Compositions and methods for enhancing immune cell therapies

Abstract

Aspects of the disclosure include modified T cells that are MED12K0 and mbIL-12+, and preferably naive-like stem cell memory T cells that are CCR7+, CD62L+, CD45RA+, enriched populations of said cells, and methods of making and using same, including to extend the persistence and / or longevity of the modified immune cells in vivo.

Description

COMPOSITIONS AND METHODS FOR ENHANCING IMMUNE CELL THERAPIESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 738,411, filed December 23, 2024, and U.S. Provisional Application No. 63 / 903,767, filed October 22, 2025. The entire contents of the above-identified applications are hereby fully incorporated herein by reference.FIELD OF DISCLOSURE

[0002] The present invention provides modified T cells expressing a naive-like stem cell memory phenotype, cell populations enriched for same, and methods of making and using same.BACKGROUND OF THE DISCLOSURE

[0003] Adoptive cellular therapy has undergone near constant iteration for more than thirty (30) years, from early days focusing on basic lymphokine activation and / or tumor infiltration to more recent strategies engineering immune cells to express genetically engineered antigen receptors, such as chimeric antigen receptors (CARs). While there have been some hints and indications of the curative potential of these approaches along the way, much still remains to be done. In particular, and regardless of whether they have been engineered or merely expanded ex vivo, infused immune cells often become functionally exhausted, and in the tumor microenvironment in particular. Gattinoni et al. Acquisition of full effector function in vitro paradoxically impairs the in vivo antitumor efficacy of adoptively transferred CD8+ T cells J. Clin Invest. 2005; 115:1616-26; Jiang et al. T cell exhaustion in the tumor microenvironment Cell Death Dis. 2015:6:el792. Accordingly, restoration and maintenance of T cell function and persistence in adoptive cell therapies represents a significant unmet need that could improve longterm outcomes for patients.

[0004] Stem cell memory T cells expressing naive T cell surface markers have been identified possessing highly proliferative and long-term survival capabilities, and producing a large number of effector T cells in response to antigen stimulation. Gattinoni et al., A human memory T cell subset with stem cell-like properties A7 / / . Med. 2011; 17:1290-1297. doi: 10.1038 / nm.2446. These11106305893\l\AMERICAScells are endowed with considerable proliferative reserves and can differentiate in vitro and in vivo to reconstitute the entire spectrum of classically delineated memory T cells, suggesting that they may be the “stem” of memory T cells. Id., Kondo et al., Generation and application of human induced-stem cell memory T cells for adoptive immunotherapy, Cancer Sci 2018; 109(7):2130-2140 doi: 10.1111 / cas.13648.

[0005] Given their superior attributes as multipotent progenitors that can both self-renew and replenish more differentiated subsets of memory T cells, these cells have become highly desirable for adoptive immune cell therapies. Indeed, the longevity exhibited by this cell population and the robust potential for immune reconstitution have been associated with improved efficacy in both adoptive T cell transfers and CAR T cell therapies. Gattinoni et al., T memory stem cells in health and disease, Nat. Med. 2017; 23:18-27; Arcangeli et al., CAR T cell manufacturing from naive / stem memory T-lymphocytes enhances antitumor responses while curtailing cytokine release syndrome, J. Clin Invest 2022; 132:el50807.

[0006] Notably, however, the extended cell culture periods typically employed in conventional CAR T manufacturing protocols can deplete this highly desired T cell subset and thereby diminish antitumor activity. Dickinson et al., A novel autologous CAR T therapy, YTB323, with preserved T-cell sternness shows enhanced CAR T cell efficacy in preclinical and early clinical development, Cancer Discov 2023; 13:1982-97. As such, myriad efforts have recently been made to reduce the manufacturing time in an effort to maintain the population of these cells. Id., Ghassemi et al., Rapid manufacturing of non-activated potent CAR T cells, Nat Biomed Eng 2022; 6:118-28. Doing so, however, can compromise the resulting cell population in other ways.

[0007] A wide variety of TCR stimulation and cytokine-based expansion protocols have also been explored in an effort to create and / or enhance this particular phenotype in immune cell populations. Kondo, supra. To date, however, none of these has proven to be reproducible or robust enough for clinical implementation. What is needed, then, are alternative compositions and methods for creating populations of naive and stem cell memory T cells. The present invention addresses this and other unmet needs.SUMMARY OF DISCLOSURE

[0008] The present disclosure derives from the surprising discovery that disrupting Mediator of RNA polymerase II transcription subunit 12 (MED12) and expressing a membrane- 21106305893\l\AMERICASbound interleukin- 12 (mbIL-12) in an immune cell, e.g. a T cell, can lead to a naive-like stem cell memory phenotype in the cell, and can dramatically increase the persistence of the resulting modified T cell in vivo. Similarly, a modified T cell comprising disruption of MED12 gene can also demonstrate increased persistence in the presence of soluble IL-12, either ex vivo or in vivo. In embodiments, the modified T cell is a naive-like stem cell memory T cell, preferably wherein the naive-like stem cell memory T cell is or comprises CCR7+, CD62L+, and CD45RA+. The naive-like stem cell memory T cell may also be CD45RO- or CD45RO+, depending on the state of T cell receptor (TCR) activation, as detailed herein. Also provided are methods of making and using same.

[0009] In one aspect, the invention provides an isolated immune cell modified to disrupt MED12 (MED12K0) and to express membrane-bound IL-12 (mbIL-12+). In embodiments, the modified immune cell is a T cell, e.g., an a[3 or a y8 T cell, preferably a yd T cell. In embodiments, the y8 T cell is a 81, a 82, a 83, or a 84 y8 T cell, preferably a 82- y8 T cell, more preferably a 81 y8 T cell. In embodiments, the modified T cell is a naive-like stem cell memory T cell, preferably wherein the modified T cell comprises, or is, CCR7+, CD62L+, CD45RA+, and CD45RO+ or CD45RO-. In embodiments, the modified T cell is CD45RO+. In embodiments, the modified T cell is CD45RO-. In embodiments, the modified T cell further comprises, or is, one or more of CD4+, CD71+, CD49b+, CD123+, CD109+, CD80+, CD86+, CD36L1+ and / or CD151+. In embodiments, the modified T cell is CD4+.

[0010] In embodiments, the immune cell displays tumor specificity. In embodiments, the immune cell has been isolated from a tumor of a subject, preferably wherein the immune cell is a tumor infiltrating lymphocyte. In embodiments, the immune cell comprises an antigen recognition moiety expressed on a surface of the cell, wherein the antigen recognition moiety comprises an affinity binding domain specific for a disease-associated antigen. In embodiments, the antigen recognition moiety is selected from the group consisting of a TCR, a[3 TCR, y8 TCR, a chimeric antigen receptor (CAR), whole antibody or their antigen-binding fragment, single-chain variable fragment (scFv), a heavy chain-only antibody (VHH), a heavy chain or a light chain single domain antibody (sdAb), a Fab, a F(ab)2, or any combination thereof that binds to: (i) a cell surface tumor antigen, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune31106305893\l\AMERICASdisease or a pathogen, or (iv) a peptide derived from an antigen associated with an autoimmune disease or a pathogen expressed on the cell surface as a peptide-MHC complex.

[0011] In embodiments, the antigen recognition moiety is a CAR. In embodiments, the CAR further comprises a transmembrane (TM) domain, a hinge domain, a co-stimulatory domain, and an intracellular signaling domain. In embodiments, the hinge domain is a CD8a hinge domain, and / or the TM domain comprises a TM domain of CD8a. In embodiments, the co-stimulatory domain is a 4- IBB domain or a CD28 domain, and / or the intracellular signaling domains is aCD3(^ intracellular signaling domain. In embodiments, the disease-associated antigen is a tumor antigen, an autoimmune disease-associated antigen, or a pathogen antigen.

[0012] In another aspect, the present disclosure provides a modified T cell comprising: a) an antigen recognition moiety expressed on a surface of the cell, wherein the antigen recognition moiety comprises an affinity binding domain specific for a disease-associated antigen; b) at least one modification to modulate cytokine activity comprising expression of a membrane-bound IL-12; and c) at least one modification to modulate T cell metabolism comprising disruption of MED12 gene. In embodiments, the modified T cell is an aP or a y8 T cell, preferably a y8 T cell. In embodiments, the y8 T cell is a 81, a 82, a 83, or a 84 y8 T cell, preferably a 82- y8 T cell, more preferably a 81 y8 T cell. In embodiments, the modified T cell is MED 12 KO and mblL-12+. In embodiments, the modified T cell further comprises, or is, CCR7+, CD62L+, CD45RA+, and CD45RO+ or CD45RO-. In embodiments, the modified T cell is CD45RO+. In embodiments, the modified T cell is CD45RO-. In embodiments, the modified T cell further comprises, or is, one or more of CD4+, CD71+, CD49b+, CD123+, CD109+, CD80+, CD86+, CD36L1+ and / or CD151+. In embodiments, the modified T cell is CD4+.

[0013] In embodiments, the modified T cell comprises an antigen recognition moiety expressed on a surface of the cell, wherein the antigen recognition moiety comprises an affinity binding domain specific for a disease-associated antigen. In embodiments, the antigen recognition moiety is selected from the group consisting of a TCR, ccfJ TCR, y8 TCR, a chimeric antigen receptor (CAR), whole antibody or their antigen-binding fragment, single-chain variable fragment (scFv), a heavy chain-only antibody (VHH), a heavy chain or a light chain single domain antibody (sdAb), a Fab, a F(ab)2, or any combination thereof that binds to: (i) a cell surface tumor antigen, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune disease or a41106305893\l\AMERICASpathogen, or (iv) a peptide derived from an antigen associated with an autoimmune disease or a pathogen expressed on the cell surface as a peptide-MHC complex.

[0014] In embodiments, the antigen recognition moiety is a CAR. In embodiments, the CAR further comprises a transmembrane (TM) domain, a hinge domain, a co-stimulatory domain, and an intracellular signaling domain. In embodiments, the hinge domain is a CD8a hinge domain, and / or the TM domain comprises a TM domain of CD8a. In embodiments, the co-stimulatory domain is a 4- IBB domain or a CD28 domain, and / or the intracellular signaling domains is aCD3(j intracellular signaling domain. In embodiments, the disease-associated antigen is a tumor antigen, an autoimmune disease-associated antigen, or a pathogen antigen.

[0015] In another aspect, the invention provides a cell population enriched for naive-like stem cell memory T cells having a CCR7+ CD62L+ CD45RA+ phenotype, wherein the T cells are genetically modified to disrupt MED12 and to express membrane-bound IL-12, or are genetically modified to disrupt MED 12 and contacted with IL-12 ex vivo or in vivo. In embodiments, the modified T cells are CD45RO+. In embodiments, the modified T cells are CD45RO-. In embodiments, the modified T cells are y8 T cells, optionally wherein the y8 T cells are 81, 82, 83, or 84 y8 T cells, preferably 82- y8 T cells, more preferably 81 y8 T cells. In embodiments, the modified T cells are a.p T cells. In embodiments, the modified T cells further comprise one or more of CD4+, CD71+, CD49b+, CD123+, CD109+, CD80+, CD86+, CD36L1+ and / or CD151+. In embodiments, the modified T cells are CD4+.

[0016] In another aspect, the invention provides a pharmaceutical composition comprising a plurality of the foregoing modified T cells, and / or or cell populations. In embodiments, the plurality of modified T cells comprise a composition that is greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90% CCR7+ T cells. In embodiments, the plurality of T cells comprises at least about 107y8 T cells, optionally from about 108to about 1010y8 T cells. In embodiments, the plurality of T cells comprises at least about 107a0 T cells, optionally from about 108to about 1010a.p T cells. In embodiments, the plurality of modified T cells are y8 T cells. In embodiments, the plurality of modified y8 T cells comprise a composition that is at least about 60%, at least about 80%, or at least about 80% to about 90% 81 y8 T cells, and a pharmaceutically acceptable carrier.

[0017] In a further aspect, methods of treating or preventing a disease in a subject, comprising administering to a subject in need thereof the foregoing pharmaceutical compositions. In51106305893\l\AMERICASembodiments, the disease is an autoimmune disease. Tn embodiments, the disease is a cancer or a precancerous condition.

[0018] In a still further aspect, methods for enriching a population of isolated T cells for naive-like stem cell memory T cells having a CCR7+ CD62L+ CD45RA+ phenotype are provided, wherein the isolated T cells are genetically modified to disrupt MED12 and to express membranebound IL-12. In embodiments, the isolated T cells are genetically modified to disrupt MED12 and contacted with IL-12 ex vivo or in vivo. In embodiments, the modified T cells are CD45RO+. In embodiments, the modified T cells are CD45RO-. In embodiments, the modified T cells are CD4+. In embodiments, the modified T cells further comprise an antigen recognition moiety expressed on a surface of the cell, wherein the antigen recognition moiety comprises an affinity binding domain specific for a disease-associated antigen. In embodiments, the modified T cells are tx|3 T cells or y5 T cells, preferably y5 T cells. In embodiments, the y5 T cells are 51, a 52, a 53, or a 54 75 T cells, preferably 52- y5 T cells, more preferably 51 y5 T cells.

[0019] In an additional aspect, methods of increasing the longevity / persistence of an immune cell are provided, comprising disrupting a MED12 gene in the cell and expressing a membranebound IL-12 in the cell, thereby increasing the persistence of the modified immune cell in vitro and / or in vivo. In embodiments, the immune cell is genetically modified to disrupt MED 12 and contacted with IL- 12 ex vivo or in vivo. In embodiments, the modified immune cell comprises an antigen recognition moiety expressed on a surface of the cell, wherein the antigen recognition moiety comprises an affinity binding domain specific for a disease-associated antigen. In embodiments, the modified immune cell can persist at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 days, from first contact with a disease cell or from a date of administration of the modified immune cell to the host organism. In embodiments, the modified immune cell is an ap T cell or a y5 T cell; preferably wherein the T cell is a y5 T cell. In embodiments, the y5 T cell is a 51, a 52, a 53, or a 5475 T cell, preferably a 82- y5 T cell, more preferably a 51 yd T cell.INCORPORATION BY REFERENCE

[0020] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.61106305893\l\AMERICASBRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 illustrates the development pathway of nai ve T cells into effector T cells, along with corresponding phenotype changes.

[0022] FIG. 2 illustrates the in vitro cytotoxicity of PSMA CAR-transduced yd T cells expressing mbIL-12 with gene-edits MED12 KO ± TGFpRII KO.

[0023] FIGS. 3A-3B illustrate the in vivo efficacy of PSMA CAR-transduced yd T cells expressing mbIL-12 with gene-edits MED 12 KO ± TGFpRII KO in a subcutaneous human xenograft PC3-PSMA model in NSG mice.

[0024] FIGS. 4A-4B illustrate and compare CCR7, CD62L, and CD45RA expression in PSMA-CAR+mb-IL-12+ yd T cells ± MED12K0.

[0025] FIGS. 5A-5C illustrate the expression of CCR7 in PSMA-CAR+mbIL-12 expressing yd T cells within ADI-212,

[0026] FIG. 6 illustrates CD45RA and CD45RO expression in ADI-212 and PSMA-CAR+mbIL-12 yd T cells.

[0027] FIG. 7 illustrates and compares CD4 expression in ADI-212 and P SMA-CAR+mb IL-12 yd T cells.

[0028] FIG. 8 illustrates and compares CD71 and CD49d expression in ADI-212 and PSMA-CAR+mbIL-12 yd T cells

[0029] FIG.9 illustrates and compares CD123 and CD109 expression in ADI-212 and PSMA-CAR+mbIL-12 yd T cells.

[0030] FIG. 10 illustrates and compares CD80 and CD86 expression in ADI-212 and PSMA-CAR+mbIL-12 yd T cells.

[0031] FIG. 11 illustrates and compares CD36L1 and CD151 expression in ADI-212 and PSMA-CAR+mbIL-12 yd T cells.

[0032] FIGS. 12A-12C illustrate and compare the gene expression profile of ADI-212 and PSMA-CAR+mbIL-12 yd T cells.71106305893\l\AMERICAS

[0033] FTG. 13 illustrates and compares the in vitro cytotoxicity of ADI-212 and PSMA-CAR+mbIL-12 y8 T cells in a repetitive tumor rechallenge assay.

[0034] FIGS. 14A-14D illustrate the in vitro expansion and cytotoxicity of ADI-212 and PSMA-CAR±mbIL-12 y8 T cells in the presence of T regulatory cells.

[0035] FIGS. 15A-15B illustrate the synergy of mbIL-12 and MED12 KO to enhance the in vitro cytotoxicity and proliferation of ADI-212.

[0036] FIGS. 16A-16D illustrate the in vivo efficacy of ADI-212 in a subcutaneous human xenograft PC3-PSMA model in NSG mice.

[0037] FIGS. 17A-17B illustrate and compare the in vitro cytotoxicity of CD4+and CD4" enriched ADI-212 cells.

[0038] FIGS. 18A-18B illustrate and compare the T cell memory phenotype of PSMA-CAR aP T cells ±mbIL-12 with and without MED 12 KO.

[0039] FIGS. 19A-19B illustrate and compare CD4 expression in PSMA-CAR aP T cells ±mbIL-12 with and without MED 12 KO.

[0040] FIGS. 20A-20B illustrate the in vitro cytotoxicity of PSMA-CAR MED 12 KO y8 T cells in the presence of exogenously added cytokines.DETAILED DESCRIPTION

[0041] The present invention provides genetically modified T cells having a naive-like stem cell memory phenotype and cell populations enriched for same, as well as methods of making and using such cells. In particular, and as demonstrated herein for the first time, combining disruption of MED 12 with expression of mbIL-12 in an immune cell provides remarkable functional advantages, including producing a naive-like stem cell memory phenotype in the modified cells. In the context of MED12K0 and mbIL-12+ y8 T cells in particular, this synergistic combination can increase tumor killing potential, enhance proliferation and survival, provide metabolic fitness advantages, and improve manufacturability. Additionally, and as further demonstrated herein, contacting a modified T cell comprising a MED12 gene disruption with IL-12 ex vivo or in vivo can also enhance T cell functionality and persistence.81106305893\l\AMERICASDEFINITIONS

[0042] For purposes of interpreting this specification, the following definitions will apply, and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0043] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0044] As used herein, “w / v” refers to the weight of the component in a given volume of solution.

[0045] “Ranges”: throughout this disclosure, various aspects of the disclosure can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0046] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal amenable to the methods described herein. In certain nonlimiting embodiments, the patient, subject or individual is a human.

[0047] As used herein, the term “agent” refers to any protein, nucleic acid molecule (including chemically modified nucleic acids), compound, antibody, small molecule, organic compound, inorganic compound, other molecule of interest, or cell (e.g., cell engineered to express a chimeric antigen receptor). Agent can include a therapeutic agent, a diagnostic agent or a pharmaceutical agent. A therapeutic or pharmaceutical agent is one that alone or together with an additional agent induces the desired response (such as inducing a therapeutic or prophylactic effect when91106305893\l\AMERICASadministered to a subject, including treating a subject suffering from cancer, or other disease / condition.

[0048] The term "therapeutically effective amount", or simply “effective amount” refers to the amount of an agent or composition (e.g., composition comprising an agent) that will elicit a biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes that amount of an agent, or a composition comprising an agent, that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease (e.g., cancer) being treated. The therapeutically effective amount will vary depending on the composition, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0049] The term “immune cell” refers to any cell that plays a role in the immune response of a subject. Immune cells may be of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, neutrophils, mast cells, basophils, and granulocytes. In embodiments, an immune cell is a T cell. In embodiments, an immune cell is a natural killer (NK) cell. In embodiments, an immune cell is a NKT (natural killer T) cell. In embodiments, an immune cell is a macrophage.

[0050] The term “T lymphocyte” or “T cell” refers to an immune cell that expresses or has expressed CD3 (CD3+) and a T Cell Receptor (TCR+). T cells play a central role in cell-mediated immunity. A T cell that “has expressed” CD3 and a TCR has been engineered to eliminate CD3 and / or TCR cell surface expression. In embodiments, a T cell is an a.p T cell. In embodiments, a T cell is an yb T cell. T cells herein include those used in adoptive cell therapies. In embodiments, T cells used in the adoptive cell therapies include tumor-infdtrating lymphocytes (TILs) (e.g., T cell comprising a tumor cell recognition moiety that is endogenously expressed by the isolated wild-type T cell such as isolated from tumor infiltrating lymphocytes of a tumor sample), virusspecific T cells (VSTs), TCR-T cells, CAR-T cells, CAR-NKT cells, and the like.

[0051] The term “yb T cells (gamma delta T cells)” as used herein refers to a subset of T cells that express a distinct T cell receptor (TCR), namely yb TCR, on their surface, composed of one y-chain and one b-chain. The term “yb T cells” specifically includes all subsets of yb T cells, including, without limitation, Vbl and Vb2, Vb3 yb T cells, as well as naive, effector memory, central memory, and terminally differentiated yb T cells. As a further example, the term “yb T101106305893\l\AMERICAScells” includes V84, V85, V87, and V88 y8 T cells, as well as Vy2, Vy3, Vy5, Vy8, Vy9, VylO, and Vyll y8 T cells. In embodiments, the y6 T cells are V81’, V62', or V81’ and V82'. Compositions and methods for making and using engineered and non-engineered y8 T cells and / or sub-types thereof include, without limitation, those described in US 2016 / 0175358; WO 2017 / 197347; US 9499788; US 2018 / 0169147; US 9907820; US 2018 / 0125889 and US 2017 / 0196910, the contents of each of which are incorporated by reference for all purposes, including the compositions and methods for making and using engineered and non-engineered y8 T cells and / or sub-types thereof. The present application further contemplates T cells, or other engineered leukocytes or lymphocytes, that express one y-chain or one 8-chain, optionally in combination with a second polypeptide to form a functional TCR. Such engineered leukocytes or lymphocytes, that express one y-chain or one 8-chain may be used in the methods or present in the compositions described herein.

[0052] The term “a0 T cell” refers to T cells expressing a and chains of the TCR as part of a complex with CD3 chain molecules. Each a and 0 chain contains one variable and one constant domain. a0 T cells primarily recognize peptide antigens presented by major histocompatibility complex (MHC) class I and class II molecules, where most of the receptor diversity is contained within the third complementarity determining region (CDR3) of the TCR a and 0 chains.

[0053] The term “TCR” or “T cell receptor” refers to a dimeric heterologous cell surface signaling protein forming an alpha-beta or gamma-delta receptor or combinations thereof. a0TCRs recognize an antigen presented by an MHC molecule, whereas y8TCR can recognize an antigen independently of MHC presentation.

[0054] The term "MHC" (major histocompatibility complex) refers to a subset of genes that encodes cell-surface antigen-presenting proteins. In humans, these genes are referred to as human leukocyte antigen (HLA) genes. Herein, the abbreviations MHC or HLA are used interchangeably.

[0055] The term “activation” refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division.

[0056] The term "chimeric antigen receptors (CARs)" refers to artificial T-cell receptors, T-bodies, single-chain immunoreceptors, chimeric T-cell receptors, or chimeric immunoreceptors, for example, and encompass engineered receptors that graft an artificial specificity onto a111106305893\l\AMERICASparticular immune effector cell. CARs may be employed to impart the specificity of a monoclonal antibody onto a T cell, thereby allowing a large number of specific T cells to be generated, for example, for use in adoptive cell therapy. In specific embodiments, CARs direct specificity of the cell to a disease associated antigen, (e.g., a tumor associated antigen, an autoimmune associated antigen, or a pathogenic antigen). In some embodiments, CARs comprise an intracellular activation domain (allowing the T cell to activate upon engagement of targeting moiety with target cell, such as a target tumor cell), a transmembrane domain, and an extracellular domain that may vary in length and comprises a disease- or disorder-associated, e.g., a tumor-antigen binding region. In embodiments, CARs comprise fusions of single-chain variable fragments (scFv) derived from monoclonal antibodies, fused to CD3-zeta a transmembrane domain and endodomain. The specificity of other CAR designs may be derived from ligands of receptors (e.g., peptides) or from pattern-recognition receptors, such as Dectins. In certain cases, the spacing of the antigenrecognition domain can be modified to reduce activation-induced cell death. In certain cases, CARs comprise domains for additional co- stimulatory signaling, such as CD3-zeta, FcR, CD27, CD28, CD137, DAP 10 / 12, and / or 0X40, ICOS, TLRs, etc. In some cases, molecules can be coexpressed with the CAR, including co-stimulatory molecules, reporter genes for imaging (e.g., for positron emission tomography), gene products that conditionally ablate the T cells upon addition of a pro-drug, homing receptors, chemokines, chemokine receptors, cytokines, and cytokine receptors.

[0057] The “costimulatory domain” in the context of a CAR enhances cell proliferation, cell survival and development of memory cells for cytotoxic cells that express the CAR. The CARs may include one or more costimulatory domains selected from the costimulatory domains of proteins in the TNFR superfamily, CD28, CD137 (4-1BB), CD134 (0X40), DaplO, CD27, CD2, CD7, CD5, ICAM-1, LFA-1 (CD1 la / CD18), Lek, TNFR-I, PD-1, TNFR-II, Fas, CD30, CD40, ICOS LIGHT, NKG2C, B7-H3, or combinations thereof. If the CAR includes more than one costimulatory domain, these domains may be arranged in tandem, optionally separated by a linker. The term “costimulatory domain” as used herein also encompasses any modifications thereof, examples of which are described in US Patent Application No. 20200129554; US Patent Application No. 20200317777; W02019010383; Li, W., et al., (2020) Immunity 53: 456-470; and Li, G., et al., (2017) J Immunol 198(1 Supplement): 198.4, the contents of each of which are incorporated by reference herein in their entirety.121106305893\l\AMERICAS

[0058] The “intracellular signaling domain” in the context of a CAR transduces the effector function signal and directs the cytotoxic cell to perform its specialized function, e.g., harming and / or destroying the target cells. Examples of suitable intracellular signaling domains include, e.g., the C, chain of the T cell receptor complex or any of its homologs, e.g., r| chain, FcsRly and p chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 chain, CD3 polypeptides (A, 5 and a), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.) and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In embodiments, the intracellular signaling domain of a chimeric receptor may be human CD3 chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (IT AM) bearing cytoplasmic receptors and combinations thereof.

