the number of cells in a treated sample increases as compared to cells in an untreated sample.

[0071] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., TCR / CD3 complex) to its cognate ligand, thereby mediating a signal transduction event, including but not limited to signal transduction via the TCR / CD3 complex. A "stimulatory molecule" refers to a molecule on a T cell that specifically binds to a cognate stimulatory ligand. As used herein, a "stimulatory ligand" means a ligand that, when presented on an antigen presenting cell (APC) (e.g., a dendritic cell, a B cell, etc.), can specifically bind to a cognate binding partner (referred to herein as a "stimulatory molecule") on a T cell, thereby mediating a primary response by the T cell, including but not limited to activation, initiation of an immune response, proliferation, etc. Stimulatory ligands include, but are not limited to, CD3 ligands (e.g., anti-CD3 antibodies such as OKT3) and CD2 ligands (e.g., anti-CD2 antibodies), peptides (e.g., cytomegalovirus (CMV) peptides, hepatitis B virus (HBV) peptides, and Epstein-Barr virus (EBV) peptides), MHC class I molecules loaded with peptides, superagonist anti-CD2 antibodies, and superagonist anti-CD28 antibodies.

[0072] A "costimulatory signal" refers to a signal that, in combination with a primary signal such as TCR / CD3 ligation, results in T cell proliferation, cytokine production, and / or up- or down-regulation of molecules (e.g., CD28).

[0073] As used herein, a “costimulatory ligand” includes a molecule on an antigen presenting cell that specifically binds to a cognate costimulatory molecule on a T cell. Binding of a costimulatory ligand provides a signal that mediates a T cell response, including but not limited to proliferation, activation, differentiation, and the like. A costimulatory ligand induces a signal in addition to the primary signal provided by a stimulatory molecule, e.g., by binding of a T cell receptor (TCR) / CD3 complex to a major histocompatibility complex (MHC) molecule loaded with a peptide. Costimulatory ligands can include, but are not limited to, 3 / TR6, 4-1BB ligand, an agonist or antibody that binds to a Toll ligand receptor, B7-1 (CD80), B7-2 (CD86), CD30 ligand, CD40, CD7, CD70, CD83, herpesvirus entry mediator (HVEM), human leukocyte antigen G (HLA-G), ILT4, immunoglobulin-like transcript (ILT) 3, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), a ligand that specifically binds to B7-H3, lymphotoxin beta receptor, MHC class I chain-related protein A (MICA), MHC class I chain-related protein B (MICB), OX40 ligand, PD-L2, or programmed death (PD) L1. Costimulatory ligands include, but are not limited to, antibodies that specifically bind to costimulatory molecules present on T cells, such as, but not limited to, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, CD7, ICOS, a ligand that specifically binds to CD83, lymphocyte function-associated antigen-1 (LFA-1), natural killer cell receptor C (NKG2C), OX40, PD-1, or tumor necrosis factor superfamily member 14 (TNFSF14 or LIGHT).

[0074] A "costimulatory molecule" is a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as but not limited to proliferation. Costimulatory molecules include, but are not limited to, "costimulatory molecule" is a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as but not limited to proliferation.Co-stimulatory molecules include, but are not limited to, 4-1BB / CD137, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD 33, CD 45, CD100 (SEMA4D), CD103, CD134, CD137, CD154, CD16, CD160 (BY55), CD18, CD19, CD19a, CD2, CD22, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 (a; b; d; e; g; z), CD30, CD37, CD4, CD4, CD40, CD49a, CD49D, CD49f, CD5, CD64, CD69, CD7, CD80, CD83 ligand, CD84, CD86, CD8a, CD8b, CD9, CD96 (Tactile), CDl-la, CDl-lb, CDl-lc, CDl-ld, CDS, CEACAM1, CRTAM, DAP-10, DNAM1 (CD226), Fcy receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, ICOS, Ig a (CD79a), IL-2R b, IL-2R g, IL-7R a, Integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, LIGHT, LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), LTBR, Ly9 (CD229), Lymphocyte function-associated antigen-1 (LFA-1 (CDlla / CD18), MHC class I molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX40, PAG / Cbp, PD-1, PSGL1, SELPLG (CD162), Signaling lymphocyte activation molecule, SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF, TNFr, TNFR2, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[0075] The term“substantially” refers to a quantity, level, value, number, frequency, percent, dimension, size, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher, for a reference quantity, level, value, number, frequency, percent, dimension, size, amount, weight, or length. In one embodiment,“substantially the same” refers to a quantity, level, value, number, frequency, percent, dimension, size, amount, weight, or length that produces approximately the same effect (e.g., a physiological effect) as a reference quantity, level, value, number, frequency, percent, dimension, size, amount, weight, or length.

[0076] As used herein,“treatment” includes any beneficial or desired effect on symptoms or pathology of a disease or pathological condition, and can include even minimal reduction of one or more measurable markers of the disease or disorder being treated (e.g., cancer). Treatment can involve, optionally, reducing or ameliorating a symptom of a disease or disorder, or delaying progression of a disease or disorder.“Treatment” does not necessarily indicate complete eradication or cure of a disease or disorder, or associated symptoms thereof.

[0077] The term“virus-induced cancer” includes any malignancy induced by a DNA virus or an RNA virus. Such viruses can include, but are not limited to, for example: cytomegalovirus (CMV), hepatitis B virus (HBV - hepatocellular carcinoma), human papillomavirus (HPV - squamous cell carcinoma, cervical epithelial carcinoma), human T-lymphotropic virus (HTLV-I - adult T-cell leukemia), and Epstein-Barr virus (EBV - Burkitt’s lymphoma, nasopharyngeal carcinoma).

[0078] A“chimeric antigen receptor” or“CAR” refers to a molecule engineered to comprise a binding motif and means of activating an immune cell (e.g., a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof) upon antigen binding. CARs are also known as artificial T-cell receptors, chimeric T-cell receptors, or chimeric immunoreceptors. In some embodiments, a CAR comprises a binding motif, an extracellular domain, a transmembrane domain, one or more costimulatory domains, and an intracellular signaling domain. A T cell that has been genetically engineered to express a chimeric antigen receptor can be referred to as a CAR T cell. An“extracellular domain” (or“ECD”) refers to the portion of a polypeptide that, when the polypeptide is present in a cell membrane, is understood to be located on the outside of the cell membrane, in the extracellular space.

[0079] The term“neutralize” refers to an antigen binding molecule, scFv, antibody, or fragment thereof that binds to a ligand and prevents or reduces the biological effect of the ligand. In some embodiments, the antigen binding molecule, scFv, antibody, or fragment thereof directly blocks the binding site on the ligand, or alters the binding ability of the ligand through indirect means (e.g., a structural or energetic change in the ligand). In some embodiments, the antigen binding molecule, scFv, antibody, or fragment thereof prevents the protein to which it binds from performing a biological function.

[0080] The term“autologous” refers to any material derived from the same individual and later reintroduced into that individual. For example, the engineered autologous cell therapy (eACT TM ) methods described herein involve collecting lymphocytes from a patient, then engineering them to express, for example, a CAR construct, followed by administration back to the same patient.

[0081] The term“allogeneic” refers to any material derived from one individual and later introduced into another individual of the same species, for example, allogeneic T cell transplantation.

[0082] The terms“transduce” and“transduced” refer to the process of introducing exogenous DNA into a cell via a viral vector (see Jones et al.,“Genetics: principles and analysis,” Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr viral vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentivirus vector, or any combination thereof.

[0083] “Transformation” refers to any process by which exogenous DNA is introduced into a host cell. Transformation can occur under natural or artificial conditions using a variety of methods. Transformation can be achieved using any known method for inserting a foreign nucleic acid sequence into a prokaryotic or eukaryotic host cell. In some embodiments, some transformation methods are selected based on the host cell being transformed and / or the nucleic acid to be inserted. Transformation methods can include, but are not limited to, viral infection, electroporation, and lipofection. In some embodiments, a“transformed” cell is stably transformed in that the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome. In some embodiments, a transformed cell can express the introduced nucleic acid.

[0084] The term "vector" refers to a recipient nucleic acid molecule that has been modified to comprise or incorporate a provided nucleic acid sequence. One type of vector is a "plasmid," which refers to a circular double stranded DNA molecule into which additional DNA can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors contain sequences that direct expression of inserted genes with which they are operatively linked. Such vectors can be referred to herein as "expression vectors." Standard techniques can be used to engineer vectors, e.g., as found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), incorporated herein by reference for any purpose.

[0085] As used herein, "anti-tumor effect" refers to a biological effect that can manifest as a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in tumor cell proliferation, a reduction in the number of metastases, an increase in overall or progression-free survival, an increase in life expectancy, or an improvement in various physiological symptoms associated with a tumor. Anti-tumor effect can also refer to the prevention of the occurrence of a tumor, e.g., a vaccine.

[0086] As used herein, “cytokine” refers to a non-antibody protein released by one cell in response to contact with a specific antigen, where the cytokine interacts with a second cell to mediate a response in the second cell. Cytokines can be expressed endogenously by a cell or administered to a subject. Cytokines can be released by immune cells, including macrophages, B cells, T cells, and mast cells, to propagate an immune response. Cytokines can induce various responses in recipient cells. Cytokines can include homeostatic cytokines, chemokines, proinflammatory cytokines, effectors, and acute phase proteins. For example, homeostatic cytokines, including interleukin (IL) 7 and IL-15, promote immune cell survival and proliferation, and proinflammatory cytokines can promote an inflammatory response. Examples of homeostatic cytokines include, but are not limited to, IL-2, IL-4, IL-5, IL-7, IL-10, IL-12p40, IL-12p70, IL-15, and interferon (IFN) gamma. Examples of proinflammatory cytokines include, but are not limited to, IL-la, IL-lb, IL-6, IL-13, IL-17a, tumor necrosis factor (TNF)-alpha, TNF-beta, fibroblast growth factor (FGF) 2, granulocyte macrophage colony-stimulating factor (GM-CSF), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), vascular endothelial growth factor (VEGF), VEGF-C, VEGF-D, and placental growth factor (PLGF). Examples of effectors include, but are not limited to, granzyme A, granzyme B, soluble Fas ligand (sFasL), and perforin. Examples of acute phase proteins include, but are not limited to, C-reactive protein (CRP) and serum amyloid A (SAA).

[0087] A “chemokine” is a cytokine that mediates chemotaxis or directed movement of cells. Examples of chemokines include, but are not limited to, IL-8, IL-16, eotaxin, eotaxin-3, macrophage-derived chemokine (MDC or CCL22), monocyte chemotactic protein 1 (MCP-1 or CCL2), MCP-4, macrophage inflammatory protein 1 alpha (MIP-1 alpha, MIP-1a), MIP-1 beta (MIP-1b), gamma-inducible protein 10 (IP-10), and thymus and activation-regulated chemokine (TARC or CCL17).

[0088] An "immune response" refers to the action of cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, and neutrophils) and soluble macromolecules produced by any of these cells or the liver (including Abs, cytokines, and complement) that result in the selective targeting, binding, impairment, destruction, and / or elimination of invading pathogens from the body of a vertebrate, cells or tissues infected with a pathogen, cancer cells or other abnormal cells, or normal human cells or tissues in the context of autoimmunity or pathological inflammation.

[0089] The term "immunotherapy" refers to the treatment of a subject afflicted with, or at risk of contracting or recurring with, a disease by a method comprising inducing, enhancing, suppressing or otherwise altering an immune response. Examples of immunotherapy include, but are not limited to, T cell therapy. T cell therapy can include adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT TM ) and allogeneic T cell transplantation. However, one skilled in the art will recognize that the conditioning methods disclosed herein will enhance the efficacy of any transplanted T cell therapy. Examples of T cell therapy are described in U.S. Patent Publication Nos. 2014 / 0154228 and 2002 / 0006409, U.S. Patent No. 5,728,388 and International Publication No. WO 2008 / 081035.

[0090] The T cells of the immunotherapy can be from any source known in the art. For example, the T cells can be differentiated in vitro from a population of hematopoietic stem cells, or the T cells can be obtained from a subject. The T cells can be obtained from, for example, peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In addition, the T cells can be derived from one or more T cell lines available in the art. The T cells can also be obtained from a blood unit collected from a subject using various techniques known to the skilled artisan, such as FICOLL TM separation and / or apheresis. Additional methods of isolating T cells for use in a T cell therapy are disclosed in U.S. Patent Publication 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0091] The term "engineered autologous cell therapy" (which can be abbreviated as "eACT TMAdoptive cell therapy (also known as adoptive cell transfer) is a process by which a patient's own T cells are collected and then genetically engineered to recognize and target one or more antigens expressed on the surface of one or more specific tumor cells or malignancies. The T cells can be engineered to express, for example, a chimeric antigen receptor (CAR) or a T cell receptor (TCR). CAR-positive (+) T cells are engineered to express an extracellular single-chain variable fragment (scFv) specific for a particular tumor antigen, linked to an intracellular signaling portion comprising at least one costimulatory domain and at least one activating domain. The costimulatory domain can be derived from a naturally occurring costimulatory domain or a variant thereof, such as a variant with a truncated hinge domain ("THD"), and the activating domain can be derived from, for example, CD3-zeta. In certain embodiments, the CAR is designed with two, three, four, or more costimulatory domains. The CAR scFv can be designed to target, for example, CD19, a transmembrane protein expressed by cells in the B cell lineage, including all normal B cells and B cell malignancies, including but not limited to NHL, CLL, and non-T cell ALL. In some embodiments, the CAR is engineered such that the costimulatory domains are expressed as separate polypeptide chains. Exemplary CAR T cell therapies and constructs are described in U.S. Patent Publications 2013 / 0287748, 2014 / 0227237, 2014 / 0099309, and 2014 / 0050708, and these references are incorporated by reference in their entireties. "Adoptive cell therapy" or "ACT" involves the transfer of immune cells with anti-tumor activity into a subject (e.g., a cancer patient). In some embodiments, ACT is a therapeutic method involving the use of lymphocytes (e.g., engineered lymphocytes) with anti-tumor activity.

[0092] "Antigen presenting cell" or "APC" refers to a cell that processes and presents antigen to T cells. Exemplary APCs include dendritic cells, macrophages, B cells, certain activated epithelial cells, and other cell types capable of TCR stimulation and appropriate T cell costimulation.

[0093] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound composed of amino acid residues connected by peptide bonds. A protein or peptide comprises at least two amino acids, and there is no limit to the maximum number of amino acids that can constitute a sequence of a protein or peptide. A polypeptide includes any peptide or protein comprising two or more amino acids connected to one another by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, e.g., peptides, oligopeptides, and oligomers) and longer chains (which are commonly referred to in the art as proteins, which can have many types). "Polypeptide" includes, e.g., biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide includes a natural, recombinant, synthetic, or a combination thereof.

[0094] The term "identity" refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods for calculating percent identity between two provided polypeptide sequences are known. For example, calculation of percent identity of two sequences can be accomplished by aligning the two nucleic acid or polypeptide sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and second sequence for optimal alignment, and nonidentical sequences can be disregarded for comparison purposes). The nucleotides or amino acids at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number of gaps, and the length of each gap, which can need to be introduced for optimal alignment of the two sequences. The comparison or alignment of sequences and the determination of percent identity between two sequences can be achieved using a mathematical algorithm such as BLAST (Basic Local Alignment Search Tool). In some embodiments, polymer molecules are considered "homologous" to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical (e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100% identical).

[0095] To calculate percent identity, the sequences in comparison are typically aligned in a manner that gives the largest matching between the sequences. One example of a computer program that can be used to determine percent identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.). The computer algorithm GAP is used to align two polypeptides or polynucleotides for which percent sequence identity is to be determined. The sequences are aligned for maximum matching of their respective amino acids or nucleotides (“span of matches,” as determined by the algorithm). In certain embodiments, the algorithm also uses a standard comparison matrix (see PAM 250 comparison matrix in Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352; BLOSUM 62 comparison matrix in Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919). Other algorithms can also be used to compare amino acid sequences or nucleic acid sequences, including those available in commercial computer programs such as BLASTN for nucleotide sequences, and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999.In addition to identifying similar sequences, the programs mentioned above often provide an indication of the degree of similarity. In some embodiments, two sequences are considered to be substantially similar if at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more of the corresponding residues in the two sequences are similar and / or identical over a stretch of related residues (e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%). In some embodiments, the stretch of related residues is the entire sequence. In some embodiments, the stretch of related residues is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 325, at least 350, at least 375, at least 400, at least 425, at least 450, at least 475, at least 500, or more residues. Sequences having substantial sequence similarity can be homologs of one another.

[0096] “Combination therapy” refers to those instances where an individual is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic moieties). In some embodiments, the two or more regimens can be administered simultaneously; in some embodiments, such regimens can be administered sequentially (e.g., all “doses” of a first regimen are administered prior to administration of any dose of a second regimen); in some embodiments, such agents are administered in overlapping dosing schedules. In some embodiments, “administering” a combination therapy can involve administering one or more agents or modalities to a subject who is receiving the other agent or modality in the combination. For clarity, combination therapy does not require that the agents be administered together in a single composition (or even necessarily simultaneously), although in some embodiments, two or more agents or their active moieties can be administered together in a composition of the combination, or even in a compound of the combination (e.g., as part of a single chemical complex or covalent entity).

[0097] The term“dosage form” can be used to refer to a physically discrete unit of an active agent (e.g., an antigen binding system or an antibody) that is administered to a subject. Generally, each such unit contains a predetermined quantity of active agent. In some embodiments, this quantity is a unit dose (or an entire portion thereof) that is suitable for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial result when administered to a relevant population. The total amount of therapeutic composition or therapeutic agent administered to a subject is determined by one or more medical practitioners and can involve administration of more than one dosage form.

[0098] The term“dosing regimen” can be used to refer to a set of one or more unit doses administered individually to a subject. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which can involve one or more doses. In some embodiments, a dosing regimen includes multiple doses, where each dose is separated in time from the others. In some embodiments, a dosing regimen includes multiple doses, and consecutive doses are separated from each other by an equal length of time period; in some embodiments, a dosing regimen includes multiple doses, and consecutive doses are separated from each other by at least two different lengths of time period. In some embodiments, all doses within a dosing regimen have the same unit dose. In some embodiments, different doses within a dosing regimen have different amounts. In some embodiments, a dosing regimen includes a first amount of a first dose, followed by one or more additional doses of a second amount that is different from the first amount. In some embodiments, a dosing regimen is adjusted periodically to achieve a desired or beneficial result.

[0099] “Effector function” refers to the biological consequences of the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-mediated cytotoxicity (CMC). Effector function can be antigen-binding dependent, antigen-binding independent, or both. ADCC refers to lysis of antibody-bound target cells by immune effector cells. Without wishing to be bound by any theory, ADCC is generally understood to involve effector cells bearing Fc receptors (FcRs) recognizing and subsequently killing antibody-coated target cells (e.g., cells expressing on their surface an antigen to which the antibody binds). Effector cells that mediate ADCC can include immune cells, including but not limited to one or more of natural killer (NK) cells, macrophages, neutrophils, eosinophils.

[0100] An "effector cell" refers to an immune system cell that expresses one or more Fc receptors and mediates one or more effector functions. In some embodiments, an effector cell can include, but is not limited to, one or more of a monocyte, a macrophage, a neutrophil, a dendritic cell, an eosinophil, a mast cell, a platelet, a large granular lymphocyte, a Langerhans cell, a natural killer (NK) cell, a T lymphocyte, and a B lymphocyte. An effector cell can be from any organism, including but not limited to a human, a mouse, a rat, a rabbit, and a monkey.

[0101] The term "excipient" refers to an agent that can be included in a composition, for example, to provide or contribute to a desired consistency or stabilizing effect. In some embodiments, suitable excipients can include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like.

[0102] A "fragment" or "portion" of a material or entity as described herein has a structure that includes a discrete portion of a whole (e.g., of a physical entity or an abstract entity). In some embodiments, a fragment lacks one or more portions present in the whole. In some embodiments, a fragment consists of, or comprises, a structural element, domain, or portion present in the whole. In some embodiments, a polymer fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) present in the whole polymer. In some embodiments, a polymer fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more (e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) of the monomeric units (e.g., residues) present in the whole material or entity. In some embodiments, the whole material or entity can be referred to as the "parent" of the fragment.

[0103] The term "fusion polypeptide" or "fusion protein" generally refers to a polypeptide comprising at least two segments. Generally, a polypeptide containing at least two such segments is considered a fusion polypeptide if the two segments are not contained in the same peptide in nature, and / or if the two segments have not previously been joined or linked to one another in a single polypeptide, and / or if the two segments have been joined or linked to one another by the hand of man.

