WO2018111340A1 - Methods for determining potency and proliferative function of chimeric antigen receptor (car)-t cells - Google Patents

Methods for determining potency and proliferative function of chimeric antigen receptor (car)-t cells Download PDF

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WO2018111340A1
WO2018111340A1 PCT/US2017/035473 US2017035473W WO2018111340A1 WO 2018111340 A1 WO2018111340 A1 WO 2018111340A1 US 2017035473 W US2017035473 W US 2017035473W WO 2018111340 A1 WO2018111340 A1 WO 2018111340A1
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cells
cell
antigen
car
described
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PCT/US2017/035473
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French (fr)
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Sadik KASSIM
Junxia Wang
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Novartis Ag
The Trustees Of The University Of Pennsylvania
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047Cells of the immune system
    • G01N33/505Cells of the immune system involving T-cells

Abstract

The invention provides methods for determining the potency and proliferative function of chimeric antigen receptor (CAR)-T cells (CART cells), as well as kits for carrying out such methods.

Description

METHODS FOR DETERMINING POTENCY AND PROLIFERATIVE FUNCTION OF CHIMERIC

ANTIGEN RECEPTOR (CAR)-T CELLS

Field of the Invention

The invention relates to methods for determining the potency and proliferative function of chimeric antigen receptor (CAR)-T cells (CART cells).

Background of the Invention

CART cells are T cells that are engineered to express tumor antigen-specific chimeric antigen receptors (CARs) that consist of intracellular T cell activation domains coupled to extracellular anti-tumor antigen single-chain antibody variable region fragments. Upon binding of a tumor antigen to the antitumor antigen antibody fragments on CART cells, the intracellular T cell activation domains induce T cell activation. The activated T cells are thus expanded, release cytokines, and can kill tumor cells in an antigen-dependent manner. There is a need for assays by which in vitro characteristics of CART cells can be correlated with in vivo clinical and efficacy parameters.

Summary of the Invention

The invention provides methods for characterizing the potency of chimeric antigen receptor (CAR)-T cells (CART cells). The methods include (a) stimulating a CART cell in an antigen-specific manner (i.e., via the CAR of the CART cell), and (b) determining the level of antigen-specific proliferation of the stimulated cell. Detection of an increase in the level of proliferation of the antigen-specific stimulated cell, as compared to that of an unstimulated CART cell or a non-specifically stimulated CART cell (i .e., a stimulated CART cells that is not stimulated in an antigen-specific manner), can optionally be used to indicate a stimulated CART cell for use in therapy. The methods can be carried out in vitro.

In some embodiments, the CART cell can be stimulated by an antigen (e.g., a tumor antigen) for which the CAR on the CART cell is specific, such as an antigen present on the surface of a cell, which optionally is fixed. In other examples, the CART cell is stimulated by an anti-idiotypic antibody that is specific for the CAR of the CART cell.

The level of antigen-specific proliferation of the stimulated CART cell can optionally be determined in an assay that detects DNA synthesis (e.g., by determining the level of incorporation of a modified nucleotide (e.g., 5-ethynyl-2'-deoxyuridine (EdU), 3H-thymidine, and 5-bromo-2'-deoxyuridine (BrdU)) into the DNA of the stimulated CART cell), a proliferation marker, dye dilution, DNA content, or cellular metabolism .

The level of antigen-specific proliferation can be calculated by determination of a Proliferation Index (PI), as follows:

PI = [(level of indicator of proliferation in antigen-specific stimulated cells) - (level of indicator of proliferation in unstimulated cells)] / %

transduction.

In examples in which the indicator of proliferation is the level of incorporation of a modified nucleotide into the DNA of the antigen-specific stimulated CART cell, the PI can be determined as follows: PI = [(% incorporation of modified nucleotide in antigen-specific

stimulated cells) - (% incorporation of modified nucleotide in

unstimulated cells)] / % transduction.

As explained elsewhere herein, "unstimulated" in the two exemplary formulas set forth above can be replaced with "stimulated, but in a non-antigen specific manner."

In some embodiments, the methods involve the use of flow cytometry.

The level of antigen-specific proliferation of the stimulated CART cell can correlate with one or more in vivo clinical parameters of the CART cell including, for example, a pharmacokinetic parameter of the stimulated CART cell, which optionally is selected from the group consisting of C max, max, and Area Under the Curve (AUC).