[0059] The intracellular signaling domains may include intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (Park et al., "Are all chimeric antigen receptors created equal?" J Clin Oncol., vol. 33, pp. 651-653, 2015). Additional intracellular signaling domains include signaling domains used by NK and NKT cells (Hermanson, et al., "Utilizing chimeric antigen receptors to direct natural killer cell activity," Front Immunol., vol. 6, p. 195, 2015) such as signaling domains of NKp30 (Zhang et al., "An NKp30-based chimeric antigen receptor promotes T cell effector functions and antitumor efficacy in vivo," J Immunol., vol. 189, pp. 2290-2299, 2012), and DAP12 (Topfer et al., "DAP12-based activating chimeric antigen receptor for NK cell tumor immunotherapy," J Immunol., vol. 194, pp. 3201-3212, 2015), NKG2D, NKp44, NKp46, DAP10, and CD3z. Additionally intracellular signaling domains also includes signaling domains of human Immunoglobulin receptors that contain immunoreceptor tyrosine based activation motif (IT AM) such as FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, FcRL5 (Gillis et al., "Contribution of Human Fc.gamma.Rs to Disease with Evidence from Human Polymorphisms and Transgenic Animal Studies," Front Immunol., vol. 5, p. 254, 2014).

[0060] In embodiments, the intracellular signaling domain includes a cytoplasmic signaling domainIn exemplary embodiments the intracellular signaling domain in the CAR includes a cytoplasmic signaling domain of human CD3 . The term “intracellular signaling domain” as used herein also131106305893\l\AMERICASencompasses any modifications thereof, examples of which are described in US Patent Application No. 2020 / 0317777, as well as Roda-Navarro, P., andReyburn, HT., (2009) J Biol Chem 284(24): 16463-16472; Giurisato, E., et al., (2007) Mol Cell Biol 27(24): 8583-8599; and Wu, J., et al., (2000) J Exp Med 192(7): 1059-1068, the contents of each of which are incorporated by reference herein in their entirety.

[0061] The terms “affinity binding domain” or “means for specifically binding” as used herein refer to a binding moiety which binds to a specific antigen with a higher affinity than to a nonspecific antigen and is endowed with an affinity of at least 10'6M, as determined by assays which are well known in the art, including surface plasmon resonance (SPR). According to a specific embodiment, the affinity is 500 nM-0.01 nM, 100 nM-0.01 nM, 50 nM-0.01 nM, 10 nM-0.01 nM, 5 nM-0.01 nM. In embodiments, the affinity binding domain or means for specifically binding recognizes a tumor antigen. In embodiments, the affinity binding domain or means for specifically binding recognizes an autoimmune disease associated antigen. In embodiments, the affinity binding domain or means for specifically binding recognizes a pathogenic antigen.

[0062] In embodiments, the affinity binding domain or means for specifically binding is an antibody. The term “antibody” is used in the broadest sense and specifically covers, for example, single monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), antibody compositions with polyepitopic specificity, polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain antibodies, and fragments of antibodies (see below), including Fab, Fab’, F(ab’)2 and Fv fragments, diabodies, heavy chain-only antibodies and single domain antibodies (sdAbs), as long as they exhibit the desired biological or immunological activity. Also included among antibodies, and among fragments in particular, are portions of antibodies (and combinations of portions of antibodies, for example, scFv) that may be used as targeting arms, directed to e.g., a target epitope (e.g., an epitope on a tumor antigen), in chimeric antigenic receptors of the present disclosure. Such fragments are not necessarily proteolytic fragments but rather portions of polypeptide sequences that can confer affinity for target. The term “immunoglobulin” (Ig) is used interchangeably with antibody herein. An antibody can be, for example, human, humanized and / or affinity matured.141106305893\l\AMERICAS

[0063] Methods of making antibodies and antibody fragments are known in the art. (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

[0064] Antibodies can be produced by the immunization of various animals, including mice, rats, rabbits, goats, primates, humans and chickens with a target antigen or peptide fragments containing the epitope of the present disclosure. Antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352: 624-8 (1991) and Marks et al., J. Mol. Biol., 222: 581-97 (1991), for example. Antibodies or antigen-binding fragment of the present invention can be purified by methods known in the art, for example, gel filtration, ion exchange, affinity chromatography, etc. Affinity chromatography or any of a number of other techniques known in the art can be used to isolate polyclonal or monoclonal antibodies from, for example, serum, ascites fluid, or hybridoma supernatants.

[0065] An “isolated antibody” is one which has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.

[0066] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to a H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for p and s isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CHI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, at page 71 and Chapter 6.151106305893\l\AMERICAS

[0067] The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated a, 5, s, y, and p, respectively. The y and a classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

[0068] The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as “VH” or “VH” The variable domain of the light chain may be referred to as “VL” or “VL”. These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0069] The term “variable” refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each about 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a P-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the P-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0070] An “intact” antibody is one which comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CHI, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.161106305893\l\AMERICAS

[0071] “Antibody fragments” comprise a portion of an intact antibody, preferably the antigen binding or one or more variable regions of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Patent No.5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-62 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen. Also included among antibody fragments are portions of antibodies (and combinations of portions of antibodies, for example, scFv) that may be used as targeting arms, directed to an epitope, in chimeric antigenic receptors of the present disclosure. Such fragments are not necessarily proteolytic fragments but rather portions of polypeptide sequences that can confer affinity for target.

[0072] Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab’ fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0073] The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

[0074] “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy-171106305893\l\AMERICASand one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two-chain Fv species. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0075] “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In embodiments, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form a desired structure for antigen binding. For a review of sFv, see, e.g., Pluckthun in The Pharmacology of Monoclonal Antibodies vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra. In one embodiment, an antibody derived scFv is used as the targeting arm of a modified T cell as disclosed herein. In terms of scFv antibody fragments, where a certain order of VH and VL region in the binding domain is explicitly or implicitly described, the present disclosure also includes the alternate embodiment in which the order of VH and VL regions are reversed, e.g., in an scFv or CAR comprising an scFv binding domain. Thus, description of a VH-VL order also describes the alternate VL-VH order, e.g., in an scFv or CAR comprising an scFv binding domain. Moreover, description of a VL-VH order also describes the alternate VH-VL order, e.g., in an scFv or a CAR comprising an scFv binding domain. VH and VL regions are either joined directly or joined by a peptide-encoding linker, which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL.

[0076] The scFv linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility. The linker can link the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain. Non-limiting examples of linkers are disclosed in Shen et al, Anal. Chem. 80(6): 1910-1917 (2008) and WO 2014 / 087010, the contents of which are hereby incorporated by reference in their entireties. Various linker sequences are known in the art, including, without limitation, glycine serine (GS) linkers such as (GS)n, (GSGGS)n (SEQ ID NO: 174), (GGGS)n (SEQ ID NO: 175), and (GGGGS)n (SEQ ID NO: 176), where n represents an integer of at least 1. Exemplary linker sequences can comprise amino acid sequences including,181106305893\l\AMERICASwithout limitation, GGSG (SEQ ID NO: 177), GGSGG (SEQ ID NO: 178), GSGSG (SEQ ID NO: 179), GSGGG (SEQ ID NO: 180), GGGSG (SEQ ID NO: 181), GSSSG (SEQ ID NO: 182), GGGGS (SEQ ID NO: 183), GGGGSGGGGSGGGGS (SEQ ID NO: 172) and the like. Those of skill in the art would be able to select the appropriate linker sequence for use in the present invention. In one embodiment, an affinity binding domain of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and VL is separated by the linker sequence having the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 172), which may be encoded by the nucleic acid sequence GGAGGCGGAGGATCTGGTGGTGGTGGATCTGGCGGCGGAGGCTCT (SEQ ID NO: 173).

[0077] The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256: 495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (e.g., U.S. Patent No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352: 624-8 (1991) and Marks et al., J. Mol. Biol., 222: 581-97 (1991), for example.

[0078] The term “hypervariable region”, “HVR”, or “HV”, when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and / or form structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health,191106305893\l\AMERICASBethesda, MD. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The end of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places the 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). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software. The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions are noted below.Loop Kabat AbM Chothia ContactLI L24-L34 L24-L34 L24-L34 L30-L36L2 L50-L56 L50-L56 L50-L56 L46-L55L3 L89-L97 L89-L97 L89-L97 L89-L96Hl H31-H35B H26-H35B H26-H32..34 H30-H35B (Kabat Numbering)Hl H31-H35 H26-H35 H26-H32 H30-H35 (Chothia Numbering)H2 H50-H65 H50-H58 H52-H56 H47-H58 H3 H95-H102 H95-H102 H95-H102 H93-H101

[0079] Hypervariable regions may comprise “extended hypervariable regions” as follows: 24-36 or 24-34 (LI), 46-56 or 50-56 (L2) and 89-97 (L3) in the VL and 26-35B (Hl), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[0080] “Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues herein defined.201106305893\l\AMERICAS

[0081] The term “variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat”, and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

[0082] The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g, Kabat et al., supra). The “EU numbering system” or “EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The “EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody. Unless stated otherwise herein, references to residue numbers in the variable domain of antibodies means residue numbering by the Kabat numbering system.

[0083] An antibody that “binds” an antigen or epitope of interest is one that binds the antigen or epitope with sufficient affinity that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity.

[0084] The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including proteins or peptides, can serve as an antigen.

[0085] The term "epitope" includes any protein determinant, lipid or carbohydrate determinant capable of specific binding to an immunoglobulin or T cell receptor. Epitopic determinants usually consist of active surface groupings of molecules such as amino acids, lipids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.211106305893\l\AMERICAS

[0086] The term "specifically binds”, as used herein refers to a receptor (which can include but is not limited to an antibody or antibody fragment) which recognizes a specific molecule / ligand, but does not substantially recognize or bind other molecules in a sample. For example, a receptor that specifically binds to a molecule from one species may also bind to that molecule from one or more other species. But, such cross-species reactivity does not itself alter the classification as specific. In another example, a receptor that specifically binds to a molecule may also bind to different allelic forms of the molecule. However, such cross reactivity does not itself alter the classification as specific. In some instances, the terms "specific binding" or "specifically binding," can be used in reference to the interaction of a protein (or a peptide) with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, a receptor recognizes and binds to a specific a structure rather than to proteins generally. If receptor is specific for epitope "A", the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled "A" and the receptor, will reduce the amount of labeled A bound to the receptor.

[0087] In embodiments, specific binding can be characterized by an equilibrium dissociation constant of at least about lxl0‘8M or less (e.g., a smaller Ka denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.

[0088] The term "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancers include solid tumors and hematologic malignancies. Examples of cancers include acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas, neuroblastoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancers, brain tumors, such as cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoma of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer,221106305893\l\AMERICASependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancers, such as non-small cell and small cell lung cancer, lymphomas, leukemias, macroglobulinemia, malignant fibrous histiocytoma of bone / osteosarcoma, medulloblastoma, melanomas, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic cancer islet cell, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasia, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas, skin cancers, skin carcinoma merkel cell, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, T-cell lymphoma, throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (gestational), cancers of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor.

[0089] The term “autoimmune disease” refers to a disorder that results from an autoimmune response. An autoimmune disease is the result of an inappropriately excessive response to an antigen associated with an autoimmune disease (e.g., a self-antigen). Examples of autoimmune diseases include rheumatoid arthritis, rheumatic fever, multiple sclerosis, experimental autoimmune encephalomyelitis, psoriasis, uveitis, diabetes mellitus, systemic lupus erythematosus (SLE), lupus nephritis, eczema, scleroderma, polymyositis / scleroderma, polymyositis / dermatomyositis, ulcerative proctitis, ulcerative colitis, severe combined immunodeficiency (SCID), DiGeorge syndrome, ataxia-telangiectasia, seasonal allergies, perennial allergies, food allergies, anaphylaxis, mastocytosis, allergic rhinitis, atopic dermatitis,231106305893\l\AMERICASParkinson’s, Alzheimer’s, hypersplenism, leukocyte adhesion deficiency, X-linked lymphoproliferative disease, X-linked agammaglobulinemia, selective immunoglobulin A deficiency, hyper IgM syndrome, HIV, autoimmune lymphoproliferative syndrome, Wiskott-Aldrich syndrome, chronic granulomatous disease, common variable immunodeficiency (CVID), hyperimmunoglobulin E syndrome, Hashimoto’s thyroiditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenia purpura, dermatomyositis, Sydenham’ a chorea, myasthenia gravis, polyglandular syndromes, bullous pemphigoid, Henoch-Schonlein purpura, poststreptococcalnephritis, erythema nodosum, erythema multiforme, gA nephropathy, Takayasu’s arteritis, Addison’s disease, sarcoidosis, ulcerative colitis, polyarteritis nodosa, ankylosing spondylitis, Goodpasture’s syndrome, thromboangitisubiterans, Sjogren’s syndrome, primary biliary cirrhosis, Hashimoto’s thyroiditis, thyrotoxicosis, chronic active hepatitis, polychondritis, pamphigus vulgaris, Wegener’s granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis, polymyalgia, peraiciousanemia, rapidly progressive glomerulonephritis, psoriasis, and fibrosing alveolitis.

[0090] The term “autologous” is meant to refer to any material derived from an individual which is later to be re-introduced into the same individual.

[0091] The term “allogeneic” refers to material derived from an animal which is later introduced into a different animal of the same species.

[0092] A “modification” of an amino acid residue / position, as used herein, refers to a change of a primary amino acid sequence as compared to a starting amino acid sequence, wherein the change results from a sequence alteration involving the amino acid residue / positions. For example, typical modifications include substitution of the residue (or at the position) with another amino acid (e.g., a conservative or non-conservative substitution), insertion of one or more (generally fewer than 5 or 3) amino acids adjacent to the residue / position, and deletion of the residue / position. An “amino acid substitution”, or variation thereof, refers to the replacement of an existing amino acid residue in a predetermined (starting) amino acid sequence with a different amino acid residue. Generally, the modification results in alteration in at least one physicobiochemical activity of the variant polypeptide compared to a polypeptide comprising the starting (or “wild type”) amino acid sequence. For example, in the case of an antibody, a physicobiochemical activity that is altered can be binding affinity, binding capability and / or binding effect upon a target molecule.241106305893\l\AMERICAS

[0093] To “treat or prevent” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. In one example, a therapy (e.g., administration of a therapeutic agent of the present disclosure) treats a disease or condition by decreasing one or more signs or symptoms associated with the disease or condition, for example as compared to the response in the absence of the therapy. For example, administration of a therapeutic agent may provide an anti-tumor effect that decreases one or more signs or symptoms associated with cancer. Treating or preventing can refer to delaying the onset of symptoms, reducing the severity of symptoms, reducing the severity of an acute episode, reducing the number of symptoms, reducing the incidence of disease-related symptoms, reducing the latency of symptoms, ameliorating symptoms, reducing secondary symptoms, reducing secondary infections, prolonging patient survival, preventing relapse to a disease, decreasing the number or frequency of relapse episodes, increasing latency between symptomatic episodes, increasing time to sustained progression, expediting remission, inducing remission, augmenting remission, speeding recovery, or increasing efficacy of or decreasing resistance to alternative therapeutics. In one embodiment, "treating" refers to both therapeutic treatment and prophylactic or preventive measures, wherein the object is to prevent or lessen the targeted pathologic condition or disorder as described herein.

[0094] The term “administration” means to provide or give a subject one or more agents, such as an agent that treats one or more signs or symptoms associated with a condition / disorder or disease including but not limited to cancer (e.g., lymphoma), autoimmune disease, viral infection, bacterial infection, etc., by any effective route. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes. Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and sequential administration in any order.

[0095] The term “pharmaceutically acceptable ”, as used herein, refers to a material, including but not limited, to a salt, carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's251106305893\l\AMERICASPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1 95), describes compositions and formulations suitable for pharmaceutical delivery of one or more agents, such as one or more modulatory agents. In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations can include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically-neutral carriers, pharmaceutical agents to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate. For example, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and one or more modified T cells. Examples of pharmaceutically acceptable carrier includes a buffer, excipient, stabilizer, or preservative. Such compositions may include 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. Cryopreservation solutions which may be used in the pharmaceutical compositions of the invention include, for example, DMSO.

[0096] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0097] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or261106305893\l\AMERICASpeptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a T cell.

[0098] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

[0099] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

[0100] “Expression cassette” refers to a nucleic acid comprising expression control sequences operatively linked to a nucleic acid encoding a transcript or polypeptide to be expressed. An expression cassette comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the T cell or in an in vitro expression system. Expression cassettes can be a component of a vector such as a cosmid, a plasmid (e.g., naked or contained in a liposome), or a virus (e.g., lentivirus, retrovirus, adenovirus, and adeno-associated virus).

[0101] The term “disruption” and grammatically equivalents as used herein refer to disturbance of the expression of a gene. Disruption of a gene includes introducing one or more mutations to the gene, deleting a fragment or the full-length of the gene, inserting one or more nucleotides to the gene, suppressing the transcription of the gene, or any combination thereof. T CELLS

[0102] The modified T cells herein may be produced by engineering T cells, e.g., T cell lines or donor T cells (e.g., allogeneic, syngeneic). The T cells can be obtained from a number of sources, including from peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. The T cells can be obtained from blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. For example, cells from the circulating blood of an individual may be obtained by apheresis. In embodiments, the T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation of the T cells can be further isolated by positive271106305893\l\AMERICASor negative selection techniques. For example, the T cells can be isolated using a combination of antibodies directed to surface markers unique to the positively selected cells, e.g., by incubation with antibody-conjugated beads for a time period sufficient for positive selection of the desired modified T cells. Alternatively, enrichment of T cell populations can be accomplished by negative selection using a combination of antibodies directed to surface markers unique to the negatively selected cells. Other specific manners of isolation and / or enrichment are disclosed herein.

[0103] In embodiments, the modified T cells have in vitro or in vivo cytotoxic activity against a disease cell (e.g., tumor cell, a cell associated with autoimmune disease, or a pathogen or pathogen-infected cell) that exhibits cell surface expression of a disease associated antigen. In some cases, the cytotoxic activity is innate activity. In some cases, the cytotoxicity is at least in part, significantly (> about 25%), or entirely, due to the presence of a CAR construct having a binding domain that specifically binds a disease associated antigen expressed on the surface of the disease cell, and / or one or more modifications described herein. In some cases, the modified T cells exhibit disease cell killing (e.g., tumor cell killing) activity that is greater than an innate level of in vitro and / or in vivo disease cell killing activity in a control cell of the same cell type. In some cases, the control cell does not comprise a CAR construct. In some cases, the control cell comprises a CAR construct lacking a binding domain described herein, a hinge region described herein, a transmembrane domain described herein, an intracellular signaling domain described herein, and / or a costimulatory domain described herein. In some cases, the control cell does not comprise the one or more modifications described herein.

[0104] In some cases, the cytotoxicity is at least in part, significantly (> about 25%), or entirely, due to the presence of a CAR construct having a binding domain that specifically binds an antigen or an epitope within an antigen, and / or the one or more modifications described herein.

[0105] In embodiments, the modified T cells can exhibit HLA-restricted (e.g., HLA class I restricted) cytotoxicity. In other embodiments, most (>50%), substantially all (>90%), or all of the cytotoxic activity is not HLA-restricted (e.g. , HLA class I restricted). HLA-restricted cytotoxic activity can be assessed by comparing in vitro cytotoxicity against an HLA (e.g., HLA class I) (null) tumor cell line versus in vitro cytotoxicity against an HLA+ (e.g., HLA class I+) tumor cell line. In embodiments, the HLA-restricted cytotoxic activity is at least in part, significantly (>25%), or entirely, provided by the use of a T cell Receptor-like binding domain. T cell receptor like binding domains are binding domains that specifically recognize the antigen when presented281106305893\l\AMERICASon the surface of a cell in complex with an MHC molecule. T cell Receptor-like binding domains are further described, e.g., in WO 2016 / 199141.

[0106] The modified T cells can exhibit robust and / or persistent disease cell (e.g., tumor cell) killing activity. In embodiments, the disease cell (e.g., tumor cell) killing activity of a modified T cell, or a progeny thereof, can persist for at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 days, from first contact with a disease cell (e.g., tumor cell), or from administration of the modified T cell. In some cases, the disease cell (e.g., tumor cell) killing activity can persist for at least about 6 days to 120 days, or for at least about 6 days to 180 days, from first contact with a tumor cell. In some cases, the disease cell (e.g., tumor cell) killing activity of a modified T cell, or a progeny thereof, can persist for at least about 6 days to 120 days, or for at least about 6 days to 180 days, from first contact with a disease cell (e.g., tumor cell), or from administration of the modified T cell. This persistent disease cell (e.g., tumor cell) killing activity can be exhibited in vitro, in vivo, or both in vitro and in vivo.

[0107] The modified T cells may proliferate in response to contact with cells that exhibit cell surface expression, or overexpression, of one or more disease associated antigens. The cells that exhibit cell surface expression, or overexpression, of one or more disease associated antigens can be tumor cells or can be non-tumor cells. In some cases, the proliferation is an innate activity. In some cases, the proliferation is at least in part, significantly (> about 20% or > about 25%), or entirely, due to the presence of a CAR construct having a binding domain that specifically binds a disease associated antigen (e.g., tumor antigen, autoimmune disease-associated antigen, or pathogenic antigen) expressed on the surface of a disease cell, and / or the one or more modifications described herein. In some cases, the modified T cell exhibits a greater level of in vitro and / or in vivo proliferation as compared to a control cell of the same type. In some cases, the control cell does not comprise a CAR construct. In some cases, the control cell comprises a CAR construct lacking a binding domain described herein, a hinge region described herein, a transmembrane domain described herein, an intracellular signaling domain described herein, and / or a costimulatory domain described herein. In some cases, the control cell does not comprise the one or more modifications described herein.

[0108] The modified T cells can exhibit robust and / or persistent proliferation in a host organism that comprises a cell, for example a tumor cell, an autoimmune disease cell, a pathogen or pathogen-infected cell, that exhibits cell surface expression, or overexpression, of one or more291106305893\l\AMERICASdisease associated antigens. Tn embodiments, the proliferation can persist at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 days, from first contact with a disease cell or from a date of administration of the modified T cell, to the host organism. In some cases, the proliferation can persist for at least about 6 days to 120 days, or for at least about 6 days to 180 days, from first contact with a disease cell or from a date of administration of the modified T cell, to the host organism. In embodiments, the proliferation of a modified T cell described herein, or a progeny thereof, in the host organism that comprises the cell that exhibits cell surface expression, or overexpression, of one or more disease associated antigens can persist for at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 days, from first contact with a disease antigen-expressing cell or from the date of first administration of a modified T cell, to the host organism. In some cases, the proliferation of a modified T cell described herein, or a progeny thereof, in the host organism that comprises the cell that exhibits cell surface expression, or overexpression, of one or more disease associated antigens can persist for at least about 6 days to 120 days, or for at least about 6 days to 180 days, from first contact with a disease antigen-expressing cell or from the date of first administration of a modified T cell, to the host organism. In some cases, the proliferation in the host organism is at least in part, significantly (> about 20% or > about 25%), or entirely, due to the presence of a CAR construct having a binding domain that specifically binds an antigen or an epitope within the antigen, and / or the one or more modifications described herein.

[0109] In embodiments, modified T cells described herein, express, or persistently express, pro-inflammatory cytokines such as tumor necrosis factor alpha or interferon gamma after contact with an antigen-expressing cell. In embodiments, the modified T cells described herein, or progeny thereof, express, or persistently express, pro-inflammatory cytokines such as tumor necrosis factor alpha or interferon gamma after contact with the antigen-expressing cell, e.g., in a host organism comprising the antigen-expressing cell.

[0110] In embodiments, the modified T cell is a y3 T cell. In embodiments, the yd T cell is a 61, a 82, a 83, or a 84 y8 T cell, preferably a 82" y8 T cell, more preferably a 81 y8 T cell. In embodiments, the modified T cell is a 81 y8 T cell. In embodiments, the modified T cell is a 82 y8 T cell. In embodiments, the modified T cell is a 83 y8 T cell. In embodiments, the modified T cell is a 84 y8 T cell. In embodiments, the modified T cell is a 82" y8 T cell. In embodiments, the modified T cell is a ot|3 T cell.301106305893\l\AMERICAS

[0111] In embodiments, the modified T cell is a naive T cell. Tn embodiments, naive T cells, which have not yet encountered antigen, are characterized by their expression of surface markers CCR7+, CD62L+, and CD45RA+, but lack CD45RO. Upon TCR activation, these antigen-experienced T cells typically lose expression of CD45RA and downregulate CCR7 and CD62L, while upregulating CD45RO as they differentiate into effector memory T cells. In embodiments, the naive-like stem cell memory T cells of the subject invention are antigen-experienced T cells, yet they retain CCR7+, CD62L+, and CD45RA+. In embodiments, such naive-like stem cell memory T cells can express CD45RO. See e.g., Daniel Powell Jr. et al, A CD45RO+ stem cell memory T cell population intermediate to canonical stem cell memory and central memory T cells in human cancer., 03 February 2022, PREPRINT (Version 1) available at Research Square [doi.org / 10.21203 / rs.3.rs-1296773 / vl],

[0112] In embodiments, the modified T cell is an aP T cell in a cell therapy. In embodiments, the modified T cell is an aP CAR T cell. Examples of suitable cell therapies that would benefit from the subject invention include ABECMA (idecabtagene vicleucel), Aucatzyl (obecabtagene autoleucel) BREYANZI (lisocabtagene maraleucel), CARVYKTI (ciltacabtagene autoleucel), KYMRIAH (tisagenlecleucel), TEC ARTUS (brexucabtagene autoleucel), YESCARTA (axicabtagene ciloleucel), as well as those described in Zugasti et al., CAR-T cell therapy for cancer: current challenges and future directions, Signal Transduct Target Ther. 2025 Jul 4; 10(l):210; Zhang et al., Onboard, tethered IL-12 boosts potency of the Tmod NOT gate and preserves selectivity, J Immunother Cancer. 2025 May 21 ; 13(5):e010976, each of which is incorporated by reference herein in its entirety.

