WO2019222642A1

WO2019222642A1 - Engineered immune cells and methods of use

Info

Publication number: WO2019222642A1
Application number: PCT/US2019/032890
Authority: WO
Inventors: Timothy Kuan-Ta Lu; Russell Morrison GORDLEY; Daniel FRIMANNSSON
Original assignee: Senti Biosciences, Inc.
Priority date: 2018-05-18
Filing date: 2019-05-17
Publication date: 2019-11-21

Abstract

The present disclosure relates to engineered immune cells. In particular, the present disclosure relates to engineered immune cells with one or more chimeric antigen receptors containing a broadly reactive extracellular target-binding moiety and methods of using same. The present disclosure relates to a pharmaceutical composition comprising the engineered immune cells and suitable pharmaceutically acceptable carriers. Compositions and methods disclosed herein may be used to treat immunodeficiencies.

Description

ENGINEERED IMMUNE CELLS AND METHODS OF USE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/673,326, filed May 18, 2018, which is hereby incorporated by reference in its entirety.

FIELD

[0002] The present disclosure relates to engineered immune cells. In particular, the present disclosure relates to engineered immune cells with one or more chimeric antigen receptors containing a broadly reactive extracellular target-binding moiety and methods of using same.

BACKGROUND

[0003] Chimeric antigen receptor (CAR) T cells are genetically-altered cells used to specifically target and kill cancer cells. These T cells are administered to cancer patients as a form of immunotherapy. Existing fixed CAR designs, however, limit flexibility with respect to antigen recognition. Generally, a new CAR T cell is engineered for each cancer antigen.

BRIEF SUMMARY

[0004] Provided herein are immune cells (e.g., T cells) engineered to comprise a chimeric receptor that can direct an immune cell not to a single antigen (e.g., cancer antigen) but rather to two or more different antigens. These chimeric receptors, in some embodiments, are described as binding to“common target sites,” for example, located on antibodies (or other molecules, such as other antigen-binding proteins) of a particular type or class, for example. Thus, a single engineered immune cell can be used to bind to many different targets (e.g., antibodies), each with different antigen-binding specificities, without reengineering the immune cell. In this way, an engineered immune cell of the present disclosure, rather than being reactive toward (e.g., specific for) only a single target, can be designed to be reactive toward (e.g., specific for) a multitude of targets (e.g., via binding to a common target site on an antibody). Thus, provided herein are immune cells (e.g., T cells) engineered to comprise multiple (e.g., two or more) chimeric receptors, each specific to a different target (e.g., different common target site located on the same or different antibody, for example, of a different type or class), thereby arming the cells with combinatorial logic (e g., AND or OR) capabilities.

[0005] For example, some aspects of the present disclosure provide immune cells designed to comprise two or more engineered chimeric receptors that each comprise an intracellular signaling domain and an extracellular target-binding moiety that binds to an antigen-binding domain of a bi-specific antibody, wherein the extracellular target-binding moieties have different target-binding specificities and/or target-binding avidities relative to each other. In some embodiments, the bi-specific antibodies differ from each other (see, e.g., FIGS. 1-3). The present disclosure is not limited by the type of antigen-binding domain present within a chimeric receptor specific for a bi-specific antibody. Indeed, any antigen-binding domain specific for a bi-specific antibody may be used in a chimeric receptor of the present disclosure. Examples of antigen binding domains specific for a particular bi-specific antibody that may be used include, but are not limited to, antigen-binding domain specific for Catumaxomab (Fresenius Biotech), antigen-binding domain specific for Lymphomun (Fresenius Biotech), antigen-binding domain specific for Ertumaxomab (Fresenius Biotech), antigen-binding domain specific for

Blinatumomab (Amgen), antigen-binding domain specific for MTl l l (Amgen), antigen-binding domain specific for MT112 BAY2010112 (Bayer) (Micromet), antigen-binding domain specific for MT110 AMG 110 (Amgen), antigen-binding domain specific for RG7221 (Roche), antigen binding domain specific for RG6013 (Chugai, Roche), antigen-binding domain specific for RG7597 (Genentech, Roche group), antigen-binding domain specific for RG7716 (Roche), antigen-binding domain specific for MM111 (Merrimack), antigen-binding domain specific for MM141 (Merrimack), antigen-binding domain specific for ABT122 (Abbvie), antigen-binding domain specific for ABT981 (Abbott), antigen-binding domain specific for MGD006

(Macrogenics and Servier), antigen-binding domain specific for MGD007 (MacroGenics and Sender), antigen-binding domain specific for BI1034020 (Ablynx, Boehringer Ingelheim), antigen-binding domain specific for ALX0761 (Ablynx, Merck Serono), antigen-binding domain specific for SAR156597 (Serono), antigen-binding domain specific for TF2 (Immunomedics), antigen-binding domain specific for IL-17/IL-34 biAb (BMS, Zymogenetics), antigen-binding domain specific for AFM13 (Affimed), antigen-binding domain specific for AFM11 (Affimed), antigen-binding domain specific for LY3164530 (Eli Lilly), or other antigen-binding domain specific for a bi-specific antibody known in the art.

[0006] In some embodiments, at least one of the extracellular target-binding moieties binds to the antigen-binding domain of a bi-specific antibody via a bridging molecule. A variety of bridging molecules may be used with the compositions and methods disclosed including, but not limited to, an extracellular molecule, secreted molecule, or small molecule drug. For example, in some aspects, a cytokine or a secreted tumor antigen (e.g., prostate specific antigen (PSA)) is used as a bridging molecule. In some embodiments, the bridging molecule is an endogenous molecule or an exogenous molecule (see, e.g., FIG. 2). In some embodiments, at least one of the extracellular target-binding moieties comprises an anti-idiotype antibody (see, e.g., FIG. 3).

[0007] Some aspects of the present disclosure provide engineered immune cells comprising two or more engineered chimeric receptors that each comprise an intracellular signaling domain and an extracellular target-binding moiety that binds to an Fc domain of an antibody of a class or subclass of immunoglobulins, wherein the extracellular target-binding moieties have different target-binding specificities and/or target-binding avidities relative to each other. In some embodiments, the antibodies of a class or subclass of immunoglobulins differ from each other (see, e.g., FIG. 4). For example, in some embodiments, the antibodies of a class of

immunoglobulins are selected from immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE), immunoglobulin G (IgG), and immunoglobulin M (IgM). In some embodiments, antibodies of a subclass of immunoglobulins are selected from IgAl and IgA2. In some embodiments, antibodies of a subclass of immunoglobulins are selected from IgGl, IgG2, IgG3, and IgG4.

[0008] Some aspects of the present disclosure provide engineered immune cells comprising two or more engineered chimeric receptors that each comprise an intracellular signaling domain and an extracellular target-binding moiety that binds to polyethylene glycol (PEG), biotin, or streptavidin, wherein the extracellular target-binding moieties have different target-binding specificities and/or target-binding avidities relative to each other (see, e g , FIG. 8). In some embodiments, at least one of the extracellular target-binding moieties is selected from PEG antibodies or fragment thereof that binds with specificity to PEG, streptavidin, and a

streptavidin-binding protein. [0009] Some aspects of the present disclosure provide immune cells designed to comprise two or more chimeric receptors that each comprise an intracellular signaling domain and an

extracellular target-binding moiety that binds to a post-translational modification (PTM) of an antibody, wherein the extracellular target-binding moieties have different target-binding specificities and/or target-binding avidities relative to each other. In some embodiments, the PTMs differ from each other (see, e.g., FIGs. 7 and 9). The present disclosure is not limited by the type of PTM recognized by an extracellular target-binding moiety of a chimeric receptor of the present disclosure. Indeed, an extracellular target-binding moiety of a chimeric receptor of the present disclosure may recognize any PTM including, but not limited to, amino acid modification, cleavage (e.g., proteolysis), addition of polypeptide(s), addition of complex molecule, and/or addition of a chemical group. Examples of addition of chemical groups include, but are not limited to, phosphorylation, hydroxylation, sulfation, acetylation, and methylation. Examples of amino acid modifications include, but are not limited to, deamidation, eliminylation, or other enzymatic modification. Examples of polypeptide additions include, but are not limited to, ubiquitination, ubiquitylation, UBL-protein conjugation (e.g., SUMO), or addition or other polypeptide. Examples of addition of complex molecules include, but are not limited to, AMPylation, ADP-ribosylation, glycosylation, prenylation, lipidation, nitrosylation, amidation, or other complex molecule.

[0010] In some embodiments, the glycosylation is selected from galactosylation, fucosylation, and sialylation. In some embodiments, at least one of the extracellular target-binding moieties does not bind the antibody that does not comprise the PTM.

[0011] Some aspects of the present disclosure provide immune cells engineered to comprise two or more chimeric receptors that each comprise an intracellular signaling domain and an extracellular target-binding moiety that binds to an Fc domain of an antibody, wherein the extracellular target-binding moieties have different target-binding specificities and/or target binding avidities relative to each other. In some embodiments, the Fc domains differ from each other (see, e.g., FIG. 10).

[0012] For example, in some embodiments, the extracellular target-binding moieties bind to different epitopes on Fc. In some embodiments, at least one of the extracellular target-binding moieties is selected from TRIM proteins (e.g., TRIM21), bacterial anti-Fc proteins, Fc receptors (e.g., CD16, CD32, or CD64), and variants thereof. In some embodiments, at least one of the extracellular target-binding moieties is a bacterial immunoglobulin-binding protein selected from Protein A, Protein G, Protein Z, Protein L, Protein Z, and immunoglobulin-binding fragments thereof.

[0013] The present disclosure is not limited to any particular intracellular signaling domain. Indeed, any intracellular signaling domain may be used in a chimeric receptor of the present disclosure including, but not limited to, CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, IT GAL, CDl la, LFA-l, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD18, LFA-l, ITGB7, TNFR2, TRAN CE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM

(SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP- 76, PAG/Cbp, NKp44, NKp30, NKp46, and/or NKG2D. In some embodiments, the intracellular signaling domain is CD28, OX40/CD134, 4-1 BB/CD 137/TNFRSF9, the high affinity immunoglobulin E receptor-gamma subunit (FcsRIy), ICOS/CD278, interleukin 2 subunit beta (ΉATb) or CD122, cytokine receptor common subunit gamma (IL-2Ry) or CD132, or CD40. In some embodiments, CD3-zeta is used as the intracellular signaling domain in a first engineered chimeric receptor and a second, different, intracellular signaling domain is used in a second engineered chimeric receptor of the engineered immune cell. The present disclosure is not limited by the type of second, different intracellular signaling domain used. In some

embodiment, the second, different intracellular signaling domain used is CD28, 0X40 (CD 134), 4-1BB (CD137), or ICOS. In some embodiments, CD3-zeta and one of CD28, 0X40 (CD134), 4-1BB (CD137), or ICOS are used in a single engineered chimeric receptor present in an engineered immune cell. [0014] In some embodiments, at least one of the extracellular target-binding moieties is an antibody, a single-chain variable fragment (scFv), an antigen binding fragment (Fab), a single domain antibody, a diabody, a VHH fragment, or a synthetic epitope.

[0015] In some embodiments, at least one of the extracellular target-binding moieties does not bind to a native antibody.

[0016] Some aspects of the present disclosure provide an engineered immune cell comprising a chimeric receptor that comprises an intracellular signaling domain, optionally an intracellular co signaling domain, and an extracellular target-binding moiety, where the extracellular target binding moiety may be a molecule that binds to an antigen-binding domain of a bi-specific antibody, an anti-hinge antibody, rheumatoid factor or IgG-binding fragments thereof (see, e.g., FIG. 5); an antibody that binds to a non-human sequence of a humanized antibody; a molecule that binds to a post-translational modification (PTM) of an antibody (e.g., a glycosylated amino acid of an antibody); a molecule that binds to polyethylene glycol (PEG); a molecule that binds an Fc domain of an antibody (e.g., a TRIM protein); and/or a molecule that binds an antibody junction (e.g., a mouse-human junction) (see, e.g., FIG. 6). In further embodiments, the engineered immune cell further comprises a second, different chimeric receptor that comprises an intracellular co-signaling domain and an extracellular target-binding moiety where the extracellular target-binding moiety may be a molecule that binds to an antigen-binding domain of a bi-specific antibody, anti-hinge antibody, rheumatoid factor or IgG-binding fragments thereof; an antibody that binds to a non-human sequence of a humanized antibody; a molecule that binds to a PTM of an antibody (e.g., a glycosylated amino acid of an antibody); a molecule that binds to PEG; a molecule that binds an Fc domain of an antibody (e.g., TRIM proteins); and/or a molecule that binds an antibody junction (e.g., mouse-human junctions), wherein the extracellular target-binding moieties of the first and second chimeric receptors bind to different targets.

[0017] A chimeric receptor of an engineered immune cell of the present disclosure may comprise a transmembrane domain derived from a natural source or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. The present disclosure is not limited to any particular transmembrane domain. Indeed, any transmembrane domain may be used including, but not limited to, the transmembrane region(s) of the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27,

CD3 epsilon, CD45, CD4, CD5, CD 8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, a transmembrane domain may include the transmembrane region(s) of, for example, KIRDS2, 0X40, CD2, CD27, LFA-l (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R .alpha., ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld,

ITGAE, CD 103, ITGAL, CDl la, LFA-l, ITGAM, CDl lb, ITGAX, CDl lc, ITGB1, CD29, ITGB2, CD 18, LFA-l, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100

(SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME

(SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, and/or NKG2C.

[0018] In some embodiments, the extracellular target-binding moiety is an antibody, a single chain variable fragment (scFv), an antigen binding fragment (Fab), a single domain antibody, a diabody, a VHH fragment, or a synthetic epitope.

[0019] In some embodiments, the molecule to which the extracellular target-binding moiety(ies) bind is a cancer-associated antigen, autoimmune-associated antigen, or an infectious disease- associated antigen. The present disclosure is not limited by the type of cancer-associated antigen recognized by an extracellular target-binding moiety of a chimeric receptor of the present disclosure. Indeed, any extracellular target-binding moiety known in the art with specificity for a cancer-associated antigen can be used in a chimeric receptor of the present disclosure including, but not limited to, those described herein. Examples of cancer-associated antigens include, but are not limited to, TSHR, CD 19, CD 123, CD22, CD30, CD171, CS-l, CLL-l, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, prostate specific membrane antigen (PSMA), ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-l3Ra2, Mesothelin, interleukin- 11 receptor a (IL-l lRa), PSCA, PRSS21, VEGFR2, LewisY, CD24, platelet-derived growth factor receptor-beta (PDGFR-beta), SSEA-4, CD20, Folate receptor alpha, ERBB2 (Her2/neu), MUC1, epidermal growth factor receptor (EGFR), NCAM, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CALX, LMP2, gplOO, bcr-abl, tyrosinase, EphA2, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD 179a, ALK, Poly sialic acid, PLAC1, GloboH, NY-BR-1, UPK2,

HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-l, LAGE-la, MAGE-A1, legumain, HPV E6,E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-l, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-l/Galectin 8, MelanA/MARTl, Ras mutant, hTERT, sarcoma translocation breakpoints, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, Androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP- 4, SSX2, RAGE-l, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxyl esterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1

[0020] The present disclosure is not limited by the type or source of immune cell engineered. In some embodiments, the engineered immune cell is an engineered T cell. In some embodiments, the immune cell is selected from CD4+ T cells, CD8+ T cells, regulatory T cells (Tregs), Natural Killer T (NKT) cells, and Natural Killer (NK) cells. In some embodiments, the T cells are alpha beta T cells. In some embodiments, the T cells are gamma delta T cells. In some embodiments, the T cells are a combination of CD4+ and CD8 T+ cells. In certain embodiments, the T cells are memory T cells. In some embodiments, the memory T cells are central memory T cells. In other embodiments, the memory T cells are effector memory T cells. In some embodiments, the T cells are tumor-infiltrating lymphocytes (TILs). In certain embodiments, the T cells are a combination of CD8+ T cells, CD4+ T cells, NK T cells, memory T cells, and/or gamma delta T cells. In some embodiments, the T cells are cytokine-induced killer cells.