The methods of the invention can further optionally include detecting one or more cellular antigens of the CART cell using one or more antibodies, which optionally enables detection of a sub- population of CART cells and/or detection of cells expressing the CAR. For example, CD4+ and/or CD8+ CART cells can be detected by use of antibodies specific for CD4 and/or CD8, respectively.

The methods of the invention can further optionally include the step of determining the level of proliferation of the CART cells in the absence of an antigen-specific for the CAR of the CART cell, or in the absence of an anti-idiotypic antibody specific for the CAR of the CART cell.

The CAR of the CART cells can optionally include, in an N-terminal to C-terminal direction, an antigen binding domain (e.g., a scFv), a transmembrane domain, and one or more signaling domains. The one or more signaling domains can include one or more primary signaling domains (e.g., a CD3-zeta stimulatory domain), and optionally one or more costimulatory signaling domains (e.g. , an intracellular domain selected from a costimulatory protein selected from the group consisting of CD27, CD28, 4-1 BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVE , ICAM-1 , lymphocyte function-associated antigen-1 (LFA-1 ), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and a ligand that specifically binds with CD83).

In various examples, the CAR is specific for an antigen selected from the group consisting of CD1 9; CD123; CD22; CD30; CD1 71 ; CS-1 ; C-type lectin-like molecule-1 , CD33; epidermal growth factor receptor variant I II (EG FRvll l); ganglioside G2 (GD2) ; ganglioside G D3; TNF receptor family member B cell maturation (BCMA) ; Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)) ; prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1 ) ; Fms-Like Tyrosine Kinase 3 (FLT3) ; Tumor-associated glycoprotein 72 (TAG72) ; CD38; CD44v6; Carcinoembryonic antigen (CEA) ; Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD1 17); lnterleukin-13 receptor subunit alpha-2; mesothelin; Interleukin 1 1 receptor alpha (IL-1 1 Ra) ; prostate stem cell antigen (PSCA) ; Protease Serine 21 ; vascular endothelial growth factor receptor 2 (VEGFR2) ; Lewis(Y) antigen ; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta) ; Stage-specific embryonic antigen-4 (SSEA-4) ; CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2/neu) ; Mucin 1 , cell surface associated (MUC1 ); epidermal growth factor receptor (EG FR); neural cell adhesion molecule (NCAM) ; Prostase; prostatic acid phosphatase (PAP) ; elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IG F-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2) ; glycoprotein 1 00 (gp100) ; oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl) ; tyrosinase; ephrin type-A receptor 2 (EphA2) ; Fucosyl GM1 ; sialyl Lewis adhesion molecule (sLe) ; ganglioside GM3; transglutaminase 5 (TGS5) ; high molecular weight- melanoma-associated antigen (HMWMAA) ; o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM1 /CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (G PRC5D); chromosome X open reading frame 61 (CXORF61 ) ; CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1 ); hexasaccharide portion of globoH glycoceramide (GloboH) ; mammary gland differentiation antigen (NY-BR-1 ); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1 ) ; adrenoceptor beta 3 (ADRB3) ; pannexin 3 (PANX3) ; G protein- coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51 E2 (OR51 E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1 ) ;

Cancer/testis antigen 1 (NY-ESO-1 ); Cancer/testis antigen 2 (LAG E-1 a); Melanoma-associated antigen 1 (MAGE-A1 ) ; ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML) ; sperm protein 1 7 (SPA1 7) ; X Antigen Family, Member 1 A (XAGE1 ) ; angiopoietin-binding cell surface receptor 2 (Tie 2) ; melanoma cancer testis antigen-1 (MAD-CT-1 ); melanoma cancer testis antigen-2 (MAD-CT-2) ; Fos- related antigen 1 ; tumor protein p53 (p53); p53 mutant; prostein ; surviving ; telomerase; prostate carcinoma tumor antigen-1 , melanoma antigen recognized by T cells 1 ; Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT) ; sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP) ; ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA1 7) ; paired box protein Pax-3 (PAX3) ; Androgen receptor; Cyclin B1 ; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) ; Ras Homolog Family Member C (RhoC) ; Tyrosinase-related protein 2 (TRP-2) ; Cytochrome P450 1 B1 (CYP1 B1 ); CCCTC-Binding Factor (Zinc Finger Protein)-Like, Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3) ; Paired box protein Pax-5 (PAX5) ; proacrosin binding protein sp32 (OY-TES1 ); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2) ; Receptor for Advanced Glycation Endproducts (RAG E-1 ) ; renal ubiquitous 1 (RU1 ) ; renal ubiquitous 2 (RU2); legumain ; human papilloma virus E6 (HPV E6) ; human papilloma virus E7 (HPV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1 ); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF) ; C-type lectin domain family 12 member A (CLEC12A) ; bone marrow stromal cell antigen 2 (BST2); EG F-like module-containing mucin-like hormone receptor-like 2 (EMR2) ; lymphocyte antigen 75 (LY75); Glypican-3 (G PC3) ; Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1 ).