[0104] The term "isolated" refers to a substance that is (1) separated from at least some components with which the substance is associated at an earlier time or with which the substance would otherwise be associated, and / or (2) present in a composition that contains a limited or defined amount or concentration of one or more known or unknown contaminants. In some embodiments, an isolated substance can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% (e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) of other non-substance components with which the substance was associated at an earlier time (e.g., other components or contaminants with which the substance was previously associated or would otherwise be associated). In certain instances, a substance is isolated if it is present in a composition that contains a limited or reduced amount or concentration of the same or similar type of molecules. For example, in certain instances, a nucleic acid, DNA, or RNA substance is isolated if it is present in a composition that contains a limited or reduced amount or concentration of non-substance nucleic acid, DNA, or RNA molecules. For example, in certain instances, a polypeptide substance is isolated if it is present in a composition that contains a limited or reduced amount or concentration of non-substance polypeptide molecules. In certain embodiments, the amount can be, for example, an amount measured relative to the amount of the desired substance present in the composition. In certain embodiments, a limited amount can be an amount that is no more than 100% of the amount of the substance in the composition (e.g., no more than 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% (e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) of the amount of the substance in the composition). In certain instances, a composition is pure or substantially pure with respect to a selected substance. In some embodiments, an isolated substance has a purity of about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% (e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%). A substance is "pure" if it is substantially free of other components or contaminants.In some embodiments, a substance can still be considered "isolated" or even "pure" after it has been mixed with certain other components, such as one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the degree of isolation or percentage of purity of the substance is calculated without including such carriers or excipients.

[0105] "Nucleic acid" refers to any polymeric chain of nucleotides. The nucleic acid can be DNA, RNA, or a combination thereof. In some embodiments, the nucleic acid comprises one or more natural nucleic acid residues. In some embodiments, the nucleic acid comprises one or more nucleic acid analogs. In some embodiments, the nucleic acid is prepared by one or more of: isolation from a natural source, enzymatic synthesis (in vivo or in vitro) by polymerization based on a complementary template, propagation in a recombinant cell or system, and chemical synthesis. In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, or more residues in length (e.g., 20 to 100, 20 to 500, 20 to 1000, 20 to 2000, or 20 to 5000 or more residues). In some embodiments, the nucleic acid is partially or entirely single-stranded; in some embodiments, the nucleic acid is partially or entirely double-stranded. In some embodiments, the nucleic acid has a nucleotide sequence comprising at least one element that encodes a polypeptide, or is a complement sequence of a sequence that encodes a polypeptide.

[0106] "Operably linked" refers to juxtaposition with respect to the components such that they function in their intended manner. For example, a control element is "operably linked" to a functional element in a manner that allows for expression and / or activity of the functional element under conditions compatible with the control element.

[0107] The term "pharmaceutically acceptable" refers to a molecular entity or composition that is not harmful to an acceptor thereof when administered to the acceptor, or any harmful effects are outweighed by the beneficial effects to the acceptor. In reference to a carrier, diluent, or excipient used in formulating a composition as disclosed herein, the pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not harmful to the acceptor thereof, or any harmful effects must be outweighed by the beneficial effects to the acceptor. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting an agent from one part of the body to another, such as from one organ to another. Each carrier present in a pharmaceutical composition must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient, or any harmful effects must be outweighed by the beneficial effects to the acceptor. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laureate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0108] The term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in unit dose form suitable for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to the relevant subject or population. In some embodiments, the pharmaceutical composition can be formulated for administration in solid or liquid form, including, but not limited to, forms suitable for oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue, parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, as, for example, a sterile solution or suspension, or sustained-release formulation, topical application, such as a cream, ointment, or controlled-release patch, or spray applied to the skin, lung, or oral cavity, intravaginally or intrarectally, such as a pessary, cream, or foam, sublingually, ocularly, transdermally, or nasally, to the lung, and to other mucosal surfaces.

[0109] The term "reference" describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested, measured, and / or assayed substantially simultaneously with the test, measurement, or assay of interest. In some embodiments, the reference or control is a historical reference or control, which is optionally embodied in a tangible medium. Generally, the reference or control is determined or characterized under conditions or circumstances comparable to those of the evaluation. There is sufficient similarity to justify reliance on and / or comparison to the selected reference or control when present.

[0110] Unless specifically noted otherwise, the present disclosure can employ chemical methods, biochemical methods, organic chemical methods, molecular biology methods, microbiology methods, recombinant DNA techniques, genetic methods, immunological methods, and cell biological methods that are within the skill of the art, many of which are described below for purposes of illustration. Such techniques are fully explained in the literature. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., 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, Volumes I and II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames and S. Higgins eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998) Current Protocols in Immunology Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, and W. Strober eds., 1991); Annual Review of Immunology; and the series Methods in Enzymology (Academic Press, Inc.); and Advances in Immunology.

[0111] The present disclosure describes engineered constructs of dominant negative TGF-β receptors designed to inhibit the immunosuppressive effects of TGF-β in the TME. These constructs can also stimulate cytokine signaling to enhance T cell function in the TME. The constructs described herein can be used alone or in combination with one another, and / or in combination with other immunotherapies, in order to inhibit TGF-β induced immunosuppression. The engineered dominant negative TGF-β receptors disclosed herein can comprise, consist essentially of, or consist of the sequences provided herein.

[0112] The wild-type TGF-β receptor type I (TGF-βRI) is the part of the TGF-β receptor complex that transmits the intracellular inhibitory pSMAD signaling program. The full-length wild-type nucleic acid sequence of TGF-βRI is a sequence of 1509 nucleotides in length set forth as SEQ ID NO: 1.

[0113]

[0114]

[0115] The full-length wild-type amino acid (aa) sequence of TGF-βRI is 503 amino acids in length set forth as SEQ ID NO: 2. The full-length TGF-βRI polypeptide of SEQ ID NO: 2 includes a signal peptide (approximately at amino acids 1-33), an extracellular domain (approximately at amino acids 34-126), a transmembrane domain (approximately at amino acids 127-147), and an intracellular domain (approximately at amino acids 148-503). The intracellular domain includes four key threonine sites located between amino acid 185 and amino acid 204 of the intracellular domain of TGF-βRI and initiates pSMAD signaling.

[0116]

[0117] Due to its role in initiating intracellular signaling cascades, modified TGF-βRI constructs can be potential therapeutic targets. Without being bound by any theory, it is hypothesized that dominant negative TGF-βRI constructs can be designed to inhibit the phosphorylation cascade, thereby limiting the immunosuppressive effects of TGF-β on T cell function. The present disclosure contemplates any truncation or modification that results in inhibition or reduction of phosphorylation such that it leads to a nonfunctional signaling pathway. In one embodiment, a DN TGF-βRI can be truncated or modified after the transmembrane domain such that the polypeptide lacks one or more amino acid residues responsible for signaling and phosphorylation and results in a nonfunctional signaling pathway compared to the wild-type receptor. In some embodiments, a DN TGF-βRI can be truncated before the threonine at amino acid 185 of the intracellular domain of SEQ ID NO: 2. In some embodiments, a DN TGF-βRI can be truncated such that it does not have an intracellular domain. In other embodiments, a DN TGF-βRI can be modified to replace the intracellular domain with another naturally or non-naturally occurring sequence that does not contain amino acid residues involved in phosphorylation signaling. In another embodiment, the polypeptide is truncated or modified in a manner that reduces phosphorylation activity. In another embodiment, the polypeptide is truncated or modified in a manner that inhibits phosphorylation signaling molecules from interacting with pSMAD molecules. The present disclosure describes truncated dominant negative TGF-βRI constructs (DN TGF-βRI) that have been engineered to remove amino acids involved in phosphorylation signaling of the intracellular domain of wild-type TGF-βRI.

[0118] In some embodiments described herein, a DN TGF-βRI comprises an extracellular ligand binding domain for binding TGF-β having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to the amino acid sequence of SEQ ID NO: 3.

[0119]

[0120] In some embodiments, the DN TGF-bRI extracellular ligand binding domain having at least 75% sequence identity (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 3 can be fused to one or more heterologous polypeptide sequences. The extracellular domain of the DN TGF-bRI can be engineered to recognize and bind to a target TGF-b molecule in order to initiate oligomerization of a complex of TGF-bRII with the DN TGF-bRI. Further, the present disclosure contemplates binding of the DN TGF-bRI to all TGF-b isoforms, including Transforming Growth Factor b Type I (TGF-bI), Transforming Growth Factor b Type II (TGF-b2), Transforming Growth Factor b Type III (TGF-b3), and Transforming Growth Factor b Type IV (TGF-b4). In some embodiments, the DN TGF-bRI can be engineered to bind TGF-bI, TGF-b2, TGF-b3, and / or TGF-b4. In one embodiment, the DN TGF-bRI binds TGF-bI.

[0121] In another embodiment described herein, the DN TGF-bRI comprises an extracellular binding domain fused to a transmembrane domain. The DN TGF-bRI can comprise a transmembrane domain comprising an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to a wild-type TGF-bRI transmembrane domain or a portion thereof. As a non-limiting example, the transmembrane domain can comprise an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 4, which includes the wild-type TGF-bRI transmembrane domain. AAVIAGPVCFVCISLMLMVYI (SEQ ID NO: 4). In other embodiments, the transmembrane domain is a heterologous transmembrane domain, including any of the various transmembrane domains described herein. The transmembrane domain of the DN TGF-bRI typically comprises a hydrophobic alpha helix that spans at least a portion of the membrane, assisting the DN TGF-bRI in anchoring to the cell membrane and facilitating dimerization of the DN TGF-bRI construct. The transmembrane domain of the DN TGF-bRI can be designed such that no intracellular signal is transmitted via the DN TGF-bRI in the cell following TGF-b binding. As a non-limiting example, the transmembrane domain of the DN TGF-bRI can be derived from any other polypeptide having a transmembrane domain (including other transmembrane domains disclosed herein) that is expressed in an immune cell or a precursor cell thereof. In another embodiment, the transmembrane domain can be derived from a polypeptide that is naturally expressed or not naturally expressed in a T cell. It will be appreciated that the portion of the polypeptide comprising the polypeptide transmembrane domain can include additional sequences from the polypeptide, e.g., additional sequences adjacent to the N-terminus or C-terminus of the transmembrane domain, or other regions of the polypeptide (as desired). In one embodiment, the DN TGF-bRI can have a transmembrane domain derived from, as non-limiting examples, TGF-bRI, TGF-bRII, PDGFR, CD4, CD8, CD28, CD127, CD132, CD3 zeta, 4-IBB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, IL-5, IL-7, IL-7R alpha, BTLA, or a mutant thereof.In one embodiment described herein, the DN TGF-bRI comprises an extracellular ligand binding domain and a transmembrane domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to the amino acid sequence of SEQ ID NO: 5.

[0122]

[0123] The DN TGF-bRI constructs described herein can be used with any suitable intracellular domain or portion thereof, or not at all. In another embodiment described herein, the DN TGF-bRI comprises an extracellular domain fused to a transmembrane domain, which is further fused to an intracellular domain. The intracellular domain of wild-type TGF-bRI is where phosphorylation signaling occurs, which inhibits T cell function through pSMAD2 / 3 activation. Thus, in one embodiment, the DN TGF-bRI described herein comprises an intracellular domain truncated after amino acid 147 of SEQ ID NO: 2, such that the intracellular signaling of the cell is reduced or inhibited.

[0124] This truncation reduces the length of the receptor protein and results in a dominant negative effect that inhibits the phosphorylation cascade that would otherwise cause T cell inhibition. Thus, the DN TGF-bRI intracellular domain can be derived from any polypeptide that is naturally or non-naturally expressed in T cells, so long as the receptor is able to anchor in the membrane and prevent any intracellular signaling from occurring. As non-limiting examples, the intracellular domain can be derived from TGF-bRI, TGF-bRII, PDGFR, CD4, CD8, CD28, CD127, CD132, CD3 zeta, 4-IBB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, IL-5, IL-7, IL-7R alpha, BTLA, or mutants thereof.

[0125] In one embodiment described herein, the DN TGF-βRI can comprise the intracellular domain of SEQ ID NO: 6. RVNRQ (SEQ ID NO: 6). In some embodiments, the DN TGF-βRI comprises the extracellular ligand binding domain of TGF-βRI, the transmembrane domain of TGF-βRI, and the intracellular binding domain of SEQ ID NO: 6 having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to the amino acid sequence of SEQ ID NO: 7.

[0126]

[0127] The engineered DN TGF-βRI described herein can also comprise an N-terminal signal peptide at the N-terminus of the extracellular ligand binding domain of the DN TGF-βRI. In one embodiment, a heterologous signal peptide can be used. The extracellular domain of the DN TGF-βRI can be fused to a leader peptide or signal peptide that directs the nascent protein into the endoplasmic reticulum and subsequent translocation to the cell surface. It will be appreciated that once the signal peptide-containing polypeptide is expressed at the cell surface, the signal peptide is typically removed proteolytically during processing of the polypeptide in the endoplasmic reticulum and translocation to the cell surface. Thus, the polypeptide, such as the DN TGF-βRI, is typically expressed at the cell surface as a mature protein lacking the signal peptide, whereas the precursor form of the polypeptide includes the signal peptide. Any suitable signal sequence can be used. In one embodiment described herein, the DN TGF-βRI comprises an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 8 or a portion thereof. MEAAVAAPRPRLLLLVLAAAAAAAAALLPGATA (SEQ ID NO: 8).

[0128] A signal peptide or leader sequence can facilitate glycosylation of the DN TGF-βRI. A signal sequence or leader sequence is a peptide sequence that is often found at the N- or C-terminus of a newly synthesized protein that directs the newly synthesized protein into the secretory pathway. In the present disclosure, the signal peptide is joined to the N-terminus of the extracellular antigen binding domain of the DN TGF-βRI as a fusion protein. In one embodiment, the DN TGF-βRI comprises: an extracellular ligand binding domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to wild-type TGF-βRI; and a signal peptide having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to the amino acid sequence of SEQ ID NO: 9 at the N-terminus of the extracellular domain TGF-βRI.

[0129]

[0130] It will be appreciated that the use of this signal peptide is exemplary. Any suitable signal peptide known in the art can be applied to the DN TGF-βRI to provide cell surface expression in an immune cell. Useful signal peptides can be derived from any of the signal peptides of the polypeptides disclosed herein, including cell surface proteins that are naturally expressed in T cells or their precursor cells. Thus, any suitable signal peptide can be utilized to direct expression of the DN TGF-βRI at the cell surface of a T cell.

[0131] Accordingly, one embodiment described herein is an engineered DN TGF-βRI comprising: a signal peptide having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to wild-type TGF-βRI; an extracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to wild-type TGF-βRI; a transmembrane domain of TGF-βRI; and an intracellular domain of SEQ ID NO: 6 having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to the amino acid sequence of SEQ ID NO: 10.

[0132]

[0133] In addition to TGF-βRI, TGF-βRII is the second member of the TGF-β receptor complex. Unlike wild-type TGF-βRI, wild-type TGF-β receptor type II (TGF-βRII) is constitutively active. Upon TGF-β ligand binding and formation of a TGF-βRI dimer- / TGF-βRII dimer-complex, the cytoplasmic domain of TGF-βRII phosphorylates TGF-βRI. Thus, TGF-βRII is responsible for activating TGF-βRI and initiating the subsequent intracellular signaling cascade that produces pSMAD signaling. The full-length wild-type nucleic acid sequence of TGF-βRII is a sequence of 1701 nucleotides in length set forth as SEQ ID NO: 12.

[0134]

[0135]

[0136]

[0137] The full-length wild-type amino acid sequence of TGF-βRII is 567 amino acids set forth as SEQ ID NO: 13. The full-length TGF-βRII polypeptide of SEQ ID NO: 13 includes a signal peptide (approximately at amino acids 1-22), an extracellular domain (approximately at amino acids 33-170), a transmembrane domain (approximately at amino acids 171-201), and an intracellular domain (approximately at amino acids 202-567).

[0138]

[0139] Upon ligand binding, the intracellular domain of wild-type TGF-βRII is responsible for interacting with the intracellular domain of wild-type TGF-βRI, phosphorylation of four key threonine sites located between amino acids 185 to amino acids 204 of the intracellular domain of wild-type TGF-βRI, and initiation of pSMAD signaling. Thus, without being bound by any theory and as described herein, it is believed that the truncated DN TGF-βRI will not be able to interact with TGF-βRII and thus inhibit pSMAD signal transduction.

[0140] Similarly, because of its role in initiating the phosphorylation cascade of DNTGF-βRI, it is hypothesized that an engineered dominant negative TGF-βRII (DN TGF-βRII) can also inhibit pSMAD signaling by virtue of omitting the amino acid sequences responsible for initiating phosphorylation of the DN TGF-βRI, thus also inhibiting pSMAD signaling. The present disclosure contemplates any truncation or modification that results in inhibition or reduction of phosphorylation such that it results in a nonfunctional signaling pathway. In one embodiment, the DN TGF-βRII can be truncated or modified after the transmembrane domain such that the polypeptide lacks one or more amino acid residues responsible for signaling and phosphorylation and results in a nonfunctional signaling pathway as compared to the wild-type receptor. In some embodiments, the DN TGF-βRII can be truncated such that it does not have an intracellular domain. In other embodiments, the DN TGF-βRII can be modified to replace the intracellular domain with another naturally or non-naturally occurring sequence that does not contain amino acid residues involved in phosphorylation signaling. In another embodiment, the polypeptide is truncated or modified in a manner that reduces phosphorylation activity. In another embodiment, the polypeptide is truncated or modified in a manner that inhibits phosphorylation signaling molecules from interacting with pSMAD molecules. Thus, one embodiment described herein is a truncated DN TGF-βRII, wherein the polypeptide is truncated after the transmembrane domain and has at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to the amino acid sequence of SEQ ID NO: 14.

[0141]

[0142] In some embodiments described herein, the DN TGF-βRII comprises an extracellular ligand binding domain for binding TGF-β to wild-type TGF-βRII that has at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to the amino acid sequence of SEQ ID NO: 15.

[0143]

[0144] In some embodiments, the DN TGF-βRII extracellular ligand binding domain having at least 75% sequence identity (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 15 can be fused to one or more heterologous polypeptide sequences. The extracellular domain of the DN TGF-βRII can be engineered to recognize and bind to a target TGF-β molecule in order to initiate oligomerization of the DN TGF-βRII complex with wild-type TGF-βRI or with a DN TGF-βRI as described herein. Further, the present disclosure contemplates binding of the DN TGF-βRII to all TGF-β isoforms, including Transforming Growth Factor Beta Type I (TGF-β1), Transforming Growth Factor Beta Type II (TGF-β2), Transforming Growth Factor Beta Type III (TGF-β3), and Transforming Growth Factor Beta Type IV (TGF-β4). In some embodiments, the DN TGF-βRII can be engineered to bind TGF-β1, TGF-β2, TGF-β3, and / or TGF-β4. In another embodiment, the DN TGF-βRII binds TGF-β1.

[0145] In another embodiment described herein, the DN TGF-bRII comprises an extracellular binding domain fused to a transmembrane domain. The DN TGF-bRII can comprise a transmembrane domain comprising an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to a wild-type TGF-bRII transmembrane domain or a portion thereof. As a non-limiting example, the transmembrane domain can comprise an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 16, which includes a wild-type TGF-bRII transmembrane domain. LLLVIFQVTGISLLPPLGVAISVIIIFYCY (SEQ ID NO: 16). In other embodiments, the transmembrane domain is a heterologous transmembrane domain, including any of the various transmembrane domains described herein. The transmembrane domain of the DN TGF-bRII typically comprises a hydrophobic alpha helix that spans at least a portion of the membrane, assisting the DN TGF-bRII in anchoring to the membrane and facilitating dimerization of the DN TGF-bRII construct. The transmembrane domain of the DN TGF-bRII can be designed such that, upon TGF-b binding, no intracellular signal is transmitted to the TGF-bRI or DN TGF-bRI via the DN TGF-bRII in the cell. In one embodiment described herein, the DN TGF-bRII comprises an extracellular ligand binding domain and a transmembrane domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to the wild-type TGF-bRII shown in the amino acid sequence of SEQ ID NO: 17.

[0146]

[0147] Without being bound by any theory, it is hypothesized that the transmembrane domain of the DN TGF-βRII can affect the ability of the receptor to interact with wild-type TGF-βRI or DN TGF-βRI and, thus, inhibit pSMAD signaling. It is further hypothesized that changes in the transmembrane domain can also stimulate beneficial cytokine expression and signaling, while inhibiting pSMAD signaling. Thus, as a non-limiting example, the transmembrane domain of the DN TGF-βRII can be derived from any other polypeptide having a transmembrane domain, including other transmembrane domains disclosed herein, expressed in an immune cell or a precursor cell thereof. In another embodiment, the transmembrane domain can be derived from a polypeptide that is naturally expressed or not naturally expressed in a T cell. It will be appreciated that the portion of the polypeptide comprising the polypeptide transmembrane domain can include additional sequence from the polypeptide, e.g., additional sequence adjacent to the N- or C-terminus of the transmembrane domain, or other regions of the polypeptide, as desired. In one embodiment, the DN TGF-βRII can have a transmembrane domain derived from, as non-limiting examples, TGF-βRI, TGF-βRII, PDGFR, CD4, CD8, CD28, CD127, CD132, CD3ζ, 4-IBB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, IL-5, IL-7, IL-7Rα, BTLA, or mutants thereof.