[0021] In some embodiments, an antibody recognized by a chimeric receptor of an engineered immune cell of the present disclosure is Revlimid® (lenalidomide), Opdivo® (nivolumab), Imbruvica® (ibrutinib), Keytruda® (pembrolizumab), Ibrance® (palbociclib), Tecentriq® (atezolizumab), Darzalex® (daratumumab), Peijeta® (pertuzumab), Xtandi® (enzalutamide), Avastin® (bevacizumab), Herceptin® (trastuzumab), Gazyva® (obinutuzumab), Jakafi® (ruxolitinib), Venclexta® (venetoclax), and/or Rituxan® (rituximab).

[0022] In another aspect, the present disclosure provides an engineered immune cell comprising an isolated nucleic acid sequence encoding a chimeric receptor of the present disclosure. For example, in one embodiment, the present disclosure provides an engineered immune cell comprising an isolated nucleic acid sequence encoding a chimeric receptor, wherein the isolated nucleic acid sequence comprises nucleic acid sequence (e.g., human, mouse, or humanized mouse nucleic acid sequence) of an extracellular target-binding moiety (e.g., a single chain variable fragment, or scFv or other extracellular target-binding moiety), the nucleic acid sequence of a transmembrane domain, and the nucleic acid sequence of one or more intracellular signaling domains (e.g., the nucleic acid sequence of a CD3 zeta signaling domain).

[0023] The present disclosure additionally includes a vector comprising an isolated nucleic acid sequence encoding a chimeric receptor of the present disclosure, wherein the isolated nucleic acid sequence comprises nucleic acid sequence (e.g., human, mouse, or humanized mouse nucleic acid sequence) of an extracellular target-binding moiety (e.g., a single chain variable fragment, or scFv), the nucleic acid sequence of a transmembrane domain, and the nucleic acid sequence of one or more intracellular signaling domains (e.g., the nucleic acid sequence of a CD3 zeta signaling domain).

[0024] In addition, the present disclosure includes methods for providing anti-cancer and/or anti tumor immunity in a subject having cancer. The methods comprise administering to the subject an effective amount of an engineered immune cell comprising an isolated nucleic acid sequence encoding a chimeric receptor of the present disclosure, wherein the isolated nucleic acid sequence comprises nucleic acid sequence (e.g., human, mouse, or humanized mouse nucleic acid sequence) of an extracellular target-binding moiety (e.g., a single chain variable fragment, or scFv), the nucleic acid sequence of a transmembrane domain, and the nucleic acid sequence of one or more intracellular signaling domains (e.g., the nucleic acid sequence of a CD3 zeta signaling domain), thereby providing anti -tumor immunity in the subject. In some embodiments, the subject is a human.

[0025] Further included in the present disclosure is a method for stimulating a beneficial and/or therapeutic T cell-mediated immune response to a cell population, tumor or tissue in a subject. The method comprises administering to the subject an effective amount of an engineered immune cell comprising an isolated nucleic acid sequence encoding a chimeric receptor of the present disclosure, wherein the isolated nucleic acid sequence comprises nucleic acid sequence (e.g., human, mouse, or humanized mouse nucleic acid sequence) of an extracellular target-binding moiety (e.g., a single chain variable fragment, or scFv), the nucleic acid sequence of a transmembrane domain, and the nucleic acid sequence of one or more intracellular signaling domains (e.g., the nucleic acid sequence of a CD3 zeta signaling domain), thereby stimulating a T cell-mediated immune response in the subject.

[0026] In another aspect, the present disclosure provides a method of treating cancer in a subject. In one embodiment, the method comprises administering to a subject having cancer a therapeutically effective amount of an engineered immune cell comprising an isolated nucleic acid sequence encoding a chimeric receptor of the present disclosure, wherein the isolated nucleic acid sequence comprises nucleic acid sequence (e.g., human, mouse, or humanized mouse nucleic acid sequence) of an extracellular target-binding moiety (e.g., a single chain variable fragment, or scFv), the nucleic acid sequence of a transmembrane domain, and the nucleic acid sequence of one or more intracellular signaling domains (e.g., the nucleic acid sequence of a CD3 zeta signaling domain), thereby treating cancer in the subject.

[0027] The present disclosure further includes a method of generating a persisting population of engineered immune cells (e.g., memory T cells) in a subject (e.g., a subject diagnosed with cancer, a subject diagnosed with autoimmune disease, or a subject with an infectious disease), the method comprising administering to the subject an effective amount of an engineered immune cell comprising an isolated nucleic acid sequence encoding a chimeric receptor of the present disclosure, wherein the isolated nucleic acid sequence comprises nucleic acid sequence (e.g., human, mouse, or humanized mouse nucleic acid sequence) of an extracellular target binding moiety (e.g., a single chain variable fragment, or scFv), the nucleic acid sequence of a transmembrane domain, and the nucleic acid sequence of one or more intracellular signaling domains (e.g., the nucleic acid sequence of a CD3 zeta signaling domain), wherein the persisting population of genetically engineered T cells persists in the subject (e.g., for weeks, a month, months, etc.) after administration. In some embodiments, the persisting population of genetically engineered T cells persists in the human for at least one month, for at least two months, for at least three months or more after administration.

[0028] In another aspect, the present disclosure provides a method of treating a disease or condition in a subject comprising administering to the subject (e.g., a patient) having a disease or condition an effective amount of engineered immune cells comprising a chimeric receptor of the present disclosure. The present disclosure is not limited by the type of disease or condition treated. Indeed, any disease or condition that is treatable (e g., for which signs or symptoms of the disease are ameliorated upon treatment) via administration of engineered immune cells can be treated in an improved and more effective manner using compositions and methods of the present disclosure. In one embodiment, the disease or condition is cancer. In another embodiment, the disease or condition is an infectious disease. The present disclosure is not limited by the type of cancer or by the type of infectious disease. Indeed, any cancer or disease known in the art for which cellular therapy (e g., CAR T cell therapy) is used for treatment may be treated with the compositions and methods of the present disclosure. For example, compositions and methods of the present disclosure can be used to modify any CAR T cell therapy known in the art via genetically introducing a chimeric receptor of the present disclosure into therapeutic CAR T cells.

[0029] In another aspect, the present disclosure provides a method of maintaining, regaining, or enhancing functionality of engineered immune cells that would otherwise experience a decrease or loss of function (e.g., antigen induced tonic signaling and/or exhaustion) in the context of treating a disease or condition. The present disclosure is not limited by the type of functionality maintained, regained or enhanced. In some embodiments, the functionality is antigen induced cytokine production. In other embodiments, the functionality is T cell cytotoxicity (e g., increased recognition of tumor targets). In yet other embodiments, the functionality is increased memory cell formation and/or enhanced proliferation in response to antigen.

[0030] In another aspect, the present disclosure provides methods of treating or delaying the progression of cancer in a patient comprising administering to the patient a therapeutically effective amount of a composition comprising engineered immune cells comprising a chimeric receptor of the present disclosure. In certain embodiments, the therapeutically effective amount of the composition comprising engineered immune cells reduces the number of cancer cells in the patient following such treatment. In certain embodiments, the therapeutically effective amount of the composition comprising engineered immune cells reduces and/or eliminates the tumor burden in the patient following such treatment. In certain embodiments, the method further comprises administering radiation therapy to the patient. In certain embodiments, the radiation therapy is administered before, at the same time as, and/or after the patient receives the therapeutically effective amount of the composition comprising engineered immune cells. In certain embodiments, the method comprises administering to the patient one or more anticancer agents and/or one or more chemotherapeutic agents. In certain embodiments, the one or more anticancer agents and/or one or more chemotherapeutic agents are administered before, at the same time as, and/or after the patient receives a therapeutically effective amount of the composition comprising engineered immune cells. In certain embodiments, treatment of a patient with a therapeutically effective amount of engineered immune cells comprising a chimeric receptor of the present disclosure and a course of an anticancer agent produces a greater tumor response and clinical benefit in such patient compared to those treated with the engineered immune cells or anticancer drugs/radiation alone. Since the doses for all approved anticancer drugs and radiation treatments are known, the present disclosure contemplates the various combinations of them with the engineered immune cells.

[0031] In certain embodiments, the present disclosure provides a therapeutically effective amount of a composition comprising engineered immune cells comprising a chimeric receptor according to the present disclosure (e.g., for use in treating or delaying the progression of cancer in a subject). As described herein, the composition may be administered before, during, or after other types of cancer treatment (e.g., chemotherapy, surgical resection of cancer, or radiation therapy). The present disclosure also provides the use of the composition to induce cell cycle arrest and/or apoptosis. The present disclosure also relates to the use of the compositions for sensitizing cells (e.g., to agent(s) such as inducers of apoptosis and/or cell cycle arrest).

Compositions of the present disclosure are useful for the treatment, amelioration, or prevention of disorders, such as any type of cancer or infectious disease and additionally any cells responsive to induction of apoptotic cell death (e.g., disorders characterized by dysregulation of apoptosis, including hyperproliferative diseases such as cancer). In certain embodiments, the compositions can be used to treat, ameliorate, or prevent a cancer that is characterized by resistance to cancer therapies (e.g., cancer that is chemoresistant, radiation resistant, hormone resistant, or the like).

[0032] These and other embodiments of the present disclosure will readily occur to those of skill in the art in view of the disclosure herein. BRIEF DESCRIPTION OF DRAWINGS

[0033] FIG. 1 shows a schematic of an example of an engineered immune cell that includes two different chimeric receptors, each of which includes an extracellular target-binding moiety that binds a different antigen-binding domain of a bi-specific antibody.

[0034] FIG. 2 shows a schematic of another example of an engineered immune cell that includes two different chimeric receptors, similar to FIG. 1, with the exception that one of the

extracellular target-binding moieties binds to the bi-specific antibody through an intermediate bridging molecule.

[0035] FIG. 3 shows a schematic of yet another example of an engineered immune cell that includes two different chimeric receptors, similar to FIG. 1, with the exception that one of the extracellular target-binding moieties is an anti-idiotype antibody (e g., anti-idiotype scFv) that binds an antigen-binding domain of the bi-specific antibody.

[0036] FIG. 4 shows a schematic of an example of an engineered immune cell that includes two different chimeric receptors, each of which includes an isotype-specific antibody that binds to a different IgG isotype (also called an IgG subclass).

[0037] FIG. 5 shows a schematic of an engineered immune cell that includes a rheumatoid factor that binds to an Fc domain of an antibody.

[0038] FIG. 6 shows a schematic of an engineered immune cell that includes an antibody that binds to a non-human sequence in a humanized antibody.

[0039] FIG. 7 shows a schematic of an engineered immune cell that includes an antibody that binds to a glycosylated antibody.

[0040] FIG. 8 shows a schematic of an engineered immune cell that includes an antibody that binds to polyethylene glycol (PEG).

[0041] FIG. 9 shows a schematic of an example of an engineered immune cell that includes two different chimeric receptors, each of which includes an extracellular target-binding moiety that binds to a different type of post-translational modifications (PTMs). [0042] FIG. 10 shows a schematic of an engineered immune cell that includes a Fc binding protein (e.g., a natural Fc binding protein or a variant thereof).

[0043] FIG. 11 shows a schematic of an example of an engineered immune cell that includes two different chimeric receptors, each of which includes an extracellular target-binding moiety that binds to a different type of IgG subclass. This example may be used, like many of the other dual-chimeric receptor cells described above, to implement combinatorial (e.g., AND/OR) logic.

DEFINITIONS

[0044] 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 below conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0045] The term“antibody,” as used herein, refers to an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies may be multimers of individual immunoglobulin molecules. Antibodies described in the present disclosure may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)₂, as well as single chain antibodies, human antibodies, and humanized antibodies (See, e.g., Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0046] The term“antibody fragment” refers to a portion of an intact antibody and preferably refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')₂, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0047] An“antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. An“antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations. Kappa and lamda light chains refer to the two major antibody light chain isotypes.

[0048] The term“scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

[0049] By the terms“synthetic antibody” and“recombinant antibody” are used interchangeably herein to mean an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.

[0050] The term“antigen” or“Ag” refers to 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 virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an“antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a“gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample, or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.

[0051] The terms“chimeric receptor,”“Chimeric Antigen Receptor” or alternatively a“CAR” refer to a recombinant polypeptide construct comprising at least an extracellular target-binding moiety, a transmembrane domain and an intracellular signaling domain (also referred to as a “cytoplasmic signaling domain”) comprising a functional signaling domain derived from a stimulatory molecule described herein.

[0052] The term“signaling domain” refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.

[0053] As used herein, the term“binding domain” or“antibody molecule” (also referred to herein as“anti-target (e.g., CD123) binding domain”) refers to a protein, e g., an

immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term“binding domain” or“antibody molecule” encompasses antibodies and antibody fragments. In an embodiment, an antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In an embodiment, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0054] The term“autoimmune disease” as used herein refers to a disorder that results from an autoimmune response, for example, .an inappropriate and excessive response to a self-antigen. Examples of autoimmune diseases include but are not limited to, Addision's disease, alopecia greata, ankylosing spondylitis, autoimmune hepatitis, autoimmune parotitis, Crohn's disease, diabetes (Type I), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, ulcerative colitis, among others.

[0055] As used herein, the term“autologous” is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.“Allogeneic” refers to a graft derived from a different animal of the same species.“Xenogeneic” refers to a graft derived from an animal of a different species.

[0056]“Co-stimulatory ligand,” as used herein, includes a molecule on an antigen presenting cell (APC) (e.g., dendritic cell, B cell, and the like) that specifically binds a cognate co stimulatory molecule on a T cell, thereby providing a signal which, in addition to the primary signal provided by, for instance, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, mediates a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A co-stimulatory ligand can include, but is not limited to, CD7, B7-1 (CD 80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducible costimulatory ligand (ICOS- L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a T cell, such as, but not limited to, CD27, CD28, 4-1BB, 0X40, CD30, CD40, PD-l, ICOS, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

[0057] A“co-stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor. [0058] A“co-stimulatory signal”, as used herein, refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to T cell proliferation and/or upregulati on or downregulation of key molecules.

[0059] The term“therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0060] The term“effective amount” or“therapeutically effective amount” are used

interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result. For example, a“therapeutically effective amount” may be the amount of a compound or composition that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. A therapeutically effective amount includes that amount of a compound or composition that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of a disorder or disease being treated. The therapeutically effective amount will vary depending on the compound or composition, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0061] To“treat” 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. For example, treat may refer to the reduction or amelioration of the progression, severity and/or duration of a proliferative disorder, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more therapies. In specific embodiments, the terms“treat”,“treatment” and“treating” refer to the amelioration of at least one measurable physical parameter of a proliferative disorder, such as growth of a tumor, not necessarily discernible by the patient. In other embodiments the terms “treat”,“treatment” and“treating”-refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e g., stabilization of a physical parameter, or both. In other embodiments the terms“treat”, “treatment” and“treating” refer to the reduction or stabilization of tumor size or cancerous cell count. [0062] As used herein“endogenous” refers to any material from or produced inside an organism, cell, tissue or system. As used herein, the term“exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0063] The term“lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.

[0064] The term“lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR.RTM. gene delivery technology from Oxford BioMedica, the LENTIMAX.TM. vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[0065]“Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0066] The term“immunoglobulin” or“Ig,” as used herein refers to a class of proteins, which function as antibodies. Antibodies expressed by B cells are sometimes referred to as the B cell receptor (BCR) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main

immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is the immunoglobulin that has no known antibody function, but may serve as an antigen receptor. IgE is the immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen.

[0067]“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 or peptide 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 host cell.

[0068] As used herein, a“substantially purified” cell is a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0069] 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. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s)

[0070] The term“operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0071]“Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.

[0072] The terms“patient,”“subject,”“individual,” and the like are used interchangeably herein and are intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof.

[0073] By the term“specifically binds,” as used herein with respect to an antibody or extracellular target-binding moiety, is meant an antibody or extracellular target-binding moiety which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms“specific binding” or“specifically binding,” can be used in reference to the interaction of an antibody, 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, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody 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 antibody, will reduce the amount of labeled A bound to the antibody.

[0074] By the term“stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-b, enhanced expression of IL-2 and/or IFN-g, and/or reorganization of cytoskeletal structures, and the like.