The methods invention can also optionally include (a) determining the number of the CART cells to administer to a subject; or (b) determining the expected level of response of a subject to the CART cell.

Further, the methods of the invention can also optionally include a step of administering the CART cells to a subject (e.g., a human subject). The invention also provides kits for determining the potency of CART cells. The kits can include (a) an agent for stimulating the CART cell in an antigen-specific manner (e.g., an antigen or an anti- idiotypic antibody), and (b) one or more reagents for detecting antigen-specific proliferation of the CART cell (e.g. , a modified nucleotide for use in detecting DNA synthesis in the CART cell).

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

The terms "a" and "an" 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.

The term "about" when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1 %, or in some instances ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.

The "potency" of a cell (e.g. , a CART cell), as referred to herein, is an indicator or measure of its efficacy or potential efficacy in achieving a desired function. In the case of a CART cell, a desired function can be targeting or killing another cell, such as a tumor cell. Potency can be assessed directly, by determination of the effect of the cell on its target (e.g., the effect of a CART cell on a tumor cell in vitro or in vivo). Alternatively, potency can be measured indirectly, as in various methods of the present invention. In particular, potency of a CART cell can be assessed by determining the level of in vitro, antigen-specific proliferation of the cell in an assay, e.g., as described herein (relative to, e.g. , the proliferation of an unstimulated CART cell as described herein). This measure of potency can then be correlated with, and thus can be considered predictive of, in vivo properties of the cell, such as PK/PD parameters as described herein (e.g., CMAX, T AX, and AUC), which can relate to the effectiveness of the cell in killing its targets. As described further herein, potency can be expressed in terms of a proliferation index, which can be normalized based on the number of cells expressing a relevant CAR.

The term "Chimeric Antigen Receptor" or alternatively a "CAR" refers to a set of polypeptides, typically two in the simplest embodiments, which when in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and with intracellular signal generation. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "an intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule and/or costimulatory molecule as defined below. In some aspects, the set of polypeptides are contiguous with each other. In some embodiments, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g. , can couple an antigen binding domain to an intracellular signaling domain. In one aspect, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below. In one aspect, the costimulatory molecule is chosen from the costimulatory molecules described herein, e.g., 4-1 BB (i.e., CD137), CD27 and/or CD28. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In one aspect the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g. , a scFv) during cellular processing and localization of the CAR to the cellular membrane.

A CAR that comprises an antigen binding domain (e.g. , a scFv, or TCR) that targets a specific tumor marker X, such as those described herein, is also referred to as XCAR. For example, a CAR that comprises an antigen binding domain that targets CD19 is referred to as CD1 9CAR.

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.

The term "antibody," as used herein, refers to a protein , or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen. Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

The term "antibody fragment" refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g. , by binding, steric hinderance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab , Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CH1 domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide brudge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g. , Hollinger and Hudson, Nature Biotechnology 23:1 126-1 1 36, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3)(see U.S. Patent No. : 6,703, 1 99, which describes fibronectin polypeptide minibodies). 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, e.g. , via a synthetic linker, e.g., 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.

The portion of the CAR of the invention comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody or bispecific antibody (Harlow et al. , 1 999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al. , 1 989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1 988, Proc. Natl. Acad. Sci . USA 85:5879-5883 ; Bird et al. , 1 988, Science 242:423-426). In one aspect, the antigen binding domain of a CAR composition of the invention comprises an antibody fragment. In a further aspect, the CAR comprises an antibody fragment that comprises a scFv. The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991 ), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1 997) JMB 273,927-948 ("Chothia" numbering scheme), or a combination thereof.