[0113] In embodiments, the modified T cell is a tumor-infiltrating lymphocyte in a cell therapy. Examples of suitable cell therapies that would benefit from the subject invention include Amtagvi (lifileucel), those studied in clinical trials numbers NCT03778814, NCT04596033, NCT04625205, NCT03610490, NCT03449108, NCT03935893, NCT03658785, NCT03991741, NCT03992326, NCT04643574, NCT01462903, NCT00002733, NCT02876510, NCT03645928, NCT05676749, NCT03997474, NCT05902520, NCT05724732, NCT05430360, NCT05729399, NCT05238818, NCT05430373, NCT05397093, NCT04426669, NCT05566223, NCT05361174, NCT03083873, NCT03108495, NCT04614103, NCT06481592, NCT05727904, NCT05468307, NCT06237881, NCT05573035, NCT05470283, NCT06060613, NCT05576077, NCT06236425, and NCT05628883, those described in Qin S et al., Adoptive T Cell Therapy for Solid Tumors:311106305893\l\AMERICASPathway to Personalized Standard of Care, Cells. 2021 Apr 5; 10(4): 808; AmariaR et al., Entering a new era of tumor-infiltrating lymphocyte cell therapy innovation, Cytotherapy, Volume 27, Issue 7, July 2025, Pages 864-873; Kumar et al., Cell Therapy With TILs: Training and Taming T Cells to Fight Cancer, Front Immunol. 2021 Jun 1:12:690499; Jones et a., Cell surface-tethered IL-12 repolarizes the tumor immune microenvironment to enhance the efficacy of adoptive T cell therapy, Sci Adv. 2022 Apr 29;8(17):eabi8075; Kim SP, et al., Drug-regulatable, inducible, and membrane-bound interleukin 12 (IL-12TM-D) for use in adoptive cell therapies against advanced cancers. Journal for ImmunoTherapy of Cancer. 2024; 12; Zhang L et al., Adoptive transfer of membrane-restricted IL-12-TCR T cells promotes antigen spreading and elimination of antigennegativetumorvariants, J Immunother Cancer. 2024 Nov 18; 12(1 l):e009868; InnamaratoP et al., IOV-5001, autologous tumor-infiltrating lymphocytes (TIL) armored with inducible membrane-tethered IL-12, shows enhanced antitumor efficacy with an improved cellular state, Cancer Res (2025) 85 (8_Supplement_l): 4863; and Ross T et al., Spatiotemporally regulated expression of membrane-bound interleukin 12 (mbIL12) for armored adoptive cell therapy (ACT) shows strong antitumor activity in syngeneic solid tumor models without overt toxicity, Cancer Res (2025) 85 (8_Supplement_2): LB025, each of which is incorporated by reference in its entirety.

[0114] In embodiments, the modified T cell is a yd T cell in a cell therapy. In embodiments, the modified T cell is a yd CAR T cell. Examples of suitable yd cell therapies that would benefit from the subject invention include clinical trials numbers NCT03849651, NCT04008381, NCT03533816, NCT04518774, NCT05015426, NCT05001451, NCT04696705, NCT04764513, NCT02656147, NCT04107142, NCT04735471, NCT04243499, NCT04887259, NCT05369000, NTR6541, NCT04165941, NCT04688853, NCT05664243, NCT05886491, NCT05358808, NCT05400603, NCT03183206, NCT03183219, NCT03183232, NCT03180437, NCT04765462, NCT06069570, NCT05546723, NCT04911478, NCT05302037, NCT06193486, NCT06150885, NCT05653271, NCT06415487, NCT04014894, NCT04864054, NCT04502082, NCT04634357, and NCT05307874, as well as those described in Saura-Esteller et al., Gamma Delta T-Cell Based Cancer Immunotherapy: Past-Present-Future, Front Immunol. 2022 Jun 16:13:915837, and Hayday et al., Cancer immunotherapy by yd T cells, Science. 2024 Oct 4;386(6717):eabq7248, each of which is incorporated by reference herein in its entirety.321106305893\l\AMERICASyd T CELLS

[0115] In embodiments, the modified T cells are modified y5 T cells. In embodiments, the modified y8 T cell, or a pharmaceutical composition containing the modified y8 T cell, exhibits essentially no, or no graft versus host response when introduced into an allogeneic host. In embodiments, the modified y8 T cell, or a pharmaceutical composition containing the modified y8 T cell, exhibits a clinically acceptable level of graft versus host response when introduced into an allogeneic host. In embodiments, a clinically acceptable level is an amount of graft versus host response that does not require cessation of a modified y8 T cell treatment to achieve a therapeutically effective treatment. In embodiments, a clinically acceptable level of graft versus host response (GvHD) is an acute response that is less severe than Grade C according to an applicable IBMTR grading scale. The severity of acute graft versus host response is determined by an assessment of the degree of involvement of the skin, liver, and gastrointestinal tract. The stages of individual organ involvement are combined to produce an overall grade, which has prognostic significance. Grade 1(A) GvHD is characterized as mild disease, grade 11(B) GvHD as moderate, grade III(C) as severe, and grade IV(D) life-threatening. The IBMTR grading system defines the severity of acute GvHD as follows (Rowlings et al., Br J Haematol 1997; 97:855):•Grade A - Stage 1 skin involvement alone (maculopapular rash over <25 percent of the body) with no liver or gastrointestinal involvement•Grade B - Stage 2 skin involvement; Stage 1 to 2 gut or liver involvement•Grade C - Stage 3 involvement of any organ system (generalized erythroderma; bilirubin 6.1 to 15.0 mg / dL; diarrhea 1500 to 2000 mL / day)•Grade D - Stage 4 involvement of any organ system (generalized erythroderma with bullous formation; bilirubin >15 mg / dL; diarrhea >2000 mL / day OR pain OR ileus). See also, Schoemans et al., Bone Marrow Transplantation volume 53, pagesl401-1415 (2018), e.g., at Tables 1 and 2, which discloses criteria for assessing and grading acute GvHD.

[0116] In embodiments, a modified y5 T cell, or a pharmaceutical composition containing the modified y5 T cell, exhibits reduced or substantially reduced graft versus host response when introduced into an allogeneic host as compared to a graft versus host response exhibited by control aP T cells, or a control pharmaceutical composition comprising the control aP T cells, administered to an allogeneic host. In some cases, the control aP T cell is an allogeneic non-engineered control331106305893\l\AMERICASa.p T cell. In some cases, the control aP T cell does not comprise a CAR or does not comprise the same CAR as a reference y8 T cell.

[0117] The y6 T cells described herein can be 61, 62, 83, or 64 y6 T cells, or combinations thereof. In some cases, the yb T cells are mostly (>50%), substantially (>90%), essentially all, or entirely 62’ y6 T cells. In some cases, the y6 T cells are mostly (>50%), substantially (>90%), essentially all, or entirely 81 y6 T cells. In some cases, the y8 T cells are mostly (>50%), substantially (>90%), essentially all, or entirely 63 y6 T cells. In embodiments, the y6 T cell is a 81 y8 T cell. In embodiments, the y8 T cell is a 82 y8 T cell. In embodiments, the y8 T cell is a 83 y8 T cell. In embodiments, the y8 T cell is a 84 y8 T cell. In embodiments, the y8 T cell is a 82" y8 T cell.

[0118] The y6 T cells herein can be obtained from an allogeneic donor. The yb T cells can be, partially or entirely purified, or not purified, and expanded ex vivo. Methods and compositions for ex vivo expansion include, without limitation, those described in WO 2017 / 197347. The expansion may be performed before or after, or before and after, a CAR polypeptide and / or one or more modifications of the present disclosure is introduced into the yb T cell(s). Other additional or alternative methods of expansion include the use of, e.g., artificial antigen-presenting cells (aAPCs), aminobisphosphonates, cytokine cocktails, and feeder cells (Cortes-Selva, D et al., (2021) Trends Pharmacol Sci. 42(1): 45-59). In embodiments, the methods of expansion comprise disrupting MED12 gene in isolated T cells and contacting the modified T cells with IL-12.

[0119] According to an aspect, provided herein is a plurality of modified y8 T cells, as herein disclosed. In embodiments, the plurality of modified yb T cells comprise a composition that is at least 60%, 80%, 90%, or 95%, or from about 60% or 80% to about 90% or 95% 81 y8 T cells. In embodiments, the plurality of modified y8 T cells comprise a composition that is at least 60%, 80%, 90%, 95%, or from about 60% or 80% to about 90% or 95% 62 y6 T cells. In embodiments, the plurality of modified y8 T cells comprise a composition that is at least 60%, 80%, 90%, 95%, or from about 60% or 80% to about 90% or 95% 83 y6 T cells. In embodiments, the plurality of modified y8 T cells comprise a composition that is at least 60%, 80%, 90%, or 95%, or from about 60% or 80% to about 90% or 95% 84 y8 T cells. In embodiments, the plurality of modified y8 T cells comprise a composition that is at least 60%, 80%, 90%, or 95%, or from about 60% or 80% to about 90% or 95% 81 or 82 y8 T cells. In embodiments, the plurality of modified y8 T cells341106305893\l\AMERICAScomprise a composition that is at least 60%, 80%, 90%, or 95%, or from about 60% or 80% to about 90% or 95% 82" y8 T cells.

[0120] In embodiments, the plurality of modified y8 T cells comprises at least about 107modified yo T cells, e.g., at least 5*107, at least 108, at least 5*108, at least 109, at least 5*109, or at least IO10modified y8 T cells. In some examples, the plurality of modified y8 T cells comprises from about 108modified y3 T cells to about 1011modified y8 T cells, e.g., from 107to 108, from 5*107to 5*108, from 108to 109, from 5*108to 5><109, or from 109to 1010modified y8 T cells.

[0121] The modified y8 T cells, as described herein, can be stored, e.g., cryopreserved, for use in adoptive cell transfer. In embodiments, the modified y8 T cells are stored prior to engineering the cells to express a CAR polypeptide and / or have one or more modifications herein. In embodiments, the y8 T are engineered to express a CAR polypeptide and / or have one or more modifications herein and then the cells are stored.MODIFICATIONS

[0122] The modified T cells provided herein comprise one or more modifications. The one or more modifications include expression of one or more exogenous proteins, disruption of one or more endogenous genes, or a combination thereof. In embodiments, the modified T cell comprises a) at least one modification to modulate T cell metabolism; and b) at least one modification to modulate cytokine activity. In preferred embodiments, the modified T cell herein comprises a disrupted MED12 gene (MED12K0). In particularly preferred embodiments, the modified T cell herein further expresses a membrane-bound interleukin- 12 (mbIL-12+). As demonstrated herein for the first time, this particular combination of modifications leads to an advantageous naive-like stem cell memory phenotype in the modified T cells and dramatically enhances their cytotoxicity and persistence. As further demonstrated herein, contacting a modified T cell comprising a MED 12 disruption with IL-12 ex vivo or in vivo can also enhance T cell functionality and persistence.

[0123] Modification(s) to modulate T cell metabolism may regulate the metabolism of molecules (e.g., nucleic acid, glucose, nucleic acid, etc.) in the T cell to enhance activity, survival, and expansion of the T cell. The preferred modification to modulate T cell metabolism comprises disruption of the MED12 gene. Mediator complex subunit 12 (MED12) is a Mediator subunit, and can regulate metabolic activity and fitness, e.g., increased glycolysis, oxidative phosphorylation, and spare respiratory capacity. Disruption of the MED12 gene may provide a functional advantage over control cells that have an intact MED 12 gene in enhancing T cell activation and effector351106305893\l\AMERICASfunction. In one example, the MED 12 gene may be disrupted as described in Katherine A Freitas et al., Enhanced T cell effector activity by targeting the Mediator kinase module, Science. 2022 Nov 1 l;378(6620):eabn5647, which is incorporated by reference in its entirety. In one example, the MED 12 gene is disrupted by gene editing using an RNA-guided nuclease system (e.g., C-RISPR-Cas system) comprising one or more guide RNAs comprising the sequence of SEQ ID NO: 353.

[0124] Modification(s) to modulate cytokine activity may enhance the modified T cell activation and / or its sensitivity to cytokines, and / or to reduce toxicity of cytokines produced by the modified T cell. The preferred modification to modulate cytokine activity comprises expression of a membrane-bound IL-12. Incorporation of a mbIL-12 in a T cell may provide a functional advantage over control cells that lack such a mbIL-12 in enhancing effector functions of the T cells and / or limiting the systemic toxicity associated with IL-12. Examples of the mbIL- 12 include those described in Hu J. et al., Cell membrane-anchored and tumor-targeted IL- 12 (attIL12)-T cell therapy for eliminating large and heterogeneous solid tumors, J Immunother Cancer. 2022 Jan;10(l):e003633; Hornbach A. et al., IL12 integrated into the CAR exodomain converts CD8+ T cells to poly-functional NK-like cells with superior killing of antigen-loss tumors, Mol Ther.2022 Feb 2;30(2):593-605; and Lee EH et al., Antigen-dependent IL-12 signaling in CAR T cells promotes regional to systemic disease targeting, bioRxiv. 2023 Jan 7;2023.01.06.522784; Jones et a., Cell surface-tethered IL-12 repolarizes the tumor immune microenvironment to enhance the efficacy of adoptive T cell therapy, Sci Adv. 2022 Apr 29;8(17):eabi8075; Kim SP, et al., Drug-regulatable, inducible, and membrane-bound interleukin 12 (IL-12TM-D) for use in adoptive cell therapies against advanced cancers. Journal for ImmunoTherapy of Cancer. 2024; 12; Zhang L et al., Adoptive transfer of membrane-restricted IL-12-TCR T cells promotes antigen spreading and elimination of antigen-negative tumor variants, J Immunother Cancer. 2024 Nov 18;12(1 l):e009868; Innamarato P et al., IOV-5001, autologous tumor-infiltrating lymphocytes (TIL) armored with inducible membrane-tethered IL-12, shows enhanced antitumor efficacy with an improved cellular state, Cancer Res (2025) 85 (8_Supplement_l): 4863; and Ross T et al., Spatiotemporally regulated expression of membrane-bound interleukin 12 (mbIL12) for armored adoptive cell therapy (ACT) shows strong antitumor activity in syngeneic solid tumor models without overt toxicity, Cancer Res (2025) 85 (8_Supplement_2): LB025, each of which is incorporated by reference herein in its entirety.361106305893\l\AMERICAS

[0125] In embodiments, the modified T cell herein expresses a soluble IL-12 and does not express membrane-bound IL-12. In embodiments, the modified T cell herein expresses a soluble IL-12 in addition to membrane-bound IL-12. In embodiments, a subject administered the modified T cell is also administered (simultaneously or sequentially) a composition for expressing soluble IL-12 in vivo. In certain examples, the subject is administered a modified T cell that has disrupted MED12 but does not express membrane-bound IL-12, and the subject is also administered exogenous IL-12 (simultaneously or sequentially), and / or a composition for expressing a soluble IL-12 in vivo. In certain examples, the subject is administered a modified T cell that has disrupted MED12 and expresses membrane-bound IL12, and the subject is also administered exogenous IL- 12 (simultaneously or sequentially) and / or a composition for expressing a soluble IL- 12 in vivo. Examples of suitable IL- 12 proteins include wild type IL- 12, modified IL- 12, or IL- 12 fused with another protein. Examples of compositions for expressing IL-12 in vivo include plasmid-based IL- 12 delivery compositions, mRNA-based IL-12 delivery compositions, virus-based IL-12 delivery compositions (e.g., adenoviruses, adeno-associated viruses, vaccinia viruses, lentivirus), chemicalbased IL- 12 delivery compositions (e.g., polymer-based), biomaterial -based IL- 12 delivery compositions (e.g., extracellular vesicles, exosomes, biomaterial depots), cell-based IL- 12 delivery compositions (e.g., dendritic cells, T cells, mesenchymal stromal cells). Examples of exogenous IL-12 and IL-12 delivery compositions include those described in Jia et al., IL12 immune therapy clinical trial review: Novel strategies for avoiding CRS-associated cytokines, Front Immunol. 2022 Sep 20:13:952231; Dong et al., Interleukin- 12 Delivery Strategies and Advances in Tumor Immunotherapy, Curr Issues Mol Biol. 2024 Oct 16;46(10): 11548-11579; Nguyen et al., Localized Interleukin- 12 for Cancer Immunotherapy, Front Immunol. 2020 Oct 15:11 :575597, each of which is incorporated by reference in its entirety.Additional Modifications

[0126] The modified T cells herein may comprise one or more modifications in addition to the modifications described above.

[0127] In embodiments, the modified T cells further comprise additional modification(s) to modulate cytokine activity. In embodiments, the modification(s) to modulate cytokine activity may further include disruption of the CISH gene, expression of a cytokine switch receptor, or combinations thereof371106305893\l\AMERICAS

[0128] In embodiments, the additional modification(s) to modulate cytokine activity includes disruption the cytokine inducible SH2-containing protein (CISH) gene. CISH is a negative regulator of TCR signaling. Disruption of the CISH gene may provide a functional advantage over control cells that have an intact CISH gene in improving the sensitivity to certain cytokines (e.g., IL-2 / IL-15), increasing T cell proliferation, and / or limiting T cell exhaustion. In some examples, the CISH gene may be disrupted by the method described in Daher M. et al, Targeting a cytokine checkpoint enhances the fitness of armored cord blood CAR-NK cells, Blood. 2021 Feb 4;137(5):624-636, which is incorporated by reference herein in its entirety. In some examples, the CISH gene is disrupted by gene editing using an RNA-guided nuclease system comprising one or more guide RNAs comprising the sequences of any one of SEQ ID NOs: 198-201 and 339-340.

[0129] In embodiments, the additional modification(s) to modulate cytokine activity includes expression of a cytokine switch receptor, e g., a chimeric switch receptor comprising an extracellular domain of a TGFp receptor for binding to TGFp (e.g., TGFpRI and / or TGFpRII), and an intracellular domain of a cytokine receptor. The chimeric switch receptors can convert a TGFP signal into a cytokine signal that promotes cytotoxicity. Examples of such chimeric switch receptors include those descried in WO2012138858, WO2016122738, WO2018094244, WO2014172584, W02019109980, and WO2022037562, each of which is incorporated by reference in its entirety.

[0130] In embodiments, the modified T cells further comprise additional modification(s) to modulate T cell metabolism. In embodiments, the additional modification(s) to modulate T cell metabolism includes disruption of the RASA2 gene.

[0131] In embodiments, the modification(s) to modulate T cell metabolism includes disruption of the RAS p21 protein activator 2 (RASA2) gene. RASA2 is a RAS GTPase-activating protein (RasGAP) that is a signaling checkpoint in T cells, which can be downregulated upon acute T cell receptor stimulation and can increase gradually with chronic antigen exposure. Disruption of RASA2 may enhance MAPK signaling and CAR T cell cytolytic activity in response to target antigen. RASA2-deficient T cells may exhibit increased activation, cytokine production and metabolic activity compared with control cells, and show a marked advantage in persistent cancer cell killing. In some examples, the RASA2 gene may be disrupted by the method described in Julia Carnevale et al., RASA2 ablation in T cells boosts antigen sensitivity and long-term function. Nature. 2022 Sep;609(7925):174-182, which is incorporated by reference herein in its entirety.381106305893\l\AMERICAS

[0132] In embodiments, the modified T cells may further comprise at least one modification to modulate disease microenvironment. Suitable modification(s) to modulate disease microenvironment may enhance the anti-disease activity of the modified T cell in the disease microenvironment. In embodiments, the at least one modification to modulate the disease microenvironment includes disruption of CBL-B gene, disruption of REGNASE-1 gene, disruption of roquin gene, disruption of TGF-0 receptor type 2 (TGF0R2) gene, expression of a dominant negative TGF-P receptor type 2 (dnTGF[3R2), or any combination thereof.

[0133] In embodiments, the modification(s) to modulate disease microenvironment includes disruption of the Cbl proto-oncogene B (CBL-B) gene. CBL-B is a negative regulator of T cell activation. Disruption of the CBL-B gene may provide a functional advantage over control cells that have an intact CBL-B gene in enhancing T cell activation. In some examples, the CBL-B gene may be disrupted by the methods described in Augustin R. et al., Targeting Cbl-b in cancer immunotherapy, J Immunother Cancer. 2023 Feb;l l(2):e006007; Hooper K. et al., Knockout of CBLB Greatly Enhances Anti-Tumor Activity of CAR T Cells, Blood (2018) 132 (Supplement 1): 338; and Guo X. et al., CBLB ablation with CRISPR / Cas9 enhances cytotoxicity of human placental stem cell-derived NK cells for cancer immunotherapy, J Immunother Cancer. 2021 Mar; 9(3 ):e001975, each of which is incorporated by reference herein in its entirety. In one example, the CBL-B gene is disrupted by gene editing using a CRISPR-Cas system comprising one or more guide RNAs comprising the sequence of any one of SEQ ID NOs: 341-344.

[0134] In embodiments, the modification(s) to modulate disease microenvironment includes disruption of the Regnase-1 gene. Regnase-1 (Regulatory RNase 1), also known as ZC3H12A or MCPIP-1, is a ribonuclease that promotes decay of target mRNA through recognition of 3’ UTR stem loop motifs. Disruption of the Regnase-1 gene may provide a functional advantage over control cells that have an intact Regnase-1 gene in enhancing T cell expansion and persistence. In one example, the Regnase-1 gene may be disrupted as described in Jun Wei et al., Targeting Regnase-1 programs long-lived effector T cells for cancer therapy. Nature. 2019 Dec; 576(7787): 471-476, which is incorporated by reference in its entirety.

[0135] In embodiments, the modification(s) to modulate disease microenvironment includes disruption of the Roquin (e.g., Roquin-1) gene. Disruption of the Roquin gene may provide a functional advantage over control cells that have an intact Roquin gene in increasing T cell proliferation and enhancing antitumor activity. In some examples, the Roquin gene may be391106305893\l\AMERICASdisrupted by the method described in Mai D et al., Combined disruption of T cell inflammatory regulators Regnase-1 and Roquin-1 enhances antitumor activity of engineered human T cells, Proc Natl Acad Sci U S A. 2023 Mar 21;120(12):e2218632120, which is incorporated by reference herein in its entirety.

[0136] In embodiments, the modification(s) to modulate disease microenvironment includes expressing a polypeptide that imparts T cells with the capability to resist disease associated antigen-specific cellular immunity, for example that mediated by transforming growth factor beta (TGF-P). For example, the modification to modulate disease microenvironment may be expressing a dominant negative receptor for TGF-beta (dnTGF0R2), e.g., as described in Foster et al., J Immunother. (2008); 31: 500-505, WO2019 / 173324A1, W02020 / 183131A1, and W02020042647A1. Incorporation of such a dominant negative receptor for TGF-beta may provide a functional advantage over control cells that lack such a dominant negative receptor for TGF-beta in the presence of a TGF-beta-secreting disease cell (e.g., tumor cell), including enhanced anti-disease (e.g., anti-tumor) activity. In embodiments, the modification to modulate disease microenvironment includes expressing a polypeptide that comprises or consists of the dominant-negative TGFp receptor II (dnTGFpR2) amino acid sequence as set forth in SEQ ID NO: 241. In embodiments, the polypeptide further comprises a signal peptide (e.g., comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 239) operably linked to SEQ ID NO: 241.

[0137] In embodiments, the modification(s) to modulate disease microenvironment includes disruption of the TGFPR2 gene. Disruption of TGFPR2 gene may mitigate the immunosuppressive effects of the TGFP signaling. In some examples, the TGFPR2 gene may be disrupted by the method described in Na Tang et al., TGF-P inhibition via CRISPR promotes the long-term efficacy of CAR T cells against solid tumors, JCI Insight. 2020 Feb 27; 5(4): el33977, which is incorporated by reference in its entirety.

[0138] In embodiments, the modified T cells may further comprise at least one alloevasion modification. Suitable alloevasion modification(s) may reduce the host v. graft allocytotoxicity. In embodiments, the at least one alloevasion modification include disruption of the ICAM-1 gene, disruption of the CD58 gene, disruption of the Fas gene, expression of dominant-negative Fas receptor (dnFas), expression of a chimeric antigen receptor (CAR) binding to CD70, or any combination thereof.401106305893\l\AMERICAS

[0139] In embodiments, the alloevasion modification(s) includes disruption of the ICAM- gene. Di sruption of the ICAM-1 gene may provide a functional advantage over control cells that have an intact ICAM-1 gene in disrupting T cell adhesion and co-stimulatory interactions to reduce Host vs Graft allocytotoxicity. In one example, the ICAM-1 gene may be disrupted as described in TeoHY etal. IL12 / 18 / 21 Preactivation Enhances the Antitumor Efficacy ofExpanded y8T Cells and Overcomes Resistance to Anti-PD-Ll Treatment, Cancer Immunol Res. 2023 Jul 5; 11(7):978-999, which is incorporated by reference in its entirety. In one example, the ICAM-1 gene is disrupted by gene editing using an RNA-guided nuclease system (e.g., CRISPR-Cas or CRISPR- Mad7 system) comprising one or more guide RNAs comprising the sequences of any one of SEQ ID NOs: 349-350.

[0140] In embodiments, the alloevasion modification(s) includes disruption of the CD58 gene. Disruption of the CD58 gene may provide a functional advantage over control cells that have an intact CD58 gene in disrupting T cell adhesion and co-stimulatory interactions to reduce Host vs Graft allocytotoxicity. In one example, the CD58 gene is disrupted by gene editing using an RNA- guided nuclease system (e.g., CRISPR-Cas or CRISPR-Mad7 system) comprising one or more guide RNAs comprising the sequences of any one of SEQ ID NOs: 351-352.

[0141] In some examples, the modified T cell comprises disruption of both the CD58 and ICAM-1 genes.

[0142] In embodiments, the alloevasion modification(s) includes disruption of the Fas (also known as CD95) gene Disruption of the Fas gene may provide a functional advantage over control cells that have an intact Fas gene in reducing T cell allocytotoxicity. In one example, the ICAM-1 gene may be disrupted as described in Jiangtao Ren et al., A versatile system for rapid multiplex genome-edited CAR T cell generation, Oncotarget. 2017 Mar 7; 8(10): 17002-17011, which is incorporated by reference herein in its entirety.

[0143] In embodiments, the alloevasion modification(s) includes expression of a dominant negative Fas (dnFas, also known as dnCD95). Incorporation of such a dominant negative Fas in a T cell may provide a functional advantage over control cells that lack such a dominant negative Fas in the prevention of Fas ligand-induced apoptosis and allowing for T cell persistence and antidisease (e.g., antitumor) efficacy. Examples of the dnFas include that described in Yamamoto TN et al., T cells genetically engineered to overcome death signaling enhance adoptive cancer411106305893\l\AMERICASimmunotherapy, J Clin Invest. 2019 Feb 25; 129(4): 1551-1565, which is incorporated by reference herein in its entirety.

[0144] In embodiments, the alloevasion modification(s) includes expression of an antibody or fragment thereof that binds to CD70. For example, the modification may be expression of a CAR comprising such antibody or fragment that binds to CD70. Incorporation of such a CD70-binding molecule in a T cell may provide a functional advantage over control cells that lack the CD70-binding molecule in reducing HvG alloreactivity by targeting CD70+ activated T cells. Examples of such CD70-binding molecules include those described in PCT / US2023 / 29047, which is incorporated by reference herein in its entirety.

[0145] In embodiments, the modified T cell further comprises disruption of Fas gene or expression of dnFas. In embodiments, the modified T cell further comprises disruption of TGF[3R2 gene or expression of dnTGF0R2.

[0146] In embodiments, the one or more additional modifications includes disruption of the ZFP91 gene. Disruption of the ZFP91 gene may provide a functional advantage over control cells that have an intact ZFP91 gene in improving T cell glycolytic fitness and effector function. In some examples, the ZFP91 gene may be disrupted by the method described in Wang F. et al., J Clin Invest. 2021 Oct 1 ; 131 (19):e 144318, which is incorporated by reference herein in its entirety.