[0075] A“stimulatory molecule,” as the term is used herein, means a molecule expressed by a T cell that provides the primary cytoplasmic signaling sequence(s) that regulate primary activation of the TCR complex in a stimulatory way for at least some aspect of the T cell signaling pathway. In one aspect, the primary signal is initiated by, for example, binding of a TCR/CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A primary cytoplasmic signaling sequence (also referred to as a“primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or IT AM. Examples of an ITAM containing primary cytoplasmic signaling sequences/domains include, but are not limited to, those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, CD278 (also known as“ICOS”), FcsRI, CD66d, DAP10 and DAP12. In a specific chimeric receptor of the present disclosure, the intracellular signaling domain in any one or more chimeric receptors of the present disclosure comprises an intracellular signaling sequence, e.g., a primary signaling sequence of CD3-zeta. In a specific chimeric receptor of the present disclosure, the primary signaling sequence of CD3-zeta is a human CD3-zeta sequence, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like. Any one of the intracellular signaling sequences described herein finds use in the chimeric receptors of the present disclosure.

[0076] A“stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a“stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

[0077] The term“antigen presenting cell” or“APC” refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC's) on its surface. T-cells may recognize these complexes using their T-cell receptors (TCRs). APCs process antigens and present them to T-cells.

[0078]“Immune effector cell,” as that term is used herein, refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response. Examples of immune effector cells include T cells, e.g., alpha/beta T cells and gamma/delta T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and myeloic-derived phagocytes.

[0079]“Immune effector function or immune effector response,” as that term is used herein, refers to function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell. E.g., an immune effector function or response refers a property of a T or NK cell that promotes killing or the inhibition of growth or proliferation, of a target cell.

In the case of a T cell, primary stimulation and co-stimulation are examples of immune effector function or response.

[0080] The term“effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines

[0081] The terms“sensitize” and“sensitizing,” as used herein, refer to making, through the administration of a first agent, an animal or a cell within an animal more susceptible, or more responsive, to the biological effects (e.g., promotion or retardation of an aspect of cellular function including, but not limited to, cell division, cell growth, proliferation, invasion, angiogenesis, necrosis, or apoptosis) of a second agent. The sensitizing effect of a first agent on a target cell can be measured as the difference in the intended biological effect (e.g., promotion or retardation of an aspect of cellular function including, but not limited to, cell growth, proliferation, invasion, angiogenesis, or apoptosis) observed upon the administration of a second agent with and without administration of the first agent. The response of the sensitized cell can be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500% over the response in the absence of the first agent.

[0082] The term“cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like. The terms“tumor” and“cancer” are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term“cancer” or“tumor” includes premalignant, as well as malignant cancers and tumors.

[0083] The terms“cancer antigen,”“cancer-associated antigen” or“tumor antigen”

interchangeably refer to a molecule (typically a protein, carbohydrate or lipid) that is expressed on the surface of a cancer cell, either entirely or as a fragment (e.g., MHC/peptide), and which is useful for the preferential targeting of a pharmacological agent to the cancer cell. In some embodiments, a tumor antigen is a marker expressed by both normal cells and cancer cells, e.g., a lineage marker, e.g., CD19 or CD123 on B cells. In some embodiments, a tumor antigen is a cell surface molecule that is overexpressed in a cancer cell in comparison to a normal cell, for instance, l-fold over expression, 2-fold overexpression, 3 -fold overexpression or more in comparison to a normal cell. In some embodiments, a tumor antigen is a cell surface molecule that is inappropriately synthesized in the cancer cell, for instance, a molecule that contains deletions, additions or mutations in comparison to the molecule expressed on a normal cell. In some embodiments, a tumor antigen will be expressed exclusively on the cell surface of a cancer cell, entirely or as a fragment (e.g., MHC/peptide), and not synthesized or expressed on the surface of a normal cell.

[0084] The term“anti-tumor effect” as used herein, refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various

physiological symptoms associated with the cancerous condition. An“anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, antibodies (or antigen-binding portions thereof), and engineered immune cells of the present disclosure in prevention of the occurrence of tumor in the first place.

[0085] The term“anti-cancer effect” refers to a biological effect which can be manifested by various means, including but not limited to, e g., a decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, decrease in cancer cell proliferation, decrease in cancer cell survival, or amelioration of various physiological symptoms associated with the cancerous condition. An“anti-cancer effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies in prevention of the occurrence of cancer in the first place.

[0086] As used herein, the term“subject suspected of having cancer” refers to a subject that presents one or more symptoms indicative of a cancer (e g., a noticeable lump or mass) or is being screened for a cancer (e.g., during a routine physical). A subject suspected of having cancer may also have one or more risk factors for developing cancer. A subject suspected of having cancer has generally not been tested for cancer. However, a“subject suspected of having cancer” encompasses an individual who has received a preliminary diagnosis (e.g., a CT scan showing a mass) but for whom a confirmatory test (e.g., biopsy and/or histology) has not been done or for whom the type and/or stage of cancer is not known. The term further includes people who previously had cancer (e.g., an individual in remission). A“subject suspected of having cancer” is sometimes diagnosed with cancer and is sometimes found to not have cancer.

[0087] As used herein, the term“subject diagnosed with a cancer” refers to a subject who has been tested and found to have cancerous cells. The cancer may be diagnosed using any suitable method, including but not limited to, biopsy, x-ray, blood test, etc.

[0088] As used herein, the term“subject at risk for cancer” refers to a subject with one or more risk factors for developing a specific cancer. Risk factors include, but are not limited to, gender, age, genetic predisposition, environmental exposure, and previous incidents of cancer, preexisting non-cancer diseases, and lifestyle.

[0089] As used herein, the term“characterizing cancer in a subject” refers to the identification of one or more properties of a cancer sample in a subject, including but not limited to, the presence of benign, pre-cancerous or cancerous tissue and the stage of the cancer. [0090]“Refractory” as used herein refers to a disease, e.g., cancer, that does not respond to a treatment. In embodiments, a refractory cancer can be resistant to a treatment before or at the beginning of the treatment. In other embodiments, the refractory cancer can become resistant during a treatment. A refractory cancer is also called a resistant cancer.

[0091]“Relapsed” or“relapse” as used herein refers to the return or reappearance of a disease (e.g., cancer) or the signs and symptoms of a disease such as cancer after a period of

improvement or responsiveness, e.g., after prior treatment of a therapy, e.g., cancer therapy. The initial period of responsiveness may involve the level of cancer cells falling below a certain threshold, e.g., below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. The reappearance may involve the level of cancer cells rising above a certain threshold, e.g., above 20%, 1%, 10%, 5%, 4%,

3%, 2%, or 1%. For example, e.g., in the context of B-ALL, the reappearance may involve, e.g., a reappearance of blasts in the blood, bone marrow (>5%), or any extramedullary site, after a complete response. A complete response, in this context, may involve <5% BM blast. More generally, in an embodiment, a response (e.g., complete response or partial response) can involve the absence of detectable MRD (minimal residual disease). In an embodiment, the initial period of responsiveness lasts at least 1, 2, 3, 4, 5, or 6 days; at least 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10, or 12 months; or at least 1, 2, 3, 4, or 5 years.

[0092] The term“derived from” as used herein refers to a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and a second molecule and does not connotate or include a process or source limitation on a first molecule that is derived from a second molecule. For example, in the case of an intracellular signaling domain that is derived from a CD3zeta molecule, the intracellular signaling domain retains sufficient CD3zeta structure such that is has the required function, namely, the ability to generate a signal under the appropriate conditions. It does not connotate or include a limitation to a particular process of producing the intracellular signaling domain, e.g., it does not mean that, to provide the intracellular signaling domain, one must start with a CD3zeta sequence and delete unwanted sequence, or impose mutations, to arrive at the intracellular signaling domain.

[0093]“Activation,” as used herein, refers to the state of an immune cell (e.g., T cell) that has been sufficiently stimulated to induce a detectable change such as, but not limited to, detectable cellular proliferation or immune response. Activation can also be associated with induced cytokine production, and/or detectable effector functions. The term“activated T cells” refers to, among other things, T cells that are undergoing cell division and/or enhanced cytokine production and/or secretion.

[0094] As used herein, the term "immune response" refers to a response by the immune system of a subject. For example, immune responses include, but are not limited to, a detectable alteration (e.g., increase) in Toll receptor activation, lymphokine (e.g., cytokine (e.g., Thl, Thl7, or Th2 type cytokines) or chemokine) expression and/or secretion, macrophage activation, dendritic cell activation, T cell activation (e.g., CD4+ or CD8+ T cells), NK cell activation, and/or B cell activation (e.g., antibody generation and/or secretion). Additional examples of immune responses include binding of an immunogen (e.g., antigen (e.g., immunogenic polypeptide)) to an MHC molecule and inducing a cytotoxic T lymphocyte ("CTL") response, inducing a B cell response (e.g., antibody production), and/or T-helper lymphocyte response, and/or a delayed type hypersensitivity (DTH) response against the antigen from which the immunogenic polypeptide is derived, expansion (e.g., growth of a population of cells) of cells of the immune system (e.g., T cells, B cells (e.g., of any stage of development (e.g., plasma cells), and increased processing and presentation of antigen by antigen presenting cells. An immune response may be to immunogens that the subject's immune system recognizes as foreign (e.g., non-self antigens from microorganisms (e.g., pathogens), or self-antigens recognized as foreign). Thus, it is to be understood that, as used herein, "immune response" refers to any type of immune response, including, but not limited to, innate immune responses (e.g., activation of Toll receptor signaling cascade), cell-mediated immune responses (e.g., responses mediated by T cells (e.g., antigen-specific T cells) and non-specific cells of the immune system), and humoral immune responses (e.g., responses mediated by B cells (e.g., via generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids)). The term "immune response" is meant to encompass all aspects of the capability of a subject's immune system to respond to antigens and/or immunogens (e.g., both the initial response to an immunogen as well as acquired (e.g., memory) responses that are a result of an adaptive immune response.

[0095] The articles“a” and“an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example,“an element” means one element or more than one element. [0096]“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 +/-l0%, 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.

[0097] Ranges: throughout this disclosure, various aspects of the present 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 present 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. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the breadth of the range.

[0098] It should also be understood that, unless clearly indicated to the contrary, in any method claimed herein that includes more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

DETAILED DESCRIPTION

[0099] Provided herein are engineered immune cells comprising one or more chimeric receptor(s) that can direct the engineered immune cells to different antigens, e.g., cancer antigens. The chimeric receptors, in some embodiments, comprise an extracellular target binding moiety that is specific to a common target site on antibodies of a particular type or class, rather than being specific for a single antigen on, for example, a cancer cell. Thus, the engineered immune cells of the present disclosure can universally target particular types and/or classes of commercially available antibodies, or antibodies that have been verified/approved for in vivo therapeutic use, which can then direct the engineered immune cell to a target (e.g., cancer) cell. In addition to the extracellular target-binding moiety, the chimeric receptor also comprises an intracellular signaling domain and a transmembrane domain linking the intracellular signaling domain to the extracellular target-binding moiety.

[0100] A chimeric receptor is an engineered receptor that grafts a selected specificity onto an engineered immune cell (e.g., an immune cell). The term“chimeric” means that the receptor is composed of parts from different sources. The chimeric receptor of the present disclosure comprises an intracellular signaling domain and an extracellular target-binding moiety. An intracellular signaling domain is a domain that, upon activation, stimulates a signaling pathway (transduces a signal) that activates and induces proliferation of the engineered immune cell (e.g., a T cell). In some embodiments, the chimeric receptor further comprises a second (co stimulatory) intracellular signaling domain that enhances signaling through the signaling pathway created by the first intracellular signaling domain. In some embodiments, the intracellular signaling domain is CD3-zeta. In some embodiments, in chimeric receptors comprising a first and a second intracellular signaling domain (comprising two so-stimulatory domains), one of the intracellular signaling domains is CD3-zeta, and the other of the intracellular signaling domains is selected from CD28, 0X40 (CD134), 4-1BB (CD137), and ICOS.

[0101] Different from a traditional chimeric antigen receptor (CAR), in which the extracellular target-binding moiety is typically an antigen-recognizing domain (e.g., a single chain fragment (scFv) of an antibody or other antibody fragment) that binds to a specific antigen (e.g., a cancer antigen), the extracellular target-binding moiety of the chimeric receptors described herein is designed to have broad target-(e.g., antibody-)binding activity, for example, and can be used to link different types and/or classes of antibodies (e.g., bi-specific antibodies, anti-cancer antibodies, anti-toxin antibodies, or other antibodies described herein) to the surface of the engineered immune cell, thus grafting the engineered immune cell with a broad range of desired specificity without the need to reengineer the immune cell.

[0102] In some aspects, an engineered immune cell comprises a single chimeric receptor comprising an intracellular signaling domain (e g., CD3-zeta) and an extracellular target-binding moiety. In some embodiments, the chimeric receptor further comprises an intracellular co signaling domain (e.g., CD28 or 4-1BB). An intracellular signaling domain and an intracellular co-signaling domain (which may be referred to collectively as two intracellular co-signaling domains) function together to fully activate an immune cell (each transduce a signal into the immune cell, both which are required to fully activate the immune cell) (see, e g., June CD et al. Mol. Cell. Biol. 1987;7:4472-4481).

[0103] It should be understood that the terms“intracellular signaling domain” and“intracellular co-signaling domain” may be used interchangeably. Throughout the disclosure, a chimeric receptor of an engineered immune cell is described as having an intracellular signaling domain. When an engineered immune cell includes two chimeric antigen receptors, for example, one of the chimeric receptors may be described as having an intracellular signaling domain, while the other may be described as having an intracellular co-signaling domain.

[0104] In other aspects, an engineered immune cell comprises two or more chimeric receptors, each comprising an intracellular signaling domain and an extracellular targeting-binding moiety. In some embodiments, in an engineered immune cell comprising two chimeric receptors, the two extracellular target-binding moieties have different target-binding specificities and/or target binding avidities relative to each other. In some embodiments, the two extracellular target binding moieties bind to different antibodies.

[0105] Two extracellular target-binding moieties have different target-binding specificities if they bind to different target sites, e.g., different target sites on different antibodies, or to different targets sites on the same antibody. For example, if one target-binding moiety binds to Antibody A and the other target-binding moiety binds to Antibody B, then the two target-binding moieties have different target-binding specificities. Likewise, if one target-binding moiety binds to Target site 1 on Antibody A and the other target-binding moiety binds to Target site 2 on Antibody A, then the two target-binding moieties have different target-binding specificities. Two

extracellular target-binding moieties have different target-binding avidities if the strength to which they bind the same target differs. For example, if one target-binding moiety binds to Antibody A with a dissociation constant (KD) of X, and the other target-binding moiety binds to Antibody A with a KD of Y (wherein X does not equal Y), then the two target-binding moieties have different target-binding avidities.

[0106] The term“specificity” refers to the number of different types of target sites to which a particular target-binding moiety can bind. The specificity of a target-binding moiety can be determined based on affinity and/or avidity. The affinity, represented by the equilibrium constant for the dissociation of a target site with an target-binding moiety (KD), is a measure for the binding strength between target site and the target-binding moiety: the lesser the value of the KD, the stronger the binding strength between an target site and the target-binding moiety (alternatively, the affinity can also be expressed as the affinity constant (KA), which is l/Ko). As will be clear to the skilled person (for example on the basis of the further disclosure herein), affinity can be determined in a manner known per se, depending on the specific target site of interest. Avidity is the measure of the strength of binding between a target-binding moiety and the pertinent target site. Avidity is related to both the affinity between a target site and the target-binding moiety and the number of pertinent binding sites present on the target-binding moiety.

[0107] In some embodiments, a target-binding moiety will bind to its target site on an antibody with a dissociation constant (KD) of l0 5 to I Q * ² moles/liter or less, for example 10 ² to 1 O * ² moles/liter or less, or 10 ⁸ to 1 O * ² moles/liter (i.e. with an association constant (KA) of 10² to 10l2 liter/ moles or more, for example 10² to l()l² liter/moles or more, or 10⁸ to 10¹² liter/moles), and/or bind to target sites as defined herein with a k_on-rate of between 10² vH s * to about 10² M ls l, for example, between 10² M ls l and 10² M ls l, more preferably between 1()4 M ls l and 10² M ls l, such as between 10² M ls l and 10² M ls l; and/or bind to target binding sites as defined herein with a k_0ff rate between ls l (t 1/2=0.69 s) and l0 6 s l

(providing a near irreversible complex with a ti/2 of multiple days), for example, between 10 ² s 1 and 10 6 s l, or between l0^~2 s l and l0^~6 s l, such as between l0 4 s l and l0 6 s l .