[0148] In one embodiment described herein, the transmembrane domain can comprise at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 18, wherein SEQ ID NO: 18 comprises an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to a wild-type IL-7Ra transmembrane domain. PILLTISILSFFSVALLVIL (SEQ ID NO: 18). In another embodiment described herein, the DNTGF-bRII comprises: an extracellular ligand binding domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to a wild-type TGF-bRII; a linker sequence; and a transmembrane domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 18 and at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to the amino acid sequence of SEQ ID NO: 19.

[0149]

[0150] In another embodiment described herein, the transmembrane domain can comprise an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO:20, wherein SEQ ID NO:20 is a wild-type IL-7Ra transmembrane domain with an amino acid insertion “CPT”. PILLTCPTISILSFFSVALLVIL (SEQ ID NO:20). In another embodiment described herein, the DNTGF-bRII comprises: an extracellular ligand binding domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to wild-type TGF-bRII; a linker sequence; and a transmembrane domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO:20 and at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to the amino acid sequence set forth in SEQ ID NO:21.

[0151]

[0152] The DN TGF-bRII constructs described herein can be used alone or with any suitable intracellular domain or portion thereof, or without an intracellular domain, and can be truncated. In one embodiment described herein, the DN TGF-bRII constructs described herein comprise an intracellular domain having at least 75% sequence identity to SEQ ID NO: 6 (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%). By way of non-limiting example, the extracellular domain of the DN TGF-bRII constructs described herein can be derived from TGF-bRI, TGF-bRII, PDGFR, CD4, CD8, CD28, CD127, CD132, CD3 zeta, 4-IBB, OX40, ICOS, CTLA-4, PD-1, LAG-3, 2B4, IL-5, IL-7, IL-7R alpha, BTLA, or a mutant of any of the foregoing. In one embodiment described herein, the DN TGF-bRII comprises an extracellular domain fused to a transmembrane domain, which is further fused to an intracellular domain. In another embodiment described herein, the DN TGF-bRII described herein comprises the intracellular domain of wild-type IL-7R alpha having at least 75% sequence identity to SEQ ID NO: 22 (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%).

[0153]

[0154] In another embodiment, the DNTGF-bRII comprises: an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to the wild-type extracellular domain of TGF-bRII; a linker sequence; a transmembrane domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 18; and an intracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 22 and at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to the amino acid sequence of SEQ ID NO: 23.

[0155]

[0156] In another embodiment described herein, the DNTGF-bRII construct comprises: an extracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to wild-type TGF-bRII; a linker sequence; a transmembrane domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 20; and an intracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 22 and at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to the amino acid sequence of SEQ ID NO: 24.

[0157]

[0158] The engineered DN TGF-bRII constructs described herein can also comprise an N-terminal signal peptide at the N-terminus of the extracellular ligand-binding domain of TGF-bRII. In one embodiment, a heterologous signal peptide can be used. The extracellular domain of the DN TGF-bRII can be fused to a leader peptide or signal peptide that directs the nascent protein into the endoplasmic reticulum and subsequent translocation to the cell surface. It will be appreciated that once the signal peptide-containing polypeptide is expressed at the cell surface, the signal peptide is typically removed proteolytically during processing of the polypeptide in the endoplasmic reticulum and translocation to the cell surface. Thus, the polypeptide, such as the DN TGF-bRII, is typically found at the cell surface as a mature protein lacking the signal peptide, whereas the precursor form of the polypeptide includes the signal peptide. Any suitable signal sequence can be used. In one embodiment described herein, the DN TGF-bRII constructs described herein comprise a signal sequence having at least 75% sequence identity (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to the amino acid sequence of SEQ ID NO: 25 or a portion thereof. MGRGLLRGLWPLHIVLWTRIAS (SEQ ID NO: 25). In another embodiment, the signal sequence is derived from colony-stimulating factor 2 receptor alpha subunit (CSF2Ra) comprising an amino acid sequence having at least 75% sequence identity (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 26 or a portion thereof. MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 26). The signal sequences described herein can also optionally be used with any suitable protein tag, including but not limited to: V5 tag, myc tag, HA tag, Spot tag, NE tag. In one embodiment described herein, the signal sequence and tag comprise an amino acid sequence having at least 75% sequence identity (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 27. MLLLVTSLLLCELPHPAFLLIPEQKLISEEDL (SEQ ID NO: 27).

[0159] A signal peptide or leader sequence can facilitate glycosylation of the DN TGF-βRII. A signal sequence or leader sequence is a peptide sequence that is often found at the N- or C-terminus of a newly synthesized protein that directs the newly synthesized protein into the secretory pathway. In the present disclosure, a signal peptide is attached to the N-terminus of the extracellular antigen binding domain of the DN TGF-βRII as a fusion protein. In one embodiment, the DN TGF-βRII comprises: an extracellular ligand binding domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to wild-type TGF-βRII; and a signal peptide having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to the amino acid sequence of SEQ ID NO: 28 at the N-terminus of the extracellular domain.

[0160]

[0161] In one embodiment, the DN TGF-βRII comprises: an extracellular ligand binding domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to wild-type TGF-βRII; a signal peptide at the N-terminus of the extracellular domain TGF-βRII; and a tag sequence, and has at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 29.

[0162]

[0163] It will be appreciated that the use of this signal peptide is exemplary. Any suitable signal peptide known in the art can be applied to the DN TGF-βRII to provide cell surface expression in an immune cell. Useful signal peptides can be derived from any of the signal peptides of the polypeptides disclosed herein, including any of the signal peptides of the polypeptides disclosed herein that are naturally expressed in a T cell or a precursor cell thereof. Thus, any suitable signal peptide can be utilized to direct expression of the DN TGF-βRII at the cell surface of a T cell.

[0164] Accordingly, one embodiment described herein is an engineered DN TGF-βRII comprising: a signal peptide; an extracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to wild-type TGF-βRII; a linker sequence; a transmembrane domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 18; and an intracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 22, the DN TGF-βRII set forth as having an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID: 30.

[0165]

[0166] In another embodiment described herein is an engineered DN TGF-βRII comprising: a signal peptide; a tag sequence; an extracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to wild-type TGF-βRII; a linker sequence; a transmembrane domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 18; and an intracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 22, the DN TGF-βRII is shown by an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID: 31.

[0167]

[0168]

[0169] Another embodiment described herein is an engineered DN TGF-βRII comprising: a signal peptide; an extracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to wild-type TGF-βRII; a linker sequence; a transmembrane domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 18; and an intracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 22, the DN TGF-βRII set forth by an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID: 32.

[0170]

[0171]

[0172] Another embodiment described herein is an engineered DN TGF-βRII comprising: a signal peptide; a tag sequence; an extracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to wild-type TGF-βRII; a linker sequence; a transmembrane domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 20; and an intracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 22, the DN TGF-βRII being set forth by an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID: 33.

[0173]

[0174] Another embodiment described herein is an engineered DN TGF-βRII comprising: a signal peptide; an extracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to wild-type TGF-βRII; a linker sequence; a transmembrane domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 20; and an intracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 22, the DN TGF-βRII being shown by an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID: 34.

[0175]

[0176] Engineered T cell receptor (TCR) therapies and chimeric antigen receptor (CAR) therapies both leverage the specificity and immunotherapeutic effects of T cells for the treatment of a wide variety of malignancies. Some studies suggest that these therapies can be susceptible to suppressive factors caused by TGF-β in the TME that inhibit T cells (Bendle et al., J Immunol, 191:3232-3239 (2013), and Vong et al., Blood, 130:1791 (2017)). The present disclosure contemplates the use of the DN TGF-β receptors described herein in combination with TCR therapies or CAR therapies as a means to maintain or in some cases restore TCR and / or CAR expansion in the presence of TGF-β inhibition.

[0177] Chimeric antigen receptor (CAR) T cell therapy provides another method of treatment against tumor progression. Clinically, researchers have demonstrated that expansion and persistence of CARs correlates with therapeutic efficacy. Without being bound by any theory, it is believed that the population of T cells found in the TME that are inhibited by TGF-b can limit the expansion and persistence of CAR T cells in patients that do not respond to CAR therapy. It is believed that the resultant inhibitory cytokines in the TME limit the function and expansion of CAR cells. Thus, TGF-b can limit the efficacy of therapeutic engineered T cells.

[0178] Since TGF-b receptors are responsible for transmitting inhibitory pSMAD signaling within the cell, therapeutic strategies focused on modified TGF-bRI and TGF-bRII constructs have potential. Furthermore, the use of such constructs in existing or new TCR and CAR therapies can enhance the ability of these cell therapies to target cancer. Thus, described herein are dominant negative TGF-b receptors that can be used to modulate TGF-b signaling. In some aspects, the dominant negative TGF-b receptors can be co-expressed with TCR or CAR constructs to limit the inhibitory effects of TGF-b in the TME.

[0179] The present disclosure contemplates the use of the DN TGF-b receptor constructs described herein with engineered T cell receptors (TCRs) in T cell immunotherapy. TCR libraries can be screened for selectivity to a target antigen. In this way, native TCRs with high avidity and reactivity to a target antigen can be selected, cloned, and subsequently introduced into a population of T cells for adoptive immunotherapy. T cells with engineered TCRs that also express the DN TGF-b receptor constructs described herein will be able to target a specific antigen not only due to TCR specificity, but will also be protected from the inhibitory effects of TGF-b. Thus, the combination of TCRs with the DN TGF-b receptor constructs described herein can provide a means to maintain the therapeutic effects of adoptive T cell immunotherapy. In one embodiment described herein, the DN TGF-B receptors described herein are co-expressed with TCRs.

[0180] In one embodiment described herein, a T cell is modified by the introduction of a polynucleotide encoding a TCR subunit, which can form a TCR conferring specificity of the T cell to tumor cells expressing a target antigen. In some embodiments, the subunit has one or more amino acid substitutions, deletions, insertions, or modifications compared to the naturally occurring subunit, so long as the subunit retains the ability to form a TCR (which confers the ability of the transfected T cell to home to target cells) and participate in signaling of immune-related cytokines. The engineered TCR can also bind to target cells displaying a relevant tumor-associated peptide with high avidity, and optionally mediate efficient killing of target cells presenting the relevant peptide in vivo.

[0181] A nucleic acid encoding an engineered TCR can be isolated from its natural context in the chromosome of a T cell (naturally occurring), and can be incorporated into a suitable vector as described elsewhere herein. Both the nucleic acid and the vector comprising the nucleic acid can be transferred into a cell, which can be a T cell. The modified T cell is then capable of expressing one or more chains (and in some aspects, both chains) of the TCR encoded by the transduced nucleic acid(s). In some embodiments, the engineered TCR is exogenous, as it is introduced into a T cell that does not normally express the introduced TCR. A key aspect of the engineered TCR is that it has high avidity for a tumor antigen presented by a major histocompatibility complex (MHC) or similar immune component. In contrast to engineered TCRs, CARs are engineered to bind to target antigens in a MHC-independent manner.

[0182] A protein encoded by a nucleic acid described herein can be expressed with an additional polypeptide attached to the amino-terminal portion or carboxy-terminal portion of the alpha chain or beta chain of the TCR, so long as the attached additional polypeptide does not hinder the ability of the alpha chain or beta chain to form a functional T cell receptor and MHC-dependent antigen recognition.

[0183] Antigens recognized by engineered TCRs contemplated herein include, but are not limited to, cancer antigens, including those for both hematological cancers and solid tumors, as well as virus-induced cancers. TCR therapy for the treatment of HPV-induced cervical epithelial cancers is a promising area of interest. The oncolytic proteins HPV-16 E6 and HPV-16 E7 can thus be potential target antigens for use with TCRs.

[0184] Other illustrative antigens include, but are not limited to, HPV oncoproteins (including HPV-16 E6 and HPV-16 E7), alpha folate receptor, 5T4, alpha vBeta 6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137 (4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, EGFR, EGFR family (including ErbB2 (HERII), EGFRvIII), EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, Fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-Al+MAGEI, HLA-A2+MAGE1, HLA A3+MAGE1, HLA-Al+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-11R alpha, IL-13R alpha 2, Lambda, Lewis-Y, Kappa, Mesothelin, Mucl, Muc16, NCAM, NKG2D ligand, NY-ES0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TAG72, TEM, and VEGFRII.

[0185] Combining any of the TCR constructs as described herein with the DNTGF-β receptor of the present disclosure can restore, maintain, or enhance the therapeutic effect of TCR therapy challenged by TGF-β inhibition. Thus, in one embodiment described herein, the DN TGF-β receptor is co-expressed in a T cell with a TCR against HPV. In another embodiment described herein, the DN TGF-β receptor is co-expressed in a T cell with a TCR against HPV-16 E6 protein. In another embodiment described herein, the DN TGF-β receptor is co-expressed in a T cell with a TCR against HPV-16 E7 protein.

[0186] T cells can also be genetically engineered with vectors designed to express CARs that redirect cytotoxicity to tumor cells. CARs are molecules that combine antibody-based specificity for a target antigen (e.g., a tumor antigen) with T cell receptor-activating intracellular domains to create a chimeric protein that exhibits specific anti-tumor cell immune activity. As used herein, the term “chimeric” describes something composed of parts of different proteins or DNA from different sources. The present disclosure contemplates the use of the DN TGF-beta receptor described herein with a chimeric antigen receptor (CAR). As with the use of TCRs, co-expression of the DN TGF-beta receptor with a CAR can facilitate CAR expansion and protect CAR therapy from the challenge of TGF-beta inhibition (in some cases restore the challenge of TGF-beta inhibition to CAR therapy). In one embodiment described herein, the DN TGF-beta receptor is co-expressed with a CAR described herein.

[0187] CARs contemplated herein comprise an extracellular domain that binds to a particular target antigen (also referred to as a binding domain or antigen-specific binding domain); a transmembrane domain; and an intracellular signaling domain. A key feature of CARs is their ability to redirect immune effector cell specificity, triggering proliferation, cytokine production, phagocytosis, or production of molecules that can mediate cell death of cells expressing the target antigen in a major histocompatibility (MHC)-independent manner, exploiting the cell-specific targeting ability of monoclonal antibodies, soluble ligands, or cell-specific co-receptors.

[0188] In some embodiments, the CAR comprises an extracellular binding domain, including but not limited to an antibody or antigen-binding fragment thereof that specifically binds to a target antigen, a tethered ligand, or an extracellular domain of a co-receptor.

[0189] As non-limiting examples, target antigens can include: HPV oncoproteins (including HPV-16 E6 and HPV-16 E7), alpha folate receptor, 5T4, alpha vB7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137 (4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, EGFR, EGFR family (including ErbB2 (HERII), EGFRvIII), EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, Fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-Al+MAGEI, HLA-A2+MAGE1, HLA A3+MAGE1, HLA-Al+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-llR a, IL-13R a2, Lambda, Lewis-Y, Kappa, Mesothelin, Mucl, Muc16, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TAG72, TEM, and VEGFRII; one or more hinge domains or spacer domains; a transmembrane domain, including but not limited to a transmembrane domain from CD8a, CD4, CD45, PD-1, and CD152; one or more intracellular costimulatory signaling domains, including but not limited to intracellular costimulatory signaling domains from CD28, CD54 (ICAM), CD134 (OX40), CD137 (41BB), CD152 (CTLA4), CD273 (PD-L2), CD274 (PD-L1), and CD278 (ICOS); and a primary signaling domain from CD3 zeta or FcR gamma. In one embodiment described herein, the CAR binds to a tumor antigen comprising CLL-1, CD19, CD20, CD28, CD137 (4-1BB), Glypican-3 (GPC3), PSCA, or PSMA.

[0190] The hinge can be of natural or synthetic origin. In some embodiments, the antigen binding systems of the present disclosure can comprise a hinge that is, is from, or is derived from (e.g., comprises all or a fragment of) CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8.alpha., CD8.beta., CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B-cell antigen receptor complex-associated alpha chain), CD79B (B-cell antigen receptor complex-associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), Inducible T-cell costimulator (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10,ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA1-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activating NK cell receptor, or Toll ligand receptor, or fragments or combinations thereof. In certain embodiments, the CAR does not comprise a CD28 hinge.

[0191] The transmembrane domain can be of natural or synthetic origin. Where the origin is natural, the domain can be derived from any membrane-bound or transmembrane protein.An exemplary transmembrane domain can be derived from (e.g., can include at least the transmembrane domain of) an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45, CD4, CD5, CD7, CD8, CD8 alpha, CD8 beta, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB / CD137, an activating NK cell receptor, an immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CDS, CEACAM1, CRT AM, a cytokine receptor, DAP-10, DNAM1 (CD226), Fcgamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, Inducible T cell costimulator (ICOS), an integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, a ligand that binds with CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), Lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, Programmed death-1 (PD-1), PSGL1, SELPLG (CD162), a signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, a TNF receptor protein, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or combination thereof. In some embodiments, the transmembrane domain can be synthetic (and may, for example, include predominantly hydrophobic residues, such as leucines and valines).In some embodiments, a triad of phenylalanine, tryptophan, and valine is included at each end of the synthetic transmembrane domain. In some embodiments, the transmembrane domain is directly linked or connected to the cytoplasmic domain. In some embodiments, a short oligopeptide or polypeptide linker (e.g., between 2 and 10 amino acids in length) can form the connection between the transmembrane domain and the intracellular domain. In some embodiments, the linker is a glycine-serine dipeptide.

[0192] In some embodiments, the signaling domain and / or activation domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAMs containing cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d (see, e.g., Love et al., Cold Spring Harb. Perspect. Biol. 2: a002485 (2010); Smith-Garvin et al., Annu. Rev. Immunol. 27: 591-619 (2009)).

[0193] In certain embodiments, suitable signaling domains include, but are not limited to: 4-1BB / CD137, Activating NK cell receptor, Immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, Cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, Inducible T cell costimulator (ICOS), Integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, Ligand that binds to CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), Ly108, Lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, Programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6, or fragments, truncations, or combinations thereof.

[0194] A CAR can include a costimulatory signaling domain, for example, to increase signaling efficacy. See U.S. Patent Nos. 7,741,465 and 6,319,494, as well as Krause et al. and Finney et al. (Id.), Song et al., Blood 119:696-706 (2012); Kalos et al., Sci Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016). Signals generated by the TCR alone can be insufficient to fully activate a T cell, and a secondary or costimulatory signal can increase activation. Thus, in some embodiments, the signaling domain further includes one or more additional signaling domains (e.g., a costimulatory signaling domain) that activates one or more immune cell effector functions (e.g., a native immune cell effector function described herein). In some embodiments, a portion of such a costimulatory signaling domain can be used so long as the portion transduces an effector function signal. In some embodiments, the cytoplasmic domain described herein comprises one or more cytoplasmic sequences of a T cell co-receptor (or fragment thereof). Non-limiting examples of such T cell co-receptors include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), MYD88, CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that binds with CD83.

[0195] In some embodiments, a CAR contemplated herein comprises an extracellular binding domain that specifically binds to a target polypeptide (e.g., a target antigen) expressed on a tumor cell. As used herein, the terms “binding domain,” “extracellular domain,” “extracellular binding domain,” “antigen-specific binding domain,” “antigen binding domain,” and “extracellular antigen-specific binding domain” are used interchangeably and provide for a CAR with the ability to specifically bind a target antigen of interest. A binding domain can include any protein, polypeptide, oligopeptide, or peptide that has the ability to specifically recognize and bind a biomolecule (e.g., a cell surface receptor or tumor protein, lipid, polysaccharide, or other cell surface target molecule, or component thereof). Binding domains include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a biomolecule of interest.

[0196] In some embodiments, the extracellular binding domain of the CAR comprises an antibody or antigen-binding fragment thereof. An "antibody" refers to a binding agent that is a polypeptide comprising at least a light or heavy chain immunoglobulin variable region that specifically recognizes and binds an epitope of a target antigen, such as a peptide, lipid, polysaccharide, or nucleic acid containing an antigenic determinant, such as those recognized by immune cells. Antibodies include antigen-binding fragments thereof. The term also includes genetically engineered forms, such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (such as bispecific antibodies), and antigen-binding fragments thereof. See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rded., W.H. Freeman & Co., New York, 1997.