[0147] In embodiments, the one or more additional modifications includes expression of an exogenous stem cell factor (SCF). The term “stem cell factor (SCF)” refers to a cytokine that that exerts its biological functions by binding to and activating the receptor tyrosine kinase c-Kit. Endogenous SCF is a stromal cell-derived cytokine synthesized by fibroblasts and other cell types. It is a glycoprotein that plays a key role in hematopoiesis acting both as a positive and negative regulator, often in synergy with other cytokines. It also plays a role in mast cell development, gametogenesis, and melanogenesis. SCF through c-Kit interaction regulates cell viability, proliferation, and differentiation both in physiological and pathological conditions (see e.g., Mazzoldi, E.L., et al. A juxtacrine / paracrine loop between C-Kit and stem cell factor promotes cancer stem cell survival in epithelial ovarian cancer. Cell Death Dis 10, 412 (2019)). In embodiments, SCF includes a soluble form SCF (e.g., comprising or consisting of EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTD LLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNR SIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAA, (human SCF based on421106305893\l\AMERICASNP_000890, first 165 aa of the mature peptide without signal peptide, SEQ ID NO: 105)). In embodiments, SCF includes a transmembrane form SCF (e.g., comprising or consisting of EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTD LLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNR SIDAFKDF VVASETSDCVVS STLSPEKDSRVSVTKPFMLPPVAAS SLRNDS S SSNRKAKN PPGD S SLHW A AM ALPALF SLIIGFAF GAL YWKKRQP SLTRA VENIQINEEDNEISMLQEK EREFQEV (human SCF based on NP_000890, mature peptide without signal peptide), see also Aderson DM et al. Cell Growth Differ. 1991 Aug;2(8):373-8), SEQ ID NO: 103), or comprising or consists of EGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTD LLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIFNR SIDAFKDFVVASETSDCVVSSTLSPEKGKAKNPPGDSSLHWAAMALPALFSLIIGFAFGA L YWKKRQP SLTRA VENIQINEEDNEISMLQEKEREFQEV (human SCF based on NP_ NP_003985, mature peptide without signal peptide), see also Johan Lennartsson et al. Physiol Rev. 2012 Oct;92(4): 1619-49, SEQ ID NO: 104). Examples of soluble and transmembrane forms of SCF also include those described in J G Flanagan et al., Transmembrane form of the kit ligand growth factor is determined by alternative splicing and is missing in the Sid mutant Cell. 1991 Mar 8;64(5): 1025-35; DM Anderson et al., Alternate splicing of mRNAs encoding human mast cell growth factor and localization of the gene to chromosome 12q22-q24, Cell Growth Differ. 1991 Aug;2(8):373-8; and Johan Lennartsson et al., Stem cell factor receptor / c-Kit: from basic science to clinical implications, Physiol Rev. 2012 Oct;92(4): 1619-49, each of which is incorporated by reference in its entirety.

[0148] In embodiments, the exogenous SCF may be a recombinant or synthetic stem cell factor, such as ancestim, a 166-amino-acid protein produced by E. coli bacteria into which a gene has been inserted for soluble human stem cell factor (MEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSL TDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFTPEEFFRIF NRSID AFKDF VVASETSDCVVS STLSPEKDSRVSVTKPFMLPPVAA, SEQ ID NO: 101) as described in McNiece IK and Briddel RA, The Cytokine Handbook (Fourth Edition), 2003); and MG da Silva et al. Ancestim (recombinant human stem cell factor, SCF) in association with fdgrastim does not enhance chemotherapy and / or growth factor-induced peripheral blood431106305893\l\AMERICASprogenitor cell (PBPC) mobilization in patients with a prior insufficient PBPC collection, Bone Marrow Transplant. 2004 Oct;34(8):683-91, each of which is incorporated by reference herein in its entirety.

[0149] Additional examples of recombinant and synthetic stem cell factor molecules include those described in WO1991005795 (titled “Stem cell factor”); US6020469 (titled “Stem cell factor formulations and methods”); US6759215 (titled “Method of preparing human stem cell factor polypeptide”); and US6218148 (titled “DNS encoding stem cell factor”), each of which is incorporated by reference herein in its entirety. In embodiments, the exogenous SCF is a SCF analog peptide, e.g., those described in US5885962 (titled “Stem cell factor analog compositions and method”); and Tilayov T. et al. Engineering Stem Cell Factor Ligands with Different c-Kit Agonistic Potencies, Molecules. 2020 Oct 21;25(20):4850, each of which incorporated by reference herein in its entirety.

[0150] In embodiments, the one or more additional modifications includes disruption of the Arid la gene. Arid la regulates the acquisition of the epigenetic state of terminal exhaustion. Disruption of the Aridla gene may provide a functional advantage over control cells that have an intact Aridla gene in improving tumor control and enhancing persistence of T cells. In one example, the Aridla gene may be disrupted as described in Julia A Belk et al., Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence, Cancer Cell. 2022 Jul 1 l;40(7):768-786.e7.

[0151] In embodiments, the one or more additional modifications includes one or more labels or markers, for example to facilitate an ability to monitor the expression level of the antigen recognition moiety (e.g., CAR), serve as an internal control, and the like. In embodiments, the one or more additional modifications includes expression of a fluorescent protein, examples of which include but are not limited to green fluorescent protein (GFP), red fluorescent protein (RFP), enhanced GFP (EGFP), enhanced cyan fluorescent protein (ECFP), enhanced yellow fluorescent protein (EYFP), and the like. Other examples can include but are not limited to chloramphenicol acetyltransferase, beta-galactosidase, beta-glucuronidase, beta-lactamase, luciferase, and the like.

[0152] In embodiments, the one or more additional modifications includes expression of a protein on a cell surface to facilitate detection and / or isolation of cells expressing the protein, e.g., via fluorescent activated cell sorting (FACS); or for enrichment through positive selection using an antibody specific to the encoded protein, e.g., use of an antibody to purify or enrich the cells441106305893\l\AMERICASproduct on a column or apparatus; or for in vivo binding of an antibody to the protein to enhance or eliminate activity, e.g., to facilitate removal of cells expressing the protein in patients as a safety consideration. Exemplary proteins useful for these purposes include, e.g., CD 19, CD20 (Rituxumab recognition domain), RQR8, LNGFR, a truncated form of the human epidermal growth factor receptor (EGFRt), and the like. By way of example, EGFRt can be targeted by a clinical stage antibody, where such treatment of a patient with the antibody results in elimination of cells containing an isolated nucleic acid encoding an antigen recognition moiety (e.g., CAR) as disclosed herein. See, e.g.,Wangel l. A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells; Blood 2011 118(5): 1255-63; Philip et al., A highly compact epitope-based marker / suicide gene for easier and safer T-cell therapy; Blood 2014 124(8): 1277-87; Smith J. et al., UCART19, an allogeneic “off-the-shelf’ adoptive T-cell immunotherapy against CD19+ B-cell leukemias, DOI: 10.1200 / jco.2015.33.15_suppl.3069 Journal of Clinical Oncology 33, no. 15_suppl (May 20, 2015) 3069-3069; Gouble A. et al., In Vivo Proof of Concept of Activity and Safety of UCART19, an Allogeneic “Off-the-Shelf’ Adoptive T-Cell Immunotherapy Against CD19+ B-Cell Leukemias, Blood (2014) 124 (21): 4689, doi.org / 10.1182 / blood.V124.21.4689.4689, each of which is incorporated by reference in its entirety.

[0153] In embodiments, the one or more additional modifications includes expression a protein that functions to increase resistance to exhaustion and activation-induced apoptosis and / or upregulate one or more proinflammatory cytokines, costimulatory molecules and / or antigen presentation machinery. A representative example includes but is not limited to lymphotoxin beta receptor (LTBR). LTBR is typically expressed in a subset of myeloid cells but is absent in lymphocytes. When expressed in T cells, LTBR may induce transcriptional remodeling that imparts the T cell with one or more of the above-mentioned advantageous functions (Legut et al., Blood. (2021); 138(1): 1726).

[0154] In embodiments, a signal peptide is operably linked to the expressed proteins to facilitate directing of the one or more proteins expressed in the modified T cell to the secretory pathway. Such proteins can be those that reside inside certain organelles, are secreted from the modified T cell, or are inserted into cellular membranes. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 170. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 196.451106305893\l\AMERICASIn embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 235. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 239. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 243. In embodiments, the signal peptide comprises or consists of the amino acid sequence set forth as SEQ ID NO: 247.

[0155] In embodiments, the one or more additional modifications includes expression a polypeptide that comprises or consists of the EGFRt amino acid sequence as set forth in SEQ ID NO: 237. In embodiments, the one or more additional modifications includes expression a polypeptide that comprises or consists of the full-length LTBR amino acid sequence as set forth in SEQ ID NO: 245. In embodiments, the signal peptide comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 243 is operably linked to SEQ ID NO: 245. In embodiments, the one or more additional modifications includes expression a polypeptide that comprises or consists of the LNGFR amino acid sequence as set forth in SEQ ID NO: 249. In embodiments, the signal peptide comprising or consisting of the amino acid sequence set forth as SEQ ID NO: 247 is operably linked to SEQ ID NO: 249. In embodiments, the one or more additional modifications includes expression a polypeptide that comprises or consists of the sIL-15 amino acid sequence as set forth in SEQ ID NO: 197. In embodiments, the signal peptide comprising or consisting of the amino acid sequence as set forth in SEQ ID NO: 196 is operably linked to SEQ ID NO: 197. In embodiments, the signal peptide comprising or consisting of the amino acid sequence MKKTQTWILTCIYLQLLLFNPLVKT is operably linked to a SCF sequence, e g., SEQ ID NO: 101, 103, 104, or 101.

[0156] In embodiments, the one or more additional modifications includes expression of a chimeric DAP10 adaptor polypeptide, which is capable of associating with a chimeric antigen receptor of the present disclosure, for example a CAR of the present disclosure may comprise a DAPlO-interacting domain. In such an example, the chimeric DAP 10 adaptor polypeptide may also associate with one or more additional endogenous or exogenous polypeptides, for example endogenous or exogenous NKG2D. In embodiments, a chimeric DAP 10 adaptor polypeptide may not associate with a CAR of the present disclosure, but instead may interact with an endogenous or exogenous polypeptide (e.g., NKG2D) that includes a DAPlO-interacting domain. Examples of a chimeric DAP 10 adaptor polypeptide and coding nucleic acid sequences include those described in U.S. Provisional Application No. 63 / 272,613 and U.S. Provisional Application No. 63 / 347,194,461106305893\l\AMERICASthe contents of each of which is hereby expressly incorporated by reference herein by reference in their entirety.

[0157] In embodiments, the one or more additional modifications includes disruption of one or more endogenous genes in addition to those describe above. For example, the one or more addition modifications includes disruption of one or more of: adenosine A2a receptor (ADORA), CD276, V-set domain containing T cell activation inhibitor 1 (VTCN1), B and T lymphocyte associated (BTLA), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), indoleamine 2,3-di oxy enase 1 (IDO1), killer cell immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1 (KIR3DL1), lymphocyte-activation gene 3 (LAG3), programmed cell death 1 (PD-1), hepatitis A virus cellular receptor 2 (HAVCR2), V-domain immunoglobulin suppressor of T-cell activation (VISTA), natural killer cell receptor 2B4 (CD244), hypoxanthine phosphoribosyltransferase 1 (HPRT), adeno-associated vims integration site (AAVS SITE (E.G. AAVS1, AAVS2, ETC.)), or chemokine (C-C motif) receptor 5 (gene / pseudogene) (CCR5), CD 160 molecule (CD 160), T-cell immunoreceptor with Ig and ITIM domains (TIGIT), CD96 molecule (CD96), cytotoxic and regulatory T-cell molecule (CRTAM), leukocyte associated immunoglobulin like receptor l(LAIRl), sialic acid binding Ig like lectin 7 (SIGLEC7), sialic acid binding Ig like lectin 9 (S1GLEC9), tumor necrosis factor receptor superfamily member 10b (TNFRSF10B), tumor necrosis factor receptor superfamily member 10a (TNFRSF10A), caspase 8 (CASP8), caspase 10 (CASP10), caspase 3 (CASP3), caspase 6 (CASP6), caspase 7 (CASP7), Fas associated via death domain (FADD), Fas cell surface death receptor (FAS), transforming growth factor beta receptor II (TGFBRII), transforming growth factor beta receptor I (TGFBR1), SMAD family member 2 (SMAD2), SMAD family member 3 (SMAD3), SMAD family member 4 (SMAD4), SKI proto-oncogene (SKI), SKI-like proto-oncogene (SKIL), TGFB induced factor homeobox 1 (TGIF1), interleukin 10 receptor subunit alpha (IL10RA), interleukin 10 receptor subunit beta (IL 1 ORB), heme oxygenase 2 (HM0X2), interleukin 6 receptor (IL6R), interleukin 6 signal transducer (IL6ST), c-src tyrosine kinase (CSK), phosphoprotein membrane anchor with glycosphingolipid microdomains 1(PAG1), signaling threshold regulating transmembrane adaptor 1 (SIT1), forkhead box P3 (FOXP3), PR domain 1 (PRDM1), basic leucine zipper transcription factor, ATF-like (BATF), guanylate cyclase 1, soluble, alpha 2 (GUCY1A2), guanylate cyclase 1, soluble, alpha 3 (GUCY1A3), guanylate cyclase 1, soluble, beta 2 (GUCY1B2), guanylate cyclase471106305893\l\AMERICAS1, soluble, beta 3 (GUCY1B3), prolyl hydroxylase domain (PHD1, PHD2, PHD3) family of proteins, or any combination thereof.

[0158] In embodiments, the one or more additional modifications includes disruption of the ZFP91 gene Disruption of the ZFP91 gene may provide a functional advantage over control cells that have an intact ZFP91 gene in improving T cell glycolytic fitness and effector function. In some examples, the ZFP91 gene may be disrupted by the method described in Wang F. et al., J Clin Invest. 2021 Oct 1 ; 131 (19):e 144318, which is incorporated by reference herein in its entirety.

[0159] In embodiments, the one or more additional modifications includes disruption of the Aridla gene. Aridla regulates the acquisition of the epigenetic state of terminal exhaustion. Disruption of the Aridla gene may provide a functional advantage over control cells that have an intact Aridla gene in improving tumor control and enhancing persistence of T cells. In one example, the Aridla gene may be disrupted as described in Julia A Belk et al., Genome-wide CRISPR screens of T cell exhaustion identify chromatin remodeling factors that limit T cell persistence, Cancer Cell. 2022 Jul 1 l;40(7):768-786.e7.

[0160] In embodiments, the one or more modifications herein increases the activity and / or function (e.g., increased cytotoxicity and / or potency when used as a T cell therapy) of the modified T cell compared with a counterpart T cell without such modification(s). In preferred embodiments, the modified T cell comprises disruption of MED12 and expression of a membrane-bound IL-12, and such modifications are demonstrated herein to dramatically increase the cytotoxicity of the modified T cell. In embodiments the modified T cell comprises a disruption in a MED12 gene, expression of a membrane-bound IL- 12, and a disruption in a Fas gene or expression of a dnFas in the modified T cell, and such modifications increase the cytotoxicity of the modified T cell. Antigen recognition moieties

[0161] The modified immune cells (e g., T cells) herein may comprise (e g., stably express) one or more antigen recognition moieties, which recognize a disease associated antigen, e.g., a tumor-associated antigen (also known as tumor antigen), an autoimmune disease-associated antigen, or an antigen associated with a pathogen (also known as pathogenic antigen). In embodiments, the modified immune cell comprises an antigen recognition moiety expressed on a surface of the cell, and the antigen recognition moiety comprises an affinity binding domain specific for a disease-associated antigen. Examples of antigen recognition moieties include a TCR, a[3 TCR, y8 TCR, a chimeric antigen receptor (CAR), whole antibody or their antigen-binding481106305893\l\AMERICASfragment, single-chain variable fragment (scFv), a heavy chain-only antibody (VHH), a heavy chain or a light chain single domain antibody (sdAb), a Fab, a F(ab)2, or any combination thereof that binds to: (i) a cell surface tumor antigen, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune disease or a pathogen, or (iv) a peptide derived from an antigen associated with an autoimmune disease or a pathogen expressed on the cell surface as a peptide-MHC complex.

[0162] A modified immune cell herein may express one, or multiple antigen recognition moieties. In embodiments, a modified immune cell expresses two or more antigen recognition moieties. In some examples, each antigen recognition moiety recognizes a different epitope of the same antigen. In some examples, each antigen recognition moiety recognizes different epitopes of different antigens.

[0163] In embodiments, two or more antigen recognition moieties may be expressed in a modified T cell from genetically different, substantially different, or substantially identical, TCR polynucleotides stably expressed from the modified T cell or from genetically distinct TCR polynucleotides stably incorporated in the modified T cell. In the case of genetically distinct TCR(s), TCR(s) recognizing different antigens associated with the same condition may be utilized. In one preferred embodiment, a modified T cell is engineered to express different TCRs, from human or mouse origin, from one or more expression cassettes that recognize the same antigen in the context of different MHC haplotypes. In another preferred embodiment, a modified T cell is engineered to express one TCR and two or more antibodies directed to the same or different peptides from a given antigen complexed with different MHC haplotypes. In some cases, expression of a single TCR by a T cell facilitates proper TCR pairing. A modified T cell that expresses different TCRs can provide a universal allogeneic modified T cell. In a second preferred embodiment, a modified T cell is engineered to express one or more different antibodies directed to peptide-MHC complexes, each directed to the same or different peptide complexed with the same or different MHC haplotypes. In some cases, an antigen recognition moiety can be an antibody that binds to peptide-MHC complexes.

[0164] A modified T cell can be engineered to express TCRs from one or more expression cassettes that recognize the same antigen in the context of different MHC haplotypes. In some cases, a modified T cell is designed to express a single TCR, or a TCR in combination with a CAR491106305893\l\AMERICASto minimize the likelihood of TCR mispairing within the engineered cell. The antigen recognition moi eties expressed from two or more expression cassettes preferably have different polynucleotide sequences, and encode antigen recognition moieties that recognize different epitopes of the same target, e.g., in the context of different HLA haplotypes. A modified T cell that expresses such different TCRs or CARs can provide a universal allogeneic modified T cell.

[0165] In some cases, a modified immune cell is engineered to express two or more antigen recognition moieties. The two or more antigen recognition moieties may be expressed from genetically identical, or substantially identical, antigen-specific chimeric (CAR) polynucleotides engineered in the modified immune cell. Two or more antigen recognition moieties may be expressed from genetically distinct CAR polynucleotides engineered in the modified immune cell. The genetically distinct CAR(s) may be designed to recognize different antigens associated with the same condition.

[0166] An antigen recognition moiety may be engineered to recognize an antigen with certain avidity. For instance, an antigen recognition moiety encoded by a TCR or CAR construct may recognize an antigen with a dissociation constant of at least at least 10 fM, at least 100 fM, at least 1 picomolar (pM), at least 10 pM, at least 20 pM, at least 30 pM, at least 40 pM, at least 50 pM, at least 60 pM, at least 7 pM, at least 80 pM, at least 90 pM, at least 100 pM, at least 200 pM, at least 300 pM, at least 400 pM, at least 500 pM, at least 600 pM, at least 700 pM, at least 800 pM, at least 900 pM, at least 1 nanomolar (nM), at least 2 nM, at least 3 nM, at least 4 nM, at least 5 nM, at least 6 nM, at least 7 nM, at least 8 nM, at least 9 nM, at least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nm, at least 60 nM, at least 70 nM, at least 80 nM, at least 90 nM, at least 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, at least 1 mM, at least 2 mM, at least 3 pM, at least 4 pM, at least 5 pM, at least 6 pM, at least 7 pM, at least 8 pM, at least 9 pM, at least 10 pM, at least 20 pM, at least 30 pM, at least 40 pM, at least 50 pM, at least 60 pM, at least 70 pM, at least 80 pM, at least 90 pM, or at least 100 pM.

[0167] In some instances, an antigen recognition moiety may be engineered to recognize an antigen with a dissociation constant of at most 10 fM, at most 100 fM, at most 1 picomolar (pM), at most 10 pM, at most 20 pM, at most 30 pM, at most 40 pM, at most 50 pM, at most 60 pM, at most 7 pM, at most 80 pM, at most 90 pM, at most 100 pM, at most 200 pM, at most 300 pM, at most 400 pM, at most 500 pM, at most 600 pM, at most 700 pM, at most 800 pM, at most 900 pM,501106305893\l\AMERICASat most 1 nanomolar (nM), at most 2 nM, at most 3 nM, at most 4 nM, at most 5 nM, at most 6 nM, at most 7 nM, at most 8 nM, at most 9 nM, at most 10 nM, at most 20 nM, at most 30 nM, at most 40 nM, at most 50 nm, at most 60 nM, at most 70 nM, at most 80 nM, at most 90 nM, at most 100 nM, at most 200 nM, at most 300 nM, at most 400 nM, at most 500 nM, at most 600 nM, at most 700 nM, at most 800 nM, at most 900 nM, at most 1 mM, at most 2 mM, at most 3 pM, at most 4 pM, at most 5 pM, at most 6 pM, at most 7 pM, at most 8 pM, at most 9 pM, at most 10 pM, at most 20 pM, at most 30 pM, at most 40 pM, at most 50 pM, at most 60 pM, at most 70 pM, at most 80 pM, at most 90 pM, or at most 100 pM.

[0168] An antigen recognition moiety may recognize a tumor associated antigen. The term “tumor associated antigen” or “tumor antigen” includes any molecule expressed on (or associated with the development of) a tumor cell that contributes to a tumorigenic characteristic of the tumor cell. Numerous tumor antigens are known in the art. Whether a molecule is a tumor antigen can also be determined according to techniques and assays well known to those skilled in the art, such as for example clonogenic assays, transformation assays, in vitro or in vivo tumor formation assays, gel migration assays, gene knockout analysis, etc. Examples of suitable tumor antigens include, but are not limited to, CD19, CD20, CD30, CD22, CD37, CD38, CD56, CD33, CD138, CD123, CD79b, CD70, CD75, CA6, GD2, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), RON, CEACAM5, CA-125, MUC-16, 5T4, NaPi2b, ROR1, ROR2, PLIF, Her2 / Neu, EGFRvIII, GPMNB, LIV-1, glycolipidF77, fibroblast activation protein (FAP), PSMA, STEAP-1, STEAP-2, mesothelin, c-Met, CSPG4, PVRL-4, VEGFR2, PSCA, CLEC12a, LI CAM, GPC2, GPC3, folate binding protein / receptor, SLC44A4, Cripto, CTAG1B, AXL, IL-13R, IL-3Ra2, SLTRK6, gplOO, MARTI, Tyrosinase, SSX2, SSX4, NYESO-1, WT-1, PRAME, epithelial tumor antigen (ETA), MAGEA family genes (such as MAGEA3. MAGEA4), KKLC1, mutated ras, VRaf, p53, MHC class I chain- related molecule A (MICA), or MHC class I chain-related molecule B (MICB), or one or more antigens of HPV, CMV, or EBV, BCMA, GPC3, TyrD, B7H6, CD70, or PSMA.

[0169] An antigen recognition moiety may recognize an antigen associated with an autoimmune disease. Such antigens include endogenous antigens that stimulate the production of an autoimmune response, such as production of autoantibodies. Antigens associated with autoimmune diseases also include antigens from a normal tissue that is the target of a cell mediated or an antibody-mediated immune response that may result in the development of an autoimmune511106305893\l\AMERICASdisease. Examples of antigens associated with autoimmune diseases include aggrecan, alanyl-tRNA syntetase (PL-12), alpha beta crystallin, alpha fodrin (Sptan 1), alpha-actinin, al antichymotrypsin, al antitripsin, al microglobulin, aldolase, aminoacyl-tRNA synthetase, an amyloid, an annexin, an apolipoprotein, aquaporin, bactericidal / permeability-increasing protein (BPI), P-globin precursor BP1, 0-actin, P-lactoglobulin A, P-2-gly coprotein I, p2-microglobulin, a blood group antigen, C reactive protein (CRP), calmodulin, calreticulin, cardiolipin, catalase, cathepsin B, a centromere protein, chondroitin sulfate, chromatin, collagen, a complement component, cytochrome C, cytochrome P4502D6, cytokeratins, decorin, dermatan sulfate, DNA topoisomerase I, elastin, Epstein-Barr nuclear antigen 1 (EBNA1), elastin, entaktin, an extractable nuclear antigen, Factor I, Factor P, Factor B, Factor D, Factor H, Factor X, fibrinogen, fibronectin, formiminotransferase cyclodeaminase (LC-1), gp210 nuclear envelope protein, GP2 (major zymogen granule membrane glycoprotein), a glutenin, glycoprotein gpIIb / IIIa, glial fibrillary acidic protein (GFAP), glycated albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), haptoglobin A2, heat shock proteins, hemocyanin, heparin, a histone, histidyl-tRNA synthetase (Jo-1), a hordein, hyaluronidase, immunoglobulins, an integrin, interstitial retinol-binding protein 3, intrinsic factor, Ku (p70 / p80), lactate dehydrogenase, laminin, liver cytosol antigen type 1 (LC1), liver / kidney microsomal antigen 1 (LKM1), lysozyme, melanoma differentiation-associated protein 5 (MDAS), Mi-2 (chromodomain helicase DNA binding protein 4), a mitochondrial protein, muscarinic receptors, myelin-associated glycoprotein, myosin, myelin basic protein, myelin proteolipid protein, myelin oligodendrocyte glycoprotein, myeloperoxidase (MPO), rheumatoid factor (IgM anti-IgG), neuron-specific enolase, nicotinic acetylcholine receptor A chain, nucleolin, a nucleoporin, nucleosome antigen, PM / ScllOO, PM / Scl 75, pancreatic 3-cell antigen, pepsinogen, peroxiredoxin 1, phosphoglucose isomerase, phospholipids, phosphatidyl inositol, platelet derived growth factors, polymerase beta (POLB), potassium channel KIR4.1, proliferating cell nuclear antigen (PCNA), proteinase-3, proteolipid protein, proteoglycan, prothrombin, recoverin, rhodopsin, ribonuclease, a ribonucleoprotein, ribosomes, a ribosomal phosphoprotein, RNA, an Sm protein, SplOO nuclear protein, SRP54 (signal recognition particle 54 kDa), a selectin, smooth muscle proteins, sphingomyelin, streptococcal antigens, superoxide dismutase, synovial joint proteins, T1F1 gamma collagen, threonyl-tRNA synthetase (PL-7), tissue transglutaminase, thyroid peroxidase, thyroglobulin, thyroid stimulating hormone receptor, transferrin, triosephosphate isomerase, tubulin, tumor necrosis factor-alpha, topoisomerase, Ul-521106305893\l\AMERICASdnRNP 68 / 70 kDa, Ul-snRNP A, Ul-snRNP C, U-snRNP B / B', ubiquitin, vascular endothelial growth factor, vimentin, and vitronectin.