[0108] Any KD value greater than 10 4 M (or any KA value lower than 10^ M l) is generally considered to indicate non-specific binding.

[0109] Chimeric Receptor Constructs: Extracellular target-binding moiety.

[0110] As detailed herein, an extracellular target-binding moiety of a chimeric receptor of the present disclosure, in some embodiments, is not limited by specificity to a single target but rather may bind with specificity to a plurality of targets. For example, some aspects of the present disclosure relate to different types of extracellular target-binding moieties that have broad antibody-binding activity. In one non-limiting example, the extracellular target-binding moiety may comprise a molecule that binds to a common target site among antibodies of a particular class or subclass of antibodies (immunoglobulins). In some embodiments, the extracellular target-binding moiety does not bind to certain antibodies, such as native (endogenous/naturally- occurring) antibodies. In some embodiments, the extracellular target-binding moiety comprises a molecule that binds to other molecules attached/conjugated to an antibody, e.g., post- translational modifications, or molecules that are attached to an antibody artificially (e.g., polyethylene glycol, FITC, or biotin). In some embodiments, the extracellular target-binding moiety is a molecule that binds to an antigen-binding domain of a bi-specific antibody (e.g., as shown in FIG. 1).

[0111] An antibody is a protein produced by B lymphocytes (B cells) of the immune system in response to an antigen. Antibodies recognize and bind to a specific antigen. Antibodies are composed of two heavy chain polypeptides and two light chains polypeptides arranged in a Y- shaped structure, wherein each arm of the Y domain is composed of one heavy chain polypeptide and one light chain polypeptide. The base of the Y-shaped structure is the fragment

crystallizable or Fc region, which binds receptor proteins on the surface of cells activated by antibodies. Both heavy and light polypeptide chains contain regions which sequences are variable (VH or VL) and constant regions which sequences are highly conserved (C). The antigen binding fragment (Fab) is the region on an antibody that binds antigens. The Fab is composed of one constant and one variable domain from each of the heavy and light chain polypeptides of the antibody. The antigen binding site is formed by the variable domains of the heavy and light chain antibodies. It should be understood that the term“antibody” includes whole antibodies and antigen-binding antibody fragments.

[0112] A bi-specific antibody is an antibody that can simultaneously bind to two different types of antigens. A bi-specific antibody can be either immunoglobulin G (IgG)-like, or non-IgG-like. An IgG-like bi-specific antibody has two Fab arms that bind different antigens and one Fc region. A non-IgG-like bi-specific antibody lack an Fc region entirely and may be composed of two chemically linked Fab arms, or various types of bivalent and trivalent single chain variable fragments (ScFvs). [0113] An antigen-binding domain of a bi-specific antibody is one of the two Fab arms of the bi- specific antibody. To bind the antigen-binding domain of the bi-specific antibody, the extracellular target-binding moiety, in some embodiments, is an epitope that is bound by one of the antigen-binding domains of the bi-specific antibody. The other antigen-binding domain of the bi-specific antibody binds the target molecule (e g., a cancer antigen on the surface of a cancer cell).

[0114] In some embodiments, the extracellular target-binding moiety binds to the antigen binding domain of the bi-specific antibody via a bridging molecule. For engineered immune cells comprising two chimeric receptors, at least one (e.g., one or both) of the extracellular target-binding moiety binds to the antigen-binding domain of the bi-specific antibody via a bridging molecule.

[0115] A bridging molecule is a molecule that binds to both the extracellular target-binding moiety of a chimeric receptor and an antigen-binding domain of a bi-specific antibody simultaneously, thus linking the two. For example, the bridging molecule may contain a region (e.g., an epitope) that is bound by the extracellular target-binding moiety and another region (e.g., an epitope) that is bound by an antigen-binding domain of the bi-specific antibody. The bridging molecule may be an endogenous molecule or an exogenous molecule (e.g., an engineered molecule). Examples of bridging molecules include, but are not limited to, extracellular molecules, secreted molecules, or small molecule drugs.

[0116] In some embodiments, the extracellular target-binding moiety is an anti-idiotype antibody. An idiotype is the unique set of antigenic epitopes that the variable portion of an antibody recognizes. An anti-idiotype antibody is an antibody that specifically binds to the antigen binding site of another antibody (e.g., the antigen-binding domain of the bi-specific antibody).

[0117] In some embodiments, the extracellular target-binding moiety binds to an Fc domain of an antibody. An Fc domain is the tail region of an antibody. In contrast to the Fab region of an antibody that contains variable regions that define the specificity of the antibody, the Fc region of all antibodies in a class are the same of each species, i.e., they are constant. Molecules that bind to the Fc domain of an antibody are known. For example, Fc binds to various cell receptors, including CD 16, CD32, CD64, TRIM family proteins, and bacterial immunoglobulin binding proteins (e.g., streptococcal M protein, fibrinogen binding protein, etc.).

[0118] In some embodiments, the extracellular target-binding moiety is an Fc receptor, or variants thereof. An Fc receptor (FcR) is a protein on the surface of immune cells, such as B lymphocytes, dendritic cells, natural killer cells, macrophages, platelets, and mast cells. FcRs bind to antibodies that are attached to the surface of infected cells. The binding of the FcR to the Fc antibody region activates the cell with the FcR. Examples of FcRs include, without limitation, CD 16, CD32, CD64, and any Fc binding variants thereof. In some embodiments, the extracellular target-binding moiety is an Fc receptor variant that has altered binding affinity (e.g., decreased or increased, for example, by at least 20%, compared to a wild-type Fc receptor) to Fc.

[0119] In some embodiments, the extracellular target-binding moiety is a tripartite motif family (TRIM) protein. TRIM proteins are induced by interferons, which are important components of resistance to pathogens and several TRIM proteins are known to be required for the restriction of infection by lentiviruses. TRIM proteins are involved in pathogen-recognition and regulation of transcriptional pathways in host defense. In some embodiments, the extracellular target-binding moiety is TRIM21, which has been identified as a cytosolic Fc receptor with broad antibody isotype specificity (e.g., as described in Foss et al, Immunol Rev. 2015 Nov; 268(1): 328-339, incorporated herein by reference). TRIM21 variants that retain the Fc binding activity but with altered affinity (e.g., decreased or increased, for example, by at least 20%, compared to a wild- type TRIM21) may also be used.

[0120] In some embodiments, the extracellular target-binding moiety is a bacterial

immunoglobulin binding protein. Bacterial immunoglobulin binding proteins are known in the art, e.g., as described in Sidorin et al., Biochemistry (Moscow), March 2011, 76:295, incorporated herein by reference. A bacterial immunoglobulin-binding protein (IBP) is capable of binding immunoglobulins without the involvement of the antigen-binding sites. These proteins are widespread in bacteria, where they are located on the surface of bacterial cells. Bacterial (IBPs) help bacteria to evade the immune system by protecting against phagocytosis by white blood cells and activation of complement. In some embodiments, the bacterial immunoglobulin binding protein is selected from Protein A, Protein G, Protein Z, Protein L, Protein Z, and immunoglobulin-binding fragments thereof. [0121] Protein A was originally discovered in the cell wall of bacteria which binds to immunoglobulins. Protein A comprises five immunoglobulin domains, each of which is able to bind the heavy chain in the Fc region of immunoglobulins and also to the Fab region in some immunoglobulins. Protein A binds to human IgA, IgD, IgG, and IgM immunoglobulins.

[0122] Protein G is an immunoglobulin-binding protein expressed on the surface of

Streptococcal bacteria. Protein G binds to the Fab and Fc regions of immunoglobulins, as well as albumin protein. Protein G binds to human IgG and IgE immunoglobulins.

[0123] Protein Z is a human protein involved in the coagulation cascade. For unknown reasons, the immune system can develop antibodies against Protein Z (autoantibodies), which destroy the protein. Acquired autoantibodies to protein Z contribute to the pathophysiology of thrombosis, fetal loss, and/or cancer.

[0124] Protein L is a bacterial protein which binds the L chain of immunoglobulin. Protein L binding is restricted to antibodies which have kappa light chains and does not bind antibodies with lambda light chains.

[0125] In some embodiments, the extracellular target-binding moiety is an epitope that can bind the Fc domain of an antibody. Such epitopes are described in the art, e.g., in Choe et al, Materials (Basel). 2016 Dec; 9(12): 994, incorporated herein by reference.

[0126] In some embodiments, the extracellular target-binding moiety binds to an Fc domain of an antibody of a class or subclass of immunoglobulins. In some embodiments, the extracellular target-binding moiety binds to an Fc domain of an antibody of a class of immunoglobulins selected from immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE), immunoglobulin G (IgG), and immunoglobulin M (IgM). In some embodiments, the extracellular target-binding moiety binds to an Fc domain of an antibody of a subclass of immunoglobulins selected from IgAl and IgA2. In some embodiments, the extracellular target binding moiety binds to an Fc domain of an antibody of a subclass of immunoglobulins selected from IgGl, IgG2, IgG3, and IgG4.

[0127] In some embodiments, the extracellular target-binding moiety is a known protein that broadly binds classes or subclasses of immunoglobulins, e g., rheumatoid factor. Rheumatoid factor (RF) is a protein produced by the immune system from healthy tissue in the body. High levels of rheumatoid factor in the blood are most often associated with autoimmune diseases, such as rheumatoid arthritis and Sjogren's syndrome. RF is known as an antibody against the Fc portion of IgG (an antibody against an antibody) and different RFs can recognize different parts of the IgG-Fc (e.g., as described in Falkenburg et al., Arthritis & Rheumatology, 67(12), incorporated herein by reference) Although predominantly encountered as IgM, rheumatoid factor can be of any isotype of immunoglobulins, e.g., IgA, IgG, IgM, IgE, IgD. In some embodiments, extracellular target-binding moiety is a RF-derived single-chain variable fragment (ScFv). Antibodies that specifically bind a class or subclass of immunoglobulins are commercially available, e.g., from Thermo Fisher (catalog # A10648, A10650, A10648,

A10631, A10651, A10663, A10654).

[0128] In some embodiments, the extracellular target-binding moiety binds to a molecule that is conjugated to an antibody. Commonly used molecules for conjugation to antibodies include, without limitation, polyethylene glycol (PEG), biotin, or streptavidin. As such, in some embodiments, the extracellular target-binding moiety binds to PEG. For example, the extracellular target-binding moiety can be a PEG antibody as described in Garay et al., Expert Opin Drug Deliv. 2012 Nov;9(l 1): 1319-23, incorporated herein by reference. PEG antibodies are commercially available, e.g., from Abeam (catalog # ab5l257). In some embodiments, the extracellular target-binding moiety binds biotin. Molecules that bind to biotin include, without limitation, avidin, streptavidin, and NeutrAvidin®. In some embodiments, the extracellular target-binding moiety binds to streptavidin. Streptavidin-binding peptides have been identified, e.g., as described in Keefe et al., Protein Expression and Purification. 23 (3): 440-6 and Wilson et al., PNAS, 98 (7): 3750-5, incorporated herein by reference. One non-limiting, exemplary streptavidin binding peptide that may be used as the extracellular target-binding moiety of the present disclosure has the amino acid sequence of

MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID NO: 1) or a fragment thereof that binds to streptavidin.

[0129] In some embodiments, the extracellular target-binding moiety binds to a post- translational modification (PTM) of an antibody. In some embodiments, the extracellular target binding moiety does not bind to the antibody without the PTM. Post-translational modifications (PTMs) are covalent additions of functional groups or proteins, proteolytic cleavage of regulatory subunits, or degradation of entire proteins following protein synthesis. PTMs can be either reversible or irreversible. Non-limiting, exemplary PTMs include phosphorylation, glycosylation (e.g., without limitation, galactosylation, fucosylation, and sialylation), acetylation, amidation, hydroxylation, methylation, sulfation or other PTM described herein or known in the art.

[0130] Phosphorylation involves the addition of phosphate groups to proteins, most commonly at a serine (S), threonine (T) or tyrosine (Y) residue by a kinase and is essential for pathway activation in cellular regulation, cell signaling and growth.

[0131] Arginine or lysine amino acids within the protein sequence represent the targets at which methylation can occur by transferring one-carbon methyl groups to nitrogen or oxygen atoms. Varying degrees of reversible methylation are mediated by peptidylarginine or lysine

methyltransferases, in which up to two or three methyl groups can be added to arginine or lysine residues respectively.

[0132] Protein acetylation occurs when an acetyl group is transferred to a nitrogen atom and is a very common co-translational modification in eukaryotes (N-acetylation) where the N-terminal methionine of a growing polypeptide chain is replaced by an acetyl group from acetyl-CoA by N-acetyltransferase (NAT) enzymes. Protein acetylation also occurs as post-translational modification and it is very well understood in histone proteins where the amino group of the lysine side chain (e-NH2) is reversibly acetylated.

[0133] Glycosylation is the reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxyl or other functional group of another molecule (a glycosyl acceptor). In biology glycosylation mainly refers to the enzymatic process that attaches glycans to proteins, or other organic molecules, to an N or O in an amino acid side chain.

[0134] Hydroxylation involves conversion of a CH group into a COH group. Hydroxylation is an oxidative process. The oxygen that is inserted into the C-H bond is usually derived from atmospheric oxygen (02).

[0135] Sulfation is the enzyme-catalyzed conjugation of a sulfo group (not a sulfate or sulfuryl group) to another molecule. Non-limiting examples of biological sulfation is in the synthesis of sulfonated glycosaminoglycans, such as heparin, heparan sulfate, chondroitin sulfate, and dermatan sulfate.

[0136] Amidation refers to the post-translational modification of the C-terminus of a protein or peptide by forming an amide bond.

[0137] In some embodiments, the extracellular target-binding moiety is an antibody or a fragment thereof that recognizes an amino acid in the antibody that is post-translationally modified. Such antibodies include, without limitation, phospho-specific antibodies, methyl- specific antibodies, acetyl-specific antibodies, and glycosylation-specific antibodies. Antibodies for detecting post translational modification are available commercially.

[0138] In some embodiment, the extracellular target-binding moiety is an anti-hinge antibody, e.g., as described in Rispens et al., J Immunol Methods 2012 Jan 31 ;375(1 -2): 93-9 and Brezski et ah, J Immunol 2008; l8l(5): 3183-3192, incorporated herein by reference. Anti-hinge antibodies (AHAs) are antibodies that, following proteolytic cleavage, recognize cryptic epitopes exposed in the hinge regions of immunoglobulins (Igs) and do not bind to the intact Ig counterpart.

[0139] In some embodiments, the extracellular target-binding moiety is a molecule that binds antibody junctions. For example, in a chimeric antibody made by fusing the antigen binding region from one species (e.g., mouse) with the Fc domain from another species (e.g., human), the junction that connects the two parts of antibodies from different species (e.g., mouse-human junction) can be specifically targeted by antibodies. These antibodies that target the junctions can be used as the extracellular target-binding moiety described herein.

[0140] In some embodiments, the extracellular target-binding moiety binds to a non-human sequence of a humanized antibody. Humanized antibodies are produced in non-human species whose protein sequences have been modified to increase their similarity to antibodies produced in humans. Humanization is often performed to reduce the immunogenicity of antibodies which will be introduced into humans. The residual non-human sequences can be targeted by antibodies, which can be used as the extracellular target-binding moiety described herein.

[0141] The extracellular target-binding moiety described herein can take various forms. For example, the extracellular target-binding moiety can be an antibody, a single-chain variable fragment (scFv), an antigen binding fragment (Fab), a single domain antibody (e.g., a VH or VHH, including modified variants thereof, such as camelized VHs and humanized VHHS), a diabody, or a synthetic epitope having the broad antibody binding activities described herein. In some embodiments, the extracellular target-binding moiety does not bind to a native antibody.