[0197] In some embodiments, the target antigen is an epitope of HPV oncoproteins (including HPV-16 E6 and HPV-16 E7), alpha folate receptor, 5T4, alpha v integrin beta 6, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137 (4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, EGFR, EGFR family (including ErbB2 (HERII), EGFRvIII), EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-Al+MAGEI, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-Al+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-llR alpha, IL-13R alpha 2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TAG72, TEM, and VEGFRII polypeptides. In one embodiment described herein, the CAR binds to a tumor antigen epitope comprising CD19, CD20, CD28, CD137 (4-1BB), CLL-1, Glypican-3 (GPC3), PSCA, or PSMA.

[0198] Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide (El-Serag, J Clin Gastroenterology 35:S72-78 (2002)). Unlike any other tumor antigen associated with hepatocellular carcinoma to date, GPC3 is a glycosylphosphatidylinositol-linked membrane-associated protein with a large extracellular domain that is attractive for antibody-directed therapy. Thus, the large extracellular domain and the relatively specific expression of GPC3 on the cell surface of malignant HCC tissue make it an attractive target for HCC tumor immunotherapy. Accordingly, one embodiment contemplated by the present disclosure is the use of a CAR directed against GPC3 together with a DN TGF-beta receptor as described herein.

[0199] In one embodiment described herein, the CAR comprises an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 37.

[0200]

[0201] A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain and in either order (e.g., VL-VH or VH-VL). Generally, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For a review of scFv see, e.g., Pluckthin, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315. In one embodiment described herein, the CAR described herein comprises a VH domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 38.

[0202]

[0203] In another embodiment described herein, the CAR described herein comprises a VL domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 39.

[0204]

[0205] In some embodiments, the CARs contemplated herein comprise an antigen- specific binding domain that can be a scFv (murine scFv, human scFv, or humanized scFv) that binds to an antigen expressed on a cancer cell. In certain embodiments, the scFv binds to: HPV oncoproteins (including HPV-16 E6 and HPV-16 E7), alpha folate receptor, 5T4, alpha v B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137 (4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, EGFR, EGFR family (including ErbB2 (HERII), EGFRvIII), EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-Al+MAGEI, HLA-A2+MAGE1, HLA A3+MAGE1, HLA-Al+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-11R alpha, IL-13R alpha 2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D ligand, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TAG72, TEM, and VEGFRII. In other embodiments described herein, the CAR comprises an antigen-specific binding domain scFv that binds to CD19, CD20, CD28, CD137 (4-1BB), CLL-1, Glypican-3 (GPC3), PSCA, or PSMA.

[0206] In certain embodiments, the CARs contemplated herein can comprise linker residues between various domains (e.g., between a VH domain and a VL domain) that are added for obtaining the proper spacing conformation of the molecule. The CARs contemplated herein can comprise one, two, three, four, or five or more linkers. In some embodiments, the linkers are about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids in length, or any intermediate length of amino acids. In some embodiments, the linkers are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more amino acids in length.

[0207] Illustrative examples of linkers include glycine polymers (G)n; glycine-serine polymers (G 1-5 S 1-5 )n, where n is an integer of at least 1, 2, 3, 4, or 5; glycine-alanine polymers; alanine-serine polymers; and other flexible linkers known in the art. Glycine and glycine-serine polymers are relatively unstructured, and thus can be able to act as neutral tethers between domains of a fusion protein, such as the CARs described herein. Glycine enters even more phi-psi space than alanine, and is subject to much less constraint than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11 173-142 (1992)). Other linkers contemplated herein include Whitlow linkers (see Whitlow, Protein Eng. 6(8):989-95 (1993)). One of ordinary skill will recognize that, in some embodiments, the design of a CAR can include all or a portion of a flexible linker, such that the linker can include a flexible linker as well as one or more portions that impart a less flexible structure, to provide a desired structure of the CAR. In one embodiment, any of the constructs described herein can comprise a “GS” linker. In another embodiment, any of the constructs described herein comprise a “GSG” linker. In another embodiment, the CARs described herein comprise an amino acid sequence having at least 75% sequence identity (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO:40. GSTSGSGKPGSGEGSTKG (SEQ ID NO:40).

[0208] In other embodiments, the CAR comprises an scFv that further comprises a variable region connecting sequence. A "variable region connecting sequence" is an amino acid sequence that connects the heavy chain variable region to the light chain variable region and provides spacer function compatible with the interaction of the two sub-binding domains, such that the resulting polypeptide retains specific binding affinity for the same target molecule as an antibody comprising the same light chain variable region and heavy chain variable region. In one embodiment, the variable region connecting sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids in length.

[0209] In other embodiments, the binding domain of the CAR is followed by one or more "spacer domains," which refers to a region that moves the antigen binding domain away from the surface of the effector cell to achieve proper cell / cell contact, antigen binding, and activation (Patel et al., Gene Therapy, 1999; 6:412-419). The spacer domain can be derived from natural sources, synthetic sources, semi-synthetic sources, or recombinant sources. In certain embodiments, the spacer domain is part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain can comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

[0210] The binding domain of the CAR can typically be followed by one or more "hinge domains," which function in positioning the antigen binding domain away from the surface of the effector cell to achieve proper cell / cell contact, antigen binding, and activation. The CAR typically comprises one or more hinge domains between the binding domain and the transmembrane domain. The hinge domain can be derived from natural sources, synthetic sources, semi-synthetic sources, or recombinant sources. The hinge domain can comprise the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

[0211] Illustrative hinge domains suitable for use in the CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8a, CD4, CD28, and CD7, which can be wild-type hinge regions from these molecules or can be altered, e.g., a truncated CD28 hinge domain. The hinge can be of natural or synthetic origin. In some embodiments, the antigen binding systems of the present disclosure can comprise a hinge that is, is from, or is derived from (e.g., comprises all or a fragment of) CD2, CD3d, CD3e, CD3g, CD4, CD7, CD8.a., CD8.b., CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B-cell antigen receptor complex-associated a chain), CD79B (B-cell antigen receptor complex-associated b chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30),CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell costimulator (ICOS), LFA-1 (CD1 la / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2Rγ, IL-7Rα, LFA1-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activating NK cell receptor, or Toll ligand receptor, or fragments or combinations thereof. In certain embodiments, the CAR does not comprise a CD28 hinge. In another embodiment, the hinge domain comprises a CD8a hinge region. In one embodiment, the CAR described herein comprises a hinge domain from CD8a having an amino acid sequence with at least 75% sequence identity (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 41. TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 41).

[0212] A "transmembrane domain" is the portion of a CAR that fuses the extracellular binding moiety to the intracellular signaling domain and anchors the CAR to the plasma membrane of the immune effector cell.An exemplary transmembrane domain can be derived from (e.g., can include at least the transmembrane domain of) an alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45, CD4, CD5, CD7, CD8, CD8 alpha, CD8 beta, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB / CD137, an activating NK cell receptor, an immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CDS, CEACAM1, CRT AM, a cytokine receptor, DAP-10, DNAM1 (CD226), Fcgamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, Inducible T cell costimulator (ICOS), an integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, a ligand that binds with CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), Lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, Programmed death-1 (PD-1), PSGL1, SELPLG (CD162), a signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Ly108), SLAMF7, SLP-76, a TNF receptor protein, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or combination thereof. In some embodiments, the transmembrane domain can be synthetic (and may, for example, include predominantly hydrophobic residues, such as leucines and valines).In some embodiments, a triad of phenylalanine, tryptophan, and valine is included at each end of the synthetic transmembrane domain. In some embodiments, the transmembrane domain is directly linked or connected to the cytoplasmic domain. In some embodiments, a short oligo- or polypeptide linker (e.g., between 2 and 10 amino acids in length) can form the connection between the transmembrane domain and the intracellular domain. In some embodiments, the linker is a glycine-serine dipeptide. In one embodiment, the CAR described herein comprises a transmembrane domain from CD8a having an amino acid sequence with at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 42. IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 42).

[0213] In some embodiments, the CARs contemplated herein comprise an intracellular signaling domain. An "intracellular signaling domain" refers to the portion of the CAR that participates in transducing the message of an effective CAR binding to a target antigen into the interior of the immune effector cell to initiate effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including release of cytotoxic factors to CAR-bound target cells, or other cellular responses elicited with antigen binding to the extracellular CAR domain. In some embodiments, the signaling domain and / or activation domain comprises an immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAMs containing cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d (see, e.g., Love et al., Cold Spring Harb. Perspect. Biol. 2: a002485 (2010); Smith-Garvin et al., Annu. Rev. Immunol. 27: 591-619 (2009)).In certain embodiments, suitable signaling domains include, but are not limited to: 4-1BB / CD137, Activating NK cell receptor, Immunoglobulin, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CDS, CEACAM1, CRT AM, Cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, Inducible T cell costimulator (ICOS), Integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, Ligand that binds to CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), Ly108, Lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, Programmed death-1 (PD-1), PSGL1, SELPLG (CD162), Signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6, or fragments, truncations, or combinations thereof.

[0214] The term "effector function" refers to a specialized function of a cell. For example, an effector function of a T cell can be cytolytic activity or activities that contribute to activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function. While it is possible to employ the entire intracellular signaling domain, in many instances it is not necessary to use the entire domain. Where a truncated portion of an intracellular signaling domain is used, such truncated portion can be used in place of the entire domain so long as it transduces the effector function signal. The term "intracellular signaling domain" is intended to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[0215] It is known that signals generated through the TCR alone are insufficient to fully activate a T cell, and that a secondary or costimulatory signal can also be required. Thus, T cell activation can be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., TCR / CD3 complex), and costimulatory signaling domains that act in an antigen-independent manner to provide a secondary or costimulatory signal. In some embodiments, the CARs contemplated herein comprise an intracellular signaling domain that includes one or more "costimulatory signaling domains" and a "primary signaling domain."

[0216] Illustrative examples of ITAM-containing primary signaling domains useful in the present disclosure include those derived from TCR zeta, FcR gamma, FcR beta, DAP12, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD22, CD79a, CD79b, and CD66d. In some embodiments, the CAR comprises a CD3 zeta primary signaling domain and one or more costimulatory signaling domains. The primary signaling domain and costimulatory signaling domains intracellularly can be linked in any serial order to the carboxy-terminal end of the transmembrane domain. In one embodiment, the CAR has a CD3 zeta domain having an amino acid sequence with at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 43.

[0217]

[0218] The CARs contemplated herein comprise one or more costimulatory signaling domains to enhance the efficacy and expansion of T cells expressing the CAR receptor. As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to an intracellular signaling domain of a costimulatory molecule. In some embodiments, the costimulatory molecule can include CD27, CD28, CD137 (4-IBB), OX40 (CD134), CD30, CD40, PD-I, ICOS (CD278), CTLA4, LFA-1, CD2, CD7, LIGHT, TRIM, LCK3, SLAM, DAPIO, LAG3, HVEM, NKD2C, and CD83. In one embodiment, the CARs described herein comprise a 4-IBB costimulatory domain having an amino acid sequence with at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO:44. KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE (SEQ ID NO:44).

[0219] The engineered CARs described herein can also comprise an N-terminal signal peptide or tag at the N-terminus of the scFv or antigen binding domain. In one embodiment, a heterologous signal peptide can be used. The antigen binding domain or scFV can be fused to a leader peptide or signal peptide that directs the nascent protein into the endoplasmic reticulum and subsequent translocation to the cell surface. It will be appreciated that once the polypeptide containing the signal peptide is expressed at the cell surface, the signal peptide is typically removed proteolytically during processing of the polypeptide in the endoplasmic reticulum and translocation to the cell surface. Thus, the polypeptide, such as the CAR constructs described herein, is typically expressed at the cell surface as a mature protein lacking the signal peptide, while the precursor form of the polypeptide includes the signal peptide. Any suitable signal sequence known in the art can be used. Similarly, any known tag sequence known in the art can also be used.

[0220] In one embodiment described herein, the CAR construct has an amino acid sequence with at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO:45.

[0221]

[0222] Combining any of the CAR constructs as described herein with any of the DN TGF-β receptors of the present disclosure can restore, maintain, or enhance the therapeutic effect of CAR T therapy challenged by TGF-β inhibition. Thus, in one embodiment described herein, a DN TGF-β receptor described herein is co-expressed in a T cell with a CAR, forming a “CAR-DN TGF-β receptor construct.” In another embodiment described herein, any one of the DN TGF-β receptors is co-expressed in a T cell with a CAR that binds to an antigen including, but not limited to, BCMA, CD19, CD20, CD28, CD137 (4-1BB), CLL-1, Glypican-3 (GPC3), PSCA, or PSMA. In another embodiment described herein, the CAR portion of the CAR-DN TGF-β receptor construct comprises an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 37 or SEQ ID NO: 45. In another embodiment described herein, the DN TGF-β receptor portion of the CAR-DN TGF-β receptor construct comprises an amino acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 10 or SEQ ID NO: 14.

[0223] To facilitate efficient expression of the CAR-DN TGF-β receptor construct in T cells as described herein, a self-cleaving sequence can be used to achieve co-expression of multiple gene constructs in equal amounts by expressing the multiple gene constructs in one open reading frame. Any suitable cleavage peptide sequence can be used with the constructs described herein, for example, a 2A cleavable peptide (e.g., T2A), a 2A-like linker, or a functional equivalent thereof and combinations thereof. In some embodiments, the linker comprises a picornavirus 2A-like linker, the CHYSEL sequence of the porcine teschovirus-1 (P2A), the foot-and-mouth disease virus (F2A), the Theilovirus (T2A), or combinations, variants, and functional equivalents thereof. In one embodiment described herein, the CAR-DN TGF-β receptor construct comprises a self-cleaving domain having an amino acid sequence with at least 75% sequence identity to SEQ ID NO: 46 (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%). EGRGSLLTCGDVEENPGP (SEQ ID NO: 46).

[0224] In one embodiment, the CAR-DN TGF-β receptor construct described herein comprises an amino acid sequence with at least 75% sequence identity to SEQ ID NO: 47 (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%).

[0225]

[0226] In one embodiment described herein, a polynucleotide encoding one or more of the DN TGF-b receptors or CAR-DN TGF-b receptor constructs polypeptides contemplated herein is provided. As used herein, the term "polynucleotide" or "nucleic acid" refers to messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive-strand RNA (RNA(+)), negative-strand RNA (RNA(-)), complementary DNA (cDNA), or recombinant DNA. Polynucleotides include single- and double-stranded polynucleotides. Polynucleotides of the present disclosure include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein (see, e.g., the Sequence Listing), typically wherein the variant retains at least one biological activity of the reference sequence. In various illustrative embodiments, the present disclosure contemplates expression vectors, viral vectors, and transfer plasmids comprising the polynucleotides, as well as compositions and cells comprising the same.

[0227] In some embodiments, provided by the present disclosure are polynucleotides encoding at least about 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 1000, 1250, 1500, 1750, or 2000 or more contiguous amino acid residues of the polypeptides of the present disclosure, as well as all intermediate lengths. It will be readily understood that "intermediate lengths" in this context means any length between the recited values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.

[0228] As used herein, the terms "polynucleotide variant" and "variant" and the like refer to a polynucleotide that exhibits substantial sequence identity to a reference polynucleotide sequence, or that hybridizes to a reference sequence under stringent conditions defined below. These terms include polynucleotides in which one or more nucleotides have been added or deleted, or in which one or more nucleotides have been replaced with a different nucleotide, as compared to a reference polynucleotide. In this regard, it is well known in the art that certain alterations can be made to a reference polynucleotide, including mutations, additions, deletions, and substitutions, whereby the altered polynucleotide expressed retains the biological function or activity of the reference polynucleotide.

[0229] As used herein, the expression "sequence identity" or for example includes "having 50% sequence identity" refers to the extent the sequence is identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a comparison window. Thus, a "percentage of sequence identity" can be calculated by comparing two optimally aligned sequences over the comparison window, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys, and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Nucleotides and polypeptides having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein are contemplated, typically wherein the polypeptide variant retains at least one biological activity of the reference polypeptide.

[0230] Regardless of the length of the coding sequence itself, the polynucleotides described herein can be combined with other DNA sequences such as promoters and / or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as disclosed elsewhere herein or as known in the art, such that their total length can vary significantly. Thus, it is contemplated that polynucleotide fragments of almost any length can be employed, where the total length can be limited by ease of preparation and use in the intended recombinant DNA protocol.

[0231] Polynucleotides can be prepared, manipulated and / or expressed using any of a variety of techniques known and available in the art. To express a desired polypeptide, a nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector. Examples of vectors are plasmids, autonomously replicating sequences, and transposable elements. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1 -derived artificial chromosomes (PAC)), bacteriophages (such as lambda phage or M13 phage), and animal viruses. Examples of classes of animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors are pClneo vectors (Promega) for expression in mammalian cells; pLenti4N5-DEST TM , pLenti6N5-DEST TM , and pLenti6.2N5-GW / lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In some embodiments, the coding sequence of a chimeric protein disclosed herein can be ligated into such an expression vector for expression of the chimeric protein in mammalian cells.

[0232] “Control elements” or “regulatory sequences” present in expression vectors are non-translated regions of the vector which are involved in promoting, either in cis or in trans, transcription and translation of proteins encoded by the vector. These include, but are not limited to, promoter sequences, enhancer sequences, transcription termination sequences, ribosome binding sites, and the like. Examples include those of a variety of promoters known and available in the art, including constitutive, inducible, and / or repressible promoters. Constitutive promoters are promoters that are active throughout the cell cycle. Inducible promoters are promoters that are active in response to an external signal, such as a chemical or physical stimulus. Repressible promoters are promoters that are active in response to an external signal, such as a chemical or physical stimulus. Examples of inducible promoters include, but are not limited to, the tetracycline-inducible promoter, the copper-inducible promoter, the cadmium-inducible promoter, the ethanol-inducible promoter, the heat-inducible promoter, the light-inducible promoter, the glucose-inducible promoter, the methanol-inducible promoter, the methionine-inducible promoter, the osmotic-inducible promoter, the phosphate-inducible promoter, the temperature-inducible promoter, the vitamin-inducible promoter, and the like. Examples of repressible promoters include, but are not limited to, the tetracycline-repressible promoter, the copper-repressible promoter, the cadmium-repressible promoter, the ethanol-repressible promoter, the heat-repressible promoter, the light-repressible promoter, the glucose-repressible promoter, the methanol-repressible promoter, the methionine-repressible promoter, the osmotic-repressible promoter, the phosphate-repressible promoter, the temperature-repressible promoter, the vitamin-repressible promoter, and the like.

[0233] In other embodiments, vectors for practicing the embodiments described herein include, but are not limited to, expression vectors and viral vectors, which will contain exogenous, endogenous, or heterologous sequences, such as promoters and / or enhancers. “Endogenous” control sequences are control sequences that are naturally linked to a given gene in the genome. “Exogenous” control sequences are control sequences that are placed in juxtaposition to a gene by way of genetic manipulation (i.e., molecular biology techniques) such that transcription of the gene is directed by the linked enhancer / promoter. “Heterologous” sequences are exogenous sequences that can be from a different protein of the same species or a different species than the protein or cell being genetically manipulated.

[0234] As used herein, the term "promoter" refers to a recognition site of a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes the polynucleotide operably linked to the promoter. In some embodiments, a promoter that functions in mammalian cells comprises an AT-rich region located approximately 25 to 30 bases upstream from the transcription initiation site, and / or another sequence, the CNCAAT region (where N can be any nucleotide), found 70 to 80 bases upstream from the transcription initiation site.

[0235] The term "enhancer" refers to a segment of DNA that contains sequences that are capable of providing increased transcription and, in some cases, can function independently of its orientation relative to another control sequence. Enhancers can act synergistically or additively with promoters and / or other enhancer elements. The term "promoter / enhancer" refers to a segment of DNA that contains sequences that are capable of providing both promoter and enhancer functions.

[0236] The term "operably linked" refers to a juxtaposition wherein the components are in a relationship permitting them to function in their intended manner. In one embodiment, this term refers to a functional linkage between an expression control sequence (such as a promoter and / or enhancer) and a second polynucleotide sequence (e.g., a polynucleotide of interest), wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.

[0237] As used herein, the term "constitutive expression control sequence" refers to a promoter, enhancer, or promoter / enhancer that allows for the transcription of an operably linked sequence constantly or continuously. A constitutive expression control sequence can be a "ubiquitous" promoter, enhancer, or promoter / enhancer that allows for expression in a wide variety of cell types and tissue types, or a "cell-specific," "cell type-specific," "cell lineage-specific," or "tissue-specific" promoter, enhancer, or promoter / enhancer that allows for expression in a limited variety of cell types and tissue types, respectively. The present disclosure contemplates the use of any suitable cell line with the various constructs described herein, including but not limited to: CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Flp-In®- cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, Jurkat cells, VERO cells, W138 cells, MRC 5 cells, A549 cells, HTI080 cells, Hep cells, HEK cells, iHPC cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, NK cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells, or any other suitable cell line.