[0170] In embodiments, an antigen recognition moiety recognizes a disease associated antigen expressed on an immune cell. In some examples, such immune cell is a B cell, including at all phases of development including terminal differentiation into plasma cells such as, e.g. pro B cells, pre B cells, transitional / immature B cells, naive B cells, GC B cells, memory B cells, plasmablasts, and plasma cell. In some examples, such immune cell is a T cell such as a Thl / Thl7 T cell, CD4+ T cell, keratinocyte, and the cells described in Lee et al., B cell depletion therapies in autoimmune disease: advances and mechanistic insights, Nat Rev Drug Discov. 2021 Mar;20(3): 179-199, which is incorporated by reference in its entirety.

[0171] In embodiments, the antigen recognition moiety binds to one or more of CD 19, CD20, CD22, CD37, CD38, CD40, CD40L, CD52, CD79b, CD123, CD138, BAFF-R, BCMA, FcRL5, GPRC50, TAC1, FcgRIIB, IL-36, IL-36R, IL-6, IL-6R, alpha4 integrin, CXCR6, DSG3, PD1, VISTA, BTLA, LAG3, ICOS, and ICOS-L. In embodiments, the antigen recognition moiety binds to a B cell antigen selected from the group comprising or consisting of CD19, CD20, CD22, CD37, CD38, CD40, CD52, CD79b, CD123, CD138, BAFF-R, BCMA, FcRL5, GPRC50, TAC1, and FcgRIIB. In embodiments, the antigen recognition moiety binds to a plasma cell antigen selected from the group comprising or consisting of CD38, CD 138, BCMA, FcRL5, GPRC5D, TACI, and FcgRIIB. In embodiments, the antigen recognition moiety binds to a T cell antigen selected from the group comprising or consisting of CD52, CD40L, alpha4 integrin, CXCR6, PD1, VISTA, BTLA, LAG3, ICOS, and ICOS-L.

[0172] An antigen recognition moiety may recognize a pathogenic antigen. A pathogenic antigen may be a bacterial, viral, or fungal molecule, such as a bacterial, viral, or fungal protein. In embodiments, an antigen presenting cell may internalize pathogenic molecules (e.g., pathogenic proteins, nucleic acids, lipids, or fragments produced by a pathogenic organi sm such as a bacterium or a virus), for instance with phagocytosis or by receptor-mediated endocytosis, and display a fragment of the antigen bound to an appropriate MHC molecule. For instance, various 9 mer fragments of a pathogenic protein may be displayed by an APC. Modified immune cells populations of the disclosure may be designed to recognize various antigens and antigen fragments of a pathogenic bacterium or a virus.531106305893\l\AMERICAS

[0173] Examples of pathogenic bacteria can be found in the: a) Bordetella genus, such as Bordetella pertussis species; b) Borrelia genus, such Borrelia burgdorferi species; c) Brucelia genus, such as Brucella abortus, Brucella canis, Brucela meliterisis, and / or Brucella suis species; d) Campylobacter genus, such as Campylobacter jejuni species; e) Chlamydia and Chlamydophila genuses, such as Chlamydia pneumonia, Chlamydia trachomatis, and / or Chlamydophila psittaci species; f) Clostridium genus, such as Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani species; g) Corynebacterium genus, such as Corynebacterium diphtheria species; h) Enterococcus genus, such as Enterococcus faecalis, and / or Enterococcus faecium species; i) Escherichia genus, such as Escherichia coli species; j) Francisella genus, such as Francisella tularensis species; k) Haemophilus genus, such as Haemophilus influenza species; 1) Helicobacter genus, such as Helicobacter pylori species; m) Legionella genus, such as Legionella pneumophila species; n) Leptospira genus, such as Leptospira interrogans species; o) Listeria genus, such as Listeria monocytogenes species; p) Mycobacterium genus, such as Mycobacterium leprae, mycobacterium tuberculosis, and / or mycobacterium ulcerans species; q) Mycoplasma genus, such as Mycoplasma pneumonia species; r) Neisseria genus, such as Neisseria gonorrhoeae and / or Neisseria meningitidia species; s) Pseudomonas genus, such as Pseudomonas aeruginosa species; t) Rickettsia genus, such as Rickettsia rickettsii species; u) Salmonella genus, such as Salmonella typhi and / or Salmonella typhimurium species; v) Shigella genus, such as Shigella sonnei species; w) Staphylococcus genus, such as Staphylococcus aureus, Staphylococcus epidermidis, and / or Staphylococcus saprophyticus spedes; x) Streptpcoccus genus, such as Streptococcus agalactiae, Streptococcus pneumonia, and / or Streptococcus pyogenes species; y) Treponema genus, such as Treponema pallidum species; z) Vibrio genus, such as Vibrio cholera; and / or aa) Yersinia genus, such as Yersinia pestis spedes.

[0174] Examples of pathogenic viruses can be found in the following families of viruses and are illustrated with exemplary spedes: a) Adenoviridae family, such as Adenovirus spedes; b) Herpesviridae family, such as Herpes simplex type 1, Herpes simplex type 2, Varicella-zoster virus, Epstein-barr virus, Human cytomegalovirus, Human herpesvirus type 8 species; c) Papillomaviridae family, such as Human papillomavirus species; d) Polyomaviridae family, such as BK virus, JC virus species; e) Poxviridae family, such as Smallpox species; f) Hepadnaviridae family, such as Hepatitis B virus species; g) Parvoviridae family, such as Human bocavirus, Parvovirus B19 species; h) Astroviridae family, such as Human astrovirus spedes; i) Caliciviridae541106305893\l\AMERICASfamily, such as Norwalk virus species; j) Flaviviridae family, such as Hepatitis C virus (HCV), yellow fever virus, dengue virus, West Nile virus species; k) Togaviridae family, such as Rubella virus species; 1) Hepeviridae family, such as Hepatitis E virus species; m) Retroviiidae family, such as Human immunodeficiency virus (HIV) species; n) Orthomyxoviridaw family, such as Influenza virus species; o) Arenaviridae family, such as Guanarito virus, Junin virus, Lassa virus, Machupo virus, and / or Sabia virus species; p) Bunyaviridae family, such as Crimean-Congo hemorrhagic fever virus species; q) Filoviridae family, such as Ebola virus and / or Marburg virus spedes; Paramyxoviridae family, such as Measles virus, Mumps virus, Parainfluenza virus, Respiratory syncytial virus, Human metapneumovirus, Hendra virus and / or Nipah virus spedes; r) Rhabdoviridae genus, such as Rabies virus species; s) Reoviridae family, such as Rotavirus, Orbivirus, Coltivirus and / or Banna virus species. In some examples, a virus is unassigned to a viral family, such as Hepatitis D.

[0175] In embodiments, one or more antigen recognition moieties in the modified immune cell is or comprises a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a bispecific antibody, and any fragment thereof. In embodiments, the antigen recognition moietyis or comprises an antibody, an antigen binding fragment (Fab), a single-chain variable fragment (scFv), a heavy chain or a light chain single domain antibody (sdAb), a F(ab)2, or any combination thereof. In embodiments, the one or more antigen recognition moieties comprises a mammalian antibody or a fragment thereof. The choice of more antigen recognition moieties may depend upon the type and number of antigens that are present on the surface of a target cell.

[0176] Additional examples of antigen binding domains included those described in PCT / US2019 / 054132, PCT / US2019 / 054144, PCT / US2023 / 034227, PCT / US2023 / 029047, PCT / US2023 / 030115, each of which is incorporated by reference in its entirety.Polyclonal Antibodies

[0177] Polyclonal antibodies may be raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen (especially when synthetic peptides are used) to a protein that is immunogenic in the species to be immunized. For example, the antigen can be conjugated to keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation551106305893\l\AMERICASthrough cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOC12, or R'N=C=NR, where R and R1 are different alkyl groups.

[0178] Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 pg or 5 pg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund’s complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with Vs to 1 / 10 the original amount of peptide or conjugate in Freund’s complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.Monoclonal Antibodies

[0179] A monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art. These include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 495-497), the human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4: 72), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy Alan R. Liss, Inc., pp.77-96). The Selected Lymphocyte Antibody Method (SLAM) (Babcook, J.S., et al., A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. Proc Natl Acad Sci U S A, 1996. 93 (15): p. 7843-8. ) and (McLean G et al., 2005, J Immunol. 174(8): 4768-78. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD and any subclass thereof. The hybridoma producing the mAbs of use in this invention may be cultivated in vitro or in vivo.

[0180] Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

[0181] In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form561106305893\l\AMERICASa hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0182] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which may contain one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[0183] Preferred fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a selective medium that selects against the unfused parental cells. Preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 andMPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Va., USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0184] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0185] The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal. Biochem. 107: 220 (1980).

[0186] Once hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPML1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal, eg., by intraperitoneal injection of the cells into mice.571106305893\l\AMERICAS

[0187] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ionexchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.

[0188] DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coll cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol. 5: 256-62 (1993) and Pliickthun, Immunol. Rev. 130: 151-88 (1992).

[0189] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348: 552-54 (1990). Clackson et al., Nature, 352: 624-28 (1991) and Marks et al., J. Mol. Biol., 222: 581-97 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10: 779-83 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21 : 2265-6 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

[0190] The DNA that encodes the antibody may be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy chain and light chain constant domain (CH and CO sequences for the homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison, et al., Broc. Natl. Acad. Sci. USA, 81: 6851 (1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence for a nonimmunoglobulin polypeptide (heterologous polypeptide). The non-immunoglobulin polypeptide sequences can substitute for the constant domains of an antibody, or they are substituted for the581106305893\l\AMERICASvariable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.Chimeric, Humanized, and Human Antibodies

[0191] In embodiments, an antigen recognition moiety is or comprises a chimeric antibody. Certain examples of chimeric antibodies designs are described, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-5 (1984)). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0192] In embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0193] An antigen recognition moiety herein may comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient591106305893\l\AMERICASantibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321: 522-5 (1986); Riechmann et al., Nature, 332: 323-9 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-6 (1992)).

[0194] A humanized antibody of the invention may comprise one or more human and / or human consensus non-hypervariable region (e.g., framework) sequences in its heavy and / or light chain variable domain. In embodiments, one or more additional modifications are present within the human and / or human consensus non-hypervariable region sequences. In one embodiment, the heavy chain variable domain of an antibody of the invention comprises a human consensus framework sequence, which in one embodiment is the subgroup III consensus framework sequence. In one embodiment, an antibody of the invention comprises a variant subgroup III consensus framework sequence modified at least one amino acid position.

[0195] As is known in the art, the amino acid position / boundary delineating a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art. Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions (as further defined below). The invention provides antibodies comprising modifications in these hybrid hypervariable positions. In one embodiment, these hypervariable positions include one or more positions 26-30, 33-35B, 47-49, 57-65, 93, 94 and 101-102 in a heavy chain variable domain. In one embodiment, these hybrid hypervariable positions include one or more of positions 24-29, 35-36, 46-49, 56 and 97 in a light chain variable domain. In one embodiment, an antibody of the invention comprises a human variant human subgroup consensus framework sequence modified at one or more hybrid hypervariable positions.

[0196] An antigen recognition moiety herein can comprise any suitable human or human consensus light chain framework sequences, provided the antibody exhibits the desired biological601106305893\l\AMERICAScharacteristics (e.g., a desired binding affinity). Tn one embodiment, an antibody of the invention comprises at least a portion (or all) of the framework sequence of human K light chain. In one embodiment, an antibody of the invention comprises at least a portion (or all) of human K subgroup I framework consensus sequence.

[0197] Methods for humanizing non-human antibodies are well known in the art. As discussed, a humanized antibody generally has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs for CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0198] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity and HAMA response (human antimouse antibody) when the antibody is intended for human therapeutic use. Reduction or elimination of a HAMA response is a significant aspect of clinical development of suitable therapeutic agents (see, e.g., Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et al., Transplantation (1986), 41 :572; Shawler et al., J. Immunol. (1985), 135: 1530; Sears et al., J. Biol. Response Mod. (1984), 3:138; Miller et al., Blood (1983), 62:988; Hakimi et al., J. Immunol. (1991), 147:1352; Reichmann et al., Nature (1988), 332: 323; Junghans et al., Cancer Res. (1990), 50: 1495). As described herein, the invention provides antibodies that are humanized such that HAMA response is reduced or eliminated. Variants of these antibodies can further be obtained using routine methods known in the art, some of which are further described below. According to the so-called “best-fit” method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human V domain sequence which is closest to that of the rodent is identified and the human framework region (FR) within it accepted for the humanized antibody (Sims et al., J. Immunol. 151: 2296611106305893\l\AMERICAS(1993); Chothiaet al., J. Mol. Biol., 196: 901 (1987)). Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol. 151: 2623 (1993)).

[0199] For example, an amino acid sequence from an antibody as described herein can serve as a starting (parent) sequence for diversification of the framework and / or hypervariable sequence(s). A selected framework sequence to which a starting hypervariable sequence is linked is referred to herein as an acceptor human framework. While the acceptor human frameworks may be from, or derived from, a human immunoglobulin (the VL and / or VH regions thereof), preferably the acceptor human frameworks are from, or derived from, a human consensus framework sequence as such frameworks that have been demonstrated to have minimal, or no, immunogenicity in human patients.

[0200] Where the acceptor is derived from a human immunoglobulin, one may optionally select a human framework sequence that is selected based on its homology to the donor framework sequence by aligning the donor framework sequence with various human framework sequences in a collection of human framework sequences, and select the most homologous framework sequence as the acceptor.

[0201] In one embodiment, human consensus frameworks herein are from, or derived from, VH subgroup III and / or VL kappa subgroup I consensus framework sequences.

[0202] While the acceptor may be identical in sequence to the human framework sequence selected, whether that be from a human immunoglobulin or a human consensus framework, the present invention contemplates that the acceptor sequence may comprise pre-existing amino acid substitutions relative to the human immunoglobulin sequence or human consensus framework sequence. These pre-existing substitutions are preferably minimal; usually four, three, two or one amino acid differences only relative to the human immunoglobulin sequence or consensus framework sequence.

[0203] Hypervariable region residues of the non-human antibody are incorporated into the VL and / or VH acceptor human frameworks. For example, one may incorporate residues corresponding to the Kabat CDR residues, the Chothia hypervariable loop residues, the Abm residues, and / or contact residues. Optionally, the extended hypervariable region residues as follows are621106305893\l\AMERICASincorporated: 24-34 (LI), 50-56 (L2) and 89-97 (L3), 26-35B (Hl), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3).

[0204] While “incorporation” of hypervariable region residues is discussed herein, it will be appreciated that this can be achieved in various ways, for example, nucleic acid encoding the desired amino acid sequence can be generated by mutating nucleic acid encoding the mouse variable domain sequence so that the framework residues thereof are changed to acceptor human framework residues, or by mutating nucleic acid encoding the human variable domain sequence so that the hypervariable domain residues are changed to non-human residues, or by synthesizing nucleic acid encoding the desired sequence, etc.

[0205] As described herein, hypervariable region-grafted variants may be generated by Kunkel mutagenesis of nucleic acid encoding the human acceptor sequences, using a separate oligonucleotide for each hypervariable region. Kunkel et al., Methods Enzymol. 154:367-382 (1987). Appropriate changes can be introduced within the framework and / or hypervariable region, using routine techniques, to correct and re-establish proper hypervariable region-antigen interactions.

[0206] Phage(mid) display (also referred to herein as phage display in some contexts) can be used as a convenient and fast method for generating and screening many different potential variant antibodies in a library generated by sequence randomization. However, other methods for making and screening altered antibodies are available to the skilled person.

[0207] Phage(mid) display technology has provided a powerful tool for generating and selecting novel proteins which bind to a ligand, such as an antigen. Using the techniques of phage(mid) display allows the generation of large libraries of protein variants which can be rapidly sorted forthose sequences that bind to a target molecule with high affinity. Nucleic acids encoding variant polypeptides are generally fused to a nucleic acid sequence encoding a viral coat protein, such as the gene III protein or the gene VIII protein. Monovalent phagemid display systems where the nucleic acid sequence encoding the protein or polypeptide is fused to a nucleic acid sequence encoding a portion of the gene III protein have been developed. (Bass, S., Proteins, 8:309 (1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology, 3:205 (1991)). In a monovalent phagemid display system, the gene fusion is expressed at low levels and wild type gene III proteins are also expressed so that infectivity of the particles is retained. Methods of generating peptide libraries and screening those libraries have been disclosed in many patents (e.g.,631106305893\l\AMERICASU.S. Pat. No. 5,723,286, U.S. Pat. No. 5,432,018, U.S. Pat. No. 5,580,717, U.S. Pat. No. 5,427,908 and U.S. Pat. No. 5,498,530).

[0208] Libraries of antibodies or antigen binding polypeptides have been prepared in a number of ways including by altering a single gene by inserting random DNA sequences or by cloning a family of related genes. Methods for displaying antibodies or antigen binding fragments using phage(mid) display have been described in U.S. Pat. Nos. 5,750,373, 5,733,743, 5,837,242, 5,969,108, 6,172,197, 5,580,717, and 5,658,727. The library is then screened for expression of antibodies or antigen binding proteins with the desired characteristics.

[0209] Methods of substituting an amino acid of choice into a template nucleic acid are well established in the art, some of which are described herein. For example, methods for introducing modifications into nucleic acid sequences can include the use of various kits available for purchase (e.g., QuickChange Site Directed Mutagenesis Kit, Agilent, Santa Clara, CA). As another example, hypervariable region residues can be substituted using the Kunkel method (e.g., Kunkel et al., Methods Enzymol. 154:367-382 (1987)).

[0210] It is important that antibodies be humanized with retention of high binding affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0211] Various forms of a humanized antibody are contemplated. For example, the humanized antibody may be an antibody fragment, such as a Fab. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgGl antibody.641106305893\l\AMERICAS

[0212] As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ -line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits et al., Proc. Natl. Acad. Set. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-8 (1993); Bruggemann et al., Year in Immuno. 7: 33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97 / 17852).

[0213] Alternatively, phage display technology (McCafferty et al., Nature 348: 552-53 (1990)) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as Ml 3 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S, and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol. 222:581-97 (1991), or Griffith et al., EMBO J. 12: 725-34 (1993) (see also, U.S. Pat. Nos. 5,565,332 and 5,573,905).

[0214] Human antibodies may also be generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275).651106305893\l\AMERICAS

[0215] Accordingly, in embodiments, human monoclonal antibodies may be generated using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system.

[0216] The HuMAb Mouse™ (Medarex, Inc.) contains human immunoglobulin gene miniloci that encode unrearranged human heavy (p and y) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous p and K chain loci (see e.g., Lonberg, et al. (1994) Nature 368(6474): 856-9). Accordingly, the mice exhibit reduced expression of mouse IgM or K, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGK monoclonal antibodies (Lonberg, N. et al. (1994), supra, reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113: 49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Set.764: 536-46). Preparation and use of the HuMAb Mouse™, and the genomic modifications carried by such mice, is further described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3720-4; Choi et al. (1993) Nature Genetics 4:117-23; Chen, J. et al. (1993) EMBO J. 12: 21-830; Tuaillon et al., (1994) J. Immunol. 152: 2912-20; Taylor, L. et al. (1994) International Immunology 6: 579-91; and Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-51, the contents of all of which are hereby specifically incorporated by reference in their entirety. See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; U.S. Pat. No. 5,545,807; PCT Publication Nos. WO 92 / 03918, WO 93 / 12227, WO 94 / 25585, WO 97 / 13852, WO 98 / 24884 and WO 99 / 45962; and PCT Publication No. WO 01 / 14424.

[0217] In another embodiment, human antibodies of this disclosure can be raised using a mouse referred to as “KM Mouse™” that carries human immunoglobulin sequences on transgenes and transchromosomes, as described in detail in PCT Publication WO 02 / 43478.

[0218] In another embodiment, an alternative transgenic system referred to as the Xenomouse (Abgenix, Inc.) can be used; such mice are described in, for example, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963.

[0219] Additional relevant transchromosomic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise the antibodies. For example,661106305893\l\AMERICASmice carrying both a human heavy chain transchromosome and a human light chain tranchromosome, referred to as “TC mice” can be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97: 722-7. As another example, cows carrying human heavy and light chain transchromosomes have been described in the art (e.g., Kuroiwa et al. (2002) Nature Biotechnology 20: 889-94 and PCT application No. WO 2002 / 092812) and can be used to raise the antibodies of this disclosure. Additional examples of transgenic animals that can be used to produce the antibodies include OmniRat™ and OmniMouse™ (see e.g., Osborn M., et al. (2013) Journal of Immunology 190: 1481-90; Ma B., et al. (2013) Journal of Immunological Methods 400-401: 78-86; Geurts A., et al. (2009) Science 325: 433, U.S. Pat. No. 8,907,157; European Pat. No. 2152880B1; European Pat. No. 2336329B1). Yet another example includes the use of VELOCIMMUNE® Technology (see, for example, U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®. Briefly, the VELOCIMMUNE® technology involves generation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antigen-binding protein, e.g., antibody, comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibody are isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody.Antibody Fragments

[0220] In embodiments, an antigen recognition moiety herein is or comprises an antibody fragment. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-7 (1992); and Brennan et al., Science, 229: 81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10: 163-7 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragment671106305893\l\AMERICASwith increased in vivo half-life comprising salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv) (see WO 93 / 16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458). Fv and sFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv (see Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a “linear antibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.

[0221] In one embodiment, an antibody derived scFv is used in a CAR of the present disclosure. Included among antibody fragments are portions of antibodies (and combinations of portions of antibodies, for example, scFv) that may be used as targeting arms, directed to an epitope. Such fragments are not necessarily proteolytic fragments but rather portions of polypeptide sequences that can confer affinity for target.Multispecific Antibodies

[0222] In embodiments, an antigen recognition moiety is or comprises a multispecific antibody, for example, a bispecific antibody. Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of a protein as described herein. Other such antibodies may combine an antigen binding site with a binding site for another protein. In some examples, an antigen-binding arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T cell receptor molecule (e.g., CD3), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD 16), so as to focus and localize cellular defense mechanisms to the antigen-expressing cell. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express athe antigen. These antibodies possess a antigen-binding arm and an arm which binds the cytotoxic agent (e.g., saporin, anti-interferon-a, vinca alkaloid, ricin A chain, methotrexate or radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0223] Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature681106305893\l\AMERICAS305: 537-9 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93 / 08829, and in Traunecker et al., EMBO J.10:3655-3659 (1991).

[0224] Other approaches for making bispecific antibodies are known. One approach is the “knobs-into-holes” or “protuberance-into-cavity” approach (see, e.g., U.S. Pat. No. 5,731,168). In this approach, two immunoglobulin polypeptides (e.g., heavy chain polypeptides) each comprise an interface. An interface of one immunoglobulin polypeptide interacts with a corresponding interface on the other immunoglobulin polypeptide, thereby allowing the two immunoglobulin polypeptides to associate. These interfaces may be engineered such that a “knob” or “protuberance” (these terms may be used interchangeably herein) located in the interface of one immunoglobulin polypeptide corresponds with a “hole” or “cavity” (these terms may be used interchangeably herein) located in the interface of the other immunoglobulin polypeptide. In embodiments, the hole is of identical or similar size to the knob and suitably positioned such that when the two interfaces interact, the knob of one interface is positionable in the corresponding hole of the other interface. Without wishing to be bound to theory, this is thought to stabilize the heteromultimer and favor formation of the heteromultimer over other species, for example homomultimers. In embodiments, this approach may be used to promote the heteromultimerization of two different immunoglobulin polypeptides, creating a bispecific antibody comprising two immunoglobulin polypeptides with binding specificities for different epitopes.

[0225] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is typical to have the first heavychain constant region (CHI) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are cotransfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three691106305893\l\AMERICASpolypeptide chains used in the construction provide the optimum yields. Tt is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

[0226] In one embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides for a facile way of separation. This approach is disclosed in WO 94 / 04690. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0227] According to another approach described in WO96 / 27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. One interface comprises at least a part of the CH 3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory “cavities” of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[0228] Bispecific antibodies include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

[0229] Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science, 229: 81 (1985) describe a procedure wherein intact antibodies are701106305893\l\AMERICASproteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was separately secreted from A. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody.

[0230] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The “diabody” technology described by Hollinger et al., Proc. Natl. Acad. Set. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy -chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the Vuand VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al, J. Immunol, 152:5368 (1994).

[0231] Another technique for making bispecific antibody fragments is the “bispecific T cell engager” orBiTE® approach (see, e.g., W02004 / 106381, W02005 / 061547, W02007 / 042261, and W02008 / 119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain includes two single chain Fv (scFv) fragments, each having a variable heavy chain (VH) and a variable light chain (VL) domain separated by a polypeptide linker of a length sufficient to allow intramolecular association between711106305893\l\AMERICASthe two domains. This single polypeptide further includes a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes may be specific for different cell types, such that cells of two different cell types are brought into close proximity or tethered when each scFv is engaged with its cognate epitope. One particular embodiment of this approach includes a scFv recognizing a cell-surface antigen expressed by an immune cell, e.g., a CD3 polypeptide on a T cell, linked to another scFv that recognizes a cellsurface antigen expressed by a target cell, such as a malignant or tumor cell.

[0232] As it is a single polypeptide, the bispecific T cell engager may be expressed using any prokaryotic or eukaryotic cell expression system known in the art, e.g., a CHO cell line. However, specific purification techniques (see, e.g., EP1691833) may be necessary to separate monomeric bispecific T cell engagers from other multimeric species, which may have biological activities other than the intended activity of the monomer. In one exemplary purification scheme, a solution containing secreted polypeptides is first subjected to a metal affinity chromatography, and polypeptides are eluted with a gradient of imidazole concentrations. This eluate is further purified using anion exchange chromatography, and polypeptides are eluted using with a gradient of sodium chloride concentrations. Finally, this eluate is subjected to size exclusion chromatography to separate monomers from multimeric species.