As used herein, the term "binding domain" or "antibody molecule" 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.

The portion of the CAR of the invention comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody, or bispecific antibody (Harlow et al., 1 999, In : Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al. , 1 989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1 988, Proc. Natl. Acad. Sci . USA 85:5879-5883 ; Bird et al. , 1 988, Science 242:423-426). In one aspect, the antigen binding domain of a CAR composition of the invention comprises an antibody fragment. In a further aspect, the CAR comprises an antibody fragment that comprises a scFv.

The term "antibody heavy chain," refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.

The term "antibody light chain," refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

The term "recombinant antibody" refers to 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.

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 invention 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.

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. The term "anti-tumor 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 tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival. The term "autologous" refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.

The term "allogeneic" refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically

The term "xenogeneic" refers to a graft derived from an animal of a different species.

The term "cancer" refers to a disease characterized by the 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.

"Derived from" as that term is used herein, indicates 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.

The phrase "disease associated with expression of a tumor antigen as described herein" includes, but is not limited to, a disease associated with expression of a tumor antigen as described herein or condition associated with cells which express a tumor antigen as described herein including, e.g., proliferative diseases such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia; or a noncancer related indication associated with cells which express a tumor antigen as described herein. In one aspect, a cancer associated with expression of a tumor antigen as described herein is a hematological cancer. In one aspect, a cancer associated with expression of a tumor antigen as described herein is a solid cancer. Further diseases associated with expression of a tumor antigen described herein include, but not limited to, e.g., atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases associated with expression of a tumor antigen as described herein. Non-cancer related indications associated with expression of a tumor antigen as described herein include, but are not limited to, e.g., autoimmune disease, (e.g. , lupus), inflammatory disorders (allergy and asthma) and transplantation. In some embodiments, the tumor antigen-expressing cells express, or at any time expressed, mRNA encoding the tumor antigen. In an embodiment, the tumor antigen -expressing cells produce the tumor antigen protein (e.g. , wild-type or mutant), and the tumor antigen protein may be present at normal levels or reduced levels. In an embodiment, the tumor antigen -expressing cells produced detectable levels of a tumor antigen protein at one point, and subsequently produced substantially no detectable tumor antigen protein.

The term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment used in the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g. , lysine, arginine, histidine), acidic side chains (e.g. , aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g. , alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta- branched side chains (e.g ., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CAR of the invention can be replaced with other amino acid residues from the same side chain family and the altered CAR can be tested using the functional assays described herein.

The term "stimulation," refers to a primary response induced by binding of a stimulatory molecule

(e.g., a TCR/CD3 complex or CAR) with its cognate ligand (or tumor antigen in the case of a CAR) thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex or signal transduction via the appropriate NK receptor or signaling domains of the CAR. Stimulation can mediate altered expression of certain molecules.

The term "stimulatory molecule," refers to a molecule expressed by an immune cell (e.g., T cell,

NK cell, B cell) that provides the cytoplasmic signaling sequence(s) that regulate activation of the immune cell in a stimulatory way for at least some aspect of the immune cell signaling pathway. In one aspect, the signal is a primary signal that is initiated by, for instance, 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 ITAM. Examples of an ITAM containing- cytoplasmic signaling sequence that is of particular use in the invention includes, but is not limited to, those derived from CD3 zeta, common FcR gamma (FCER1 G), Fc gamma Rlla, FcR beta (Fc Epsilon R1 b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP1 0, and DAP12. In a specific CAR of the invention, the intracellular signaling domain in any one or more CARS of the invention comprises an intracellular signaling sequence, e.g., a primary signaling sequence of CD3-zeta. In a specific CAR of the invention, the primary signaling sequence of CD3-zeta is the sequence provided as SEQ ID NO:18, or the equivalent residues from a non-human species, e.g. , mouse, rodent, monkey, ape and the like. In a specific CAR of the invention, the primary signaling sequence of CD3-zeta is the sequence as provided in SEQ I D NO:20, or the equivalent residues from a non-human species, e.g. , mouse, rodent, monkey, ape and the like.

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.