[0238] Illustrative ubiquitous expression control sequences suitable for use in some embodiments of the present disclosure include, but are not limited to: cytomegalovirus (CMV) immediate early promoter, viral simian virus 40 (SV40) (e.g., early or late), Moloney murine leukemia virus (MoMLV) LTR promoter, Rous sarcoma virus (RSV) LTR, herpes simplex virus (HSV) (thymidine kinase) promoter, HS, P7.5, and Pll promoters from vaccinia virus, elongation factor 1-alpha (EF1a) promoter, early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4Al (EIF4Al), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa beta member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), beta-kinesin (beta-KIN), human ROSA 26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase-I (PGK) promoter, cytomegalovirus enhancer / chicken beta-actin (CAG) promoter, beta-actin promoter, and myeloproliferative sarcoma virus enhancer, negative control region deleted dl587rev primer binding site substituted (MND) promoter (Challita et al., J Viral. 69(2):748-55 (1995)).

[0239] In another embodiment described herein, it can be desirable to co-express a polynucleotide comprising a DN TGF-beta receptor described herein with an engineered TCR or CAR to redirect T cells to cells expressing a target antigen from a promoter that provides stable and long-term expression in T cells at sufficient levels.

[0240] As used herein, "conditionally expressed" can refer to any type of conditional expression, including but not limited to: inducible expression; repressible expression; expression in a cell or tissue having a physiological, biological, or disease state, and the like. This definition is not intended to exclude cell type or tissue specific expression. Certain embodiments described herein provide for conditional expression of a polynucleotide of interest, e.g., by subjecting a cell, tissue, organism, etc. to a treatment or condition that causes expression of the polynucleotide or causes an increase or decrease in expression of a polynucleotide encoded by the polynucleotide of interest.

[0241] Illustrative examples of inducible promoters / systems include, but are not limited to: steroid-inducible promoters, such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone); metallothionein promoters (inducible by treatment with various heavy metals); MX-1 promoters (inducible by interferons); the "GeneSwitch" mifepristone regulatable system (Sirin et al., 2003, Gene, 323:67), the cumate inducible gene switch (WO 2002 / 088346), the tetracycline-dependent regulation system, and the like.

[0242] Conditional expression can also be achieved using site-specific DNA recombinases. According to certain embodiments of the present disclosure, the vector comprises at least one (typically two) recombination sites mediated by a site-specific recombinase. As used herein, the term "recombinase" or "site-specific recombinase" includes excision or integration proteins, enzymes, cofactors, or related proteins involved in recombination reactions involving one or more recombination sites (e.g., two, three, four, five, seven, ten, twelve, fifteen, twenty, thirty, fifty, etc.), which can be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), or mutants, derivatives (e.g., fusion proteins containing a recombination protein sequence or fragments thereof), fragments, and variants thereof. Illustrative examples of recombinases suitable for use in some embodiments of the present disclosure include, but are not limited to: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCEl, and ParA.

[0243] The present disclosure contemplates co-expression of polynucleotides comprising engineered DN TGF-beta receptors described herein with engineered TCR and CAR polypeptide constructs, including CAR-DN TGF-beta receptor constructs and fragments thereof as described herein, cells and compositions comprising the co-expression, and vectors expressing the polypeptides. Unless specified to the contrary, “polypeptide,” “polypeptide fragment,” “peptide,” and “protein” are according to their conventional meaning, i.e., as an amino acid sequence. Polypeptides are not limited to a particular length, e.g., they can include full-length protein sequences or fragments of full-length proteins, and can include post-translational modifications of the polypeptides, e.g., glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art, both naturally occurring and non-naturally occurring. In various embodiments, the polypeptides contemplated herein include signal (or leader) sequences at the N-terminus of the protein that direct the transfer of the protein upon or after translation. Illustrative examples of suitable signal sequences that can be used in the present disclosure include, but are not limited to, IgG 1 heavy chain signal sequence and CD8a signal sequence. The polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. The polypeptides contemplated herein specifically include engineered DN TGF-beta receptors, engineered TCRs, and CARs of the present disclosure, or sequences having deletions, additions, and / or substitutions of one or more amino acids of the polypeptides as contemplated herein.

[0244] A polypeptide includes "polypeptide variants." A polypeptide variant can differ from a naturally occurring polypeptide by virtue of having one or more substitutions, deletions, additions and / or insertions. Such variants can be naturally occurring or can be artificially produced, e.g., by modifying one or more of the polypeptide sequences described above. For example, in some embodiments, it can be desirable to improve the binding affinity and / or other biological properties of an engineered DNTGF-β receptor, TCR, or CAR by introducing one or more substitutions, deletions, additions and / or insertions. Preferably, a polypeptide of the present disclosure includes a polypeptide having at least about 50%, 60%, 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% amino acid identity thereto. A polypeptide of the present disclosure includes a variant having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein (see, e.g., the Sequence Listing), typically wherein the variant retains at least one biological activity of the reference sequence. A polypeptide includes "polypeptide fragments." A polypeptide fragment refers to a monomeric or multimeric polypeptide that can have an amino-terminal deletion, a carboxy-terminal deletion, and / or internal deletions or substitutions as compared to a naturally occurring or recombinantly produced polypeptide. In certain embodiments, a polypeptide fragment can comprise an amino acid chain that is at least 5 to about 500 amino acids in length. It will be appreciated that in certain embodiments, the fragment is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids in length.

[0245] The polypeptide can also be fused in frame to or conjugated to a linker or other sequence to facilitate synthesis, purification, or identification of the polypeptide (e.g., polyhistidine), or to enhance binding of the polypeptide to a solid support.

[0246] As described above, the polypeptides of the present disclosure can be altered in various ways including amino acid substitutions, deletions, truncations and insertions. Methods for such manipulations are generally known in the art. For example, reference to a polypeptide's amino acid sequence variant can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, e.g., Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al. (1987, Methods in Enzymol, 154:367-382), U.S. Patent No. 4,873,192, Watson, J.D. et al. (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987), and references cited therein. Guidance in the choice of appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the models of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).

[0247] In certain embodiments, the variant will contain conservative substitutions. "Conservative substitutions" are those substitutions that do not significantly affect the secondary structure and the hydrophilicity of the polypeptide, as will be understood by those skilled in the art of peptide chemistry. Modifications can be made in the structure of the polynucleotides and polypeptides of the present disclosure, and still obtain a functional molecule that encodes a variant or derivative polypeptide having the desired properties.

[0248] Polypeptide variants also include glycosylated forms, aggregated conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (e.g., PEGylated molecules). Covalent variants can be prepared by attaching functional groups to groups found in the amino acid chain or at the N-terminal residue or C-terminal residue, as is known in the art. Variants also include allelic variants, species variants, and muteins. Truncations or deletions of regions that do not affect the functional activity of the protein are also variants.

[0249] Where it is desired to express two or more polypeptides, the polynucleotide sequences encoding these polypeptides can be separated by an IRES sequence as discussed elsewhere herein. In another embodiment, two or more polypeptides can be represented as a fusion protein comprising one or more self-cleaving polypeptide sequences.

[0250] The polypeptides of the present disclosure include fusion polypeptides. In some embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to polypeptides having at least two, three, four, five, six, seven, eight, nine, or ten or more polypeptide segments. Fusion polypeptides will typically join C-termini to N-termini, however they can also join C-termini to C-termini, N-termini to N-termini, or N-termini to C-termini. The polypeptides of the fusion protein can be in any order or in a specified order. Fusion polypeptides or fusion proteins can also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, so long as the desired transcriptional activity of the fusion polypeptide is retained. Fusion polypeptides can be produced by chemical synthesis methods or by chemical linkages between two moieties, or can generally be prepared using other common techniques. The linked DNA sequences comprising the fusion polypeptides are operably linked to suitable transcriptional or translational control elements as discussed elsewhere herein.

[0251] In one embodiment, the fusion partner comprises a sequence that aids in expressing the protein at a higher yield than the native recombinant protein (expression enhancer). Other fusion partners can be selected in order to increase the solubility of the protein, or to enable the protein to be targeted to a desired intracellular compartment, or to facilitate transport of the fusion protein across a cell membrane.

[0252] The fusion polypeptides can further include a polypeptide cleavage signal between each of the polypeptide domains described herein. In addition, a polypeptide site can be placed in any of the linker peptide sequences. Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26).

[0253] Suitable protease cleavage sites and self-cleaving peptides are known to the skilled artisan (see, e.g., Ryan et al., 1997. J Gener. Viral. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Exemplary protease cleavage sites include, but are not limited to, cleavage sites for the following: a potyvirus Nia protease (e.g., tobacco etch virus protease), a potyvirus HC protease, a potyvirus Pl (P35) protease, a byovirus Nia protease, a protease encoded by byovirus RNA-2, a foot-and-mouth disease virus L protease, an enterovirus 2A protease, a rhinovirus 2A protease, a picoma 3C protease, a comovirus 24K protease, a nematode-transmitted polyhedral virus 24K protease, an RTSV (Rice tungro spherical virus) 3C-like protease, a PYVF (pepoysi virus F) 3C-like protease, heparin, thrombin, factor Xa, and enterokinase. A TEV (tobacco etch virus) protease cleavage site can be used due to its high cleavage stringency. In other embodiments, the self-cleaving peptide can include those polypeptide sequences obtained from the following: potyvirus and cardiovirus 2A peptides, FMDV (foot-and-mouth disease virus), equine rhinitis A virus, eastern equine encephalitis virus, and porcine teschovirus. In other embodiments, the self-cleaving polypeptide site includes a 2A or 2A-like site, sequence, or domain (Donnelly et al., 2001. J Gen. Viral. 82: 1027-1041).

[0254] In general, it will be appreciated that any suitable viral vector can be used to transduce the engineered constructs described herein. In one embodiment described herein, a cell (e.g., a T cell) is transduced with a retroviral vector (e.g., a lentiviral vector) encoding an engineered DN TGF-beta receptor construct and an engineered TCR or CAR construct as described herein. These transduced T cells elicit a stable, long-term, and persistent T cell response.

[0255] As used herein, the term “retrovirus” refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Illustrative retroviruses suitable for use in some embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), foamy virus, Friend murine leukemia virus, murine stem cell virus (MSCV), Rous sarcoma virus (RSV), and lentivirus.

[0256] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna-maedi virus (VMV); caprine arthritis encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0257] The term "vector" is used herein to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is typically linked to, e.g., inserted into, the vector nucleic acid molecule. Vectors can include sequences that directly self-replicate in a cell, or can include sequences sufficient to permit integration into the host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-defective retroviruses and lentiviruses.

[0258] As will be apparent to those skilled in the art, the term "viral vector" is used broadly to refer to a nucleic acid molecule that includes a virus-derived nucleic acid element that typically facilitates transfer or integration of a nucleic acid molecule into a cell genome (e.g., a transfer plasmid), or a viral particle that mediates nucleic acid transfer. Viral particles will typically include various viral components, and sometimes host cell components in addition to nucleic acid.

[0259] The term "viral vector" can refer to a virus or viral particle that is capable of transferring a nucleic acid into a cell or to the transferred nucleic acid itself. Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are derived primarily from a virus. The term "retroviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof that are derived primarily from a retrovirus. The term "lentiviral vector" refers to a viral vector or plasmid that contains structural and functional genetic elements or portions thereof (including LTRs) that are derived primarily from a lentivirus. The term "hybrid vector" refers to a vector, LTR, or other nucleic acid that contains both retroviral (e.g., lentiviral) sequences and non-retroviral sequences. In one embodiment, a hybrid vector refers to a vector or transfer plasmid that includes retroviral (e.g., lentiviral) sequences for reverse transcription, replication, integration, and / or packaging.

[0260] In some embodiments, the terms “lentiviral vector,” “lentiviral expression vector” can be used to refer to both lentiviral transfer plasmids and / or infectious lentiviral particles. Where reference is made herein to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is understood that the sequence of these elements exists in the lentiviral particles of the disclosure in RNA form and in the DNA plasmids of the disclosure in DNA form. In one embodiment described herein, the expression vector is a lentiviral expression vector.

[0261] “long terminal repeat sequence” or “LTR” is a structural term referring to base pair domains located at the ends of the retroviral DNA that are direct repeats in the context of their native sequence and contain a U3 region, an R region, and a U5 region. LTRs generally provide functions that are essential for expression of retroviral genes (e.g., initiation, start, and polyadenylation of the gene transcript) and viral replication. The LTR contains a number of regulatory signals, including transcription control elements, polyadenylation signals, and sequences required for replication and integration of the viral genome. The viral LTR is divided into three regions, called U3, R, and U5. The U3 region contains enhancer elements and promoter elements. The U5 region is the sequence that intervenes between the primer binding site and the R region, and contains the polyadenylation sequence. The R (repeat) region flanks the U3 and U5 regions. The LTR is composed of a U3 region, an R region, and a U5 region, and occurs both at the 5’ end of the viral genome and at the 3’ end of the viral genome. Adjacent to the 5’ LTR are sequences necessary for reverse transcription of the genome (tRNA primer binding site), and sequences necessary for efficient packaging of the viral RNA into a particle (Psi site).

[0262] As used herein, the term “packaging signal” or “packaging sequence” refers to sequences located within the retroviral genome that are required for insertion of the viral RNA into the viral capsid or particle, see, e.g., Clever et al., 1995. J of Virology, vol. 69, no. 4; pp. 2101-2109. Several retroviral vectors use the minimal packaging signal (also known as the psi ['P] sequence) required for encapsidation of the viral genome. Thus, as used herein, the terms “packaging sequence,” “packaging signal,” “psi,” and the symbol “'P” are used to refer to the non-coding sequence required for encapsidation of the retroviral RNA strand during viral particle formation.

[0263] In various embodiments, the vector includes a modified 5' LTR and / or 3' LTR. Either or both of the LTRs can include one or more modifications, including but not limited to one or more deletions, insertions, or substitutions. Modifications to the 3' LTR are typically made to increase the safety of the lentiviral or retroviral system by making the system replication defective for the virus. As used herein, the term "replication defective" refers to a virus that is unable to complete efficient replication such that infectious virions are not produced (e.g., replication defective lentiviral progeny). The term "replication competent" refers to a wild-type or mutant virus that is able to replicate such that viral replication of the virus is able to produce infectious virions (e.g., replication competent lentiviral progeny).

[0264] A "self-inactivating" (SIN) vector refers to a replication defective vector (e.g., a retroviral vector or a lentiviral vector) in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the right (3') LTR U3 region is used as a template for the left (5') LTR U3 region during viral replication, and thus, without the U3 enhancer-promoter, viral transcripts cannot be produced. In another embodiment of the disclosure, the 3' LTR is modified such that the U5 region is replaced, e.g., with a desirable polyadenylation sequence. It is noted that modifications to the LTRs are also contemplated herein, such as modifications to the 3' LTR, the 5' LTR, or both the 3' LTR and the 5' LTR.

[0265] Additional safety enhancements are provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during production of the viral particle. Examples of heterologous promoters that can be used include, for example, a simian virus 40 (SV40) (e.g., early or late) promoter, a cytomegalovirus (CMV) (e.g., immediate early) promoter, a Moloney murine leukemia virus (MoMLV) promoter, a Rous sarcoma virus (RSV) promoter, and a herpes simplex virus (HSV) (thymidine kinase) promoter. A typical promoter is able to drive high levels of transcription in a Tat-independent manner. This replacement reduces the possibility of recombination to produce replication-competent virus, as there is no complete U3 sequence in the viral production system. In certain embodiments, the heterologous promoter has additional advantages in the way it controls transcription of the viral genome. For example, the heterologous promoter can be inducible, such that transcription of all or part of the viral genome occurs only when an inducing factor is present. Inducing factors include, but are not limited to, one or more chemical compounds or physiological conditions (such as temperature or pH) under which the host cells are cultured.

[0266] In some embodiments, the viral vector comprises a TAR element. The term "TAR" refers to the "trans-activation response" genetic element (e.g., HIV) LTR located in the R region of a lentivirus. This element interacts with the lentivirus trans-activator (tat) genetic element to enhance viral replication.

[0267] The "R region" refers to the region within a retroviral LTR that begins at the start of the capping group (i.e., the transcription start site) and ends immediately prior to the start of the polyadenylation segment. The R region is also defined as flanking the U3 region and the U5 region. The R region serves a role during reverse transcription to allow the transfer of nascent DNA from one end of the genome to the other.

[0268] As used herein, the term "FLAP element" refers to a nucleic acid whose sequence includes a central polypurine tract and central termination sequence (cPPT and CTS) of a retrovirus (e.g., HIV-I or HIV-2). Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou et al., 2000, Cell, 101 : 173. During HIV-I reverse transcription, the central initiation of plus-strand DNA at the central polypurine tract (cPPT) and the central termination at the central termination sequence (CTS) results in the formation of a triple-stranded DNA structure: the HIV-I central DNA flap. While not wishing to be bound by any theory, the DNA flap can act as a cis-active determinant for nuclear import of the lentiviral genome and / or can increase the titer of the virus.

[0269] In one embodiment, the retroviral or lentiviral transfer vector comprises one or more export elements. The term "export element" refers to a cis-acting post-transcriptional regulatory element that modulates the transport of RNA transcripts from the nucleus to the cytoplasm. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see, e.g., Cullen et al., 1991. J Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423), and the hepatitis B virus post-transcriptional regulatory element (HPRE). Generally, the RNA export element is placed within the 3' UTR of the gene, and can be inserted as one or more copies.

[0270] In other embodiments, expression of the heterologous sequence in the viral vector is increased by the incorporation of a post-transcriptional regulatory element, a highly efficient polyadenylation site, and optionally a transcription termination signal into the vector. A variety of post-transcriptional regulatory elements can increase expression of a heterologous nucleic acid at the protein level, for example, the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999, J Virol., 73:2886); the post-transcriptional regulatory element found in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and others (Liu et al., 1995, Genes Dev., 9:1766).

[0271] In some embodiments, the vector can comprise a regulatory oligonucleotide having transcriptional or translational regulatory activity. Such oligonucleotides can be used in a variety of gene expression configurations for modulating control of expression. Transcriptional regulatory oligonucleotides can increase (enhance) or decrease (silence) the expression level of a recombinant expression construct. Regulatory oligonucleotides can selectively modulate expression in an environmentally specific manner, including, for example, for conferring tissue-specific expression, developmental stage-specific expression, or similar expression, including constitutive or inducible expression, of a polynucleotide. The regulatory oligonucleotides of the present disclosure can also be components of an expression vector or recombinant nucleic acid molecule, including regulatory oligonucleotides operably linked to an expressible polynucleotide. Regulatory elements can have a variety of lengths from a few nucleotides to several hundred nucleotides.

[0272] Elements that direct efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are typically found downstream of the polyadenylation signal. In some embodiments, the vector comprises a polyadenylation sequence located at the 3' end of a polynucleotide encoding a polypeptide to be expressed. As used herein, the term "polyadenylation site" or "polyadenylation sequence" represents a DNA sequence that directs termination and polyadenylation of nascent RNA transcripts by RNA polymerase II. A polyadenylation sequence can promote mRNA stability by adding a poly(A) tail to the 3' end of a coding sequence, and thus helps to increase translation efficiency. Efficient polyadenylation of a recombinant transcript is desirable because transcripts lacking a poly(A) tail are unstable and rapidly degraded. Illustrative examples of polyadenylation signals that can be used in the vectors of the present disclosure include ideal polyadenylation sequences (e.g., AATAAA, ATTAAA, AGTAAA), the bovine growth hormone polyadenylation sequence (BGH pA), the rabbit beta-globin polyadenylation sequence (rβgpA), or another suitable heterologous or endogenous polyadenylation sequence known in the art.

[0273] In one embodiment described herein, the vector described herein comprises a promoter operably linked to a polynucleotide encoding an engineered DN TGF-beta receptor and encoding a TCR or CAR polypeptide. In one embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 11, which encodes a polypeptide that expresses an extracellular domain having at least 75% sequence identity (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 3; and a transmembrane domain having at least 75% sequence identity (such as, at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 4.

[0274]

[0275] In another embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 11 operably linked to a nucleic acid sequence encoding an engineered TCR. In another embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 11 operably linked to a nucleic acid sequence encoding an engineered CAR. In another embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 11 under the control of the same promoter as a nucleic acid sequence encoding an engineered TCR or an engineered CAR.

[0276] In another embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 48 as shown in the figure encoding a polypeptide that expresses an extracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 15 and a transmembrane domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 16.