[0233] Other relevant bispecific antibody fragment formats include but are not limited to dualaffinity re-targeting proteins (DARTs) and Tandem diabodies (TandAbs). A DART is composed of two Fv fragments, with two unique antigen-binding sites formed when two Fv fragments heterodimerize (Holliger et al., Proc. Natl. Acad. Sci. USA. 90:6444-6448 (1993). Specifically, Fvl consists of a VH from antibody “A” and a VL from antibody “B”, while Fv2 is made from a VH from antibody “B” and VL from antibody “A”. Unlike BiTE antibodies which are connected by a polypeptide linker, this combination allows DART to mimic natural interaction within an IgG molecule. Adding another cysteine residue to the end of each heavy-chain improves stability by forming a C-terminal disulfide bridge. TandAbs are tetravalent bispecific antibodies provide two binding sites for each antigen to maintain the avidity of a natural bivalent antibody. Moreover, TandAbs have a molecular weight (approximately 105 kDa) exceeding the first-pass renal clearance threshold, thus offering a longer half-life compared to smaller antibody constructs (Reusch et al., Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res. 22:5829-5838 (2016); Reusch et al., MAbs. 6:728-739 (2014); Compte et al., Oncoimmunology. 3:e28810 (2014). For a recent721106305893\l\AMERICASreview of common formats of bispecific antibodies including scFv-based and full-length IgG-like asymmetric antibodies, including methods of production thereof, see Wang et al., Antibodies (Basel), 8(3): 43 (2019).Antibody Variants and ModificationsSubstitution, Insertion, and Deletion Variants

[0234] In embodiments, an antigen recognition moiety is or comprises an antibody variant. Antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and / or by synthesis of the desired antibody or polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the antibody, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.

[0235] Variations in the antibodies described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Patent No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence antibody or polypeptide. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the antibody. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the antibody with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

[0236] Antibody fragments are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full-length native antibody or protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity of the antibody.731106305893\l\AMERICAS

[0237] Antibody fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating antibody or polypeptide fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired antibody or polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR. Preferably, antibody fragments share at least one biological and / or immunological activity with the native antibody disclosed herein.

[0238] In particular embodiments, conservative substitutions of interest are shown in Table 1 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 1, or as further described below in reference to amino acid classes, are introduced and the products screened.Table 1Original Exemplary PreferredResidue Substitutions SubstitutionsAla (A) val; leu; ile valArg (R) lys; gin; asn lysAsn (N) gin; his; lys; arg ginAsp (D) glu gluCys (C) ser serGin (Q) asn asnGlu (E) asp aspGly (G) pro; ala alaHis (H) asn; gin; lys; arg arglie (I) leu; val; met; ala; phe;norleucine leuLeu (L) norleucine; ile; val;met; ala; phe ile741106305893\l\AMERICASLys (K) arg; gin; asn argMet (M) leu; phe; ile leuPhe (F) leu; val; ile; ala; tyr leuPro (P) ala alaSer (S) thr thrThr (T) ser serTrp (W) tyr; phe tyrTyr (Y) trp; phe; thr; ser pheVal (V) ile; leu; met; phe;ala; norleucine leu

[0239] Substantial modifications in function or immunological identity of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common sidechain properties:(1) hydrophobic: norleucine, met, ala, val, leu, ile;(2) neutral hydrophilic: cys, ser, thr;(3) acidic: asp, glu;(4) basic: asn, gin, his, lys, arg;(5) residues that influence chain orientation: gly, pro; and(6) aromatic: trp, tyr, phe.

[0240] Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.

[0241] The variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13: 4331 (1986); Zoller et al., Nucl. Acids Res., 10: 6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34: 315 (1985)), restriction selection751106305893\l\AMERICASmutagenesis (Wells et al., Philos. Trans. R. Soc. London Ser A, 317: 415 (1986)) or other known techniques can be performed on the cloned DNA to produce the antibody variant DNA.

[0242] Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant (Cunningham and Wells, Science, 244: 1081-5 (1989)). Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150: 1 (1976)). If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.

[0243] Any cysteine residue not involved in maintaining the proper conformation of the antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

[0244] A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and antigen polypeptide. Such contact residues and neighboring residues are761106305893\l\AMERICAScandidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

[0245] Nucleic acid molecules encoding amino acid sequence variants of the antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared variant or a non-variant version of the antibody.Modifications

[0246] Covalent modifications of antibodies are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of an antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the antibody. Derivatization with bifunctional agents is useful, for instance, for crosslinking an antibody to a water-insoluble support matrix or surface for use in a method for purifying antibodies, and vice-versa. Commonly used crosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.

[0247] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0248] Another type of covalent modification of the antibody included within the scope of this invention comprises altering the native glycosylation pattern of the antibody or polypeptide. “Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence antibody (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and / or enzymatic means), and / or adding one or more glycosylation sites that are not present in the native sequence antibody. In771106305893\l\AMERICASaddition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.

[0249] Glycosylation of antibodies and other polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0250] Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites). The antibody amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the antibody at preselected bases such that codons are generated that will translate into the desired amino acids.

[0251] Another means of increasing the number of carbohydrate moieties on the antibody is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87 / 05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0252] Removal of carbohydrate moieties present on the antibody may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).781106305893\l\AMERICASFc Region Variants

[0253] It may be desirable to modify the antibody of the invention with respect to effector function, e.g., so as to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an Fc region of the antibody. Alternatively or additionally, cysteine residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-mediated cell killing and antibodydependent cellular cytotoxicity (ADCC) (see Caron et al., J. Exp Med. 176: 1191-5 (1992); Shopes, B. J. Immunol. 148: 2918-22 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53: 2560-5 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3: 219-30 (1989). To increase the serum half life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Patent 5,739,277, for example. As used herein, the term “salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.Cysteine Engineered Antibody Variants

[0254] In certain embodiments, it may be desirable to create cysteine engineered antibodies, e.g.,“thioMAbs,” in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. Cysteine engineered antibodies can be generated as described, e.g, in U.S. Patent No.7,521,541.Chimeric Antigen Receptor (CAR)

[0255] In embodiments, the antigen recognition moiety in the modified immune cell (e.g., T cell) is a CAR. The CAR may be expressed on the surface of the modified immune cell and791106305893\l\AMERICAScomprise an affinity binding domain specific for a disease associated antigen (e.g., tumor antigen, autoimmune disease-associated antigen, or pathogenic antigen), or a means for specifically binding a disease associated antigen. The present disclosure further provides the nucleic acid encoding the CAR, and the construct and vector containing such nucleic acid. The present disclosure further provides an isolated polynucleotide (used exchangeable with “nucleic acid” herein) comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) and a nucleic acid sequence encoding at least one c-kit agonist. In some cases, the nucleic acid is a, e.g., heterologous, component of an expression cassette. In embodiments, the nucleic acid is a, e.g., heterologous, component of a retroviral vector. In embodiments, the nucleic acid is a, e.g., heterologous, component of a modified immune cell. In embodiments, the nucleic acid is a, e.g., heterologous, component of a modified y5 T cell. In some embodiments, the nucleic acid is a, e.g., heterologous, component ofay T cell and / or a 8+T cell.

[0256] A CAR may comprise an affinity binding domain or means for specifically binding operably linked to another domain of the CAR, for example a transmembrane domain, a costimulatory domain and / or an intracellular signaling domain, as described herein. The affinity binding domains or means for specifically binding described herein can be combined with any of the transmembrane, costimulatory, and / or intracellular signaling domain(s) described herein, and / or any of the other domains described herein that may be included in a CAR herein. A CAR may also include a hinge domain as described herein. A CAR may also include at least one spacer domain as described herein.

[0257] Aspects of the invention include nucleic acids encoding CARs, and constructs and vectors containing such nucleic acids. In some cases, the nucleic acid is a, e.g., heterologous, component of an expression cassette. In embodiments, the nucleic acid is a, e.g., heterologous, component of a retroviral vector. In embodiments, the nucleic acid is a, e.g., heterologous, component of a T cell. In embodiments, the nucleic acid is a, e.g., heterologous, component of a 78 T cell. In embodiments, the nucleic acid is a, e.g., heterologous, component of an+T cell and / or a 6+T cell. In embodiments, the nucleic acid is a, e.g., heterologous, component of an a T cell and / or a 0" T cell.Affinity Binding Domain / Means for Specifically Binding

[0258] The affinity binding domain can include any domain that binds to a disease associated antigen (e.g., the antigens associated with tumors and autoimmune diseases and pathogenic801106305893\l\AMERICASantigens described hereinabove). Similarly, the means for specifically binding can interact with any disease associated antigen, e.g. associated with tumors and autoimmune diseases and pathogenic antigens. In embodiments, the affinity binding domain or means for specifically binding binds to a tumor antigen. For example, the affinity binding domain or means for specifically binding may bind to CD20, BCMA, GPC3, TyrD, B7H6, CD70, or PSMA.

[0259] The affinity binding domain or means for specifically binding may comprise an antibody, fragment thereof, or variant thereof described above. In embodiments, the affinity binding domain or means for specifically binding comprises a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a bispecific antibody, and any fragment thereof. In embodiments, the affinity binding domain or means for specifically binding may be an antibody, an antigen binding fragment (Fab), a singlechain variable fragment (scFv), a heavy chain-only antibody (VHH), a heavy chain or a light chain single domain antibody (sdAb), aF(ab)2, or any combination thereof. In embodiments, the affinity binding domain or means for specifically binding comprises a mammalian antibody or a fragment thereof. The choice of affinity binding domain or means for specifically binding may depend upon the type and number of antigens that are present on the surface of a target cell.

[0260] The present disclosure provides antibodies and CARs with “substantial identity” or “substantial similarity” to the sequences provided herein in the CDR or framework regions. The term "substantial identity" or "substantially identical," when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with another nucleic acid (or the complementary strand of the other nucleic acid), there is nucleotide sequence identity in %, for example, at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, or 100% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0261] As applied to polypeptides, the term "substantial similarity" or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or811106305893\l\AMERICASBESTFIT using default gap weights, share at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 99.5%, or 100% sequence identity. In some aspects, residue positions, which are not identical, differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, which is herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalaninetyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet el al. (1992) Science 256: 1443 45, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0262] Sequence identity and / or similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as GAP and BESTFIT which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG821106305893\l\AMERICASVersion 6.1. Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Sequences also can be compared using the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. Another preferred algorithm when comparing a sequence disclosed herein to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g., Altschul el al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389-3402, each of which is herein incorporated by reference.Transmembrane Domain

[0263] The CAR may comprise a transmembrane domain that couples the affinity binding domain of the CAR to one or more intracellular domains of the CAR. The transmembrane domain of a CAR of the present disclosure is a region that is capable of spanning the plasma membrane of a cell (e.g., a T cell). In embodiments, the transmembrane domain is interposed between the affinity binding domain / means for specifically binding and the one or more intracellular domains of a CAR.

[0264] In embodiments, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In embodiments, the transmembrane domain can be selected or modified by one or more amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, to minimize interactions with other members of the receptor complex.

[0265] For example and without limitation, a transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in this invention may be derived from (i.e. comprise at least the transmembrane region(s) of) 4-1BB / CD137, activating NK cell receptors, an Immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD3 zeta, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7,831106305893\l\AMERICASCD84, CD8, CD8alpha, CD8beta, CD96 (Tactile), CDl la, CDl lb, CDl lc, CDl ld, CDS, CEACAM1, CRT AM, cytokine receptor, DAP10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell costimulator (ICOS), integrins, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, a ligand that specifically binds with CD83, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen- 1 (LFA-1; CDlla / 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 Lymphocytic Activation Molecules (SLAM proteins), SLAM (SLAMF1; CD 150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A; Lyl08), SLAMF7, SLP-76, TNF receptor proteins, TNFR2, TNFSF14, a Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or a fragment, truncation, or a combination thereof. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

[0266] In certain embodiments, the transmembrane domain comprises a transmembrane domain of CD8. In certain embodiments, the transmembrane domain of CD8 is a transmembrane domain of CD8a. In certain embodiments, the transmembrane domain of CD8 comprises the amino acid sequence set forth in SEQ ID NO: 211. In certain embodiments, the transmembrane domain comprises a transmembrane domain of CD28. In certain embodiments, the transmembrane domain of CD28 comprises the amino acid sequence set forth in SEQ ID NO: 215. In certain embodiments, the transmembrane domain comprises a transmembrane domain of ICOS. In certain embodiments, the transmembrane domain of ICOS comprises the amino acid sequence set forth in SEQ ID NO: 216.

[0267] The transmembrane domains described herein can be combined with any of the affinity binding domains described herein, any of the intracellular domains described herein, or any of the other domains described herein that may be included in a subject CAR.

[0268] In embodiments, the transmembrane domain further comprises a hinge region. A subject CAR of the present invention may also include a hinge region. The hinge region of the CAR is a hydrophilic region which is located between the affinity binding domain / means for specifically binding and the transmembrane domain. In embodiments, this domain facilitates841106305893\l\AMERICASproper protein folding for the CAR. The hinge region is an optional component for the CAR. The hinge region may include a domain selected from Fc fragments of antibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof. Examples of hinge regions include, without limitation, a CD8a hinge, CD8P hinge, CD28 hinge, 4- IBB hinge, CD7 hinge, artificial hinges made of polypeptides which may be as small as, three glycines (Gly), as well as CHI and CH3 domains of IgGs (such as human IgG4). Naturally-occurring hinge domains may be used as wild-type hinge regions or the molecules may be altered.

[0269] In embodiments, a subject CAR of the present disclosure includes a hinge region that couples the affinity binding domain / means for specifically binding with the transmembrane domain, which, in turn, couples to one or more intracellular domain(s). The hinge region is preferably capable of supporting the affinity binding domain / means for specifically binding to recognize and bind to the target antigen on the target cells (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In embodiments, the hinge region is a flexible domain, thus allowing the affinity binding domain / means for specifically binding to have a structure to optimally recognize the specific structure and density of the target antigens on a cell such as tumor cell (Hudecek et al., supra). The flexibility of the hinge region permits the hinge region to adopt many different conformations. In embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region).

[0270] The hinge region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa. In embodiments, the hinge region can have a length of greater than 5 aa, greater than 10 aa, greater than 15 aa, greater than 20 aa, greater than 25 aa, greater than 30 aa, greater than 35 aa, greater than 40 aa, greater than 45 aa, greater than 50 aa, greater than 55 aa, or more.

[0271] Suitable hinge regions can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can851106305893\l\AMERICASbe 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can have a length of greater than 20 amino acids (e.g., 30, 40, 50, 60 or more amino acids).

[0272] For example, hinge regions include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 174) and (GGGS)n (SEQ ID NO: 175), where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge regions can comprise amino acid sequences including, but not limited to, GGSG (SEQ ID NO: 177), GGSGG (SEQ ID NO: 178), GSGSG (SEQ ID NO: 179), GSGGG (SEQ ID NO: 180), GGGSG (SEQ ID NO: 181), GSSSG (SEQ ID NO: 182), and the like.

[0273] In embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et ah, Proc. Natl. Acad. Sci. USA (1990) 87(1): 162-166; and Huck et ah, Nucleic Acids Res. (1986) 14(4): 1779-1789. As non-limiting examples, an immunoglobulin hinge region can include one of the following amino acid sequences: DKTHT (SEQ ID NO: 217); CPPC (SEQ ID NO: 218); CPEPKSCDTPPPCPR (SEQ ID NO: 219) (see, e g., Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); ELKTPLGDTTHT (SEQ ID NO: 220); KSCDKTHTCP (SEQ ID NO: 221); KCCVDCP (SEQ ID NO: 222); KYGPPCP (SEQ ID NO: 223); EPKSCDKTHTCPPCP (SEQ ID NO: 224) (human IgGl hinge); ERKCCVECPPCP (SEQ ID NO: 225) (human IgG2 hinge); ELKTPLGDTTHTCPRCP (SEQ ID NO: 226) (human IgG3 hinge); SPNMVPHAHHAQ (SEQ ID NO: 227) (human IgG4 hinge); and the like.

[0274] The hinge region can comprise an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region. In one embodiment, the hinge region can include one or more amino acid substitutions and / or insertions and / or deletions compared to a wild-type (naturally-occurring) hinge region. For example, His229 of human IgGl hinge can be substituted with Tyr, so that the hinge region comprises the sequence EPKSCDKTYTCPPCP (SEQ ID NO: 228); see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897.861106305893\l\AMERICAS

[0275] In certain embodiments, the hinge region can comprise an amino acid sequence derived from human CD8, or a variant thereof. In certain embodiments, the CAR comprises a CD8 alpha hinge sequence comprising the amino acid sequence set forth in SEQ ID NO: 209. In certain embodiments, the CAR comprises a hinge and transmembrane domain sequence comprising the amino acid sequence set forth in SEQ ID NO: 213.Costimulatory Domain

[0276] In embodiments, a CAR comprises at least one costimulatory domain, wherein the costimulatory domain comprises functional costimulatory signaling domain derived from e.g., a MHC class I molecule, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, and the like. For example, it is within the scope of this disclosure that the CAR can include 2, 3, 4 or more costimulatory domains. It is also within the scope of this disclosure that when more than one costimulatory domain is included, the costimulatory domains may be the same, or they may be different. In embodiments, the costimulatory domains are derived from one or more of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, B7-H3, CEACAM1, CRTAM, CD2, CD3C, CD4, CD7, CD8a, CD80, CDlla, CDllb, CDllc, CDlld, lL2Rp, !L2y, lL7Ra, 1L4R, 1L7R, 1L15R, 1L21R, CD18, CD19, CD19a, CD27, CD28, CD29, CD30, CD40, CDS, CD49a, CD49D, CD49f, CD54 (ICAM), CD69, CD70, CD80, CD83, CD84, CD86, CD96 (Tactile), CD100 (SEMA4D), CD103, CD134 (0X40), CD137 (4-1BB), CD152 (CTLA-4), CD160 (BY55), CD162 (SELPLG), CD244 (2B4), CD270 (HVEM), CD226 (DNAM1), CD229 (Ly9), CD278 (ICOS), ICAM-1, LFA-1 (CDlla / CD18), FcR, FcyRI, FcyRII, FcyRI 11, LAT, NKG2C, SLP76, TRIM, ZAP70, GITR, BAFFR, LTBR, LAT, GADS, LIGHT, HVEM (LIGHTR), KIRDS2, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB1, ITGB2, ITGB7, NKG2C, NKG2D, IA4, VLA-1, VLA-6, SLAM (SLAMF1, CD150, IPO-3), SLAMF4, SLAMF6 (NTB-A, Lyl08), SLAMF7, SLAMF8 (BLAME), SLP-76, PAG / Cbp, NKp80 (KLRF1), NKp44, NKp30, NKp46, BTLA, JAML, CD150, PSGL1, TSLP, TNFR2, and TRANCE / RANKL, or a portion thereof, and combinations thereof.

[0277] In embodiments, a nucleic acid encoding a CAR encodes at least one 4- IBB costimulatory domain, and optionally a second costimulatory domain selected from 4- IBB, 2B4, ICOS, CD28, 0X40, and CD27 costimulatory domains, or any of the above-mentioned871106305893\l\AMERICAScostimulatory domains. Tn embodiments, the nucleic acid encodes at least two 4-1BB costimulatory domains, or at least two 4-1BB costimulatory domains in combination with one, two, three, or four, or more, costimulatory domains selected from 4- IBB, ICOS, CD28, 0X40, and CD27, or any of the above-mentioned costimulatory domains. In embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence set forth as SEQ ID NO: 202. In embodiments, the 4-1BB costimulatory domain comprises an amino acid sequence having at least one, at least two, or at least three or more modifications of an amino acid sequence of SEQ ID NO: 202. In embodiments, the 4-1BB costimulatory domain is substantially similar to the 4-1BB costimulatory domain comprising SEQ ID NO: 202.

[0278] In embodiments, a nucleic acid encoding a CAR encodes at least one CD27 costimulatory domain, and optionally at least one second costimulatory domain selected from 4-1BB, ICOS, CD28, 0X40, 2B4, and CD27 costimulatory domains, or any of the above-mentioned costimulatory domains. In embodiments, the nucleic acid encodes at least one CD27 costimulatory domain, and a 4-IBB costimulatory domain. In embodiments, the nucleic acid encodes two CD27 costimulatory domains, and at least one second costimulatory domain selected from a 4-IBB, ICOS, CD28, and CD27. In embodiments, the CD27 costimulatory domain comprises SEQ ID NO: 208. In embodiments, the CD27 costimulatory domain comprises an amino acid sequence having at least one, at least two, at least three or more modifications of an amino acid sequence of SEQ ID NO: 208. In embodiments, the CD27 costimulatory domain is substantially similar to the CD27 costimulatory domain comprising SEQ ID NO: 208.

[0279] In embodiments, a nucleic acid encoding a CAR encodes at least one CD28 costimulatory domain, and optionally a second costimulatory domain selected from 4-IBB, 2B4, ICOS, CD28, 0X40, and CD27 costimulatory domains, or any of the above-mentioned costimulatory domains. In embodiments, the nucleic acid encodes at least two CD28 costimulatory domains, or at least two CD28 costimulatory domains in combination with one, two, three, or four, or more, costimulatory domains selected from a 4- IBB, ICOS, CD28, 0X40, and CD27, or any of the above-mentioned costimulatory domains. In embodiments, the CD28 costimulatory domain comprises SEQ ID NO: 204. In embodiments, the CD28 costimulatory domain comprises SEQ ID NO: 205. Included in SEQ ID NO: 204 and SEQ ID NO: 205 are three subdomains YMNM, PRRP, and PYAP, that are capable to regulate signaling pathways. In embodiments, a disclosed CAR comprises mutation or deletion of one or more of the subdomains (seee.g., W02019010383).881106305893\l\AMERICASIn embodiments, the CD28 costimulatory domain comprises an amino acid sequence having at least one, at least two, at least three or more modifications of an amino acid sequence of SEQ ID NO: 204, or an amino acid sequence of SEQ ID NO: 205. In embodiments, the CD28 costimulatory domain is substantially similar to the CD28 costimulatory domain comprising SEQ ID NO: 204. In embodiments, the CD28 costimulatory domain is substantially similar to the CD28 costimulatory domain comprising SEQ ID NO: 205.

[0280] In embodiments, a nucleic acid encoding a CAR encodes at least one ICOS costimulatory domain, and optionally a second costimulatory domain selected from 4- IBB, 2B4, ICOS, CD28, 0X40, and CD27 costimulatory domains, or any of the above-mentioned costimulatory domains. In embodiments, the nucleic acid encodes at least two ICOS costimulatory domains, or at least two ICOS costimulatory domains in combination with one, two, three, or four, or more, costimulatory domains selected from 4-1BB, ICOS, CD28, 0X40, and CD27, or any of the above-mentioned costimulatory domains. In embodiments, the ICOS costimulatory domain comprises SEQ ID NO: 206. In embodiments, the ICOS costimulatory domain comprises an amino acid sequence having at least one, at least two, at least three or more modifications of an amino acid sequence of SEQ ID NO: 206 (see e.g., US20170209492). In embodiments, the ICOS costimulatory domain is substantially similar to the ICOS costimulatory domain comprising SEQ ID NO: 206.

[0281] In embodiments, a nucleic acid encoding a CAR encodes at least one 0X40 costimulatory domain, and optionally a second costimulatory domain selected from 4-1BB, 2B4, ICOS, CD28, 0X40, and CD27 costimulatory domains, or any of the above-mentioned costimulatory domains. In embodiments, the nucleic acid encodes at least two 0X40 costimulatory domains, or at least two 0X40 costimulatory domains in combination with one, two, three, or four, or more, costimulatory domains selected from 4-1BB, ICOS, CD28, 0X40, and CD27, or any of the above-mentioned costimulatory domains. In embodiments, the 0X40 costimulatory domain comprises SEQ ID NO: 207. In embodiments, the 0X40 costimulatory domain comprises an amino acid sequence having at least one, at least two, at least three or more modifications of an amino acid sequence of SEQ ID NO: 207. In embodiments, the 0X40 costimulatory domain is substantially similar to the 0X40 costimulatory domain comprising SEQ ID NO: 207.891106305893\l\AMERICASIntracellular Signaling Domain

[0282] In embodiments, a CAR comprises at least one intracellular signaling domain. In embodiments, the at least one intracellular signaling domain is additional to one or more costimulatory domains. In embodiments, the one or more intracellular signaling domains are included to increase proliferation, persistence, and / or cytotoxic activity of the modified yd cell, harboring the CAR as herein disclosed. For example, in some embodiments, the intracellular signaling domain(s) comprise CD3(^, repeat (e.g., 2-5) DAP10 YINM motifs, signaling domains derived from LFA-1, DAP12, FcRy, FcR[3, CD3y, CD35, CD3e, CD79a, CD79b, CD5, CD22, FcsRI, CD66d, and the like. It is within the scope of this disclosure that the endodomain of a disclosed CAR can include a plurality (e.g., 2, 3, 4, or more) of intracellular signaling domains. In a case where more than one intracellular signaling domain is included, the intracellular signaling domains may be the same, or they may be different.

[0283] In embodiments, an intracellular signaling domain of a disclosed CAR is or comprises a CD3^ signaling domain. In embodiments, a CD3(^ signaling domain is or comprises the amino acid sequence set forth in SEQ ID NO: 229, 231, or 232.Additional Components of CAR

[0284] In embodiments, an isolated nucleic acid encoding a CAR can also encode for one or more multi-cistronic linker region(s) configured to facilitate translation of the CAR polypeptide and one or more additional polypeptides. In embodiments, nucleic acids encoding the one or more additional polypeptides and associated linker region can be positioned at the 3’ end of the isolated nucleic acid, or at the 5’ end of the isolated nucleic acid, or in some examples at both the 5’ end and the 3’ end of the isolated nucleic acid. In some examples, the linker region(s) can encode a self-cleavage and / or a cleavage polypeptide sequence. In some examples, the self-cleavage sequence is a 2A self-cleaving sequence (e.g., T2A, P2A, E2A, F2A) which can induce ribosomal skipping during translation of the CAR. In embodiments, the cleavage sequence is a furin sequence. In some examples, the cleavage sequence (e.g., furin cleavage sequence as set forth in SEQ ID NO: 190) is amino terminal to a self-cleavage sequence, for example furin-P2A (FP2A). In embodiments, the multi-cistronic linker region encodes an internal ribosome entry site. In embodiments, the addition of an optional linker “GSG” or “SGSG” and the like can improve cleavage efficiency. In this way, the one or more additional polypeptides can be release from the CAR and directed to the secretory pathway.901106305893\l\AMERICAS

[0285] In embodiments, the cleavage sequence is the FP2A amino acid sequence as set forth in SEQ ID NO: 184. In embodiments, the cleavage sequence is a P2A amino acid sequence as set forth in SEQ ID NO: 186, or SEQ ID NOs: 188-189. In embodiments, the cleavage sequence is a furin amino acid sequence as set forth in SEQ ID NO: 190. In embodiments, the cleavage sequence is a F2A amino acid sequence as set forth in SEQ ID NO: 191. In embodiments, the cleavage sequence is a E2A amino acid sequence as set forth in SEQ ID NO: 192. In embodiments, the cleavage sequence is a T2A amino acid sequence as set forth in SEQ ID NO: 193. In certain aspects, multiple cleavage and / or self-cleavage sequences can be encoded carboxy-terminal to signaling and / or costimulatory domain(s) and amino-terminal to an encoded one or more additional polypeptide. In certain aspects, one or more self-cleavage sequences and one or more sequences cleaved by an endogenous protease are encoded in a construct described herein. In certain embodiments, an endogenous protease recognition site is encoded amino terminal to a selfcleavage sequence.