[0142] An antigen binding fragment (Fab) is the region on an antibody that binds antigens. The Fab is composed of one constant and one variable domain from each of the heavy and light chain polypeptides of the antibody. The antigen binding site is formed by the variable domains of the heavy and light chain antibodies.

[0143] A single-chain variable fragment (scFv) is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins, connected with a short peptide linker comprising 10-25 amino acids. The linker peptide is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and connects the N-terminus of the VH chain with the C-terminus of the VL chain, or vice versa. The scFv retains the specificity of the original immunoglobulin, despite the addition of the linker and removal of the constant regions.

[0144] A single domain antibody is an antibody fragment consisting of a monomeric VH or VL domain which retains selective binding to a specific antigen. Single domain antibodies are small (-12-15 kilodaltons), readily cross the blood-brain barrier, have improved solubility, and are thermostable relative to full-length antibodies.

[0145] A diabody is a dimeric antibody fragment designed to form two antigen binding sites. Diabodies are composed of two single-chain variable fragments (scFvs) in the same polypeptide connected by a linker peptide which is too short (-3-6 amino acids) to allow pairing between the two domains on the same chain, forcing the domains to pair with complementary domains of another chain to form two antigen binding sites. Alternately, the two scFvs can also be connected with longer linkers, such as leucine zippers.

[0146] A VHH fragment (e.g., NANOBODY^®) is a recombinant, antigen-specific, single domain, variable fragment derived from camelid heavy chain antibodies. Although they are small, VHH fragments retain the full antigen-binding capacity of the full antibody.

[0147] The extracellular target-binding moiety of a chimeric receptor of the present disclosure can be any domain that binds to a target (e.g., Fc portion of an immunoglobulin) including but not limited to monoclonal antibodies, polyclonal antibodies, synthetic antibodies, human antibodies, humanized antibodies, and fragments thereof. In some instances, it is beneficial for the extracellular target-binding moiety to be derived from the same species in which the chimeric receptor will ultimately be used in. For example, for use in humans, it may be beneficial for the extracellular target-binding moiety of the chimeric receptor to comprise a human antibody or fragment thereof. Thus, in one embodiment, the extracellular target-binding moiety portion comprises a human antibody or a fragment thereof.

[0148] For in vivo use of antibodies in humans, it may be desirable to use human antibodies. Completely human antibodies may be desirable for therapeutic treatment of human subjects. Human antibodies can be made by a variety of methods known in the art including phage display methods using antibody libraries derived from human immunoglobulin sequences, including improvements to these techniques. See, also, U.S. Pat. Nos. 4,444,887 and 4,716, 111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, W098/16654, WO 96/34096, WO 96/33735, and WO 91/10741 ; each of which is incorporated herein by reference in its entirety. A human antibody can also be an antibody wherein the heavy and light chains are encoded by a nucleotide sequence derived from one or more sources of human DNA.

[0149] Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human

immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. 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. The modified embryonic stem cells are expanded and microinj ected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT Publication Nos. WO 98/24893, WO 96/34096, and WO 96/33735; and U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, each of which is incorporated by reference herein. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above. For a specific discussion of transfer of a human germ-line immunoglobulin gene array in germ-line mutant mice that will result in the production of human antibodies upon antigen challenge see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA,

90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993); and Duchosal et al., Nature, 355:258 (1992).

[0150] Human antibodies can also be derived from phage-display libraries (Hoogenboom et al.,

J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Vaughan et al., Nature Biotech., 14:309 (1996)). Phage display technology (McCafferty et al., Nature, 348:552- 553 (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; for their review see, 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 unimmunized 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., 222:581-597 (1991), or Griffith et al., EMBO I, 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905, each of which is incorporated herein by reference.

[0151] Human antibodies may also be generated by in vitro activated B cells (see, U.S. Pat. Nos. 5,567,610 and 5,229,275, each of which is incorporated herein by reference). Human antibodies may also be generated in vitro using hybridoma techniques such as, but not limited to, that described by Roder et al. (Methods Enzymol., 121 : 140-167 (1986)).

[0152] Alternatively, in some embodiments, a non-human antibody is humanized, where specific sequences or regions of the antibody are modified to increase similarity to an antibody naturally produced in a human. In one embodiment, the antigen-binding domain portion is humanized.

[0153] A humanized antibody can be produced using a variety of techniques known in the art, including but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400;

International Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated herein by reference), veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, PNAS, 91 :969-973, each of which is incorporated herein by reference), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein by reference), and techniques disclosed in, e.g., U.S. Patent Application Publication No US2005/0042664, U S Patent Application Publication No. US2005/0048617, U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO 9317105, Tan et al., J. Immunol., 169: 1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16): 10678-84 (1997), Roguska et al., Protein Eng., 9(l0):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8): 1717-22 (1995), Sandhu J S, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol.

Biol., 235(3):959-73 (1994), each of which is incorporated herein by reference. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen-binding. These framework substitutions are identified by methods well-known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen-binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference).

[0154] A humanized antibody has one or more amino acid residues introduced into it from a source which is nonhuman. These nonhuman amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Thus, humanized antibodies comprise one or more CDRs from nonhuman immunoglobulin molecules and framework regions from human. Humanization of antibodies is well-known in the art and can essentially be 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 or CDR sequences for the corresponding sequences of a human antibody, i.e., CDR-grafting (EP 239,400; PCT Publication No. WO 91/09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference). In such humanized chimeric antibodies, substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some framework (FR) residues are substituted by residues from analogous sites in rodent antibodies. Humanization of antibodies can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498; Studnicka et al., Protein Engineering, 7(6):805- 814 (1994); and Roguska et al., PNAS, 91 :969-973 (1994)) or chain shuffling (U.S. Pat. No. 5,565,332), the contents of which are incorporated herein by reference.

[0155] In some instances, a human scFv may also be derived from a yeast display library.

[0156] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is to reduce antigenicity 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 sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151 :2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference). Another method uses a particular framework 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), the contents of which are incorporated herein by reference).

[0157] Antibodies can be humanized with retention of high affinity for the target antigen and other favorable biological properties. According to one aspect of the present disclosure, 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 the target 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, is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen-binding.

[0158] A“humanized” antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody for its target may be increased using methods of“directed evolution,” as described by Wu et al., J. Mol. Biol., 294: 151 (1999), the contents of which are incorporated herein by reference.

[0159] In some embodiments, the antibodies bound by the extracellular target-binding moiety described herein recognizes and binds to a cancer-associated antigen or an autoimmune- associated antigen. A cancer-associated antigen is an antigenic substance produced by a cancer cell and triggers an immune response in the host. In some embodiments, the cancer-associated antigen is a protein that specifically expresses or is upregulated in a cancer cell, as compared to a non-cancerous cell. Exemplary cancer-associated antigens include, without limitation: MAGE family members, NY-ESO-l, tyrosinase, Melan-A/MART-l, prostate cancer antigen, Her-2/neu, Survivin, Telomerase, WT1, CEA, gplOO, Pmell7, mammaglobin-A, NY-BR-l, ERBB2, OA1, PAP, RAB38/NY-MEL-1, TRP-l/gp75, TRP-2, CD33, BAGE-l, D393-CD20n, cyclin-Al, GAGE-l, GAGE-2, GAGE-8, GnTVf, HERV-K-MEL, KK-LC-l, KM-HN-l, LAGE-l, LY6K, MAGE-A1, MAGE-A2, MAGE- A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE- A 10, MAGE- A12, MAGE-C1, MAGE-C2, mucink, NA88-A, SAGE, spl7, SSX-2, SSX-4, surviving, TAG-l, TAG-3, TRAG-3, XAGE-lb, BCR-AB1, adipophiln, AIM-2, ALDH1A1, BCLX(L), BING-4, CALCA, CD45, CD274, CPSF, cyclin Dl, DKK1, ENAH, EpCAM, EphA3, EZH2, FGF5, glypican-3, G250, HER-2, HLA-DOB, hepsin, IDOl, IGF2B3, IL12Ralpha2, intestinal carboyxyl esterase, alpha-foetoprotein, kallikrein 4, KIF20A, Lengsin, M-CSF, M-CSP, mdm-2, Meloe, midkine, MMP-2, MMP-7, MUC1, MUC5AC, p53, PAX5, PBF, PRAME, PSMA, RAGE-l, RGS5, RhoC, RNF43, RU2AS, secerinel, SOXIO, STEAP1, telomerase, TPBG, mesothelin, Axl, VEGF or other cancer antigens described herein. An autoimmune-associated antigen refers to an antigen that is derived from one’s own body (a self-antigen).

[0160] Examples of antibodies that may be bound by the extracellular target-binding moiety of the chimeric receptor described herein include, without limitation: bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), alemtuzumab (CAMPATH®, indicated for B cell chronic lymphocytic leukemia,), gemtuzumab (MYLOTARG®, hP67.6, anti-CD33, indicated for leukemia such as acute myeloid leukemia), rituximab (RITUXAN®), tositumomab (BEXXAR®, anti-CD20, indicated for B cell malignancy), MDX-210 (bi-specific antibody that binds simultaneously to HER-2/neu oncogene protein product and type I Fc receptors for

immunoglobulin G (IgG) (Fc gamma RI)), oregovomab (OVAREX®, indicated for ovarian cancer), edrecolomab (PANOREX®), daclizumab (ZENAPAX®), palivizumab (SYNAGIS®, indicated for respiratory conditions such as RSY infection), ibritumomab tiuxetan (ZEVALIN®, indicated for Non-Hodgkin’s lymphoma), cetuximab (ERBITUX®), MDX-447, MDX-22, MDX-220 (anti-TAG-72), IOR-C5, IOR-T6 (anti-CDl), IOR EGF/R3, celogovab

(ONCOSCINT® OV103), epratuzumab (LYMPHOCIDE®), pemtumomab (THERAGYN ® ) and Gliomab-H (indicated for brain cancer, melanoma). In some embodiments, the antibodies that may be bound by the extracellular target-binding moiety of the chimeric receptor described herein is selected from: Revlimid® (lenalidomide), Opdivo® (nivolumab), Imbruvica®

(ibrutinib), Keytruda® (pembrolizumab), Ibrance® (palbociclib), Tecentriq® (atezolizumab), Darzalex® (daratumumab), Perjeta® (pertuzumab), Xtandi® (enzalutamide), Avastin®

(bevacizumab), Herceptin® (trastuzumab), Gazyva® (obinutuzumab), Jakafi® (ruxolitinib), Venclexta® (venetoclax), Rituxan® (rituximab), or any of the bi-specific antibodies described herein.

[0161] Chimeric Receptor Constructs: Transmembrane Domains.

[0162] The present disclosure is not limited by the type of transmembrane domain used in a chimeric receptor. In some embodiments, when an engineered immune cell of the present disclosure is designed to contain two or more different, chimeric receptors, the same or different transmembrane domain may be used in each chimeric receptor. The transmembrane domain of a chimeric receptor may be a transmembrane domain derived from a natural source or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein.

[0163] The transmembrane domain of a chimeric receptor may be a transmembrane domain that naturally is associated with one of the other domains in the chimeric receptor (e.g., naturally associated with an intracellular signaling domain). In some embodiments, the transmembrane domain is selected or modified by amino acid substitution (e.g., to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, for example, in order to minimize interactions with other members of the receptor complex).

[0164] Transmembrane regions that find particular use in the present disclosure include, but are not limited to, a transmembrane region derived from (e.g., comprises all or a portion of the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD 8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, a transmembrane domain may include the transmembrane region(s) of, for example, KIRDS2, 0X40, CD2, CD27, LFA-l (CDl la, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R .alpha., ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld,

(SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, NKG2C. In some embodiments, a variety of human hinges are utilized including, but not limited to, the human Ig

(immunoglobulin) hinge.

[0165] In some embodiments, the transmembrane domain is synthetic, in which case it comprises predominantly hydrophobic residues such as leucine and valine. In some

embodiments, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length forms a linkage between the transmembrane domain and the cytoplasmic signaling domain.

[0166] Chimeric Receptor Constructs: Intracellular Signaling Domains .

[0167] The present disclosure is not limited by the type of intracellular domain used in a chimeric receptor. In some embodiments, when an engineered immune cell of the present disclosure is designed to contain two or more different, chimeric receptors, the same or different intracellular domain is used in each chimeric receptor. In some embodiments, a chimeric receptor contains a signaling domain and a co-signaling domain.

[0168] The intracellular signaling domain of a chimeric receptor of the present disclosure is responsible for activation of at least one of the effector functions of the immune cell (e.g., T cell) in which the chimeric receptor has been placed. The term“effector function” refers to a specialized function of a differentiated cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Effector function in a naive, memory, or memory -type T cell includes antigen-dependent proliferation. Accordingly, in some embodiments, the term“intracellular signaling domain” refers to the portion of a protein that transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain is employed, in many cases it will not be necessary to use the entire intracellular polypeptide. To the extent that a truncated portion of the intracellular signaling domain may find use, such truncated portion may be used in place of the intact chain as long as it still transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal. Examples of intracellular signaling domains include, but are not limited to, cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[0169] Cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.

[0170] Examples of IT AM containing cytoplasmic signaling sequences that are of particular use in the present disclosure include those derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signaling molecule in the CAR of the present disclosure comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0171] In some embodiments, the cytoplasmic domain of a chimeric receptor is designed to comprise the CD3-zeta signaling domain by itself. In other embodiments, the cytoplasmic domain of a chimeric receptor is designed to comprise the CD3-zeta signaling domain and a second desired cytoplasmic domain(s). For example, in some embodiments, the cytoplasmic domain of a chimeric receptor is designed to comprise a CD3-zeta chain portion and a costimulatory signaling region or domain. The costimulatory signaling region refers to a portion of the chimeric receptor comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include, but are not limited to, CD27, CD28, 4- IBB (CD 137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, and the like.

[0172] Examples of intracellular signaling domains that find use in a chimeric receptor of the present disclosure include, but are not limited to, CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-l, ICOS, lymphocyte function-associated antigen-l (LFA-l), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-l, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDl ld, ITGAE, CD103, IT GAL, CDl la, LFA-l, ITGAM, CDl lb, ITGAX, CDl lc, ITGB 1, CD29, ITGB2, CD 18, LFA-l, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, and/or NKG2D. In some embodiments, the intracellular signaling domain is CD28, OX40/CD134, 4-1BB/CD137/TNFRSF9, the high affinity immunoglobulin E receptor-gamma subunit (FcsRIy), ICOS/CD278, interleukin 2 subunit beta (ILR ) or CD122, cytokine receptor common subunit gamma (IL-2RY) or CD132, or CD40.

[0173] In some embodiments, CD3-zeta is used as the intracellular signaling domain in a first engineered chimeric receptor and a second, different, intracellular signaling domain is used in a second engineered chimeric receptor of an engineered immune cell described herein. The present disclosure is not limited by the type of second, different intracellular signaling domain used. In some embodiment, the second, different intracellular signaling domain used is CD28, 0X40 (CD134), 4-1BB (CD137), ICOS, or other intracellular signaling domain described herein or known in the art. In some embodiments, CD3-zeta and one of CD28, 0X40 (CD134), 4-1BB (CD 137), or ICOS are used in a single engineered chimeric receptor present in an engineered immune cell. For example, in one embodiment, the cytoplasmic domain of a chimeric receptor is designed to contain a CD3ITAM-containing CD3-zeta domain in combination with CD28 or 4- 1BB as the co-stimulatory signaling element. The cytoplasmic signaling sequences within a signaling portion of a single cytoplasmic domain of a chimeric receptor of the present disclosure may be linked to each other in any order. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form a linkage. For example, a glycine- serine doublet may be used as a linker.

[0174] An example of a CD3-zeta signaling domain useful in the present disclosure is the protein sequence provided as GenBan Acc. No. BAG36664.1, or the equivalent residues from a non human species, e.g., mouse, rodent, monkey, ape and the like. In one aspect the cytoplasmic domain of CD3-zeta comprises residues 52 through 164 of GenBank Acc. No. BAG36664.1 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like, that are functional orthologs thereof. An example of a 4-1BB/CD137 domain useful in the present disclosure is the protein sequence provided as GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.

[0175] Source and Type of Engineered Immune Cells.