An "intracellular signaling domain," as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the CAR containing cell , e.g., a CART cell. Examples of immune effector function, e.g. , in a CART cell, include cytolytic activity and helper activity, including the secretion of cytokines.

In an embodiment, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In an embodiment, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation. For example, in the case of a CART, a primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule.

A primary intracellular signaling domain can comprise a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1 G), Fc gamma Rlla, FcR beta (Fc Epsilon R1 b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12.

The term "zeta" or alternatively "zeta chain", "CD3-zeta" or "TCR-zeta" is defined as the protein 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, and a "zeta stimulatory domain" or alternatively a "CD3-zeta stimulatory domain" or a "TCR-zeta stimulatory domain" is defined as the amino acid residues from the cytoplasmic domain of the zeta chain, or functional derivatives thereof, that are sufficient to functionally transmit an initial signal necessary for T cell activation. In one aspect the cytoplasmic domain of zeta comprises residues 52 through 1 64 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. In one aspect, the "zeta stimulatory domain" or a "CD3-zeta stimulatory domain" is the sequence provided as SEQ I D NO:1 8. In one aspect, the "zeta stimulatory domain" or a "CD3-zeta stimulatory domain" is the sequence provided as SEQ ID NO:20.

The term a "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation . Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are contribute to an efficient immune response. Costimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor, as well as OX40, CD27, CD28, CDS, ICAM-1 , LFA-1 (CD1 1 a/CD1 8), ICOS (CD278), and 4-1 BB (CD137). Further examples of such costimulatory molecules include CDS, ICAM-1 , GITR, BAFFR, HVEM

(LIGHTR), SLAMF7, NKp80 (KLRF1 ), NKp44, NKp30, NKp46, CD1 60, CD1 9, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6, CD49f, ITGAD, CD1 1 d, ITGAE, CD1 03, ITGAL, CD1 1 a, LFA-1 , ITGAM, CD1 1 b, ITGAX, CD1 1 c, ITGB1 , CD29, ITGB2, CD1 8, LFA-1 , ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RAN KL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1 , CD1 50, I PO-3), BLAME (SLAMF8), SELPLG (CD1 62), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD1 9a, and a ligand that specifically binds with CD83.

A costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. A costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1 BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1 , lymphocyte function-associated antigen-1 (LFA-1 ), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like.

The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment or derivative thereof.

The term "4-1 BB" refers to a member of the TNFR superfamily with an amino acid 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; and a "4-1 BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Acc. No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like. In one aspect, the "4-1 BB costimulatory domain" is the sequence provided as SEQ ID NO:14 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.

"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 myeloid-derived phagocytes.

"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.

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

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 a RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

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.

The term "endogenous" refers to any material from or produced inside an organism, cell, tissue or system.

The term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

The term "expression" refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.

The term "transfer vector" refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non- plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system . Expression vectors include all those known in the art, including cosmids, plasmids (e.g. , naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

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. 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. 1 7(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® gene delivery technology from Oxford BioMedica, the LENTIMAX™ 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.

The term "homologous" or "identity" refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g. , if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g. , if half (e.g. , five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of 1 0), are matched or homologous, the two sequences are 90% homologous.

"Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementary-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non- human residues. Furthermore, a humanized antibody/antibody fragment can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance. In general, the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al. , Nature, 321 : 522-525, 1 986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol. , 2: 593-596, 1 992.

"Fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.

The term "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.

In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

The term "operably linked" or "transcriptional control" 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. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.

The term "parenteral" administration of an immunogenic composition includes, e.g. ,

subcutaneous (s.c), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, intratumoral , or infusion techniques.

The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) , alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991 ); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1 985) ; and Rossolini et al. , Mol. Cell. Probes 8:91 -98 (1 994)).

The terms "peptide," "polypeptide," and "protein" are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

The term "promoter" refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. The term "promoter/regulatory sequence" refers to a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter/regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter/regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

The term "constitutive" promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

The term "inducible" promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide which encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer which corresponds to the promoter is present in the cell.