[0277]

[0278]

[0279] In another embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 48 operably linked to a nucleic acid sequence encoding an engineered TCR. In another embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 48 operably linked to a nucleic acid sequence encoding an engineered CAR. In another embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 48 under the control of the same promoter as the nucleic acid sequence encoding the engineered TCR or engineered CAR.

[0280] In another embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 35 encoding a polypeptide that expresses an extracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 15 and a transmembrane domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 18.

[0281]

[0282] In another embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 35 operably linked to a nucleic acid sequence encoding an engineered TCR. In another embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 35 operably linked to a nucleic acid sequence encoding an engineered CAR. In another embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 35 under the control of the same promoter as a nucleic acid sequence encoding an engineered TCR or an engineered CAR.

[0283] In another embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 36 encoding a polypeptide that expresses an extracellular domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 15 and a transmembrane domain having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 20.

[0284]

[0285] In another embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 49, which encodes a polypeptide of a CAR construct targeting GPC3 (“GPC3-CAR”).

[0286]

[0287]

[0288] In another embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 49 operably linked to a nucleic acid sequence encoding a DN TGF-beta receptor. In another embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 49 operably linked to a nucleic acid sequence encoding an engineered CAR. In another embodiment described herein, the expression vector comprises a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 49 under the control of the same promoter as a nucleic acid sequence encoding an engineered DN TGF-beta receptor. In one embodiment described herein, the expression vector comprises a nucleic acid sequence of a CAR-DN TGF-beta receptor construct having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 50.

[0289]

[0290]

[0291]

[0292] "codon-optimized" nucleic acid is one that has been altered so that codons are optimal for expression in a particular system, such as a particular species or group of species. For example, a nucleic acid sequence can be optimized for expression in mammalian cells or a particular mammalian species, such as human cells, by replacing at least one, more than one, or a substantial number of codons of the native sequence with codons that are more frequently or most frequently used in the genes of that species. Codon optimization does not change the amino acid sequence of the encoded protein.

[0293] The codon-optimized nucleotide sequences presented in the present disclosure can exhibit improved properties related to expression efficacy. In some embodiments, the DNA sequence to be transcribed can be optimized to promote more efficient transcription and / or translation. In some embodiments, the DNA sequence can be optimized in terms of cis-regulatory elements (e.g., TATA box, termination signal, and protein binding sites), artificial recombination sites, chi sites, CpG dinucleotide content, negative CpG islands, GC content, polymerase slippage sites, and / or other elements related to transcription; the DNA sequence can be optimized in terms of cryptic splice sites, mRNA secondary structure, stable free energy of the mRNA, repetitive sequences, RNA instability motifs, and / or other elements related to mRNA processing and stability; the DNA sequence can be optimized in terms of codon usage bias, codon adaptiveness, internal chi sites, ribosome binding sites (e.g., IRES), immature polyadenylation sites, Shine-Dalgarno (SD) sequences, and / or other elements related to translation; and / or the DNA sequence can be optimized in terms of codon context, codon-anti-codon interactions, translation pausing sites, and / or other elements related to protein folding.

[0294] In another embodiment described herein, the expression vector comprises a codon-optimized nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 51, which encodes a polypeptide that expresses a GPC3-CAR construct ("CAR 1").

[0295]

[0296]

[0297] In another embodiment described herein, the expression vector comprises a codon-optimized nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 51 operably linked to a nucleic acid sequence encoding a DN TGF-beta receptor as described herein. In another embodiment described herein, the expression vector comprises a codon-optimized nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 51 operably linked to a nucleic acid sequence encoding an engineered CAR. In another embodiment described herein, the expression vector comprises a codon-optimized nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 51 under the control of the same promoter as a nucleic acid sequence encoding an engineered DN TGF-beta receptor. In one embodiment described herein, the expression vector comprises a codon-optimized nucleic acid sequence of a CAR-DN TGF-beta receptor construct having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%) to SEQ ID NO: 52 (“CAR DNR A”).

[0298]

[0299]

[0300]

[0301] The vector can have one or more LTRs, with any LTR comprising one or more modifications, such as one or more nucleotide substitutions, additions, or deletions. The vector can further comprise one or more accessory elements to increase transduction efficiency (e.g., cPPT / FLAP); viral packaging (e.g., Psi (Ψ) packaging signal, RRE); and / or other elements to increase expression of the therapeutic gene (e.g., polyadenylation sequence), and can optionally comprise a WPRE or HPRE. The skilled artisan will appreciate that many other different embodiments can be made from the existing embodiments of the present disclosure.

[0302] A“host cell” includes a cell that is transfected, infected, or transduced in vivo, ex vivo, or in vitro with a recombinant vector or polynucleotide of the present disclosure. Host cells can include packaging cells, producer cells, and cells infected with a viral vector. In some embodiments, a host cell infected with a viral vector of the present disclosure is administered to a subject in need of treatment. In certain embodiments, the term“target cell” is used interchangeably with host cell and refers to a transfected, infected, or transduced cell of a desired cell type. In some embodiments, the target cell is a T cell.

[0303] Large-scale viral particle production is generally necessary to obtain reasonable viral titers. Viral particles are produced by transfecting a transfer vector into a packaging cell line that contains viral structural and / or accessory genes (e.g., gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes or other retroviral genes).

[0304] As used herein, the term“packaging vector” refers to an expression vector or viral vector that lacks a packaging signal and comprises a polynucleotide encoding one, two, three, four, or more viral structural and / or accessory genes. Typically, a packaging vector is introduced into a cell via transfection, transduction, or infection. Methods for transfection, transduction, or infection are well known to those of skill in the art. A retroviral / lentiviral transfer vector of the present disclosure can be introduced into a packaging cell line via transfection, transduction, or infection to produce a producer cell or cell line. A packaging vector of the present disclosure can be introduced into a human cell or cell line by common methods, including, for example, calcium phosphate transfection, lipofection, or electroporation. In some embodiments, a packaging vector is introduced into a cell along with a primary selectable marker, such as neomycin, hygromycin, puromycin, blasticidin, bleomycin, thymidine kinase, DHFR, Gln synthetase, or ADA, followed by selection in the presence of the appropriate drug, and isolation of a clone. The selectable marker gene can be physically linked to the gene encoded by the packaging vector (e.g., by an IRES or self-cleaving viral peptide).

[0305] The viral envelope protein (env) determines the range of host cells that can ultimately be infected and transformed by recombinant retroviruses produced by the cell line. In the case of lentiviruses, such as HIV-1, HIV-2, SIV, FIV, and EIV, the env proteins include gp41 and gpl20. In some embodiments, the viral env proteins expressed by the packaging cells of the present disclosure are encoded on separate vectors from the viral gag and pol genes, as has been previously described.

[0306] Illustrative examples of retrovirus-derived env genes that can be employed in the embodiments described herein include, but are not limited to: MLV envelope, IOAI envelope, BAEV, FeLV-B, RDI14, SSAV, Ebola, Sendai, FPV (avian plague virus), and influenza virus envelope. Similarly, genes encoding envelopes from RNA viruses (e.g., the following RNA virus families: Picomaviridae, Calciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Bimaviridae, Retroviridae), as well as from DNA viruses (the following virus families: Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxyiridae, and Iridoviridae) can be utilized. Representative examples include FeLV, VEE, HFVW, WDSV, SFV, Rabies, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV SMRV, RAV, FuSV, MH2, AEV, AMV, CTIO, and EIAV.

[0307] In other embodiments, the envelope protein used to pseudotype a virus of the present disclosure includes, but is not limited to, any of the following viruses: influenza A (such as H1N1, H1N2, H3N2, and H5N1 (avian influenza)), influenza B, and influenza C viruses, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, any of the Norwalk virus group, enteric adenovirus, parvovirus, dengue virus, monkeypox virus, Mononegavirales, Lyssavirus (such as rabies virus), Lagos bat virus, Mokola virus, Duvenhage virus, European bat viruses 1 and 2, and Australian bat virus, Ephemerovirus, Vesiculovirus, vesicular stomatitis virus (VSV), herpes viruses (such as herpes simplex virus types 1 and 2), varicella-zoster virus, cytomegalovirus, Epstein-Barr virus (EBV), human herpes virus (HHV), human herpes virus types 6 and 8, human immunodeficiency virus (HIV), papillomavirus, murid gammaherpesvirus, Arenaviruses (such as Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-related hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, lymphocytic choriomeningitis virus (LCMV)), Bunyaviridae (such as Crimean-Congo hemorrhagic fever virus), hantavirus, nephropathia syndrome hemorrhagic fever-causing virus, Rift Valley fever virus, Filoviridae (filovirus) including Ebola hemorrhagic fever and Marburg hemorrhagic fever, Flaviviridae including Kaysanur Forest disease virus, Omsk hemorrhagic fever virus, tick-borne encephalitis-causing virus, and Paramyxoviridae (such as Hendra virus and Nipah virus), variola major and minor (smallpox virus), alphavirus (such as Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, SARS- related coronavirus (SARS-Co V), West Nile virus, or any encephalitis-causing virus.

[0308] As used herein, the term "pseudotype" or "pseudotyped" refers to a virus whose viral envelope protein has been replaced with that of another virus with different characteristics. For example, HIV can be pseudotyped with vesicular stomatitis virus G-protein (VSV-G) envelope protein, which allows HIV to infect a wider range of cells because the HIV envelope protein (encoded by the env gene) normally targets the virus to CD4 + presenting cells.

[0309] As used herein, the term "packaging cell line" is used in reference to a cell line that does not contain a packaging signal, but which stably or transiently expresses viral structural proteins and replicase (e.g., gag, pol, and env) that are essential for the proper packaging of viral particles. Any suitable cell line can be employed to make the packaging cells of the present disclosure. Generally, the cells are mammalian cells. In another embodiment, the cells used to generate the packaging cell line are human cells. Suitable cell lines that can be used to generate packaging cell lines include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC 5 cells, A549 cells, HTI080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells.

[0310] As used herein, the term "producer cell line" refers to a cell line capable of producing recombinant retroviral particles, including packaging cell lines and transfer vector constructs comprising a packaging signal. Production of infectious viral particles and viral stock solutions can be performed using conventional techniques. Methods of preparing viral stock solutions are known in the art and are illustrated by, for example, Y. Soneoka et al. (1995) Nucl. Acids Res. 23:628-633 and N.R. Landau et al. (1992) J Virol. 66:5110-5113. Infectious viral particles can be collected from packaging cells using conventional techniques. For example, infectious particles can be collected by cell lysis or by collecting the supernatant of a cell culture, as is known in the art. Optionally, if desired, the collected viral particles can be purified. Suitable purification techniques are well known to those of skill in the art.

[0311] Delivery of a gene or other polynucleotide sequence using a retroviral or lentiviral vector by means of viral infection rather than by transfection is referred to as “transduction.” In one embodiment, a retroviral vector transduces into a cell by infection and proviral integration. In certain embodiments, a target cell (e.g., a T cell) is “transduced” if it comprises a gene or other polynucleotide sequence that was delivered to the cell by infection using a viral or retroviral vector. In some embodiments, a transduced cell comprises one or more genes or other polynucleotide sequences in its cellular genome that were delivered by a retroviral or lentiviral vector. In one embodiment described herein, T cells are transduced with an expression vector comprising a nucleic acid sequence having at least 75% sequence identity (such as at least 75%, at least 80%, at least 90%, at least 95%, or 100% identity; e.g., 85% to 90%, 85% to 95%, 85% to 100%, 90% to 95%, 90% to 100%, or 95% to 100%) to SEQ ID NO: 11.

[0312] In some embodiments, host cells co-expressing a DN TGF-beta receptor construct of the disclosure with an engineered TCR and / or CAR are provided. The host cells can be transduced with one or more viral vectors comprising nucleic acid sequences encoding one or more polypeptides expressing a DN TGF-beta receptor construct of the disclosure and an engineered TCR and / or CAR. The host cells can be administered to a subject to treat and / or prevent a T cell malignancy. Other methods related to the use of viral vectors in gene therapy that can be utilized in accordance with certain embodiments of the disclosure can be found in, for example, Kay, M. A. (1997) Chest 111 (6th Supplement): 138S-142S; Ferry, N. and Heard, J. M. (1998) Hum. Gene Ther. 9: 1975-81; Shiratory, Y. et al. (1999) Liver 19: 265-74; Oka, K. et al. (2000) Curr. Opin. Lipidol. 11: 179-86; Thule, P. M. and Liu, J. M. (2000) Gene Ther. 7: 1744-52; Yang, N. S. (1992) Crit. Rev. Biotechnol. 12: 335-56; Alt, M. (1995) J Hepatol. 23: 746-58; Brody, S. L. and Crystal, R. G. (1994) Ann. N Y Acad. Sci. 716: 90-101; Strayer, D. S. (1999) Expert Opin. Investig. Drugs 8: 2159-2172; Smith-Arica, J. R. and Bartlett, J. S. (2001) Curr. Cardiol. Rep. 3: 43-49; and Lee, H. C. et al. (2000) Nature 408: 483-8.

[0313] The compositions described herein can comprise one or more polynucleotides, polypeptides, vectors comprising the same, and T cell compositions, as contemplated herein. One embodiment described herein is a composition comprising a modified T cell that co-expresses one or more engineered DN TGF-beta receptors described herein with an engineered TCR and / or CAR. Compositions include, but are not limited to, pharmaceutical compositions. A "pharmaceutical composition" refers to a composition formulated to be administered to a cell or an animal, either alone, or in combination with one or more other modalities of treatment, in a pharmaceutically- or physiologically- acceptable solution. It will also be appreciated that the compositions of the disclosure can also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutics, prodrugs, drugs, antibodies, or other various pharmaceutically active agents, if desired. There is little restriction in the additional components that can also be included in the compositions, as long as the additional agents do not adversely affect the ability of the composition to deliver the intended therapy.

[0314] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0315] As used herein, "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any auxiliary agent, carrier, excipient, glidant, sweetening, diluting, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, surface active agent or emulsifying agent, which has been approved by the U.S. Food and Drug Administration as being acceptable for use in humans or domestic animals. Exemplary pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; gum tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, paraffin, silicone, bentonite, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laureate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and any other nontoxic compatible substances used in pharmaceutical formulations.

[0316] In one embodiment described herein, the composition disclosed herein comprises a certain amount of the modified T cells contemplated herein. It can generally be noted that a pharmaceutical composition comprising the T cells contemplated herein can be administered in the following dosage: 10 2 One to 10 10 Cells / kg body weight, 10 5 One to 10 9 Cells / kg body weight, 10 5 One to 10 8 Cells / kg body weight, 10 5 One to 10 7 Cells / kg body weight, 10 7 One to 10 9 Cells / kg body weight, or 10 7 One to 10 8 Cells / kg body weight, including all integer values ​​within these ranges. The number of cells will depend on the intended end use of the composition, and the type of cells contained therein will also depend on that end use. T cells modified to express engineered TCRs or CARs can be administered multiple times at doses within these ranges. The cells can be allogeneic, homologous, xenogeneic, or autologous cells from a patient receiving therapy. If desired, treatment may also include administration of mitogens (e.g., PHAs) or lymphokines, cytokines, and / or chemokines (e.g., IFN-γ, IL-2, IL-7, IL-15, IL-12, TNF-α, IL-18 and TNF-β, GM-CSF, IL-4, IL-13, Flt3-L, RANTES, MIP1α, etc.) as described herein to enhance the transplantation and function of the infused T cells.

[0317] Generally, compositions comprising activated and expanded cells, as described herein, can be used to treat and prevent diseases occurring in immunocompromised or immunosuppressed individuals. In some cases, compositions contemplated herein comprising modified T cells are intended for the treatment of cancer. The modified T cells described herein can be administered alone or in combination with a carrier, diluent, excipient, and / or other components (such as IL-2, IL-7, and / or IL-15, or other cytokines or cell populations) as a pharmaceutical composition. In some embodiments, the pharmaceutical compositions contemplated herein comprise a quantity of genetically modified T cells in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0318] The pharmaceutical compositions contemplated herein comprising modified T cells can also comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the disclosure can be formulated for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal or intramuscular administration.

[0319] Liquid pharmaceutical compositions (whether in solution, suspension, or other like form) can include one or more of the following: sterile diluent solutions such as water for injection; saline solution; Ringer's solution; isotonic sodium chloride; fixed oils such as synthetic mono- or diglycerides, which can serve as the solvent or suspending medium; polyethylene glycols; glycerin; propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Sterile injectable solutions are also contemplated.

[0320] In some embodiments, the compositions contemplated herein include an effective amount of an expanded modified T cell composition alone or in combination with one or more therapeutic agents. Thus, the T cell composition can be administered alone or in combination with other known cancer treatments such as radiation therapy, chemotherapy, transplantation, immunotherapy, hormone therapy, photodynamic therapy, etc. The composition can also be administered in combination with antibiotics and antiviral agents. Such therapeutic agents can be accepted in the art as being useful for the treatment of disease states such as cancer as described herein. In one embodiment, the compositions contemplated herein can also be administered with an inhibitor of TGF-β (e.g., the small molecule inhibitor LY55299). Exemplary therapeutic agents contemplated include cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory agents, chemotherapeutic agents, radiation therapy, therapeutic antibodies, or other active agents and adjunct agents.

[0321] In certain embodiments, the compositions comprising T cells contemplated herein can be administered in combination with any number of chemotherapeutic agents. Illustrative examples of chemotherapeutic agents include, but are not limited to: alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN® TMalkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodepa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine resume; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as aclacinomycin, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thiopurine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogucal; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., paclitaxel (TAXOL®; Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®, Aventis); Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®, Aventis); Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RPS 2000; difluoromethylornithine (DMFO); retinoic acid derivatives such as Targretin TM (pegaspargase), Panitumumab TM (Alitretinoin); ONTAK TM (Denileukin diftitox); esperamicin; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are anti-hormonal agents that act to regulate or inhibit hormone action on tumors, such as anti-estrogens which include, for example, tamoxifen, raloxifene, 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, keoxifene citrate, LY117018, onapristone, and toremifene (fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0322] A variety of other therapeutic agents can be used in conjunction with the compositions described herein. In one embodiment, the composition comprising T cells is administered with an anti-inflammatory agent. Anti-inflammatory agents or anti-inflammatory drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone); non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide, and mycophenolate.

[0323] In some embodiments, the NSAID is selected from the group consisting of ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors such as (Vioxx) and (Celebrex), and salicylates. Exemplary analgesics are selected from the group consisting of acetaminophen, oxycodone, tramadol, or propoxyphene hydrochloride. Exemplary glucocorticoids are selected from the group consisting of cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules against cell surface markers (e.g., CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (e.g., etanercept adalimumab and infliximab Chemokine inhibitors and adhesion molecule inhibitors. Biologic response modifiers include monoclonal antibodies as well as recombinant forms of the molecules. Exemplary disease-modifying antirheumatic drugs (DMARDs) include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, gold preparations (oral (auranofin) and intramuscular), and minocycline.

[0324] In other embodiments, therapeutic antibodies suitable for use in combination with the CAR-modified T cells contemplated herein include, but are not limited to: abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farietuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab,Lexatumumab, Lintuzumab, Lorvotuzumab, Lucatumumab, Mapatumumab, Matuzumab, Milatuzumab, Minretumomab, Mitumomab, Moxetumomab, Namatumab, Naptumomab, Necitumumab, Nimotuzumab, Nofetumomab, Ocaratuzumab, Ofatumumab, Olaratumab, Onartuzumab, Oportuzumab, Oregovomab, Panitumumab, Parsatuzumab, Patritumab, Pemtumomab, Pertuzumab, Pintumomab, Pritumumab, Racotumomab, Radretumab, Rilotumumab, Rituximab, Robatumumab, Satumomab, Sibrotuzumab, Siltuximab, Simtuzumab, Solitomab, Tacatuzumab, Taplitumomab, Tenatumomab, Teprotumumab, Tigatuzumab, Tositumomab, Trastuzumab, Tucotuzumab, Ublituximab, Veltuzumab, Vorsetuzumab, Votumumab, Zalutumumab, CC49, and 3F8.

[0325] In some embodiments, the compositions described herein are administered in combination with a cytokine. As used herein, the term "cytokine" means a generic term for proteins released by one cell population that act on another cell to elicit a specific cellular response. Examples of such cytokines are lymphokines, monokines, chemokines, and traditional polypeptide hormones. Cytokines include growth hormone such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; fibroblast growth factor; prolactin; placental lactogen; tumor necrosis factor-alpha and -beta; Mullerian-inhibiting substance; mouse gonadotropin-associated peptide; activin; inhibin; vascular endothelial growth factor; integrin; thrombopoietin (TPO); nerve growth factors such as NGF-beta; platelet-growth factor; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta; insulin-like growth factor-I and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as IL-1, IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and kit ligand (KL). As used herein, the term cytokine includes proteins from natural sources or from recombinant cell culture and biologically active equivalents of the native sequence cytokine.