[0176] A variety of different types and/or sources of immune cells may be engineered to contain a chimeric receptor of the present disclosure. The engineered immune cell of the present disclosure can be an engineered mammalian cell (e.g., human cell). An immune cell is a cell that plays a role in the immune system. Exemplary immune cells include, without limitation, granulocytes, mast cells, monocytes, neutrophils, dendritic cells, natural killer cells, B cells, and T cells. In some aspects, the immune cells are T cells. In some embodiments, the T cells are CD3+ T cells (e.g., CD4+ and/or CD8+ T cells). In certain embodiments, the T cells are CD8+

T cells. In other embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are natural killer (NK) T cells. In some embodiments, the T cells are alpha beta T cells. In some embodiments, the T cells are gamma delta T cells. In some embodiments, the T cells are a combination of CD4+ and CD8 T+ cells. In certain embodiments, the T cells are memory T cells. In some embodiments, the memory T cells are central memory T cells. In some embodiments, the memory T cells are effector memory T cells. In some embodiments, the T cells are tumor infdtrating lymphocytes. In certain embodiments, the T cells are a combination of CD8+ T cells, CD4+ T cells, Natural Killer T cells, Natural Killer cells, memory T cells, and/or gamma delta T cells. In some embodiments, the T cells are cytokine-induced killer cells.

[0177] A CD4+ T cell (helper T cell) instigates the adaptive immune responses by recognizing antigen peptides presented on major histocompatibility complex (MHC) Class-II molecules found on antigen presenting cells (APCs).

[0178] A CD8+ T cell (cytotoxic T cell) is a T lymphocyte that kills damaged cells, such as cancer cells or infected cells. Damaged cells present MHC Class-I molecules on their cell surface, which are recognized by CD8 T cells, which are then activated to kill the damaged cell.

[0179] Regulatory T cells (Treg) are CD4+ T cells which suppress potentially deleterious activities of helper T cells. Among these suppressed activities are: maintaining self-tolerance, suppression of allergy or asthma, and/or suppression of T cell activation triggered by weak stimuli. Regulatory T cells are essential in the activation and growth of cytotoxic T cells.

[0180] Natural killer (NK) cells have features of both innate and adaptive immunity. They are important for recognizing and killing virus-infected cells or tumor cells. They contain intracellular compartments called granules, which are filled with proteins that can form holes in the target cell and also cause apoptosis, the process for programmed cell death. Apoptosis, unlike necrosis, does not release danger signals that can lead to greater immune activation and inflammation. Through apoptosis, immune cells can discreetly remove infected cells and limit bystander damage. Recently, researchers have shown in mouse models that NK cells, like adaptive cells, can be retained as memory cells and respond to subsequent infections by the same pathogen.

[0181] Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties of both T cells and natural killer cells. Many of these cells recognize the non-polymorphic CDld molecule, an antigen-presenting molecule that binds self and foreign lipids and glycolipids.

[0182] In some embodiments, the T cell is an anti -tumor T cell (e.g., a T cell with activity against a tumor (e.g., an autologous tumor) that becomes activated and expands in response to antigen). Anti-tumor T cells (e.g., useful for adoptive T cell transfer) include, in one embodiment, peripheral blood derived T cells genetically modified with receptors that recognize and respond to tumor antigens. Such receptors are generally composed of extracellular domains comprising a single-chain antibody (scFv) specific for a tumor antigen, linked to intracellular T cell signaling motifs (See, e.g., Westwood, J. A. et al, 2005, Proc. Natl. Acad. Sci., USA, 102(52): l905l-l9056). Other anti-tumor T cells include T cells obtained from resected tumors or tumor biopsies (e.g., tumor infiltrating lymphocytes (TILs). In another embodiment, the T cell is a polyclonal or monoclonal tumor-reactive T cell (e.g., obtained by apheresis, expanded ex vivo against tumor antigens presented by autologous or artificial antigen-presenting cells). In another embodiment, the T cell is engineered to express a T cell receptor of human or murine origin that recognizes a desired target (e.g., an Fc region of an antibody or a tumor/cancer antigen). The present disclosure is not limited by the type of tumor antigen so recognized. Indeed, any T cell containing a receptor that recognizes a tumor antigen finds use in the compositions and methods of the present disclosure. Examples include, but are not limited to, T cells expressing a receptor (e.g., a native or naturally occurring receptor, or a receptor engineered to express a synthetic receptor such as an engineered T cell receptor or a CAR) that recognize an antigen selected from CD 19, CD20, CD22, receptor tyrosine kinase-like orphan receptor 1 (ROR1), disialoganglioside 2 (GD2), Epstein-Barr Virus (EBV) protein or antigen, folate receptor, mesothelin, human carcinoembryonic antigen (CEA), CD33/IL3Ra, tyrosine protein kinase Met (c-Met) or hepatocyte growth factor receptor (HGFR), prostate-specific membrane antigen (PSMA), Glycolipid F77, epidermal growth factor receptor variant III (EGFRvIII), NY- ESO-l, melanoma antigen gene (MAGE) Family Member A3 (MAGE-A3), melanoma antigen recognized by T cells 1 (MART-l), GP1000, p53, or other tumor antigen described herein.

[0183] Engineered immune cells of the present disclosure may serve as a type of vaccine for ex vivo immunization and/or in vivo therapy. With respect to ex vivo immunization, at least one of the following occurs in vitro prior to administering the cell to the subject: i) expansion of the cells, ii) introducing a nucleic acid encoding a chimeric receptor to the cells, and/or iii) cryopreservation of the cells.

[0184] Ex vivo procedures are well known in the art. Briefly, cells are isolated from a mammal (preferably a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a chimeric receptor disclosed herein. The genetically-modified cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the engineered immune cell can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.

[0185] A procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described in El.S. Pat. No. 5,199,942, incorporated herein by reference and can be applied to the cells of the present disclosure. The present disclosure is not limited to any particular method of ex vivo expansion of the cells. Briefly, ex vivo culture and expansion of T cells comprises: (1) collecting CD34+hematopoietic stem and progenitor cells from a mammal from peripheral blood harvest or bone marrow explants; and (2) expanding such cells ex vivo. In addition to the cellular growth factors described in El.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-l, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells. [0186] In addition to using a cell-based vaccine in terms of ex vivo immunization, the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response in a patient.

[0187] For example, in some aspects, the present disclosure provides a method of generating a persisting population of engineered immune cells (e.g., T cells) in a subject (e.g., a subject diagnosed with cancer, a subject diagnosed with autoimmune disease, a subject with an infectious disease, or other type of subject), the method comprising administering to the subject an effective amount of an engineered immune cell comprising an isolated nucleic acid sequence encoding a chimeric receptor of the present disclosure. In some embodiments, the nucleic acid sequence encoding a chimeric receptor of the present disclosure comprises nucleic acid sequence (e.g., human, mouse, or humanized mouse nucleic acid sequence) of an extracellular target binding moiety (e.g., that binds an Fc of an antibody (e.g., of a class or subclass of

immunoglobulins) a PTM on an antibody, an antibody without a PTM, a molecule (e.g., PEF, biotin, streptavidin) bound to an antibody, a hinge region, or other non-specific or specific target described herein), the nucleic acid sequence of a transmembrane domain (e.g., transmembrane domain described herein), and the nucleic acid sequence of one or more intracellular signaling domains (e.g., the nucleic acid sequence of a CD3 zeta signaling domain and/or other signaling domain described herein), wherein the persisting population of genetically engineered T cells persists in the subject (e.g., for weeks, a month, two months, three months, or longer) after administration.

[0188] The present disclosure is not limited by the means of expressing an isolated nucleic acid sequence encoding a chimeric receptor of the present disclosure. In some embodiments, an isolated nucleic acid sequence is expressed constitutively. In other embodiments, an isolated nucleic acid sequence is expressed in a regulated fashion (e.g., using a system to regulate expression via a small molecule or using an endogenously regulated system). An isolated nucleic acid sequence, in another embodiment, is genetically integrated into the cellular DNA using a retroviral, lentiviral or other viral vector or via CRISPR/Cas9 based system. In yet another embodiment, an isolated nucleic acid sequence is expressed via RNA or an oncolytic virus or other transient expression system known in the art. Isolated nucleic acid sequences can be delivered ex vivo into T cells for adoptive transfer, or delivered via in vivo genetic transfer. [0189] Chimeric Receptor Constructs. Functionality and Logic.

[0190] In some embodiments, the engineered immune cell described herein comprises one chimeric receptor and the extracellular target-binding moiety of the chimeric receptor is any of the extracellular target-binding moieties described herein. In some embodiments, the engineered immune cell described herein comprises two chimeric receptors and at least one (e.g., one or both) of the extracellular target-binding moiety is any of the extracellular target-binding moieties described herein. For example, at least one (e.g., one or both) of the extracellular target-binding moiety may be a molecule that binds to an antigen-binding domain of a bi-specific antibody, may bind to an Fc domain of an antibody or an Fc domain of an antibody of a class or subclass of immunoglobulins, may bind to a molecule that is conjugated to an antibody (e.g., PEG, biotin, or streptavidin), may bind to a PTM on an antibody, may be an anti-hinge antibody, and may bind to a non-human sequence in a humanized antibody.

[0191] In some embodiments, the two extracellular target-binding moieties have different target binding specificities and/or target-binding avidities relative to each other. For example, the two extracellular target-binding moieties may have different target-binding specificities relative to each other, i.e., bind to different molecules or two different epitopes on the same molecule.

[0192] In some embodiments, one extracellular target-binding moiety binds an antigen-binding domain of a bi-specific antibody and the other extracellular target-binding moiety binds an antigen-binding domain of a different bi-specific antibody, an Fc of an antibody, an Fc of an antibody of a class or subclass of immunoglobulins, a PTM on an antibody, a molecule conjugated to an antibody (e.g., PEG, biotin, or streptavidin), a hinge region of a chimeric antibody, or a non-human sequence of a humanized antibody.

[0193] In some embodiments, one extracellular target-binding moiety binds an Fc of an antibody, and the other extracellular target-binding moiety binds an antigen-binding domain of a bi-specific antibody, a different epitope on the Fc of the antibody, an Fc of a different antibody, an Fc of an antibody of a class or subclass of immunoglobulins, a PTM on an antibody, a molecule conjugated to an antibody (e.g., PEG, biotin, or streptavidin), a hinge region of a chimeric antibody, or a non-human sequence of a humanized antibody. [0194] In some embodiments, one extracellular target-binding moiety binds an Fc of an antibody of a class or subclass of immunoglobulins, and the other extracellular target-binding moiety binds an antigen-binding domain of a bi-specific antibody, an Fc of an antibody, an Fc of an antibody of a different class or different subclass of immunoglobulins, a PTM on an antibody, a molecule conjugated to an antibody (e g., PEG, biotin, or streptavidin), a hinge region of a chimeric antibody, or a non-human sequence of a humanized antibody.

[0195] In some embodiments, one extracellular target-binding moiety binds an Fc of an antibody of a class or subclass of immunoglobulins, and the other extracellular target-binding moiety binds a PTM on an antibody, an Fc of an antibody, an Fc of an antibody of a different class or different subclass of immunoglobulins, a different PTM on an antibody, an antibody without a PTM, a molecule conjugated to an antibody (e.g., PEG, biotin, or streptavidin), a hinge region of a chimeric antibody, or a non-human sequence of a humanized antibody.

[0196] In some embodiments, one extracellular target-binding moiety binds an Fc of an antibody of a class or subclass of immunoglobulins, and the other extracellular target-binding moiety binds a molecule conjugated to an antibody (e.g., PEG, biotin, or streptavidin), an Fc of an antibody, an Fc of an antibody of a different class or different subclass of immunoglobulins, a PTM on an antibody, a different molecule conjugated to an antibody (e.g., PEG, biotin, or streptavidin), a hinge region of a chimeric antibody, or a non-human sequence of a humanized antibody.

[0197] In some embodiments, one extracellular target-binding moiety binds an Fc of an antibody of a class or subclass of immunoglobulins, and the other extracellular target-binding moiety binds a hinge region of a chimeric antibody, an Fc of an antibody, an Fc of an antibody of a different class or different subclass of immunoglobulins, a PTM on an antibody, a different molecule conjugated to an antibody (e.g., PEG, biotin, or streptavidin), a hinge region of a different chimeric antibody, or a non-human sequence of a humanized antibody.

[0198] In some embodiments, one extracellular target-binding moiety binds an Fc of an antibody of a class or subclass of immunoglobulins, and the other extracellular target-binding moiety binds a non-human sequence of a humanized antibody, an Fc of an antibody, an Fc of an antibody of a different class or different subclass of immunoglobulins, a PTM on an antibody, a different molecule conjugated to an antibody (e.g., PEG, biotin, or streptavidin), a hinge region of a chimeric antibody, a different non-human sequence of a humanized antibody, or a non human sequence of a different humanized antibody.

[0199] In some embodiments, the two extracellular target-binding moieties may bind to the same target but have different target-binding avidities relative to each other. For example, the difference of the target-binding avidities between the two extracellular target-binding moieties may be at least 20%, at least 50%, or at least 90%. In some embodiments, the difference of the target-binding avidities between the two extracellular target-binding moieties is 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more.

[0200] When the two extracellular target-binding moieties have different target-binding specificity and/or affinities, the immune cell can be engineered to comprise two chimeric receptors such that the engineered immune cell is activated (e.g., induces a desired immune response) only when each of the extracellular target-binding moieties is bound to its respective antibody target and the antibodies are bound to their respective targets (e.g., different cancer antigens on the surface of a cancer cell).

[0201] Engineered immune cells that include two different chimeric receptors may be used, in some embodiments, to implement Boolean logic functions, such as AND functions and OR functions. For example, two chimeric receptors of an engineered immune cell may be designed to bind to different antibodies such that activation (e.g., full activation of a desired immune response) of the immune cell occurs only when both chimeric receptors are bound to their respective targets This may be achieved, for example, by including one intracellular signaling domain (e.g., CD3-zeta) on one receptor and another intracellular co-signaling domain (e.g., CD28 or 4-1BB) on the other receptor. Likewise, two chimeric receptors of an engineered immune cell may be designed to bind to different antibodies such that full activation of the engineered immune cell occurs when either (or both) of the chimeric receptors are bound to their respective targets. This may be achieved, for example, by including an intracellular signaling domain (e.g., CD3-zeta) and an intracellular co-signaling domain (e.g., CD28 or 4-1BB) on each of the two receptors.

[0202] Therapeutic Applications. [0203] Some aspects of the present disclosure provide methods of treating a disease or condition comprising administering (e.g., intravenous administration, e.g., transfusion) to a subject (e.g., human subject) in need of such treatment a therapeutically effective amount of engineered immune cells of the present disclosure. In some embodiments, the disease is cancer. In other embodiments, the disease is an autoimmune disease. In still further embodiments, the disease is an infectious disease. In some embodiments, the condition is sepsis.

[0204] The present disclosure is not limited by the type of cancer treated. The cancer may be a hematological cancer or malignancy, a solid tumor cancer or malignancy, or other type of cancer or malignancy. Hematologic cancers include but are not limited to leukemia (such as acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphoid leukemia, chronic lymphoid leukemia and myelodysplastic syndrome) and malignant lymphoproliferative conditions including lymphoma (such as multiple myeloma, Hodgkin's lymphoma, non- Hodgkin's lymphoma, Burkitf s lymphoma, and small cell- and large cell-follicular lymphoma). Hematological cancer conditions are the types of cancer such as leukemia and malignant lymphoproliferative conditions that affect blood, bone marrow and the lymphatic system.

Examples of hematologic cancers include but are not limited to myeloid leukemias, acute myelogenous leukemia (AML) and its subtypes, chronic myeloid leukemia (CML),

myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), histiocytic disorders, and mast cell disorders.

[0205] Leukemia can be classified as acute leukemia and chronic leukemia. Acute leukemia can be further classified as acute myelogenous leukemia (AML) and acute lymphoid leukemia (ALL). Chronic leukemia includes chronic myelogenous leukemia (CML) and chronic lymphoid leukemia (CLL). Other related conditions include myelodysplastic syndromes (MDS, formerly known as "preleukemia") which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells and risk of transformation to AML.