The term "tissue-specific" promoter refers to a nucleotide sequence which, when operably linked with a polynucleotide encodes or specified by a gene, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

The terms "cancer associated antigen" or "tumor antigen" interchangeably refers 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 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, 1 -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. In some embodiments, the CARs of the present invention includes CARs comprising an antigen binding domain (e.g., antibody or antibody fragment) that binds to a MHC presented peptide. Normally, peptides derived from endogenous proteins fill the pockets of Major histocompatibility complex (MHC) class I molecules, and are recognized by T cell receptors (TCRs) on CD8 + T lymphocytes. The MHC class I complexes are constitutively expressed by all nucleated cells. In cancer, virus-specific and/or tumor-specific peptide/MHC complexes represent a unique class of cell surface targets for immunotherapy. TCR-like antibodies targeting peptides derived from viral or tumor antigens in the context of human leukocyte antigen (HLA)-A1 or HLA-A2 have been described (see, e.g. , Sastry et al. , J Virol. 201 1 85(5):1 935-1942; Sergeeva et al., Blood, 201 1 1 17(16):4262-4272; Verma et al. , J Immunol 2010 184(4):21 56-21 65; Willemsen et al., Gene Ther 2001 8(21 ):1 601 -1608 ; Dao et al. , Sci Transl Med 2013 5(1 76) :176ra33; Tassev et al. , Cancer Gene Ther 2012 19(2):84-1 00). For example, TCR-like antibody can be identified from screening a library, such as a human scFv phage displayed library. The term "tumor-supporting antigen" or "cancer-supporting antigen" interchangeably refer to a molecule (typically a protein, carbohydrate or lipid) that is expressed on the surface of a cell that is, itself, not cancerous, but supports the cancer cells, e.g., by promoting their growth or survival e.g., resistance to immune cells. Exemplary cells of this type include stromal cells and myeloid-derived suppressor cells (MDSCs). The tumor-supporting antigen itself need not play a role in supporting the tumor cells so long as the antigen is present on a cell that supports cancer cells.

The term "flexible polypeptide linker" or "linker" as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and/or serine residues used alone or in combination, to link variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly/Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser)n, where n is a positive integer equal to or greater than 1 . For example, n=1 , n=2, n=3, n=4, n=5 and n=6, n=7, n=8, n=9 and n=1 0 (SEQ I D NO:28). In one embodiment, the flexible polypeptide linkers include, but are not limited to, (Gly4 Ser)4 (SEQ ID NO:29) or (Gly4 Ser)3 (SEQ I D NO:30). In another embodiment, the linkers include multiple repeats of (Gly2Ser), (GlySer) or (Gly3Ser) (SEQ ID NO:31 ). Also included within the scope of the invention are linkers described in WO2012/1 38475, incorporated herein by reference).

As used herein, a 5' cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified guanine nucleotide that has been added to the "front" or 5' end of a eukaryotic messenger RNA shortly after the start of transcription. The 5' cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNases. Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other. Shortly after the start of transcription, the 5' end of the mRNA being synthesized is bound by a cap-synthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction . The capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.

As used herein, "in vitro transcribed RNA" refers to RNA, preferably mRNA, which has been synthesized in vitro. Generally, the in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA.

As used herein, a "poly(A)" is a series of adenosines attached by polyadenylation to the mRNA. In the preferred embodiment of a construct for transient expression, the polyA is between 50 and 5000, preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. Poly(A) sequences can be modified chemically or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.

As used herein, "polyadenylation" refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3' poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre-m RNA through the action of an enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3' end at the cleavage site.

As used herein, "transient" refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.

As used herein, the terms "treat", "treatment" and "treating" 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 (e.g., one or more therapeutic agents such as a CAR of the invention). 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.

The term "signal transduction pathway" refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase "cell surface receptor" includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.

The term "subject" is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human).

The term , a "substantially purified" cell refers to 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 aspects, the cells are cultured in vitro. In other aspects, the cells are not cultured in vitro.

The term "therapeutic" as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.

The term "prophylaxis" as used herein means the prevention of or protective treatment for a disease or disease state. In the context of the present invention, "tumor antigen" or "hyperprol iterative disorder antigen" or "antigen associated with a hyperproliferative disorder" refers to antigens that are common to specific hyperproliferative disorders. In certain aspects, the hyperproliferative disorder antigens of the present invention are derived from, cancers including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.

The term "transfected" or "transformed" or "transduced" refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A "transfected" or "transformed" or

"transduced" cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

The term "specifically binds," refers to an antibody, or a ligand, which recognizes and binds with a binding partner (e.g., a tumor antigen) protein present in a sample, but which antibody or ligand does not substantially recognize or bind other molecules in the sample.