[0326] The present disclosure contemplates, in part, genetically modified T cells redirected to target cells (e.g., tumor cells or cancer cells) and include engineered DN TGF-beta receptors described herein and engineered TCRs or CARs with binding domains that bind to target antigens on cells, including CAR-DN TGF-beta receptor constructs described herein. Cancer cells can also spread to other parts of the body through the bloodstream and lymphatic system. There are several main types of cancer. Carcinomas start in the skin or in tissues that line or cover internal organs. Sarcomas start in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemias start in blood-forming tissue such as the bone marrow and cause large numbers of abnormal blood cells to be produced and enter the blood. Lymphomas and multiple myelomas start in cells of the immune system. Central nervous system cancers start in brain and spinal cord tissue.

[0327] In one embodiment, the target cell expresses an antigen, e.g., a target antigen. In one embodiment, the target cell is a pancreatic parenchymal cell, pancreatic ductal cell, hepatocyte, cardiomyocyte, skeletal muscle cell, osteoblast, skeletal muscle myoblast, neuron, vascular endothelial cell, chromatic cell, smooth muscle cell, glial cell, adipocyte, bone cell, chondrocyte, islet cell, CNS cell, PNS cell, liver cell, adipocyte, hepatocyte, kidney cell, lung cell, skin cell, ovarian cell, follicle cell, epithelial cell, immune cell, or endothelial cell.

[0328] In certain embodiments, the target cell is part of pancreatic tissue, neural tissue, cardiac tissue, bone marrow, muscle tissue, bone tissue, skin tissue, liver tissue, hair follicle, vascular tissue, adipose tissue, lung tissue, and kidney tissue.

[0329] In one embodiment, the target cell is a tumor cell. In another embodiment, the target cell is a cancer cell, such as a cell in a patient having cancer. Exemplary cells that can be killed with the methods disclosed herein include cells of the following tumors: liquid tumors, such as leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic and erytholeukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphomas, Hodgkin's disease, non-Hodgkin's lymphoma, multiple myeloma, macroglobulinemia, and heavy chain disease.

[0330] In another embodiment, the cell is a solid tumor cell, such as sarcomas and epithelial carcinomas, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon epithelioma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, hepatocellular epithelioma, lung cancer, colorectal cancer, squamous cell epithelioma, basal cell epithelioma, adenocarcinoma (e.g., adenocarcinoma of the pancreas, colon, ovary, lung, breast, stomach, prostate, cervix, or esophagus), sweat gland carcinoma, sebaceous gland carcinoma, papillary epithelioma, papillary adenocarcinoma, medullary epithelioma, bronchogenic carcinoma, renal cell epithelioma, hepatoma, bile duct epithelioma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, urothelial carcinoma, CNS tumors such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0331] In one embodiment, the cancer can include Wilms' tumor, Ewing's sarcoma, neuroendocrine tumor, glioblastoma, neuroblastoma, melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, kidney cancer, pancreatic cancer, lung cancer, bile duct cancer, cervical cancer, endometrial cancer, esophageal cancer, stomach cancer, head and neck cancer, medullary thyroid epithelioma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and bladder cancer.

[0332] In one embodiment, the target cell is a malignant cell of the liver, pancreas, lung, breast, bladder, brain, bone, thyroid, kidney, skin, and hematopoietic system. In another embodiment, the target cell is a cell in a liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, skin cancer, or blood cancer. In another embodiment, the target cell is a cell (e.g., a cancer cell) infected with a virus, including but not limited to CMV, HPV, and EBV.

[0333] In one embodiment, the target antigen is directed against or is an epitope of HPV oncoprotein (including HPV-16 E6 and HPV-16 E7), alpha folate receptor, 5T4, alpha vBeta6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD28, CD30, CD33, CD44, CD44v6, CD44v7 / 8, CD70, CD79a, CD79b, CD123, CD137 (4-1BB), CD138, CD171, CEA, CSPG4, CLL-1, EGFR, EGFR family (including ErbB2 (HERII), EGFRvIII), EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRa, GD2, GD3, Glypican-3 (GPC3), HLA-Al+MAGEI, HLA-A2+MAGE1, HLA A3+MAGE1, HLA-Al+NY-ES0-1, HLA-A2+NY-ES0-1, HLA-A3+NY-ES0-1, IL-llR alpha, IL-13R alpha 2, Lambda, Lewis-Y, Kappa, Mesothelin, Mucl, Muc16, NCAM, NKG2D ligands, NYE-S0-1, PRAME, PSCA, PSMA, RORI, SSX, Survivin, TAG72, TEM, and VEGFRII.

[0334] The modified T cells contemplated herein provide improved adoptive immunotherapy for the treatment of various disorders, including but not limited to cancer, infectious diseases, autoimmune diseases, inflammatory diseases, and immunodeficiencies. In some embodiments, the specificity of primary T cells is redirected to tumor or cancer cells by genetically modifying the primary T cells to co-express a TCR or CAR contemplated herein with an engineered DN TGF-beta receptor. For example, in the case of using a CAR-DN TGF-beta receptor construct described herein, the antigen binding domain of the scFv of the CAR directs the T cell to cells expressing the tumor antigen, and the TGF-beta receptor construct described herein protects the population of such T cells by inhibiting the suppressive effects of TGF-beta.

[0335] In one embodiment of the disclosure, includes a type of cell therapy in which T cells are modified to co-express an engineered DN TGF-beta receptor with an engineered TCR or CAR, including a CAR-DN TGF-beta receptor construct described herein that targets cancer cells expressing a target antigen, and the modified T cells are infused into a recipient in need thereof. Thus, the infused cells are able to kill tumor cells in the recipient while being minimally affected by TGF-beta inhibition.

[0336] Any cell can be used as a host cell for the polynucleotides, vectors, or polypeptides of the present disclosure. In some embodiments, the cell can be a prokaryotic cell, a fungal cell, a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Suitable prokaryotic cells include, but are not limited to, eubacteria, such as gram-negative or gram-positive organisms, for example, Enterobactehaceae, such as Escherichia, for example, E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, for example, Salmonella typhimurium; Serratia, for example, Serratia marcescans, and Shigella; Bacilli such as B. subtilis and B. licheniformis; Pseudomonas, such as P. aeruginosa; and Streptomyces. In some embodiments, the cell is a human cell. In some embodiments, the cell is an immune cell. In some embodiments, the immune cell is selected from the group consisting of a T cell, a B cell, a tumor infiltrating lymphocyte (TIL), a TCR-expressing cell, a natural killer (NK) cell, a dendritic cell, a granulocyte, an innate lymphoid cell, a megakaryocyte, a monocyte, a macrophage, a platelet, a thymocyte, and a myeloid cell. In one embodiment, the immune cell is a T cell. In another embodiment, the immune cell is a NK cell. In certain embodiments, the T cell is a tumor infiltrating lymphocyte (TIL), an autologous T cell, an engineered autologous T cell (eACT TM ), an allogeneic T cell, a heterologous T cell, or any combination thereof. Unlike antibody therapy or independent TCR- or CAR-modified T cells, T cells modified to co-express an engineered DN TGF-β receptor described herein with an engineered TCR or CAR are not only able to replicate in vivo and thus contribute to long-term persistence (which can lead to sustained cancer therapy), but also have the additional advantage of avoiding the inhibitory effects of TGF-β. Thus, one embodiment described herein is a method of inhibiting TGF-β activity, comprising administering to a subject in need thereof a therapeutically effective amount of a modified T cell co-expressing a DN TGF-β receptor described herein and a TCR or CAR as described herein.

[0337] Without being bound by any theory, it is understood that administration of the modified T cells described herein results in inhibition of the phosphorylation cascade of the DNTGF-β receptor (despite the binding of the TGF-β ligand to the TGF-βRI dimer-DN TGF-βRII dimer complex, which still initiates intracellular pSMAD signaling), but still allows the T cell to retain specificity for a target antigen. In another embodiment, the method of inhibiting TGF-β activity comprises reducing the levels of pSMAD2 and pSMAD3 compared to the levels of pSMAD2 and pSMAD3 in a non-transduced T cell.

[0338] In one embodiment described herein, administration of the modified T cells described herein results in a reduction in the levels of pSMAD2 and pSMAD3 of between about 10% to about 100%, between about 20% to about 90%, between about 30% to about 80%, between about 40% to about 70%, or between about 50% to about 60%. In another embodiment described herein, the reduction in pSMAD2 can be about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. In another embodiment described herein, the reduction in pSMAD3 can be about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0339] In another embodiment described herein, administration of the modified T cells described herein results in an increase in cytokine secretion in the presence of TGF-β1 of between about 10% to about 100%, between about 20% to about 90%, between about 30% to about 80%, between about 40% to about 70%, or between about 50% to about 60%. In another embodiment described herein, administration of the modified T cells described herein results in an increase in cytokine secretion in the presence of TGF-β1 of about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%.

[0340] In another embodiment, the T cells co-expressing a DN TGF-β receptor and an engineered TCR or CAR as described herein can undergo T cell expansion such that the therapeutic T cell population can be maintained or sustained for an extended period of time. Accordingly, another embodiment described herein is a method of expanding a T cell population comprising administering to a subject in need thereof a therapeutically effective amount of the T cells described herein.

[0341] In one embodiment described herein, the population of T cells retains between about 50% to about 100% after 7 days, retains between about 60% to about 90% after 7 days, or retains between about 70% to about 80% after 7 days. In another embodiment described herein, the population of T cells retains about 50% after 7 days, retains about 60% after 7 days, retains about 70% after 7 days, retains about 80% after 7 days, retains about 90% after 7 days, or retains about 100% after 7 days.

[0342] In another embodiment described herein, administering the modified T cells described herein results in transduced T cells expanding by about 10% to about 100%, about 20% to about 90%, or about 30% to about 80% in the presence of TGF-βΙ. In another embodiment described herein, administering the modified T cells described herein results in transduced T cells expanding by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% in the presence of TGF-βΙ.

[0343] In one embodiment described herein, administering the modified T cells from the codon-optimized sequences as described herein results in an increase in expression efficiency of about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In another embodiment described herein, administering the modified T cells from the codon-optimized sequences as described herein results in an increase in transduction efficiency of about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0344] Without being bound by any theory, it is believed that despite the promise of CAR T cell therapy, constitutive tonic signaling in the absence of tumor antigen can lead to reduced efficacy, poor CAR T cell survival, and toxicity (Ajina et al., Mol Cancer Ther., 17(9): 1795-1815 (2018)). Thus, CAR T cell therapy with reduced tonic signaling produces superior performance. In one embodiment described herein, administration of modified T cells from a codon-optimized sequence as described herein in the absence of tumor antigen results in a reduction of cytokine release of about 10%, about 20%, about 30%, about 40%, about 50%, 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In one embodiment described herein, administration of modified T cells from a codon-optimized sequence in the absence of tumor antigen results in a reduction of cytokine release of about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0345] In another embodiment, administration of modified T cells from a codon-optimized sequence as described herein results in a reduction in tumor volume of about 50% to about 10% to about 100%, about 20% to about 90%, or about 30% to about 80%. In another embodiment, tumor volume is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.

[0346] Another embodiment described herein is a method of treating cancer in a subject in need thereof comprising administering an effective amount (e.g., a therapeutically effective amount) of a composition comprising T cells co-expressing a DN TGF-beta receptor and a TCR or CAR as described herein. The amount and frequency of administration will be determined by such factors as the condition of the patient and the type and severity of the patient's disease, although an appropriate dosage can be determined through clinical trials.

[0347] Another embodiment described herein is a method of treating a liver cancer in a subject in need thereof comprising administering an effective amount (e.g., a therapeutically effective amount) of a composition comprising T cells co-expressing a DN TGF-beta receptor and a TCR or CAR as described herein, including the CAR-DN TGF-beta receptor constructs described herein. The amount and frequency of administration will be determined by factors such as the condition of the patient and the type and severity of the patient’s disease, although an appropriate dosage can be determined through clinical trials.

[0348] In other embodiments, compositions comprising T cells genetically modified with a vector containing a promoter operably linked to a polynucleotide encoding a DN TGF-beta receptor and a polynucleotide encoding a TCR or CAR, including the CAR-DN TGF-beta receptor constructs described herein, are used to treat solid tumors or cancers, including but not limited to liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, bone cancer, thyroid cancer, kidney cancer, skin cancer, or a virus-induced cancer.

[0349] In some embodiments, compositions comprising T cells genetically modified with a vector containing a promoter operably linked to a polynucleotide encoding a DN TGF-beta receptor and a polynucleotide encoding a TCR or CAR, including the CAR-DN TGF-beta receptor constructs described herein, are used to treat various cancers, the TCR or CAR comprising an antigen-specific binding domain that binds to an epitope BCMA, CD19, CD20, CD28, CD137 (4-1BB), CLL-1, GPC3, PCMA, or PSMA.

[0350] In other embodiments, methods are provided comprising administering a therapeutically effective amount of a modified T cell contemplated herein, or a composition comprising the T cell, alone or in combination with one or more therapeutic agents, to a patient in need thereof. In certain embodiments, the cells of the disclosure are used to treat a patient at risk of developing a cancer. Accordingly, the disclosure provides methods for treating or preventing a cancer, comprising administering to a subject in need thereof a therapeutically effective amount of a modified T cell of the disclosure.

[0351] One of ordinary skill in the art will recognize that multiple administrations of the compositions of the disclosure can be required to achieve a desired therapy. For example, the compositions can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times over a span of 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years, or more.

[0352] In certain embodiments, it can be desirable to administer activated T cells to a subject, then redraw blood (or perform apheresis), activate T cells therefrom according to the present disclosure, and reinfuse these activated and expanded T cells to the patient. This process can be performed multiple times every few weeks. In certain embodiments, T cells can be activated from 10 cc to 400 cc of drawn blood. Without being bound by theory, using this multiple draw blood / multiple reinfusion protocol can be used to select for certain T cell populations.

[0353] Administration of the compositions contemplated herein can be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, infusion, implantation or transplantation. In some embodiments, the compositions are administered parenterally. As used herein, the phrases "parenterally administered" and "administered parenterally" refer to modes of administration other than enteral and topical administration, generally by injection, and include, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion. In one embodiment, the compositions contemplated herein are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

[0354] In one embodiment, an effective amount of a composition is administered to a subject in need thereof to increase the cellular immune response to a cancer in the subject. The immune response can include a cellular immune response mediated by cytotoxic T cells capable of killing infected cells, regulatory T cells, and helper T cell responses. A humoral immune response can also be induced, which is mediated primarily by helper T cells capable of activating B cells leading to antibody production. A variety of techniques can be used to analyze the type of immune response induced by the compositions of the present disclosure, which are well described in the art; for example, Current Protocols in Immunology, eds.: John E. Coligan, Ada M. Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strober, (2001) John Wiley & Sons, NY, N.Y.

[0355] In terms of T cell-mediated killing, CAR-ligand binding initiates CAR signaling to the T cell, leading to activation of multiple T cell signaling pathways that induce the T cell to produce or release proteins that can induce target cell apoptosis through various mechanisms. These T cell-mediated mechanisms include, but are not limited to, transfer of intracellular cytotoxic granules from the T cell into the target cell, T cell secretion of pro-inflammatory cytokines that can directly (or indirectly via recruitment of other killer effector cells) induce target cell killing, and upregulation of death receptor ligands (e.g., FasL) on the T cell surface that, upon binding to their cognate death receptors (e.g., Fas) on the target cell, induce target cell apoptosis.

[0356] One embodiment described herein is a method of treating a subject diagnosed with a cancer, comprising removing T cells from the subject, genetically modifying the T cells with a vector comprising a nucleic acid encoding an engineered DN TGF-beta receptor and a TCR or CAR as contemplated herein, including the CAR-DN TGF-beta receptor constructs described herein, thereby producing a population of modified T cells, and administering the population of modified T cells to the same subject.

[0357] In certain embodiments, the present disclosure also provides a method for stimulating an effector cell-mediated immune modulator response to a target cell population in a subject, comprising the step of: administering to the subject a population of immune effector cells expressing a nucleic acid construct encoding an engineered DN TGF-beta receptor and a TCR or CAR molecule, including the CAR-DN TGF-beta receptor constructs described herein.

[0358] Methods for administering the cell compositions described herein include any method that is effective to result in the reintroduction of an ex vivo genetically modified immune effector cell directly expressing an engineered TCR or CAR, or a genetically modified progenitor cell that differentiates into a mature immune effector cell expressing an engineered TCR or CAR in a subject. One method includes transducing an ex vivo peripheral blood T cell with a nucleic acid construct according to the present disclosure, and returning the transduced cell to the subject.

[0359] While the foregoing disclosure has been described in some detail for purposes of clarity and the preambles of the appended claims, certain changes and modifications might naturally occur to one skilled in the art, to which the teachings of this disclosure are susceptible. The following examples are provided by way of illustration and not by way of limitation. One skilled in the art will readily recognize a variety of noncritical parameters that can be changed or modified to yield essentially the same results.

[0360] EMBODIMENTS

[0361] EMBODIMENT 1

[0362] An engineered dominant-negative TGF-β receptor type 1 (DN TGF-βRI) construct was designed and synthesized based on the sequence SEQ ID NO:10. This construct is 152 amino acids in length and includes: a signal peptide domain of amino acids 1 to 33 from wild-type TGF-βRI; an extracellular domain of amino acids 34 to 126 from wild-type TGF-βRI; a transmembrane domain of amino acids 127 to 147 from wild-type TGF-βRI; and an intracellular domain of amino acids 148 to 152 from TGFβ-RII. The construct was designed to omit key phosphorylation sites for intracellular pSMAD signaling. The intracellular domain is specifically derived from TGFβ-RII and contains five amino acids.

[0363] In addition, engineered truncated dominant-negative TGF-β receptor type 2 (DN TGF-βRII) was designed and synthesized based on sequence SEQ ID NO:14.

[0364]

[0365] The TCR and CAR constructs used in the following examples were designed and manufactured using common techniques known in the art. Two different CAR constructs with two different co-stimulatory domains (CD28 and 4-1BB) are shown below:

[0366] a.CD19: (FMC63 scFv + CD28 intracellular domain + CD3ζ intracellular domain)

[0367] b.PSMA: (PSMA scFv+4-1BB intracellular domain+CD3ζ intracellular domain).

[0368] Lentiviral vectors were used for all T-cell transductions. The EF1A promoter was used with all constructed constructs and tested.

[0369] STEMCELL cells were isolated from peripheral blood mononuclear cells obtained from healthy donors. TM CD3 from Technologies(Vancouver,Canada) + Cells, and Cell cryopreservation medium (Sigma) Frozen in lentivirus. CD3 pan-T cells were thawed prior to lentiviral transduction and used for CD3 / CD28. (ThermoFisher Scientific) were activated according to the manufacturer’s recommendations and then rested overnight. The next day, cells were transduced with lentivirus containing myc-tagged CAR constructs with and without a dominant negative receptor (DNR) as described herein. Cells were grown in TC media (X-VIVO 15, Lonza) with 5% human serum for 14 days, supplemented with 100 international units / ml of interleukin-2 (IL-2) three times per week. After 14 days of cell culture, cells in all experiments were approximately 50-80% positive for the transgene as measured by flow cytometry. TM ,Lonza) for 14 days, supplemented with 100 international units / ml of interleukin-2 (IL-2) three times per week. After 14 days of cell culture, cells in all experiments were approximately 50-80% positive for the transgene as measured by flow cytometry.

[0370] All flow cytometry data were collected on a LSR-Fortessa (BD LSR Fortessa TM ) with BD FACSDiva TM software and analyzed using FlowJo (all software from BD Sciences). All antibody stainings were performed in PBS with 1% BSA at 4°C. Reagents from BioLegend and Cell Signaling Technology were used to assess cell viability, CD3 + , CD4 + , CD8 + , and anti-myc activity to determine % CAR positive using common techniques. For expansion assays, CAR cells were counted with counting beads from ThermoFisher Scientific.

[0371] Equal numbers of T cells were cultured in serum-free X-VIVO TM 15 media for two hours, after which they were stimulated with 1 or 5 ng / mL soluble recombinant TGF-β1 reconstituted in acid for 30 minutes as recommended by the manufacturer (R&D Systems). Cells were then collected by centrifugation and lysed with RIPA buffer + protease and phosphatase inhibitors (ThermoFisher Scientific) before being analyzed for pSMAD2 and pSMAD3.