[0286] In embodiments, the multi-cistronic linker region encodes an internal ribosome entry site. An exemplary internal ribosome entry site is encoded by the nucleotide sequence set forth in SEQ ID NO: 194. Another exemplary internal ribosome entry site is encoded by the nucleotide sequence set forth in SEQ ID NO: 195. Further suitable internal ribosome entry sites include, but are not limited to, those disclosed e.g., in Nucleic Acids Res. 2010 Jan;38(Database issue):D131-6. doi: 10.1093 / nar / gkp981. Epub 2009 Nov 16, those described at iresite.org, those described in WO 2018 / 215787, the sequence described in GenBank accession No. KP019382.1, and the IRES element disclosed in GenBank accession No. LT727339.1. Additional multi-cistronic linker regions, including cleavage self-cleavage, and IRES elements, are disclosed in US 2018 / 0360992 and U.S. 8,865,467.

[0287] In some examples, a modified immune cell herein comprises a chimeric antigen receptor (CAR) expressed on a surface of the cell and comprising an affinity binding domain specific for a tumor antigen, and expresses an exogenous stem cell factor.

[0288] In some examples, a modified immune cell herein comprises a chimeric antigen receptor (CAR) expressed on a surface of the cell and comprising an affinity binding domain specific for a disease associated antigen (e.g., CD20, CD70, or PSMA), has disruption of CISH gene, disruption of CBL-B gene, disruption of ICAM-1 gene, disruption of CD58 gene, disruption911106305893\l\AMERICASof Roquin-1 gene, disruption of Regnase-1 gene, disruption of TGF[3R2 gene, disruption of RASA2 gene, disruption of MED12 genes, and optionally expresses dnTGFpR2 and dnFas. Vectors

[0289] In embodiments, the modified immune cells (e.g., T cells) provided herein comprise one or more nucleic acid constructs encoding one or more modifications (e.g., exogenous proteins), and / or an antigen recognition moiety (e.g., CAR).

[0290] The nucleic acid sequences encoding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

[0291] The present invention provides vectors in which a DNA of the present invention is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve longterm gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0292] In another embodiment, the vector comprising the nucleic acid is an adenoviral vector (A5 / 35). In another embodiment, the expression of nucleic acids can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases.

[0293] In brief, the expression of natural or synthetic nucleic acids is typically achieved by operably linking a nucleic acid or portions thereof to a promoter and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0294] The expression constructs of the present invention may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art (e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties). In another embodiment, the invention provides a gene therapy vector.921106305893\l\AMERICAS

[0295] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0296] Further, the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193). A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

[0297] Additional promoter elements, e g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0298] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-la (EF-la). However, other931106305893\l\AMERICASconstitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0299] In order to assess the expression of a polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the modified immune cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[0300] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 EEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter941106305893\l\AMERICASgene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[0301] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0302] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0303] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0304] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0305] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector951106305893\l\AMERICASassociated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0306] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20 °C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology’ 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[0307] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs961106305893\l\AMERICASand Western blots) or by assays described herein to identify agents falling within the scope of the invention.Gene editing

[0308] In embodiments, gene editing is used to introduce one or more modifications and / or one or more antigen recognition moieties into a modified immune cell (e.g., T cell). In some examples, one or more endogenous genes in the modified immune cell may be disrupted by gene editing. In some examples, one or more exogenous genes encoding one or more proteins may be introduced to the modified immune cell by gene editing. In some examples, gene editing may be used to simultaneously introduce one or more exogenous genes and disrupt one or more genes in the modified immune cell.

[0309] Gene editing is a type of genetic engineering in which nucleotide(s) / nucleic acid(s) is / are inserted, deleted, and / or substituted in a DNA sequence, such as the genome of a modified immune cell. Targeted gene editing enables insertion, deletion, and / or substitution at pre-selected sites in the genome of a targeted cell. When a sequence of an endogenous gene is edited, for example by deletion, insertion, or substitution of nucleotide(s) / nucleic acid(s), the endogenous gene comprising the affected sequence may be knocked-out or knocked-down due to the sequence alteration. Therefore, targeted editing may be used to disrupt endogenous gene expression. Discussed herein, a “disrupted gene” refers to a gene comprising an insertion, deletion or substitution relative to an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited. As used herein, “disrupting a gene” refers to a method of inserting, deleting, or substituting at least one nucleotide / nucleic acid in an endogenous gene such that expression of a functional protein from the endogenous gene is reduced or inhibited. Methods of disrupting a gene are known to those of skill in the art, and described, e.g., in United States Patent No. 11254912, incorporated herein by reference in its entirety.

[0310] In embodiments, a nuclease-dependent approach can be used to conduct targeted gene editing of a T cell. Such a nuclease-dependent approach can achieve targeted editing through the specific introduction of double strand breaks (DSBs) by specific endonucleases. Such nucleasedependent targeted editing utilizes DNA repair mechanisms, for example, non-homologous end joining (NHEJ), which occurs in response to DSBs. DNA repair by NHEJ often leads to random insertions or deletions (indels) of a small number of endogenous nucleotides. In contrast to NHEJ mediated repair, repair can also occur by a homology directed repair (HDR). When a donor971106305893\l\AMERICAStemplate containing exogenous genetic material flanked by a pair of homology arms is present, the exogenous genetic material can be introduced into the genome by HDR, which results in targeted integration of the exogenous genetic material. Available endonucleases capable of introducing specific and targeted DSBs include, but are not limited to, zine-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and RNA-guided CRISPR-Cas9 nuclease (CRISPR / Cas9; Clustered Regular Interspaced Short Palindromic Repeats Associated 9). Discussed herein, a CRISPR system, or CRISPR nuclease system can include a non-coding RNA molecule (e.g., guide RNA) that binds DNA and Cas proteins (e.g., Cas9) with nuclease functionality (Sander et al., Nature Biotechnology (2014); 32:347-355; Hsu et al., Cell (2014); 157(6): 1262-1278).Pharmaceutical Compositions

[0311] The modified immune cells (e.g., T cells) of the present disclosure can be administered to a subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients. In another aspect, the present disclosure provides pharmaceutical compositions comprising a plurality of modified immune cells herein and one or more pharmaceutically acceptable carriers. In embodiments, the pharmaceutical composition comprises a cell population or cell culture, in which at least 50%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 100% cells in the cell population or cell culture are the modified immune cells herein. In embodiments, the modified immune cell is a yd T cell. In embodiments, the yd T cell is a dl , a 82, a 63, or a d4 yd T cell, preferably a 82" yd T cell, more preferably a dl yd T cell. In embodiments, the cell population or cell culture are free of other cells. In embodiments, the modified immune cell is an a.p T cell.

[0312] The modified immune cells may be administered by any route appropriate to the condition to be treated. Administration is typically parenterally, e.g., infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural.

[0313] The pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient. In some embodiments, the composition includes at least one additional therapeutic agent.

[0314] The choice of carrier may be determined in part by the particular cell, binding molecule, and / or antibody, and / or by the method of administration. Accordingly, there are a variety of suitable formulations. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th981106305893\l\AMERICASedition, Osol, A. Ed. (1980) . Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed.

[0315] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulm onary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. I n some embodiments, the cell populations are administered parenterally. The term “parenteral, ” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperito neal administration. In some embodiments, the cell populations are administered to a subject usin g peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.Methods of Treatment

[0316] Pharmaceutical compositions containing the modified immune cells (e.g., T cells) herein may be administered for prophylactic and / or therapeutic treatments. For example, the compositions can be administered to a subject already suffering from a disease or condition in an amount sufficient to cure or at least partially arrest the symptoms of the disease or condition. The compositions can also be administered to lessen a likelihood of developing, contracting, or worsening a condition. Effective amounts of the modified immune cells for therapeutic use can vary based on the severity and course of the disease or condition, previous therapy, the subject’s health status, weight, and / or response to the drugs, and / or the judgment of the treating physician.

[0317] The modified immune cells can be used to treat a subject in need of treatment for a condition. Examples of conditions include cancer, infectious disease, autoimmune disorder and sepsis, such as the cancer, autoimmune diseases, and infection caused by the bacteria and viruses disclosed herein. Subjects can be humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. A subject can be of any age. Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants.

[0318] The modified immune cells can also be administered to lessen a likelihood of developing, contracting, or worsening a condition. Effective amount of the modified immune cells for therapeutic use can vary based on the severity and course of the disease or condition, previous therapy, the subject’s health status, weight, and / or response to various drugs, and / or the judgement of a treating physician.991106305893\l\AMERICAS

[0319] In embodiments, a pharmaceutical composition comprising the modified immune cells may be administered in a first regimen. The subject may be monitored, for example by a healthcare provider (e.g., treating physician or nurse). In some examples, the subject is monitored to determine or gauge an efficacy of the modified immune cells in treating the condition of the subject. In some situations, the subject may also be monitored to determine the in vivo expansion of the modified immune cells in the subject. Another pharmaceutical composition comprising the modified immune cells may be administered to the subject in a second regimen. The pharmaceutical composition administered in the second regimen may comprise a same type of modified immune cells as that administered to the subject in the first regimen. However, it is within the scope of this disclosure that the pharmaceutical composition administered in the second regimen may comprise a different type of modified immune cells, e.g., expressing a different CAR. In some examples, the second regimen is not performed, for example if the first regimen is found to be effective (e.g., a single round of administration may be sufficient to treat the condition). In embodiments, the modified immune cells can be administered to various subjects (e.g., where the T cell has universal donor characteristics).

[0320] The modified immune cells can be administered to a subject in any order or simultaneously. If simultaneously, the modified immune cells can be provided in a single, unified form, such as an intravenous injection, or in multiple forms, for example, as multiple intravenous infusions, s.c, injections or pills. The modified immune cells can be packed together or separately, in a single package or in a plurality of packages. The modified immune cells can be given in multiple doses. If not simultaneous, the timing between the multiple doses may vary to as much as about a week, a month, two months, three months, four months, five months, six months, or about a year. In some cases, the modified immune cells can proliferate within a subject's body, in vivo, after administration to a subject. The modified immune cells can be frozen to provide cells for multiple treatments with the same cell preparation. The modified immune cells and pharmaceutical compositions comprising the same, can be packaged as a kit. A kit may include instructions (e.g., written instructions) on the use of the one or multiple modified immune cells and compositions comprising the same.

[0321] The modified immune cells can be administered before, during, or after the occurrence of a disease or condition, and the timing of administering the modified immune cells can vary. For example, the modified immune cells can be used as a prophylactic and can be administered1001106305893\l\AMERICAScontinuously to subjects with a propensity to conditions or diseases in order to lessen a likelihood of the occurrence of the disease or condition. The initial administration can be via any route practical, such as by any route described herein using any formulation described herein. In some examples, the administration of the modified immune cells is an intravenous administration. One or multiple dosages of the modified immune cells can be administered as soon as is practicable after the onset of a particular condition (e.g., cancer) and for a length of time necessary for the treatment of the disease / condition, such as, for example, from about 24 hours to about 48 hours, from about 48 hours to about 1 week, from about 1 week to about 2 weeks, from about 2 weeks to about 1 month, from about 1 month to about 3 months. In embodiments, one or multiple dosages of the modified immune cells can be administered years after onset of the disease / condition (e.g., cancer) and before or after other treatments. In some examples, one or more compositions described herein can be administered for at least about 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years at least 3 years, at least 4 years, or at least 5 years. The length of treatment can vary for each subject.

[0322] The modified immune cells can also be advantageously administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities (e.g., for treating cancer) including, e.g., chemotherapy, radiation therapy, or immunotherapy. A patient can also be preconditioned with a therapeutically effective amount of the modified immune cells prior to receiving the chemotherapy, radiation therapy, or immunotherapy (e.g., cell therapy). Suitable immunotherapies for use in combination with the modified immune cells include autologous and allogeneic cell therapies, engineered T and NK cells, immune engagers, fusion proteins, or other immune-oncology agents.

[0323] In embodiments, the modified immune cells can be administered in conjunction with another cellular immunotherapy for treating a disease such as cancer (e.g. CAR T or CAR NK cells, or Treg therapy). For example, a method of treatment (e.g., for treating cancer) can include a conditioning step, e.g. a preconditioning step, of administering a therapeutically effective amount of the modified immune cells to a subject simultaneously or sequentially with administration of1011106305893\l\AMERICASanother cellular immunotherapy directed against the disease (e.g., cancer). In embodiments, the cellular immunotherapy can comprise further administering an engineered T cell or NK cell therapy comprising a CAR binding to any tumor associated antigen of interest.

[0324] In embodiments, the modified immune cells may be advantageously administered in conjunction with additional adoptive cell therapies (ACT) (for reviews of HSCT and adoptive cell therapy approaches, see, Rager & Porter, Ther Adv Hematol (2011) 2(6) 409-428; Roddie & Peggs, Expert Opin. Biol. Ther. (2011) 11(4):473-487; Wang et al. Int. J. Cancer: (2015)136, 1751-1768; and Chang, Y. J. and X. J. Huang, Blood Rev, 2013. 27(1): 55-62), each of which is incorporated by reference herein in its entirety. Such adoptive cell therapies include, but are not limited to, allogeneic and autologous hematopoietic stem cell transplantation, donor leukocyte (or lymphocyte) infusion (DLI), adoptive transfer of tumor infiltrating lymphocytes, or adoptive transfer of T cells orNK cells (including recombinant cells, e.g., CAR T, CARNK). Beyond the necessity for donor-derived cells to reconstitute hematopoiesis after radiation and chemotherapy, immunologic reconstitution from transferred cells is important for the elimination of residual tumor cells. The efficacy of ACT as a curative option for malignancies is influenced by a number of factors including the origin, composition and phenotype (lymphocyte subset, activation status) of the donor cells, the underlying disease, the pre-transplant conditioning regimen and post-transplant immune support (e.g., IL-2 therapy) and the graft-versus-tumor (GVT) effect mediated by donor cells within the graft. Additionally, these factors may be balanced against transplant-related mortality, typically arising from the conditioning regimen and / or excessive immune activity of donor cells within the host (i.e., graft-versus-host disease, cytokine release syndrome, etc.).

[0325] In some aspects, the present disclosure provides methods of increasing the activity and / or function of a T cell by introducing the one or more modifications herein. In embodiments, the method increases the potency of a T cell therapy using the modified immune cells. In embodiments, the method enhances the cytotoxicity of the modified immune cells. In preferred embodiments, the method comprises disrupting a MED 12 gene in the modified immune cell and expressing a membrane-bound IL- 12 in the modified immune cell. In some examples, the method comprises disrupting a MED12 gene in the modified immune cell, expressing a membrane-bound IL-12 in the modified immune cell, and disrupting a Fas gene or expressing a dnFas in the modified immune cell.1021106305893\l\AMERICAS

[0326] In further aspects, the present disclosure provides methods of increasing the longevity / persistence of an immune cell comprising disrupting a MED12 gene in the cell and expressing a membrane-bound IL- 12 in the cell, thereby increasing the persistence of the modified immune cell in vitro and / or in vivo. In embodiments, the methods comprise disrupting a MED12 gene in the cell and contacting the cell with IL- 12 ex vivo or in vivo. In embodiments, the immune cell comprises an antigen recognition moiety expressed on a surface of the cell, wherein the antigen recognition moiety comprises an affinity binding domain specific for a disease-associated antigen. In embodiments, the immune cell is a T cell; preferably wherein the T cell is a yb T cell. In embodiments, the yb T cell is a bl, a b2, a b3, or a b4 yb T cell, preferably a b2- yb T cell, more preferably a bl yb T cell. In embodiments, the T cell is an 0 T cell.EXAMPLES

[0327] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.Example 1: In vitro activity of PSMA CAR-transduced yb T cells expressing mbIL-12 with gene-edits

[0328] PSMA-CAR or PSMA-CAR+mbIL 12 transduced yb T cells with or without gene edits (circle=unedited, square=MED12 knockout, triangle=MED12 knockout and TGFpRII knockout) were cocultured with PC3-PSMA expressing tumor cells at a ratio of 3:1 in the presence of 20ng / mL TGFpi. PC3-PSMA survival was monitored on the Incucyte platform. Every 3 days the effector yb T cells were restimulated with PC3-PSMA tumor cells by transferring half of the effector cells from the existing coculture to a new plate of tumor targets and replenishing the media with TGFp. % Cytotoxicity was calculated based on the Cytotoxicity Index (CI) at the final timepoint of each stimulation round. The CI was calculated by dividing the Total Integrated Intensity of NucNlR signal of each time point by the value at the beginning of each new round of stimulation. % Cytotoxicity was then calculated as (ClTumoraione-CLocuiture) / ClTumor alone *100. Once1031106305893\l\AMERICASthe effector y5 T cells were no longer able to suppress tumor cells, they were removed from future analysis and reported as 0% Cytotoxicity. The results shown in FIG. 2 support that the combination of mbIL-12 armoring and MED 12 KO significantly enhanced the cytotoxicity of PSMA-CAR transduced y8 T cells, compared with each modification applied separately.Example 2: In vivo activity of PSMA CAR-transduced y6 T cells expressing mbIL-12 with gene-edits.

[0329] To evaluate the impact of gene edits (MED12 KO ± TGFpRII KO) combined with mbIL-12 on the efficacy of PSMA CAR-transduced y8 T cells, an in vivo efficacy study was performed utilizing a subcutaneous human prostate PC3 tumor cell (ATCC) model, which was modified to express PSMA (PC3-PSMA). Female NSG mice (The Jackson Laboratory) were implanted subcutaneously with le6 PC3-PSMA cells, mixed with Matrigel (1:1 ratio) (Corning). Mice were randomized into study cohorts when tumor volumes were approximately 150 mm3(n=5 mice per group). A suboptimal treatment of le6 CAR+ yd T cells was administered as a single bolus dose via intravenous (IV) injection per mouse on Day 0. The cohorts included PSMA-CAR, PSMA-CAR MED12 KO, PSMA-CAR MED12 KO+TGF0RII KO, PSMA-CAR+mbIL12, PSMA-CAR+mblL-12 MED12 KO, and PSMA-CAR+mblL-12 MED12 KO+TGF R11 KO y<5 T cells. Throughout the experiment, tumor volume and mouse weight were closely monitored. The results are shown in FIGS. 3A and 3B. FIG. 3A shows the mean tumor volumes (MTV) with standard error bars for each group over the duration of the study until the endpoint was reached on Day 36, when MTVs in the tumor alone cohort reached approximately 1000 mm3. Statistical analysis of Day 36 tumor volume data (end-of-study) was performed using one-way ANOVA, with Dunnett’s multiple comparisons test. **** represents a p value of <0.0001, ** represents a p value of <0.01. At a suboptimal dose level, PSMA-CAR+mbIL12 and PSMA-CAR+mbIL12 MED12 KO (± TGFPRII KO) y<5 T cells showed statistically significant tumor control. Notably, the PSMA-CAR+mblL-12 MED12 KO (± TGF RII KO) y8 T cells demonstrated a strong capacity to inhibit tumor growth. This finding confirms the synergistic effect of combining MED12 KO (± TGFpRII KO) with mb IL-12 expression, which enhances the efficacy of PSMA-CAR V61 T cells.

[0330] FIG. 3B shows the % Tumor Growth Inhibition (%TGI) of each treated group compared to the Tumor Alone control group, which was calculated using the formula %TGI = (1-(MTV of treated group < MTV of tumor alone group)) x 100. Further statistical analysis with Bliss1041106305893\l\AMERICASindependence model (e.g., the model described in Duarte, Diana, and Nuno Vale. Evaluation of synergism in drug combinations and reference models for future orientations in oncology. Current Research in Pharmacology and Drug Discovery, vol. 3 100110. 12 May. 2022) was performed to evaluate the synergy of MED12 KO (± TGF0RII KO) in combination with mbIL-12. In this approach, MED 12 KO (± TGFpRII KO) yb T cells were defined as treatment A (EA), mbIL-12 was defined as treatment B (EB), and MED12 KO (± TGFPRII KO) with mbIL-12 group was defined as the combination of treatment A and B (EAB). The %TGI represents the treatment effect. The expected combined effect (EAB) (shown as the dotted line in FIG. 3B) was calculated using the formula EAB = EA + EB x (1-EA), where EA and EB represent the observed effects of treatments A and B, respectively. Synergism is indicated when the observed combined effect (OCE) is greater than EAB. In conclusion, the Bliss Model confirmed that MED12 KO (± TGFpRII KO) in combination with mbIL-12 armoring synergistically enhances the efficacy of PSMA-CARVbl T cells.Example 3: MED12 KO Promotes the Expression of highly potent Naive-like / Stem Cell Memory T Cell Markers

[0331] T cell memory phenotypes are characterized by the expression of specific surface markers that distinguish naive T cells from more differentiated subsets, such as central and effector memory T cells, as shown in FIG. 1. To assess the T cell memory phenotype of yb T cells expressing PSMA-CAR+mbIL12 with or without MED12 KO, cells were stained with fluorophore-conjugated antibodies (BioLegend) to detect surface expression of CCR7, CD62L, and CD45RA. The stained samples were acquired on the Novocyte (Agilent) flow cytometer and analyzed using FlowJo™ software (BD Biosciences). FIG. 4A displays a representative flow cytometric contour plot of CCR7 (x-axis) and CD62L (y-axis) double staining from PSMA CAR+mbIL-12 (unedited, no MED12 KO) and PSMA-CAR+mbIL12 MED12 KO (ADI-212) yb T cells. ADI-212 shows an increased % of CCR7 and CD62L double-positive cells (upper right quadrant - highlighted by a blue box) compared to PSMA-CAR+mbIL-12 yb T cells. Across multiple donors (n=5), ADI-212 exhibited a statistically significant increase in the expression of CCR7 and CD62L compared to donor-matched PSMA-CAR-mbIL12 yb T cells, with no change in CD45RA, as shown in FIG. 4B. The expression of CCR7, CD62L, and CD45RA supports a1051106305893\l\AMERICASnaive-like stem cell memory T cell phenotype associated with ADI-212. Statistical analysis was performed using Two-way Anova, ****p value<0.0001.

[0332] FIG. 5A compares the surface expression of the naive-like stem cell memory T cell-associated marker CCR7 between PSMA-CAR+mbIL-12 (unedited), PSMA-CAR MED12 KO (no mbIL-12), and ADI-212. The MED 12 KO in PSMA-CAR y5 T cells increased CCR7 expression compared to the PSMA-CAR+mbIL-12 unedited control, but having both mbIL-12 armoring and MED12 KO (ADI-212) was associated with the highest % of CCR7+y8 T cells (Oneway ANOVA, **p<0.01, ****p<0.0001). These data support that the combination of MED12 KO and mbIL-12 armoring increases CCR7 expression in y8 T cells. To determine if CCR7 expression is increased on ADI-212 cells that are positive for the PSMA-CAR, cells were stained with an antibody (Cell Signaling Technology) against the G4S linker protein sequence expressed within the extracellular domain of the CAR. Flow cytometry histogram plots of gated CAR-positive y8 T cells revealed an increase in both expression level and % of CCR7+y6 T cells compared to the CAR-negative group from 3 different donors, as shown in FIG. 5B, with statistical significance indicated in FIG.5C (unpaired t-test, **p<0.01). In summary, combining MED12 KO with mblL-12 armoring increases CCR7 expression in ADI-212, a key marker associated with naive-like stem cell memory T cells.Example 4: MED12 KO maintains the expression of CD45RA and CD45RO

[0333] Naive-like stem cell memory T cells can be identified by their expression of CCR7, CD62L, CD45RA, and CD45RO, FIG. 1. ADI-212 exhibits high levels of CCR7 and CD62L surface expression (see FIG. 4 and FIG. 5). To assess the expression of CD45RA and CD45RO in ADI-212, cells were stained using antibodies (BioLegend) against these markers. The stained samples were acquired on the Novocyte flow cytometer and analyzed using FlowJo™ software. ADI-212 and PSMA-CAR+mbIL12 (unedited) y8 T cells showed a high % of CD45RA and CD45RO, with no significant differences in expression (unpaired t-test), FIG. 6. The CD45RA / CD45RO ratio was calculated using the following formula: Ratio = % CD45RA+cells - % CD45RO+cells. These results indicate that the combination of MED12 KO and mbIL-12 armoring does not significantly change the percent of CD45RA and CD45RO in ADI-212, further supporting a naive-like stem cell memory T cell phenotype.1061106305893\l\AMERICASExample 5: MED12 KO is associated with increased CD4+ y6 T cells in ADI-212

[0334] To identify surface marker phenotype changes associated with MED 12 KO in ADI-212 compared to PSMA-CAR+mbIL-12 (unedited) y8 T cells, the LEGENDScreen™ Human Kit (BioLegend) was used to screen 354 cell surface markers following the manufacturer’s instructions. The stained samples were acquired on the Novocyte flow cytometer and analyzed using FlowJo™ software. The screening revealed that CD4 was a surface marker that was highly expressed in ADI-212. When compared to PSMA-CAR+mbIL-12 y8 T cells, ADI-212 was associated with a statistically significant increase in the % of CD4+y8 T cells across 3 different donors (unpaired t-test, ***p<0.001), FIG. 7. Representative flow cytometry histogram plots of gated y8 T cells highlight the increased expression of CD4 with ADI-212 compared to PSMA-CAR+mbIL-12 y8 T cells (red=isotype stain, blue=CD4 stain). These results indicate that MED12 KO increases the presence of a CD4+y8 T cell phenotype in ADI-212.Example 6: MED12 KO is associated with increased CD71+, CD49d+, CD123+ and CD109+ 76 T cells in ADI-212