"Membrane anchor" or "membrane tethering domain", as that term is used herein, refers to a polypeptide or moiety, e.g., a myristoyl group, sufficient to anchor an extracellular or intracellular domain to the plasma membrane.

"Refractory" as used herein refers to a disease, e.g., cancer, which 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.

"Relapsed" as used herein refers to the return of a disease (e.g., cancer) or the signs and symptoms of a disease such as cancer after a period of improvement, e.g., after prior treatment of a therapy, e.g. , cancer therapy

Ranges: throughout this disclosure, various aspects of the invention 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 invention. 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.

Brief Description of the Drawings

Figure 1 is a plot showing FSC-A (x-axis/linear) vs. SSC-A (y-axis/linear), (WBC). Set "WBC Gate" to gate out debris and encompass the white blood cell populations.

Figure 2 is a plot showing SSC-H (x-axis/linear) vs. SSC-W (y-axis/linear), (Single). Gated off the "WBC Gate" set the "Single" gate to exclude doublets and encompass single white blood cells. Figure 3 is a plot showing DNA (x-axis/linear) vs. CD3 (y-axis/log), (Viable CD3). Gated off the "Single" set the "Viable" gate to exclude the CD3 DNAl0W/ dead or dying cells.

Figure 4 is a plot showing CD8 (x-axis/log) vs. CD4 (y-axis/log). Gated off the "Viable" gate set the "CD37CD4+" and "CD37CD8+" gate to encompass the CD3 positive, CD4 or CD8 positive cells. Further gating on CAR+ and CAR- populations in CD3, CD4, and CD8 T cell compartments can also be done.

Figure 5 is an illustration showing the design and development of an in vitro proliferation assay for CART cells. The workflow for an EdU-based proliferation assay is shown. On day 0, CART cells are co-cultured with K562 or K562-tumor antigen at a K562-to-T cell ratio of 1 :1 . K562 cells are killed by fixation with paraformaldehyde to provide solely a stimulation on CAR receptor without influencing CART cell proliferation. On day 2, EdU is added one hour before the incubation time period endpoint. Cells are harvested for surface staining and Click-iT® reaction analysis.

Figure 6 is a set of plots showing Total DNA (x-axis/linear) vs. EdU (y-axis/log). Gated off the "CD37CD4+" and "CD37CD8+" gate; set the "%EdU+" gate to detect EdU+ cells.

Figure 7 is a series of plots showing that in vitro proliferation correlates with in vivo PK parameters. Measurements for CD1 9-specific proliferation were performed using CTL019 final products in two replicates. Linear regression is shown (dark lines) along with the 95% confidence interval (shading) ; each dot or circle represents an individual patient. Detailed Description of the Invention

The invention provides methods for determining the potency and proliferative function of CART cells. Generally, the methods include antigen-specific stimulation of the CAR on a CART cell, followed by quantification of antigen-specific CART cell proliferation. The measure of CART cell potency can be used as an in vitro indication of the expected in vivo pharmacokinetics of a CART cell therapy product. It can further be used to determine whether a CART cell product is suitable for clinical use, to assess potential efficacy of the CART cell product, to determine dosage of CART cells administered, and/or to characterize new manufacturing approaches for CART cell therapy products. Use of the CART cell proliferation assays of the invention provide for the enhancement of safety and efficacy of CART cell therapy products for use in immunotherapy. Once the potency of a CART cell preparation is determined, the methods of the invention can optionally further include a step of administering the CART cells to a patient for therapy, as described herein.

CART cells that are assessed using the methods of the invention can be generated using any of a number of different methods effective to lead to the expression of a CAR on the surface of a T cell. Typically, T cells used in the generation of CART cells are autologous patient T cells, which can be obtained from peripheral blood mononuclear cells of a patient by plasmapheresis. The methods of the invention can also be used in connection with T cells obtained from healthy donors. Stable expression of CARs in T cells can be achieved using, for example, viral vectors (e.g. , lentiviral vectors or γ-retroviruses) or transposon/transposase systems. Other approaches include the use of mRNA transfer-mediated gene expression. Examples of methods for making CART cells that can be assessed using the methods of the present invention, as well as CARs that can be expressed on the CART cells, are provided in detail below, after a description of the following steps of the methods of the invention: (i) CAR stimulation on CART cells, (ii) detection of CART cell proliferation, and (iii) determination of CART cell potency based on their proliferation. CAR Stimulation