[0206] Lymphoma is a group of blood cell tumors that develop from lymphocytes. Exemplary lymphomas include non-Hodgkin lymphoma and Hodgkin lymphoma. [0207] AML has a number of subtypes that are distinguished from each other by morphology, immunophenotype, and cytochemistry. Five classes are described, based on predominant cell type, including myeloid, myeloid-monocytic, monocytic, erythroid, and megakaryocytic.

[0208] Chronic myelogenous (or myeloid) leukemia (CML) is also known as chronic granulocytic leukemia, and is characterized as a cancer of the white blood cells.

[0209] Myelodysplastic syndromes (MDS) are hematological medical conditions characterized by disorderly and ineffective hematopoiesis, or blood production. Thus, the number and quality of blood-forming cells decline irreversibly. Some patients with MDS can develop severe anemia, while others are asymptomatic. The classification scheme for MDS is known in the art, with criteria designating the ratio or frequency of particular blood cell types, e.g., myeloblasts, monocytes, and red cell precursors. MDS includes refractory anemia, refractory anemia with ring sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, chronic myelomonocytic leukemia (CML).

[0210] The present disclosure is not limited by the type of cancer treated. Examples of cancers that can be treated with compositions and methods of the present disclosure include, but are not limited to, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, Prolymphocytic leukemia, aids-related cancers, Kaposi sarcoma (soft tissue sarcoma), AIDS-related lymphoma (lymphoma), primary CNS lymphoma (lymphoma), anal cancer, gastrointestinal carcinoid tumors, astrocytomas (brain cancer), atypical teratoid/rhabdoid tumor, basal cell carcinoma of the skin, bile duct cancer, bladder cancer, bone cancer (includes Ewing sarcoma and osteosarcoma and malignant fibrous histiocytoma), brain tumors, breast cancer, bronchial tumors, Burkitt lymphoma, carcinoid tumor (gastrointestinal), carcinoma of unknown primary, cardiac (heart) tumors, atypical teratoid/rhabdoid tumor, embryonal tumors, germ cell tumor, cervical cancer, cholangiocarcinoma, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative neoplasms, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer (uterine cancer), ependymoma, esophageal cancer, esthesioneuroblastoma, extracranial germ cell tumor, extragonadal germ cell tumor, eye cancer, intraocular melanoma, retinoblastoma, fallopian tube cancer, fibrous histiocytoma of bone, malignant, and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumors (gist) (soft tissue sarcoma), germ cell tumors, extragonadal germ cell tumors, ovarian germ cell tumors, testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, heart tumors, hepatocellular (liver) cancer, histiocytosis, langerhans cell, Hodgkin lymphoma, hypopharyngeal cancer, islet cell tumors, pancreatic neuroendocrine tumors, kidney (renal cell) cancer, langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (non-small cell and small cell), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma, melanoma, intraocular (eye), Merkel cell carcinoma (skin cancer), mesothelioma, malignant, metastatic cancer, metastatic squamous neck cancer with occult primary, midline tract carcinoma with nut gene changes, mouth cancer, multiple endocrine neoplasia syndromes multiple myeloma/plasma cell neoplasms, mycosis fungoides (lymphoma), myelodysplastic syndromes, myelodysplastic/myeloproliferative neoplasms, myelogenous leukemia, myeloproliferative neoplasms, chronic, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, lip and oral cavity cancer and oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary peritoneal cancer, prostate cancer, rectal cancer, recurrent cancer, renal cell (kidney) cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, osteosarcoma (bone cancer), soft tissue sarcoma, uterine sarcoma, Sezary syndrome (lymphoma), skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma of the skin - see skin cancer, squamous neck cancer with occult primary, metastatic, stomach (gastric) cancer, t-cell lymphoma, testicular cancer, throat cancer, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, ureter and renal pelvis, transitional cell cancer, urethral cancer, uterine cancer, endometrial, uterine sarcoma, vaginal cancer, vascular tumors (soft tissue sarcoma), vulvar cancer, Wilms tumor and other kidney tumors. [0211] In some embodiments, the present disclosure provides methods of treating or delaying the progression of cancer in a patient comprising administering to the patient a therapeutically effective amount of a composition comprising engineered immune cells comprising a chimeric receptor described herein. In certain embodiments, the therapeutically effective amount of the composition comprising engineered immune cells reduces the number of cancer cells in the patient following such treatment. In certain embodiments, the therapeutically effective amount of the composition comprising engineered immune cells reduces and/or eliminates the tumor burden in the patient following such treatment. In certain embodiments, the method further comprises administering radiation therapy to the patient. In certain embodiments, the radiation therapy is administered before, at the same time as, and/or after the patient receives the therapeutically effective amount of the composition comprising engineered immune cells. In certain embodiments, the method further comprises administering to the patient one or more anticancer agents and/or one or more chemotherapeutic agents. In certain embodiments, the one or more anticancer agents and/or one or more chemotherapeutic agents are administered before, at the same time as, and/or after the patient receives the therapeutically effective amount of the composition comprising engineered immune cells. In certain embodiments, treatment of a patient with a therapeutically effective amount of engineered immune cells comprising a chimeric receptor of the present disclosure and a course of an anticancer agent produces a greater tumor response and clinical benefit in such patient compared to those treated with the engineered immune cells or anticancer drugs/radiation alone. Since the doses for all approved anticancer drugs and radiation treatments are known, the present disclosure contemplates the various combinations of them with the engineered immune cells.

[0212] The present disclosure is not limited by the type of anticancer agent used. A number of suitable anticancer agents are contemplated for use in the methods of the present disclosure. Indeed, the present disclosure contemplates, but is not limited to, administration of numerous anticancer agents such as: agents that induce apoptosis; polynucleotides (e.g., anti-sense, ribozymes, siRNA); polypeptides (e.g., enzymes and antibodies); biological mimetics; alkaloids; alkylating agents; antitumor antibiotics; antimetabolites; hormones; platinum compounds;

monoclonal or polyclonal antibodies (e.g., antibodies conjugated with anticancer drugs, toxins, defensins), toxins; radionuclides; biological response modifiers (e.g., interferons (e.g., IFN-a) and interleukins (e.g., IL-2)); adoptive immunotherapy agents; hematopoietic growth factors; agents that induce tumor cell differentiation (e.g., all-trans-retinoic acid); gene therapy reagents (e.g., antisense therapy reagents and nucleotides); tumor vaccines; angiogenesis inhibitors;

proteasome inhibitors: NF-KB modulators; anti-CDK compounds; HDAC inhibitors; and the like. Numerous other examples of chemotherapeutic compounds and anticancer therapies suitable for co-administration with the disclosed compounds are known to those skilled in the art.

[0213] In certain embodiments, anticancer agents comprise agents that induce or stimulate apoptosis. Agents that induce apoptosis include, but are not limited to, radiation (e.g., X-rays, gamma rays, UV); tumor necrosis factor (TNF)-related factors (e.g., TNF family receptor proteins, TNF family ligands, TRAIL, antibodies to TRAIL-R1 or TRAIL-R2); kinase inhibitors (e.g., epidermal growth factor receptor (EGFR) kinase inhibitor, vascular growth factor receptor (VGFR) kinase inhibitor, fibroblast growth factor receptor (FGFR) kinase inhibitor, platelet- derived growth factor receptor (PDGFR) kinase inhibitor, and Bcr-Abl kinase inhibitors (such as GLEEVEC)); antisense molecules; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN, and AVASTIN); anti-estrogens (e.g., raloxifene and tamoxifen); anti-androgens (e.g., flutamide, bicalutamide, finasteride, aminoglutethamide, ketoconazole, and corticosteroids);

cyclooxygenase 2 (COX-2) inhibitors (e.g., celecoxib, meloxicam, NS-398, and non-steroidal anti-inflammatory drugs (NSAIDs)); anti-inflammatory drugs (e.g., butazolidin, DECADRON, DELTASONE, dexamethasone, dexamethasone intensol, DEXONE, HEXADROL,

hydroxychloroquine, METICORTEN, ORADEXON, ORASONE, oxyphenbutazone,

PEDIAPRED, phenylbutazone, PLAQUENIL, prednisolone, prednisone, PRELONE, and TANDEARIL); and cancer chemotherapeutic drugs (e.g., irinotecan (CAMPTOSAR), CPT-l l, fludarabine (FLUDARA), dacarbazine (DTIC), dexamethasone, mitoxantrone, MYLOTARG, VP- 16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, bevacizumab, TAXOTERE or TAXOL); cellular signaling molecules; ceramides and cytokines; staurosporine, and the like.

[0214] In still other embodiments, the compositions and methods of the present disclosure are used together with at least one anti-hyperproliferative or antineoplastic agent selected from alkylating agents, antimetabolites, and natural products (e.g., herbs and other plant and/or animal derived compounds). [0215] In some embodiments, the compositions and methods of the present disclosure are used together with alkylating agents including, but not limited to: nitrogen mustards (e g.,

mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin); and chlorambucil); ethylenimines and methylmelamines (e.g., hexamethylmelamine and thiotepa); alkyl sulfonates (e g., busulfan); nitrosoureas (e g., carmustine (BCNU); lomustine (CCNU); semustine (methyl- CCNU); and streptozocin (streptozotocin)); and triazenes (e.g., dacarbazine (DTIC;

dimethy ltri azenoimi d-azol ecarb oxami de) .

[0216] In some embodiments, the compositions and methods of the present disclosure are used together with antimetabolites including, but not limited to: folic acid analogs (e.g., methotrexate (amethopterin)); pyrimidine analogs (e.g., fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorode-oxyuridine; FudR), and cytarabine (cytosine arabinoside)); and purine analogs (e.g., mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2’- deoxycoformycin)).

[0217] In still further embodiments, chemotherapeutic agents suitable for use with the compositions and methods of the present disclosure include, but are not limited to: vinca alkaloids (e.g., vinblastine (VBL), vincristine); epipodophyllotoxins (e.g., etoposide and teniposide); antibiotics (e.g., dactinomycin (actinomycin D), daunorubicin (daunomycin;

rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)); enzymes (e.g., L-asparaginase); biological response modifiers (e.g., interferon-alfa);

platinum coordinating complexes (e.g., cisplatin (cis-DDP) and carboplatin); anthracenediones (e.g., mitoxantrone); substituted ureas (e.g., hydroxyurea); methylhydrazine derivatives (e.g., procarbazine (N-methylhydrazine; MIH)); adrenocortical suppressants (e.g., mitotane (o,p’- DDD) and aminoglutethimide); adrenocorticosteroids (e.g., prednisone); progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); estrogens (e.g., diethylstilbestrol and ethinyl estradiol); antiestrogens (e.g., tamoxifen); androgens (e.g., testosterone propionate and fluoxymesterone); antiandrogens (e.g., flutamide): and

gonadotropin-releasing hormone analogs (e.g., leuprolide).

[0218] Any oncolytic agent that is routinely used in a cancer therapy context may also be used with the compositions and methods of the present disclosure. For example, the U.S. Food and Drug Administration maintains a formulary of oncolytic agents approved for use in the United States. International counterpart agencies to the U.S. F.D.A. maintain similar formularies.

[0219] Anticancer agents further include compounds which have been identified to have anticancer activity. Examples include, but are not limited to, 3-AP, 12-O-tetradecanoylphorbol- 13 -acetate, 17AAG, 852A, ABI-007, ABR-217620, ABT-751, ADI-PEG 20, AE-941, AG- 013736, AGRO100, alanosine, AMG 706, antibody G250, antineoplastons, AP23573, apaziquone, APC8015, atiprimod, ATN-161, atrasenten, azacitidine, BB-10901, BCX-1777, bevacizumab, BG00001, bicalutamide, BMS 247550, bortezomib, bryostatin-l, buserelin, calcitriol, CCI-779, CDB-2914, cefixime, cetuximab, CG0070, cilengitide, clofarabine, combretastatin A4 phosphate, CP-675,206, CP-724,714, CpG 7909, curcumin, decitabine, DENSPM, doxercalciferol, E7070, E7389, ecteinascidin 743, efaproxiral, eflomithine, EKB-569, enzastaurin, erlotinib, exisulind, fenretinide, flavopiridol, fludarabine, flutamide, fotemustine, FR901228, G17DT, galiximab, gefitinib, genistein, glufosfamide, GTI-2040, histrelin, HKI-272, homoharringtonine, HSPPC-96, hul4. l8-interleukin-2 fusion protein, HuMax-CD4, iloprost, imiquimod, infliximab, interleukin-l2, IPI-504, irofulven, ixabepilone, lapatinib, lenalidomide, lestaurtinib, leuprolide, LMB-9 immunotoxin, lonafarnib, luniliximab, mafosfamide, MB07133, MDX-010, MLN2704, monoclonal antibody 3F8, monoclonal antibody J591, motexafm, MS- 275, MVA-MUC1-IL2, nilutamide, nitrocamptothecin, nolatrexed dihydrochloride, nolvadex, NS-9, 06-benzylguanine, oblimersen sodium, ONYX-015, oregovomab, OSI-774,

panitumumab, paraplatin, PD-0325901, pemetrexed, PHY906, pioglitazone, pirfenidone, pixantrone, PS-341, PSC 833, PXD101, pyrazoloacridine, Rl 15777, RAD001, ranpirnase, rebeccamycin analogue, rhuAngiostatin protein, rhuMab 2C4, rosiglitazone, rubitecan, S-l, S- 8184, satraplatin, SB-, 15992, SGN-0010, SGN-40, sorafenib, SR31747A, ST1571, SU011248, suberoylanilide hydroxamic acid, suramin, talabostat, talampanel, tariquidar, temsirolimus, TGFa-PE38 immunotoxin, thalidomide, thymalfasin, tipifarnib, tirapazamine, TLK286, trabectedin, trimetrexate glucuronate, TroVax, UCN-l, valproic acid, vinflunine, VNP40101M, volociximab, vorinostat, VX-680, ZD1839, ZD6474, zileuton, and zosuquidar trihydrochloride.

[0220] The present disclosure provides methods for administering compositions and methods of the present disclosure with (e.g., before, during, or after) radiation therapy. The present disclosure is not limited by the types, amounts, or delivery and administration systems used to deliver the therapeutic dose of radiation. For example, a subject may receive photon

radiotherapy, particle beam radiation therapy, other types of radiotherapies, and combinations thereof. In some embodiments, the radiation is delivered using a linear accelerator. In still other embodiments, the radiation is delivered using a gamma knife. The source of radiation can be external or internal.

[0221] External radiation therapy is most common and involves directing a beam of high-energy radiation to a tumor site through the skin using, for instance, a linear accelerator. While the beam of radiation is localized to the tumor site, it is nearly impossible to avoid exposure of normal, healthy tissue. However, external radiation is usually well tolerated. Internal radiation therapy involves implanting a radiation-emitting source, such as beads, wires, pellets, capsules, particles, and the like, inside the body at or near the tumor site including the use of delivery systems that specifically target cancer cells (e.g., using particles attached to cancer cell binding ligands). Such implants can be removed following treatment, or, left in the body inactive. Types of internal radiation therapy include, but are not limited to, brachytherapy, interstitial irradiation, intracavity irradiation, radioimmunotherapy, and the like.

[0222] A subject may optionally receive radiosensitizers (e.g., metronidazole, misonidazole, intra-arterial Budr, intravenous iododeoxyuridine (IudR), nitroimidazole, 5-substituted-4- nitroimidazoles, 2H-isoindolediones, [[(2-bromoethyl)-amino]methyl]-nitro-lH-imidazole-l- ethanol, nitroaniline derivatives, DNA-affmic hypoxia selective cytotoxins, halogenated DNA ligand, 1,2,4 benzotriazine oxides, 2-nitroimidazole derivatives, fluorine-containing nitroazole derivatives, benzamide, nicotinamide, acridine-intercalator, 5-thiotretrazole derivative, 3-nitro- 1,2, 4-triazole, 4,5-dinitroimidazole derivative, hydroxylated texaphrins, cisplatin, mitomycin, tiripazamine, nitrosourea, mercaptopurine, methotrexate, fluorouracil, bleomycin, vincristine, carboplatin, epirubicin, doxorubicin, cyclophosphamide, vindesine, etoposide, paclitaxel, heat (hyperthermia), and the like), radioprotectors (e.g., cysteamine, aminoalkyl dihydrogen phosphorothi oates, amifostine (WR 2721), IL-1, IL-6, and the like). Radiosensitizers enhance the killing of tumor cells. Radioprotectors protect healthy tissue from the harmful effects of radiation.