[0372] To investigate whether expression of a truncated form of TGF-βRI (DN TGF-βRI) could rescue T cells from the inhibitory effects of TGF-β, a construct was designed to encode a DN TGF-βRI of SEQ ID NO: 10 with a CAR with CD19 and PSMA-specific scFv followed by a T2A self-cleaving peptide. A DN TGF-βRII of SEQ ID NO: 14 served as a positive control. ​

[0373] By eliminating the intracellular portion of the TGF-βRI molecule that inhibits pSMAD signaling, it was shown that the truncated form of TGF-βRI (DN TGF-βRI) outcompetes the binding of endogenous wild-type TGF-βRI to TGF-βRII, thereby blocking signal transduction and limiting the inhibitory pSMAD signaling induced by TGF-β1, thus rescuing CAR cell function. TGF-β mediates inhibitory signaling in T cells via the pSMAD signaling pathway. To determine whether the truncated dominant negative receptors described herein can limit pSMAD induction, CD3 + Cells were isolated from primary human peripheral blood mononuclear cells (PBMCs) and transduced with the following constructs: CD19 CAR (FMC63 scFv + CD28 intracellular domain + CD3ζ intracellular domain) only, CD19 CAR + DN TGF-βRI, or CD19 CAR + DN TGF-βRII. Next, total T cells were stimulated with TGF-β1 at 1 ng / mL for 30 minutes, whole cell lysates were prepared, and pSMAD2 and pSMAD3 were quantified. The results are described in Table 4a.

[0374]

[0375] The background levels of pSMAD2 and pSMAD3 levels in all engineered T cells are shown in Table 4a. CD19 CAR-expressing T cells stimulated with TGF-β1 induced pSMAD2 and pSMAD3 10,000-fold and 2,500-fold, respectively, while TGFβ1 -stimulated cells transduced with CD19 CAR + DN TGF-βRI or CD19 CAR + DN TGF-βRII induced less pSMAD2 and pSMAD3 (pSMAD2 induction was less than about 4-fold, and pSMAD3 induction was less than about 2-fold, p-value < 0.01). In addition, the levels of pSMAD2 and pSMAD3 induction in TGFβ1 -treated T cells transduced with CD19 CAR + DN TGF-βRI or CD19 CAR + DN TGF-βRII were equivalent to the levels of pSMAD2 and pSMAD3 in control transduced T cells co-treated with TGFβ1 and the small molecule inhibitor TGF-βRI LY57299. This data confirms that DN TGF-βRI and DN TGF-βRII limit the inhibitory pSMAD signaling induced by TGF-β1 in T cells.

[0376] A second construct expressing a PSMA CAR (PSMA scFv + 4-1BB intracellular domain + CD3 zeta intracellular domain) followed by DN TGF-βRI or DN TGF-βRII was made. To determine if the truncated dominant negative receptor could limit pSMAD induction in this CAR context, CD3 + Cells were isolated from primary human peripheral blood mononuclear cells (PBMCs) and transduced with the following constructs: containing only a PSMA CAR, containing PSMA CAR + DN TGF-βRI or PSMA CAR + DN TGF-βRII. Total T cells were stimulated with TGF-β1 (5 ng / mL for 30 minutes), whole cell lysates were prepared, and pSMAD2 and pSMAD3 were quantified by pSMAD2 and pSMAD3 were quantified. Results are described in Table 4b.

[0377]

[0378] TGF-β1 stimulated PSMA CAR only expressing T cells induced pSMAD2 and pSMAD3 8,500-fold and 2,200-fold, respectively, while TGF-β1 stimulated cells transduced with PSMA CAR + DN TGF-βRI or PSMA CAR + DN TGF-βRII induced less pSMAD2 and pSMAD3 (less than about 2-fold induction, p-value < 0.05). Thus, the data in Table 4a and Table 4b confirm that DN TGF-βRI and DN TGF-βRII limit TGF-β1 mediated pSMAD signaling in CAR T cells expressing two different scFv and combined with costimulation of both CD28 and 4-1BB.

[0379] EMBODIMENT 2

[0380] CD19 CAR positive T cells were co-cultured with Nalm6 target cells from American Type Culture Company (ATCC, Manassas, VA) treated with mitomycin (Sigma Aldrich) at a 1 : 1 ratio for seven days. The number of CAR + T cells was measured by counting beads and flow cytometry every other day with target cells added at a 1 : 1 ratio. For groups treated with TGFβ1, acid-activated TGFβ1 (5 ng / mL) was added to the co-culture media (RPMI + 10% FBS) every other day.

[0381] Although in vivo CAR T-cell efficacy in mouse tumor models is generally considered the gold standard for asserting clinical efficacy, it has been established that repeated stimulation with the target in long-term in vitro killing assays often correlates with in vivo results. Therefore, a series of stimulation assays were used to measure CD19 CAR T-cell expansion and to test whether DN TGF-βRI and DN TGF-βRII could limit the inhibitory effect of TGF-β1 in this assay.

[0382] To determine the effect of TGF-β1 on CD19 CAR T cell function, CD19 was used over a week. + Nalm6 target cells stimulated CD19 CAR T cells three times, and target-mediated CAR T cell expansion was measured. The results are described in Table 5.

[0383]

[0384] The results showed that TGF-β1 inhibited CD19CAR T cell expansion by approximately 7-fold in this assay compared to treatment with the mediator alone. To determine whether DN TGF-βRI or DN TGF-βRII could rescue CD19 CAR T cell function in this repetitive stimulation assay using TGF-β1, T cells were transduced with CD19 CAR + TGF-βRI or CD19 CAR + TGF-βRII, and their expansion was measured seven days after a series of target stimulations in both the presence and absence of TGF-β1. The results indicated that TGF-βRI rescued the inhibitory effect of TGF-β1 on CD19 CAR T cell expansion.

[0385] EMBODIMENT 3

[0386] CAR-positive T cells were co-cultured with K562 target cells obtained from American Type Culture Company (ATCC, Manassas, VA), which were engineered to express prostate-specific membrane antigen (PSMA). After 96 hours of co-culture, the cells were centrifuged and the supernatant was collected. Interferon-γ (IFNγ) was analyzed in the supernatant during multiplexing assays, and then... Analysis was performed on a ThermoFisher Scientific system. For some groups, acid-activated TGF-β1 was added to the culture medium at 5 ng / mL at the start of the experiment.

[0387] CAR T cells mediate their cytolytic activity against tumor cells by secreting cytotoxic molecules, including IFNγ. The effects of exogenous TGF-β1 on PSMA were tested. +The impact of PSMA CAR T cells cultured with target cells on IFNy production. Results are described in Table 6.

[0388]

[0389] It was found that exogenous TGF-β1 administered at 5 ng / mL every two days reduced the IFNy production by CAR T cells to less than one third over 96 hours. In addition, it was found that TGF-β treated T cells transduced with PSMA CAR+DN TGF-βRI or PSMA CAR+DN TGF-βRII produced more IFNy in response to PSMA+target cells + IFNy production was not reduced in response to target cells and produced more IFNy than PSMA CAR T cells treated with TGF-β1 and co-cultured with PSMA+target cells. This data indicates that the DN TGF-β receptors restored the functional properties of both CD19 CAR T cells and PSMA CAR T cells challenged by TGF-β1 inhibition.

[0390] EMBODIMENT 4

[0391] Additional experiments were performed using DN TGF-βRI of SEQ ID NO: 10 and DN TGF-βRII of SEQ ID NO: 14, each with the CAR construct shown below:

[0392] a. GPC3: (GPC3 scFV + CD28 intracellular domain + CD3ζ intracellular domain); and

[0393] b. GPC3: (GPC3 scFV + 4-1BB intracellular domain + CD3ζ intracellular domain), SEQ ID NO: 45. Similar to the previous experiments, CD3 +Cells were isolated from primary human peripheral blood mononuclear cells (PBMCs) and transduced with the GPC3 constructs described above. Background levels of pSMAD2 levels and pSMAD3 levels in all engineered T cells are shown in Table 7. TGF-βΙ -stimulated GPC3 CAR-expressing T cells induced pSMAD2 and pSMAD3 by 10,000-fold and 700-fold, respectively, while TGF-βΙ -stimulated cells transduced with GPC3 CAR + DN TGF-βRII (shown in SEQ ID NO: 47) induced pSMAD2 and pSMAD3 significantly less (pSMAD2 induction was approximately less than 20-fold, and pSMAD3 induction was approximately less than 4-fold, p-value < 0.01). GPC3 CAR + DN TGF-βRII did limit induction of pSMAD2 and pSMAD3 (pSMAD2 induction was approximately less than 3-fold when co-stimulated with 4-1BB; and pSMAD3 induction was approximately less than 1.3-fold when co-stimulated with 4-1BB). Results are described in Table 7.

[0394]

[0395] In addition, GPC3 CAR-positive T cells were co-cultured with Hep3B target cells from American Type Culture Company (ATCC, Manassas, VA). Target cells were added every three to four days twice a week and the number of CAR + T cells was measured by counting beads and flow cytometry. For groups treated with TGF-βΙ, acid-activated TGF-βΙ (5 ng / mL) was added to the co-culture media (RPMI + 10% FBS) every three to four days twice a week. Results are described in Table 8.

[0396]

[0397]

[0398] EMBODIMENT 5

[0399] In addition to the experiments described above, DN TGF-βRI and DN TGF-βRII were also evaluated in the CD19, PSMA, and GPC3 constructs described herein in an in vivo animal model. This study evaluated the receptors in pSMAD reduction, CAR cell expansion, and cytokine production, as described herein.

[0400] EMBODIMENT 6

[0401] An engineered dominant negative TGF-beta receptor type 2 (DN TGF-betaRII) construct was made according to sequence SEQ ID NO:33. The construct has a length of 394 amino acids and comprises: a signal peptide domain at amino acids 1 to 22; a tag sequence at amino acids 23 to 32; an extracellular domain at amino acids 33 to 169, which is wild-type TGF-betaRII; a linker sequence at amino acids 170 to 171; a transmembrane domain from IL-7Ra at amino acids 172 to 194, which is SEQ ID NO:20 with a "CPT" insertion; and an intracellular domain from IL-7Ra at amino acids 195 to 394.

[0402] CD3 + cells isolated from human PBMCs were transduced with the construct comprising the amino acid sequence SEQ ID NO:33. T cells were cultured for two hours in X-VIVO TM medium without serum, after which they were stimulated with 1 ng / mL soluble recombinant TGF-beta 1 reconstituted in acid for 30 minutes as recommended by the manufacturer (R&D Systems). Cells were then collected by centrifugation and lysed with RIPA buffer + protease and phosphatase inhibitors (ThermoFisher Scientific) before being analyzed by Western blot. pSMAD2, pSMAD3, pERK, and pSTAT5 were analyzed. Background levels of pSMAD2 and pSMAD3 levels were very low in all engineered T cells. TGF-beta 1 -stimulated T cells induced pSMAD2 and pSMAD3 4,000-fold and 1,500-fold, respectively, while TGF-beta 1 -stimulated cells transduced with the DN TGF-betaRII construct induced significantly less pSMAD2 and pSMAD3 (pSMAD2 was induced by less than about 25-fold and pSMAD3 was induced by less than about 11 -fold, p-value < 0.01 ), while pERK and pSTAT5 signaling in chimerically transduced T cells was elevated (pERK was elevated by 28-fold and pSTAT5 was elevated by almost 80-fold). Increased pERK and pSTAT signaling was independent of TGF-beta 1 contact and was elevated in both with and without contact due to constitutive signaling mediated by IL-7Ra. Results are described in Table 9.

[0403]

[0404] EMBODIMENT 7

[0405] The codon-optimized sequence of the CAR construct targeting GPC3, SEQ ID NO: 51 (CAR 1), was compared to two additional CAR constructs targeting GPC3. The experiment was performed by generating the CAR constructs shown below:

[0406] a. Construct CAR 1: (GPC3 scFv + 4-1BB intracellular domain + CD3 zeta intracellular domain), shown in codon-optimized sequence SEQ ID NO: 51.

[0407] b. Construct CAR 2: (GPC3 scFv + 4-1BB intracellular domain + CD3 zeta intracellular domain, as described in Li, Gastroenterology, 2020 and WO2019094482A1), having the nucleotide sequence shown in SEQ ID NO: 53.

[0408]

[0409]

[0410] c. Construct CAR 3: (construct with transgene having the same amino acid sequence as CAR 2, but where the nucleotide sequence has been optimized to minimize splicing and repeats), shown in SEQ ID NO: 54.

[0411]

[0412]

[0413] Similar to the previous experiment, CD3 + cells were isolated from primary human peripheral blood mononuclear cells (PBMCs) and transduced with the GPC3 CAR constructs described above at a multiplicity of infection of 2 in the presence of CS5 and stored in liquid nitrogen until thawed. After thawing, the cells were rested in T cell media with 100 UI / mL IL-2 overnight, washed, and stained in PBS with Live / Dead Violet diluted 1:1000, recombinant human GPC3 (rhGPC3) conjugated to DyLight 650 diluted 1:200, and anti-CD3 BV650 diluted 1:100 at 4°C for 30 minutes. DNA pellets were extracted in QuickExtract Buffer and 1 x 10 TM TM TM 5 ​​​Cells were frozen and used to measure the average vector copy number (VCN) by quantifying the frequency of viral integration in each cell via droplet digital PCR. + The percentage of cells was defined as Live / Dead TM Negative, CD3 + The percentage of positive cells stained with rhGPC3, and CAR is given in this example. + The mean fluorescence intensity (MFI) and vector copy number (VCN) were also considered. As shown in Table 10, higher VCN and %CAR values ​​correlated with higher CAR values. + Construct CAR 1 showed higher transduction efficiency than constructs CAR2 and CAR 3. Construct CAR 1 also exhibited higher CAR efficiency. + Cellular MFI, which can indicate that the CAR is expressed more efficiently and has a higher affinity for rhGPC3.

[0414]

[0415] EMBODIMENT 8

[0416] CARs that have been manufactured, frozen, left to stand, and stained as previously described + T cells (5×10) 4 (5 × 10) and HCC target cells (5 × 10) 4 (Number of cells) were co-cultured for 48 hours to induce cytokine release, and the supernatant was frozen at -80°C. The supernatant was then diluted 500-fold in PBS and analyzed using Millipore... The Map Human High Sensitivity T cellPanel (catalog number HSCMAG-28PMX13BK) was used to measure interferon-γ (IFNγ) concentration. The data in Table 11 show that, in the presence of GPC3-cell lines Hep3B GPC3 KO and Sk-Hep1, the CAR 1 construct secreted less IFNγ compared to the CAR 2 and CAR 3 constructs. This data indicates that the CAR 1 construct induced less tetanic activation compared to the CAR 2 and CAR 3 constructs.

[0417]

[0418] EMBODIMENT 9

[0419] As described above, T cells transduced with or without CAR 1, CAR 2, and CAR 3 constructs were manufactured and tested in vivo in a Hep3B xenograft model in NSG mice. On day one of the experiments, 2 × 10⁶ cells were... 6Hep3B 2.1-7 cells were implanted into NSG mice. Thereafter, every three to four days, tumor volume was measured using calipers. At day 14, when tumors reached an average size of 149 mm 3 6 + 6 + 6 6

[0420]

[0421]

[0422]

[0423]

[0424]

[0425]

[0426]

[0427]

[0428]

[0429]

[0430]

[0431] EMBODIMENT 10

[0432] The GPC3 CAR-DN TGF-βRII construct was manufactured as set forth in SEQ ID NO: 47 and tested on Hep3B tumor cells. On the first day of the experiment, 2 x 10 6 null Hep3B 2.1-7 cells were implanted into NOD-SCID IL-2Rγ​​​​​​​(NSG) mice. Tumor volume was measured every three to four days thereafter using calipers. At day 14, when tumors reached an average size of 150 mm 3 6 + 6 + 3 6 +

[0433]

[0434]

[0435]

[0436]

[0437]

[0438]

[0439]

[0440]

[0441]

[0442] EMBODIMENT 11

[0443] On the last day of the study (day 33) for all cohorts, the significance of anti-tumor efficacy was evaluated by one-way ANOVA and Tukey’s multiple comparison test using the constructs of Example 10 and is included in Table 26. Long-term efficacy and tumor growth delay (TGD) was determined by progression to a tumor volume endpoint set at four times the initial volume (Table 27), with statistical significance determined by the log-rank (Mantel-Cox) test (Table 28). FIG. 1

[0444] ​​​​​​​​​

[0445] Statistical significance of tumor volume data from Table 10 through Table 15 at day 33 was evaluated in all groups using analysis of variance and using Tukey’s post-hoc test that crossed between all groups. Comparisons were indicated as having significance (p < 0.05) with “yes” and as having no significance with “ns”.

[0446]

[0447] Statistical survival to the endpoint of tumor reaching 4 times the initial volume was indicated by the number of animals that reached this and the day of study. Long-term efficacy (defined by failure to reach this endpoint) was depicted by NR (not reached).

[0448]

[0449] Statistical significance of survival data from Table 16 was evaluated using log-rank (Mantle-Cox) pairwise comparisons. Comparisons were indicated as having significance (p < 0.05) with “yes” and as having no significance with “ns”.

[0450] At day 33, GPC3 CARs were effective at controlling tumor volume and had a dose-dependent effect on efficacy, as shown in Table 26. At 1 x 10 6 The addition of DN TGF-βRII had a significant impact on tumor efficacy under the CAR. At 4 x 10 6 This effect was not seen at day 33 at the 4 x 10 6 and 4 x 10 6 both had a significant impact on long-term efficacy.

[0451] EMBODIMENT 12

[0452] Additional experiments were performed to compare CAR-DN TGF-βRII constructs generated from codon-optimized sequences to similar constructs generated from non-codon-optimized sequences. The following CAR-DN TGF-βRII constructs were generated:

[0453] a. Construct CAR DNR A: (GPC3 scFv + 4-1BB intracellular domain + CD3 zeta intracellular domain, T2A self-cleaving motif, and DN TGF-βRII), which is set forth in SEQ ID NO: 52.

[0454] b. Construct CAR DNR B: (GPC3 scFv + 4-1BB intracellular domain + CD3 zeta intracellular domain as described in Li, Gastroenterology, 2020 + WO2019094482A1, T2A and DN TGF-beta RII), which is SEQ ID NO: 55.

[0455]

[0456]

[0457] Similar to previous experiments, CD3 + cells were isolated from primary human peripheral blood mononuclear cells (PBMCs) and transduced with the GPC3 CAR constructs described above at a multiplicity of infection of 2 in the presence of polybrene and frozen at the end of manufacture in CS5 and stored in liquid nitrogen until thawed. After thawing, cells were rested overnight in T cell media with 100 UI / mL IL-2, washed, and stained in PBS with Live / Dead TM Violet at 1:1000 dilution, recombinant human GPC3 (rhGPC3) at 1:200 dilution conjugated to DyLight TM 650 fluorescence, and anti-TGF-beta RII-PE and anti-CD3 BV650 at 1:100 dilution for 30 minutes at 4°C. DNA pellets were extracted in QuickExtract TM buffer and 1 x 10 5 cells were frozen and used to measure average vector copy number (VCN) by droplet digital PCR quantifying the frequency of integrated virus in each cell. The percentage of CAR + cells was defined as the percentage of CD3 TM cells that were Live / Dead + negative and stained with rhGPC3, and the mean fluorescence intensity (MFI) for CAR + is given in this example, as well as the vector copy number (VCN). Construct CAR DNR A had a higher CAR + cell MFI, which can indicate that this CAR is more efficiently expressed and has a higher affinity for rhGPC3.

[0458]

[0459] EMBODIMENT 13

[0460] CAR+ T cells (5 x 10 4 cells were co-cultured with HCC target cells (5 x 10 4 cells for 48 hours to induce cytokine release, and supernatants were frozen at -80°C. After dilution of the supernatants 500-fold in PBS, interferon gamma (IFNy) concentrations were measured using Map Human High Sensitivity T cell Panel (Catalog No. HSTCMAG-28PMX13BK). The data in Table 30 demonstrate that construct CAR DNR A secretes less IFNy than construct CAR DNR B in the presence of GPC3-cell...

Claims

1. A recombinant nucleic acid comprising a sequence encoding a chimeric antigen receptor (CAR), wherein the sequence encoding the CAR consists of the nucleotide sequence of SEQ ID NO:

51.

2. The recombinant nucleic acid of claim 1, further comprising a sequence encoding a DN TGF-βRII, wherein the sequence encoding the DN TGF-βRII consists of the nucleotide sequence of SEQ ID NO:

48.

3. The recombinant nucleic acid of claim 2, which encodes a polypeptide consisting of the amino acid sequence of SEQ ID NO:

47.

4. An expression vector comprising the recombinant nucleic acid of any one of claims 1-3.

5. A T cell transduced with the recombinant nucleic acid of any one of claims 1-3 or with the expression vector of claim 4.

6. Use of the T cell of claim 5 in the manufacture of a medicament for treating a liver cancer.

7. The use of claim 6, wherein the liver cancer is a hepatocellular carcinoma.

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