[0335] To identify surface marker phenotype changes associated with MED 12 KO in ADI-212 compared to PSMA-CAR+mbIL-12 (unedited) y8 T cells, the LEGENDScreen™ Human Kit (BioLegend) was used to screen 354 cell surface markers following the manufacturer’s instructions. The stained samples were acquired on the Novocyte flow cytometer and analyzed using FlowJo™ software. The screening results showed that surface markers CD71, CD49d, CD123, and CD109 were upregulated in ADL212 compared to PSMA-CAR+mbIL-12 y8 T cells (n=2 different donors, unpaired t-test, *p<0.05, ***<p0.001), FIG. 8 and FIG. 9. Representative flow cytometry histogram plots of gated y8 T cells show the increased expression of CD71, CD49d, CD123, and CD109 with ADL212 compared to PSMA-CAR+mbIL-12 y8 T cells (red=isotype stain, blue=Target stain). CD71, transferrin receptor, is involved in iron uptake of transferrin-bound iron. CD49d, integrin alpha 4, dimerizes with CD29 (highly expressed on V81 T cells) forming VLA-4 to promote extravasation (binds to VCAM-1 and fibronectin). CD 123, IL3R alpha chain, binds to IL-3 and is associated with hematopoietic cell proliferation and differentiation. Not normally expressed on T cells. CD 109 is a coreceptor for TGFB and has been associated with TGFBR degradation to reduce TGFB-mediated signaling.1071106305893\l\AMERICASExample 7: MED12 KO is associated with increased costim markers CD80 and CD86 y3 T cells in ADI-212

[0336] To identify surface marker phenotype changes associated with MED 12 KO in ADI-212 compared to PSMA-CAR+mbIL-12 (unedited) yb T cells, the LEGENDScreen™ Human Kit (BioLegend) was used to screen 354 cell surface markers following the manufacturer’s instructions. The stained samples were acquired on the Novocyte flow cytometer and analyzed using FlowJo™ software. The screening results showed that surface markers CD80 and CD86 were upregulated in ADI-212 compared to PSMA-CAR+mbIL-12 yb T cells (n=2 different donors, unpaired t-test, *p<0.05), FIG. 10. Representative flow cytometry histogram plots of gated yb T cells show the increased expression of CD80 and CD86 with ADI-212 compared to PSMA-CAR+mbIL-12 yb T cells (red=isotype stain, blue=target stain).Example 8: MED12 KO is associated with increased CD36L1+ and CD151+ y3 T cells in ADI-212

[0337] To identify surface marker phenotype changes associated with MED 12 KO in ADI-212 compared to PSMA-CAR+mbIL-12 (unedited) yb T cells, the LEGENDScreen™ Human Kit (BioLegend) was used to screen 354 cell surface markers following the manufacturer’s instructions. The stained samples were acquired on the Novocyte flow cytometer and analyzed using FlowJo™ software. The screening results showed that surface markers CD36L1 and CD151 were upregulated in ADI-212 compared to PSMA-CAR+mbIL-12 yb T cells (n=2 different donors, unpaired t-test, *p<0.05), FIG. 11. Representative flow cytometry histogram plots of gated yb T cells show the increased expression of CD36L1 and CD151 with ADI-212 compared to PSMA-CAR+mbIL-12 yb T cells (red=isotype stain, blue=target stain). CD36L1, scavenger receptor class B type I (SCARB1), is involved in fatty acid uptake. CD151, a tetraspanin, is associated with T cell activation and hyperproliferation.Example 9: mbIL-12 Armoring and MED12 KO Promote Increased Metabolic-associated Transcriptional Pathways

[0338] To identify differences in T cell-related gene expression profdes between ADI-212 and PSMA-CAR+mbIL-12 (unedited) yb T cells, RNA was extracted from cell pellets using the RNeasy Mini Kit (QIAGEN). Gene expression levels were quantified using the nCounter® CAR1081106305893\l\AMERICAST Characterization Panel (NanoString) on the nCounter® SPRINT Profiler (NanoString), following the manufacturer's instructions. The nSolver™ Advanced Analysis software (NanoString) was used to normalize transcript expression levels and identify differentially expressed genes (DEGs). A volcano plot showing the number of DEGs that were upregulated (35 genes, blue triangle) and downregulated (87 genes, red triangle) in ADI-212 compared to PSMA-CAR+mbIL-12 yb T cells, which served as the baseline (n=3 different donors), FIG. 12A. DEGs were identified as -LoglO(p-value adjusted) >1.3 and Log2(fold change) >1 or < -1. A gene ontology analysis was performed using ShinyGO 0.85 (https: / / bioinformatics.sdstate.edu / go / ) to identify pathways associated with the upregulated DEGs in ADI-212. The analysis revealed pathways related to metabolism, including glycolytic and phosphorylation of nucleoside and nucleotide diphosphates processes, FIG. 12B. Selected upregulated DEGs associated with metabolic processes are displayed in a hierarchical clustering heatmap of Z-scores comparing ADI-212 and PSMA-CAR+mbIL-12 yb T cells from 3 different donors, FIG. 12C. These results highlight the impact of MED 12 KO on gene expression profiles, including the upregulation of genes associated with metabolic pathways in ADI-212 compared to PSMA-CAR+mbIL-12 yb T cells.Example 10: ADI-212 displayed robust inhibition of tumor growth through six rounds of stimulation

[0339] The in vitro cytotoxic potential of ADI-212 and PSMA-CAR+mbIL-12 (unedited) yb T cells was evaluated against PSMA-expressing PC3 tumor cells using the repetitive stimulation Incucyte killing assay in cytokine-free conditions at an effector to target (E:T) ratio of 1:2. T cell effectors were cocultured with target cells for three days, then all the surviving cells were transferred into a new vessel with freshly plated target cells for an additional three days. This process was repeated for a total of six rounds. The cytotoxicity index (CI) was calculated by dividing the total NIR object area (pm2 / well) of all time points by the value at the time of tumor challenge. The results showed ADI-212 (represented by the green circle) demonstrated greater tumor control compared to PSMA-CAR+mbIL-12 yb T cells (represented by the blue circle), as indicated by a lower CI at the end of each tumor challenge (the target alone is shown by the black circle), FIG. 13. These findings support the robust cytotoxicity and sustained persistence associated with ADI-212.1091106305893\l\AMERICASExample 11: PSMA CAR mbIL-12 y8 T cells with MED12 KO maintain in vitro functionality in the presence of immunosuppressive T regulatory cells (Tregs)

[0340] The potential impact of mbIL-12 armoring on ADI-212 in the presence of immunosuppressive cells such as macrophages and T regulatory cells (Tregs) is illustrated in FIG.14A, (created using BioRender, https: / / biorender.com). To evaluate the change in expression of Foxp3, a key transcription factor involved in the development and function of Tregs, Foxp3+human Tregs (iQ Biosciences) were cocultured with PSMA-CAR (unedited), PSMA-CAR+mb IL-12 (unedited), and ADI-212 y8 T cells in the presence of the PSMA-expressing 22Rvl prostate tumor cell line (ATCC) for 6 days at an E:T:Treg ratio of 1 : 1 : 1. The cells were harvested, stained with anti-human TCRa[3 FITC-conjugated antibody (BD Biosciences), fixed and permeabilized with Foxp3 Fix / Perm buffer (BioLegend), and then subsequently stained with anti-human Foxp3 PE-conjugated antibody (ThermoFisher). The stained samples were acquired on the Novocyte flow cytometer and analyzed using FlowJo™ software. The results showed a decrease in % Foxp3 within the TCRa[3+T cell gated population (pink colored bar) in the presence of effector y8 T cells expressing mbIL-12 armoring (PSMA-CAR+mbIL-12 and ADI-212) compared to PSMA-CAR y8 T cells without mbIL-12, FIG. 14B. These data suggest that mbIL12 armoring on PSMA-CAR y8 T cells has the potential to reduce Foxp3 expression in Tregs.

[0341] Tregs can suppress the proliferative effector functions of T cells. From the cocultures described in FIG. 14B, the absolute counts of y8 T cells were additionally measured through staining with an anti-human V81 PE-CY7-conjugated antibody (Adicet Bio). The % differential proliferation from each condition was calculated based on counts obtained without Tregs added to the culture using the following formula: (-[(V81 cell counts from tumor culture without Tregs)-(V81 cell counts from tumor culture with Tregs) / (V81 cell counts from tumor culture without Tregs)])* 100. PSMA-CAR y8 T cells without mbIL-12 armoring exhibited the largest % decrease in differential proliferation in the presence of Tregs (gray bar), followed by a minor decrease with the PSMA-CAR+mbIL-12 condition (light blue bar), FIG. 14C. However, the combination of MED 12 KO and mbIL-12 armoring incorporated into ADI-212 was associated with an increased % differential proliferation with Tregs present compared to without Tregs in culture (dark blue bar), FIG. 14C. The results suggest mbIL-12 armoring can reduce the inhibitory effects on T cell proliferation caused by Tregs.1101106305893\l\AMERICAS

[0342] Tumor killing of the 22RV1 PSMA-expressing cells by PSMA-CAR, PSMA-CAR+mbIL-12, and ADI-212 yb T cells in the presence of Tregs was evaluated in an Incucyte killing assay at an E:T:Treg ratio of 1:1:2 for 6 days. Cytotoxicity Index (CI) was calculated by dividing the total NIR-object area (mm2 / well) of all time points by the value at time zero. % Cytotoxicity was calculated by [(CI of target alone)-(CI of target+effector) / (CI of target alone)]xl00. The yb T cell cultures expressing mbIL-12 armoring (PSMA-CAR+mbIL-12, light blue squares and ADI-212, dark blue triangle) compared to PSMA-CAR yb T cells without mb IL-12 (gray circles), were not associated with decreased cytotoxicity in the presence of Tregs (Oneway Anova, ****p<0.0001), FIG. 14 D. Overall, these data support that mbIL-12 armoring has the potential to overcome Treg-mediated immunosuppressive effects.Example 13: mbIL-12 armoring and MED12 KO synergistically improve ADI-212 proliferation and in vitro tumor cytotoxicity

[0343] The cytotoxic potential of ADI-212 or effector controls was assessed against PSMA-expressing PC3 tumor cells using the repetitive stimulation Incucyte killing assay in cytokine-free conditions at a 3 : 1 E:T ratio. CAR Vbl T cells were cocultured with target cells for three days then all the surviving cells were transferred into a new vessel with freshly plated target cells for an additional three days. This process was repeated for six total rounds. The cytotoxicity index (CI) was calculated by dividing the total NIR object area (pm2 / well) of all time points by the value at the time of tumor challenge. ADI-212 (dark blue circle) demonstrated robust in vitro tumor control across the 6 tumor challenges compared to PSMA-CAR±mbIL-12 (light green and light blue circles) and PSMA-CAR MED12 KO (dark green circle) yb T cell conditions, FIG. 15A. The Cell Trace Violet dye (CTV, ThermoFisher) was used to label effector T cells to assess their proliferation potential when cocultured with PC3-PSMA tumor cells at an E:T ratio of 1:2 for 7 days. The samples were acquired on the Novocyte flow cytometer and analyzed using FlowJo™ software. The Proliferation Ratios (PR) were calculated for both stimulated and unstimulated conditions using the geometric mean of CTV fluorescence at day 7 divided by the geometric mean of CTV fluorescence on day 0. The Proliferation Index (PI) was calculated using the following formula: PI=(PRstimuiated-PRunstimuiated). ADI-212 (dark blue bar) was observed to have the highest PI compared to PSMA-CAR±mbIL-12 (light green and light blue bars), PSMA-CAR MED12 KO (dark green bar), and non-PSMA targeting CAR (gray and black bars) yb T cell conditions, FIG.1111106305893\l\AMERICAS15B. Representative flow cytometric histogram plots show ADI-212 is associated with the greatest decrease in CTV fluorescent intensity compared to PSMA-CAR+mbIL-12 and non-PSMA targeting CAR unedited yd T cells. These data support that the combination of MED 12 KO and mbIL-12 armoring in ADI-212 has a synergistic effect to enhance proliferation and cytotoxicity in the presence of tumor cells expressing PSMA.Example 14: ADI-212 demonstrates potent and durable tumor growth control in vivo

[0344] FIG. 16A, an illustration that outlines the study design evaluating ADI-212 at a stresstest treatment dose of 5e5 CAR+cells in a PC3-PSMA subcutaneous model using NSG mice. Female NSG mice were implanted subcutaneously with le6 PC3-PSMA cells, mixed with Matrigel (1:1 ratio). Mice were randomized into study cohorts when tumor volumes were approximately 150 mm3(n=5 mice per group). A stress-test treatment dose of 5e5 CAR+ADI-212 cells was administered as a single bolus dose via IV injection per mouse on Day 0. FIG. 16B shows the mean tumor volumes (MTV) with standard error bars for each group over the duration of the study. When the MTVs in the tumor alone cohort reached the end point of approximately 1,200 mm3(day 39), there was significant tumor control with mice receiving ADI-212 (Two-way ANOVA, ****p<0.0001), FIG. 16B, and this robust tumor control extended out to day 81 posttreatment, supporting ADI-212’s durable in vivo tumor control.

[0345] FIG. 16C, an illustration that outlines a tumor rechallenge study design evaluating ADI-212 at a 3e6 CAR+dose in a PSMA-expressing PC3 subcutaneous model with a second flank rechallenge 15 days post-initial tumor inoculation (n=5 per group). Mice were randomized into study cohorts when tumor volumes were approximately 150 mm3(n=5 mice per group). A treatment dose of 3e6 CAR+ADI-212 cells was administered as a single bolus dose via IV injection per mouse on Day 0. At 15 days post-initial tumor implant, NSG mice received a second challenge of tumors implanted subcutaneously with le6 PC3-PSMA cells, mixed with Matrigel at the opposite flank. FIG. 16D shows the primary and rechallenge MTVs with standard error bars for each group over the duration of the study. A single treatment of ADI-212 demonstrated robust tumor regression and prolonged tumor growth inhibition for both the initial tumor implant and rechallenged tumor in NSG mice compared to the untreated controls. Two-way ANOVA, ****p<0.0001. These findings highlight the ability of ADI-212 to both persist in the mouse following tumor eradication and mount a secondary response at a distant location.1121106305893\l\AMERICASExample 15: Enhanced in vitro cytotoxicity of CD4+sorted compared to CD4’ sorted ADI-212 cell populations

[0346] MED12 KO increased CD4 expression in ADI-212 (FIG. 7). To determine if there are functional differences between the subsets of CD4+and CD4’ cell populations, ADI-212 cells were stained with an anti-CD4 antibody conjugated to PE (BioLegend) and cell sorted to obtain purified CD4+and CD4' populations using a cell sorter (MA900, Sony Biotechnology), FIG. 17A.Unsorted, CD4+sorted, and CD4’ sorted ADI-212 cell populations were cocultured with PC3-PSMA expressing tumor cells at an E:T ratio of 1:2 for 48 hours. The survival of PC3-PSMA tumor cells was monitored and imaged every 4 hours on the Incucyte platform (Sartorius). The Cytotoxicity Index (CI) was calculated by dividing the Total Area (|jm2 / well) of the NucNIR signal at each time point by the value at the start of the coculture (time 0). % Cytotoxicity was then calculated as (Cliumor alone - CIcocuiture) / ClTumor al...

Claims

CLAIMS:What is claimed is:

1. A modified T cell comprising disruption of the Mediator of RNA polymerase II transcription subunit 12 (MED12K0) gene and expression of a membrane-bound interleukin- 12 (mbIL-12+).

2. The modified T cell of claim 1, wherein the modified cell is an a0 T cell or a y8 T cell, preferably a y8 T cell; optionally wherein the y8 T cell is a 81, a 82, a 83, or a 84 y8 T cell, preferably a 82' y8 T cell, more preferably a 81 y8 T cell.

3. The modified T cell of claim 1 or 2, wherein the modified T cell is a naive-like stem cell memory T cell, preferably wherein the modified cell comprises, or is, CCR7+, CD62L+, and CD45RA+.

4. The modified T cell of claim 3, wherein the modified T cell is or comprises CD45RO+.

5. The modified T cell of claim 3, wherein the modified T cell is or comprises CD45RO-.

6. The modified T cell of any one of claims 1-5, wherein the modified T cell further comprises, or is, one or more of CD4+, CD71+, CD49b+, CD123+, CD109+, CD80+, CD86+, CD36L1+ and / or CD151+; preferably wherein the modified T cell is CD4+.

7. The modified T cell of any preceding claim, wherein the immune cell displays tumor specificity.

8. The modified T cell of claim 7, wherein the immune cell has been isolated from a tumor of a subject, preferably wherein the immune cell is a tumor infiltrating lymphocyte.1271106305893\l\AMERICAS9. The modified T cell of claim 7, wherein the immune cell comprises an antigen recognition moiety expressed on a surface of the cell, wherein the antigen recognition moiety comprises an affinity binding domain specific for a disease-associated antigen.

10. The modified T cell of claim 9, wherein the antigen recognition moiety is selected from the group consisting of a TCR, a0 TCR, y5 TCR, a chimeric antigen receptor (CAR), whole antibody or their antigen-binding fragment, single-chain variable fragment (scFv), a heavy chain-only antibody (VHH), a heavy chain or a light chain single domain antibody (sdAb), a Fab, a F(ab)2, or any combination thereof that binds to: (i) a cell surface tumor antigen, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune disease or a pathogen, or (iv) a peptide derived from an antigen associated with an autoimmune disease or a pathogen expressed on the cell surface as a peptide-MHC complex.

11. The modified T cell of claim 10, wherein the antigen recognition moiety is a CAR.

12. The modified T cell of claim 11, wherein the CAR further comprises a transmembrane (TM) domain, a hinge domain, a co- stimulatory domain, and an intracellular signaling domain.

13. The modified T cell of claim 12, wherein the hinge domain is a CD8a hinge domain, and / or wherein the TM domain comprises a TM domain of CD8a.

14. The modified T cell of claim 12 or 13, wherein the co- stimulatory domain is a 4-1BB domain or a CD28 domain, and / or wherein the intracellular signaling domains is a CD3(^ intracellular signaling domain.

15. The modified T cell of any one of claims 9-14, wherein the disease-associated antigen is a tumor antigen, an autoimmune disease-associated antigen, or a pathogen antigen.1281106305893\l\AMERICAS16. A modified T cell comprising: a) an antigen recognition moiety expressed on a surface of the cell, wherein the antigen recognition moiety comprises an affinity binding domain specific for a disease-associated antigen; b) at least one modification to modulate cytokine activity comprising expression of a membrane-bound IL-12; and c) at least one modification to modulate T cell metabolism comprising disruption of MED12 gene.

17. The modified T cell of claim 16, wherein the modified T cell is an aP T cell or a y5 T cell, preferably a yb T cell; optionally wherein the y5 T cell is a 51, a 52, a 53, or a 54 yb T cell, preferably a 52- yb T cell, more preferably a 51 yb T cell.

18. The modified T cell of claim 16 or 17, wherein the modified T cell is a naive-like stem cell memory T cell, preferably wherein the modified cell comprises, or is, CCR7+, CD62L+, and CD45RA+.

19. The modified T cell of claim 18, wherein the modified T cell is or comprises CD45RO+.

20. The modified T cell of claim 18, wherein the modified T cell is or comprises CD45RO-.

21. The modified T cell of any one of claims 16-20, wherein the modified T cell further comprises, or is, one or more of CD4+, CD71+, CD49b+, CD123+, CD109+, CD80+, CD86+, CD36L1+ and / or CD151+; preferably wherein the modified T cell is CD4+.

22. The modified T cell of claim 21, wherein the disease-associated antigen is a tumor antigen, an autoimmune disease-associated antigen, or a pathogen antigen.

23. The modified T cell of claim 22, wherein the antigen recognition moiety is selected from the group consisting of a TCR, a TCR, yb TCR, a chimeric antigen receptor (CAR), whole antibody or their antigen-binding fragment, single-chain variable fragment (scFv), a heavy chain-only antibody (VHH), a heavy chain or a light chain single domain antibody (sdAb), a Fab, a F(ab)2, or any combination thereof that binds to: (i) a cell surface tumor antigen, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune disease or a pathogen, or (iv) a peptide derived from an1291106305893\l\AMERICASantigen associated with an autoimmune disease or a pathogen expressed on the cell surface as a peptide-MHC complex.

24. The modified T cell of claim 23, wherein the antigen recognition moiety is a CAR.

25. The modified T cell of claim 24, wherein the CAR further comprises a transmembrane (TM) domain, a hinge domain, a co-stimulatory domain, and an intracellular signaling domain.

26. The modified T cell of claim 25, wherein the hinge domain is a CD8a hinge domain, and / or wherein the TM domain comprises a TM domain of CD8a.

27. The modified T cell of claim 25 or 26, wherein the co-stimulatory domain is a 4-1BB domain or a CD28 domain, and / or wherein the intracellular signaling domains is a CD3(^ intracellular signaling domain.

28. A cell population enriched for naive-like stem cell memory T cells having a CCR7+ CD62L+ CD45RA+ phenotype, wherein the T cells are genetically modified to disrupt MED12 and to express membrane-bound IL-12, or wherein the T cells are modified to disrupt MED12 and contacted with IL-12 ex vivo or in vivo.

29. The cell population of claim 28, wherein the modified T cells are CD45RO+.

30. The cell population of claim 28, wherein the modified T cells are CD45RO-.

31. The cell population of any one of claims 28-30, wherein the modified T cells are ct|3 T cells or y8 T cells, preferably y8 T cells, optionally wherein the y8 T cells are 31, 82, 83, or 84 y8 T cells, preferably 82- y8 T cells, more preferably 81 y8 T cells.

32. The cell population of any one of claims 28-31, wherein the modified T cells further comprise one or more of CD4+, CD71+, CD49b+, CD123+, CD109+, CD80+, CD86+, CD36L1+ and / or CD151+; preferably wherein the modified T cells are CD4+.1301106305893\l\AMERICAS33. The cell population of any one of claims 28-32 wherein the immune cell displays tumor specificity.

34. The cell population of claim 33, wherein the immune cell has been isolated from a tumor of a subject, preferably wherein the immune cell is a tumor infiltrating lymphocyte.

35. The cell population of claim 33, wherein the immune cell comprises an antigen recognition moiety expressed on a surface of the cell, wherein the antigen recognition moiety comprises an affinity binding domain specific for a disease-associated antigen.

36. The cell population of any one of claims 28-35, wherein the antigen recognition moiety is selected from the group consisting of a TCR, a0 TCR, yo TCR, a chimeric antigen receptor (CAR), whole antibody or their antigen-binding fragment, single-chain variable fragment (scFv), a heavy chain-only antibody (VHH), a heavy chain or a light chain single domain antibody (sdAb), a Fab, a F(ab)2, or any combination thereof that binds to: (i) a cell surface tumor antigen, (ii) a peptide derived from a tumor antigen expressed on the cell surface as a complex with MHC (peptide-MHC complex), (iii) a cell surface antigen associated with an autoimmune disease or a pathogen, or (iv) a peptide derived from an antigen associated with an autoimmune disease or a pathogen expressed on the cell surface as a peptide-MHC complex.

37. The cell population of claim 36, wherein the antigen recognition moiety is a CAR.

38. The cell population of claim 37, wherein the CAR further comprises a transmembrane (TM) domain, a hinge domain, a co-stimulatory domain, and an intracellular signaling domain.

39. The cell population of claim 38, wherein the hinge domain is a CD8a hinge domain, and / or wherein the TM domain comprises a TM domain of CD8a.1311106305893\l\AMERICAS40. The cell population of claim 38 or 39, wherein the co-stimulatory domain is a 41 -BB domain or a CD28 domain, and / or wherein the intracellular signaling domains is a CD3(^ intracellular signaling domain.

41. The cell population of any one of claims 28-40, wherein the disease-associated antigen is a tumor antigen, an autoimmune disease-associated antigen, or a pathogen antigen.

42. A pharmaceutical composition comprising a plurality of the modified T cells of any one of claims 1-27, or the cell population of any one of claims 28-41.

43. The pharmaceutical composition of claim 42, wherein the plurality of modified T cells are y8 T cells, preferably wherein the plurality of y8 T cells comprise a composition that is at least about 60%, at least about 80%, or at least about 80% to about 90% 81 y8 T cells, and a pharmaceutically acceptable carrier.

44. The pharmaceutical composition of claim 42 or 43, wherein the plurality of modified T cells comprise a composition that is greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, or greater than 90% CCR7+ T cells.

45. The pharmaceutical composition of any one of claims 42-44, wherein the plurality of T cells comprises at least about 107y8 T cells, optionally from about 108to about 1010y8 T cells.

46. A method for treating or preventing a disease in a subject, comprising administering to a subject in need thereof the pharmaceutical composition of any one of claims 42-45.

47. The method of claim 46, wherein the disease is an autoimmune disease.

48. The method of claim 46, wherein the disease is a cancer or a precancerous condition.

49. A method for enriching a population of isolated T cells for naive-like stem cell memory T cells having a CCR7+ CD62L+ CD45RA+ phenotype, wherein the T cells are genetically1321106305893\l\AMERICASmodified to disrupt MED12 and to express membrane-bound IL-12, or wherein the T cells are genetically modified to disrupt MED12 and contacted with IL-12 ex vivo or in vivo.

50. The method of claim 49, wherein the modified T cells are CD45RO+.

51. The method of claim 49, wherein the modified T cells are CD45RO-.

52. The method of any one of claims 49-51, wherein the modified T cells further comprise an antigen recognition moiety expressed on a surface of the cells, wherein the antigen recognition moiety comprises an affinity binding domain specific for a disease-associated antigen.

53. The method of any one of claims 49-52, wherein the modified T cells are y5 T cells.

54. The method of claim 53, wherein the y5 T cells are 51, 52, 53, or 54 y5 T cells, preferably 52‘ y5 T cells, more preferably 51 y5 T cells.

55. The method of any one of claims 49-54, wherein the modified T cells further comprise one or more of CD4+, CD71+, CD49b+, CD123+, CD109+, CD80+, CD86+, CD36L1+ and / or CD151+; preferably wherein the modified T cells are CD4+.

56. A method of increasing the longevity / persistence of a T cell comprising disrupting a MED 12 gene in the cell and expressing a membrane-bound IL- 12 in the cell, or disrupting a MED12 gene in the cell and contacting the modified T cell with IL-12 ex vivo or in vivo, thereby increasing the persistence of the modified T cell.

57. The method of claim 56, wherein the modified T cell comprises an antigen recognition moiety expressed on a surface of the cell, wherein the antigen recognition moiety comprises an affinity binding domain specific for a disease-associated antigen.

58. The method of claim 56 or 57, wherein the modified T cell is a y5 T cell.1331106305893\l\AMERICAS59. The method of claim 58, wherein the y5 T cell is a 51, a 52, a 53, or a 54 y5 T cell, preferably a 52" 75 T cell, more preferably a 51 y5 T cell.

60. The method of any one of claims 56-59, wherein the modified T cell further comprises one or more of CD4+, CD71+, CD49b+, CD123+, CD109+, CD80+, CD86+, CD36L1+ and / or CD151+; preferably wherein the modified T cell is CD4+.1341106305893\l\AMERICAS

Images