[0223] Any type of radiation can be administered, so long as the dose of radiation is tolerated without unacceptable negative side-effects. Suitable types of radiotherapy include, for example, ionizing (electromagnetic) radiotherapy (e.g., X-rays or gamma rays) or particle beam radiation therapy (e.g., high linear energy radiation). Ionizing radiation is defined as radiation comprising particles or photons that have sufficient energy to produce ionization, i.e., gain or loss of electrons (as described in, for example, U.S. 5,770,581 incorporated herein by reference). The effects of radiation can be at least partially controlled by the clinician In one embodiment, the dose of radiation is fractionated for maximal target cell exposure and reduced toxicity.

[0224] In some embodiments, engineered immune cells and one or more therapeutic agents or anticancer agents are administered to an animal under one or more of the following conditions: at different periodicities, at different durations, at different concentrations, by different

administration routes, etc. In some embodiments, engineered immune cells are administered prior to the therapeutic or anticancer agent, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, 18 hours or more, 1, 2,

3, 4, 5, 6 or more days, or 1, 2, 3, 4, 5, 6 or more weeks prior to the administration of the therapeutic or anticancer agent. In some embodiments, engineered immune cells are

administered after the therapeutic or anticancer agent, e.g., 0.5, 1, 2, 3, 4, 5, 10, 12, 18 or more hours, 1, 2, 3, 4, 5, 6 or more days, or 1, 2, 3, 4, 5, 6, or more weeks after the administration of the anticancer agent. In some embodiments, engineered immune cells and the therapeutic or anticancer agent are administered concurrently but on different schedules, e.g., engineered immune cells are administered daily while the therapeutic or anticancer agent is administered once a week, once every two weeks, once every three weeks, once every four weeks, or more. In other embodiments, engineered immune cells are administered once a week while the therapeutic or anticancer agent is administered daily, once a week, once every two weeks, once every three weeks, once every four weeks, or more.

[0225] Compositions within the scope of the present disclosure include all compositions wherein the engineered immune cells are contained in an amount which is effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art. In one non-limiting example, engineered immune cells may be administered to a subject, e.g. human patient, in order to provide the human between 1000 and 10¹⁰ engineered immune cells per day (e.g., for treating cancer). In another embodiment, between 1000 and 10¹⁰ engineered immune cells are administered to treat, ameliorate, or prevent cancer (e.g., prevent metastasis, recurrence, and/or progression of cancer). The unit dose may be administered in one or more administrations one or more times daily (e.g., for 1, 2, 3, 4, 5, 6, or more days or weeks).

[0226] Engineered immune cells may be administered as part of a pharmaceutical preparation containing suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing and/or administration of the engineered immune cells into preparations which can be used pharmaceutically. Engineered immune cells and/or

pharmaceutical preparations containing the same may be administered intravenously (e.g., via intravenous infusion). Alternative routes of administration may be used. Administration may be carried out in any convenient manner, including by injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient trans arterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. Engineered immune cells may be injected directly into a tumor, lymph node, or site of infection.

[0227] Compositions and methods disclosed herein may be used to treat immunodeficiencies including, but not limited to, primary immunodeficiency disease (PIDD),T-cell deficiencies, combined T-cell and B-cell deficiencies, phagocyte disorders, immune dysregulation diseases, innate immune deficiencies, ataxia telangiectasia, DiGeorge syndrome, severe combined immunodeficiency (SCID), Wiskott-Aldrich syndrome, Kostmann syndrome, Shwachman- Diamond syndrome, Griscelli syndrome, and NF-Kappa-B Essential Modulator (NEMO) deficiency.

[0228] The present disclosure will be more fully understood by reference to the following Examples. They should not, however, be construed as limiting the scope of the present disclosure.

EXAMPLES

[0229] Examples of chimeric receptor designs are provided. Different molecules that have broad antibody binding activity can be used as the extracellular target-binding moiety of the chimeric receptor.

[0230] FIG. 1 shows a schematic of an engineered immune cell that comprises two different chimeric receptors. One of the two chimeric receptors comprise an extracellular target-binding moiety that that binds to an antigen-binding domain of a bi-specific antibody, a transmembrane domain and an intracellular signaling domain. The other chimeric receptor comprises an extracellular target-binding moiety that that binds to an antigen-binding domain of a different bi- specific antibody, a transmembrane domain and an intracellular co-signaling domain. Activation of both the intracellular signaling domain and co-signaling domain is required for activation of the engineered immune cell. Alternatively, the engineered immune cell can comprise a single chimeric receptor that binds to an antigen-binding domain of a bi-specific antibody, and the single chimeric receptor includes both a signaling domain and a co-signaling domain. In each instance, the other antigen-binding domain of the bi-specific antibody recognizes and binds a target molecule, for example, a cancer antigen on the surface of a cancer cell.

[0231] FIG. 2 shows a schematic of a split chimeric receptor design similar to the one in FIG. 1, except that the binding of one of the extracellular target-binding moiety to the bi-specific antibody is mediated by a bridging molecule. The bridging molecule may be an exogenous molecule or an endogenous molecule.

[0232] FIG. 3 shows a schematic of yet another example of an engineered immune cell that includes two different chimeric receptors, similar to FIG. 1, with the exception that one of the extracellular target-binding moieties is an anti-idiotype antibody (e.g., anti-idiotype scFv) that binds an antigen-binding domain of the bi-specific antibody.

[0233] FIG. 4 shows a schematic of an example of an engineered immune cell that includes two different chimeric receptors, each of which includes an isotype-specific antibody that binds to a different IgG isotype (also called an IgG subclass).

[0234] FIG. 5 shows a schematic of an engineered immune cell that includes a rheumatoid factor that binds to an Fc domain of an antibody. The extracellular domain can be a molecule that binds to antibodies universally, for example, rheumatoid factor (RF), which binds to most IgG isotypes in the Cy2-C 3 region. Further, low levels of RF that bind to IgM are found at low levels in healthy individuals, indicating that it is tolerated to some extent. RF has a lower affinity to antibodies than CD32 can be blocked by CD32.

[0235] FIG. 6 shows a schematic of an engineered immune cell that includes an antibody that binds to a non-human sequence in a humanized antibody. Most therapeutic antibodies are selected in a non-human context and then grafted into a humanized antibody scaffold. Non human sequences created by this common process (e.g., junctions between mouse Fv and human Fc) can serve as epitopes for the extracellular target-binding moiety (e.g., a ScFv).

[0236] FIG. 7 shows a schematic of an engineered immune cell that includes an antibody that binds to a glycosylated antibody. Certain therapeutic antibodies are glycosylated, or otherwise modified in a way that distinguishes them from native human antibodies. The extracellular target-binding moiety can bind to the specific modifications of therapeutic antibodies (e.g., glycosylation) to distinguish them from native ones.

[0237] FIG. 8 shows a schematic of an engineered immune cell that includes an antibody that binds to polyethylene glycol (PEG). Certain therapeutic antibodies are conjugated to other molecules, e.g., polyethylene glycol (PEG), biotin, FITC. The extracellular target-binding moiety can bind to molecule conjugated to the therapeutic antibodies, for example, a PEG antibody.

[0238] FIG. 9 shows a schematic of an example of an engineered immune cell that includes two different chimeric receptors, each of which includes an extracellular target-binding moiety that binds to a different type of post-translational modifications (PTMs).

[0239] FIG. 10 shows a schematic of an engineered immune cell that includes a Fc binding protein (e.g., a natural Fc binding protein or a variant thereof). The extracellular target-binding moiety can be a natural Fc binding protein and their variants. For example, one Fc binding protein that may be used is are TRIM family members, e.g., TRIM 21 or its variants, which binds to a region that interfaces between CH2 and CH3 in the Fc domain. TRIM21 has very broad antibody specificity, binding to IgG, IgM, and IgA isotypes. Additional Fc binding proteins can also be used, such as Fc receptors (CD16, CD32, CD64 and their variants) and bacterial immunoglobulin binding proteins (e.g., Protein A, Protein G).

[0240] FIG. 11 shows a schematic of an example of an engineered immune cell that includes two different chimeric receptors, each of which includes an extracellular target-binding moiety that binds to a different type of IgG subclass. This example may be used, like many of the other dual-chimeric receptor cells described above, to implement combinatorial (e.g., AND/OR) logic. For example, an anti-Fc that does not bind IgG4 can be paired with an ScFv that selectively binds IgG4 to form an AND logic gate.

[0241] Various modifications of the present disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass their entirety.

Claims

CLAIMS What is claimed is:

1. An engineered immune cell comprising

two chimeric receptors that each comprise an intracellular signaling domain and an extracellular target-binding moiety that binds to an antigen-binding domain of a bi-specific antibody, wherein the extracellular target-binding moieties have different target-binding specificities and/or target-binding avidities relative to each other, and optionally wherein the bi-specific antibodies differ from each other.

2. The engineered immune cell of claim 1, wherein at least one of the extracellular target binding moieties binds to the antigen-binding domain of a bi-specific antibody via a bridging molecule, optionally wherein the bridging molecule is an endogenous molecule or an exogenous molecule.

3. The engineered immune cell of claim 1 or 2, wherein at least one of the extracellular target-binding moieties comprises an anti-idiotype antibody.

4. An engineered immune cell comprising

two chimeric receptors that each comprise an intracellular signaling domain and an extracellular target-binding moiety that binds to an Fc domain of an antibody of a class or subclass of immunoglobulins,

wherein the extracellular target-binding moieties have different target-binding specificities and/or target-binding avidities relative to each other, and optionally wherein the antibodies of a class or subclass of immunoglobulins differ from each other.

5. The engineered immune cell of claim 4, wherein the antibodies of a class of

immunoglobulins are selected from immunoglobulin A (IgA), immunoglobulin D (IgD), immunoglobulin E (IgE), immunoglobulin G (IgG), and immunoglobulin M (IgM).

6. The engineered immune cell of claim 4, wherein antibodies of a subclass of

immunoglobulins are selected from IgAl and IgA2.

7. The engineered immune cell of claim 4, wherein antibodies of a subclass of

immunoglobulins are selected from IgGl, IgG2, IgG3, and IgG4.

8. An engineered immune cell comprising

two chimeric receptors that each comprise an intracellular signaling domain and an extracellular target-binding moiety that binds to polyethylene glycol (PEG), biotin, or streptavidin, wherein the extracellular target-binding moieties have different target-binding specificities and/or target-binding avidities relative to each other.

9. The engineered immune cell of claim 8, wherein at least one of the extracellular target binding moieties is selected from PEG antibodies, streptavidin, and streptavidin-binding proteins.

10. An engineered immune cell comprising

two chimeric receptors that each comprise an intracellular signaling domain and an extracellular target-binding moiety that binds to a post-translational modification (PTM) of an antibody, wherein the extracellular target-binding moieties have different target-binding specificities and/or target-binding avidities relative to each other, and optionally wherein the PTMs differ from each other.

11. The engineered immune cell of claim 10, wherein the PTM is selected from

phosphorylation, glycosylation, acetylation, amidation, hydroxylation, methylation, and sulfation.

12. The engineered immune cell of claim 11, wherein the glycosylation is selected from galactosylation, fucosylation, and sialylation.

13. The engineered immune cell of any one of claims 10-12, wherein at least one of the extracellular target-binding moieties does not bind the antibody that does not comprise the PTM.

14. An engineered immune cell comprising

two chimeric receptors that each comprise an intracellular signaling domain and an extracellular target-binding moiety that binds to an Fc domain of an antibody,

wherein the extracellular target-binding moieties have different target-binding specificities and/or target-binding avidities relative to each other, and optionally wherein the Fc domains differ from each other.

15. The engineered immune cell of claim 14, wherein the extracellular target-binding moieties bind to different epitopes on Fc.

16. The engineered immune cell of claim 14 or claim 15, wherein at least one of the extracellular target-binding moieties is selected from TRIM proteins (e.g., TRIM21), bacterial anti-Fc proteins, Fc receptors (e.g., CD16, CD32, or CD64), and variants thereof.

17. The engineered immune cell of claim 14 or 15, wherein at least one of the extracellular target-binding moieties is selected from Protein A, Protein G, Protein Z, Protein L, Protein Z, and immunoglobulin-binding fragments thereof.

18. The engineered immune cell of any one of claims 1-17, wherein one of the intracellular signaling domains is CD3-zeta, and the other of the intracellular signaling domains is selected from CD28, 0X40 (CD134), 4-1BB (CD137), and ICOS.

19. The engineered immune cell of any one of claims 1-18, wherein at least one of the extracellular target-binding moieties is an antibody, a single-chain variable fragment (scFv), an antigen binding fragment (Fab), a single domain antibody, a diabody, a VHH fragment, or a synthetic epitope.

20. The engineered immune cell of any one of claims 1-19, wherein at least one of the extracellular target-binding moieties does not bind to a native antibody.

21. An engineered immune cell comprising

a chimeric receptor that comprises an intracellular signaling domain, optionally an intracellular co-signaling domain, and an extracellular target-binding moiety selected from: molecules that bind to an antigen-binding domain of a bi-specific antibodies, anti-hinge antibodies, rheumatoid factor or IgG-binding fragments thereof; antibodies that bind to a non-human sequence of a humanized antibody; molecule that binds to a post translational modification (PTM) of an antibody, optionally a glycosylated amino acid of an antibody; molecules that bind to polyethylene glycol (PEG); molecules that bind an Fc domain of an antibody (e g , TRIM proteins); and molecules that bind antibody junctions (e.g., mouse-human junctions).

22. The engineered immune cell of claim 21 further comprising another chimeric receptor that comprises an intracellular co-signaling domain and an extracellular target-binding moiety selected from: molecules that bind to an antigen-binding domain of a bi-specific antibody, anti hinge antibodies, rheumatoid factor or IgG-binding fragments thereof; antibodies that bind to a non-human sequence of a humanized antibody; molecule that binds to a PTM of an antibody, optionally a glycosylated amino acid of an antibody; molecules that bind to PEG; molecules that bind an Fc domain of an antibody (e.g., TRIM proteins); and molecules that bind antibody junctions (e.g., mouse-human junctions),

wherein the extracellular target-binding moieties of the chimeric receptors bind to different molecules.

23. The engineered immune cell of claim 21 or 22, wherein the intracellular signaling domain is CD3-zeta, and the intracellular co-signaling domains is selected from CD28, 0X40 (CD134), 4- IBB (CD 137), and ICOS.

24. The engineered immune cell of any one of claims 21-23, wherein the extracellular target binding moiety is an antibody, a single-chain variable fragment (scFv), an antigen binding fragment (Fab), a single domain antibody, a diabody, a VHH fragment, or a synthetic epitope.

25. The engineered immune cell of any one of claims 21-24, wherein the extracellular target binding moiety does not bind to a native antibody.

26. The engineered immune cell of any one of claims 1-25, wherein the molecule to which the extracellular target-binding moieties bind recognizes a target selected from cancer antigens and autoimmune antigens.

27. The engineered immune cell of any one of claims 1-26, wherein the engineered immune cell is an engineered T cell.

28. The engineered immune cell of claim 27, wherein the engineered immune cell is selected from CD4+ T cells, CD8+ T cells, regulatory T cells (Tregs), Natural Killer T (NKT) cells, and Natural Killer (NK) cells.

29. The engineered immune cell of any one of the preceding claims, wherein the antibody is selected from Revlimid® (lenalidomide), Opdivo® (nivolumab), Imbruvica® (ibrutinib), Keytruda® (pembrolizumab), Ibrance® (palbociclib), Tecentriq® (atezolizumab), Darzalex® (daratumumab), Perjeta® (pertuzumab), Xtandi® (enzalutamide), Avastin® (bevacizumab), Herceptin® (trastuzumab), Gazyva® (obinutuzumab), Jakafi® (ruxolitinib), Venclexta® (venetoclax), and Rituxan® (rituximab).

30. A method of treating a disease comprising

administering to a subject in need of such treatment an engineered immune cell of any one of the preceding claims in an amount effective to treat the disease.

31. The method of claim 30, wherein the disease is cancer.

32. The method of claim 30, wherein the disease is an autoimmune disease.