In a first step of the methods of the invention, the CAR of a CART cell is stimulated in an antigen- specific manner. This can be achieved by contacting the cell with the antigen for which the CAR on the CART cell is specific (e.g. , the antigen against which the single chain antibody portion of a CAR is directed), using any of a number of methods that are known in the art. In one approach, the antigen is expressed on the surface of another cell (e.g., a K562 cell), and the CART cells are cultured in the presence of the cells expressing the antigen. Optionally, the cells expressing the antigen are fixed, to ensure that the only stimulation of the CART cells is due to the antigen. In a variation of this approach, the antigen is present on the surface of a bead, which is contacted with a CART cell culture.

In other examples, the CART cells are stimulated by the use of anti-idiotypic antibodies which, in being specific for, e.g., the single chain antibody portion of the CAR, provide antigen-specific stimulation of the CART cell. Anti-idiotypic antibodies can optionally be present on the surfaces of beads, which are contacted with the CART cells to achieve antigen-specific stimulation of the cells.

To facilitate determination of antigen-specific proliferation, appropriate controls are selected, as can be determined by those of skill in the art. For example, in the case of cells (e.g., K562 cells) expressing a target antigen, an appropriate control can be the same cells, but lacking expression of the antigen. Thus, a sample of CART cells similar to that cultured in the presence of the cells expressing the antigen can be cultured in the presence of the control cells, which lack the antigen, as a control . Other controls include beads lacking antigen and/or anti-idiotypic antibody, or beads including non-specific antibodies. Further controls include non-antigen-specific approaches to T cell stimulation such as, for example, antibodies against CD3 and CD28, which optionally may be present on the surfaces of beads.

Detection of CART Cell Proliferation

After antigen-specific stimulation, the CART cells are permitted to proliferate (for, e.g., 24-72 hours or about 48 hours (e.g. , 48 hours, ±2 hours)) , and then after the proliferation period, the level of proliferation is assessed. Any of a number of well-known methods can be used to assess the level of proliferation of an antigen-specific stimulated CART cell (and corresponding controls). In various examples, the methods are high-throughput, single cell-based in vitro functional tests which optionally employ approaches including flow cytometry. The use of flow cytometry readily permits the detection of CART cells based on a number of different parameters that can be helpful in their assessment. For example, as described further below, a feature directly related to showing proliferation level can be detected. Furthermore, by using antibodies for detection of particular cellular antigens (e.g., CD4 and CD8), the frequency of proliferating CAR-T cells in unique populations within the CD3 T cell compartment, including CD4+ and CD8+ T cells, can be determined. In addition, the proportion of CAR-expressing T cells can further be assessed by use of antibodies specific for the CAR. As is explained further below, determining the proportion of CAR-expressing T cells can be useful in the determination of a proliferation index for the cells.

The level of proliferation of CART cells according to the methods of the invention can be determined by any of a number of known proliferation assays. These assays may fall within one of the following categories, which include assays involving (i) measurement of DNA synthesis, (ii) detection of proliferation-specific cell markers, (iii) measurement of successive cell divisions by the use of cell membrane binding dyes, (iv) measurement of cellular DNA content, and (v) measurement of cellular metabolism. For any of these methods, CART cell proliferation induced by CAR stimulation using a CAR- specific tumor antigen is typically compared to an unstimulated control to determine the proliferative capacity of the CART cells, as is described above.

As noted above, DNA synthesis assays can be used to determine proliferation levels of CART cells, according to the methods of the invention. In one example of such methods, incorporation of a nonradioactive, modified nucleotides into the DNA of dividing cells is detected as a measure of proliferation. As one example, 5-ethynyl-2'-deoxyuridine (EdU), a thymidine analog, can be used to assess active DNA synthesis. Such an analog (e.g., EdU) can be added to proliferating cells prior to the end of the proliferation period noted above (e.g., 4 - ½ hours, or about 1 hour (±1 5 minutes), prior to the end of the proliferation period). The incorporated analog (e.g. , EdU) can be detected by, for example, a click reaction (a copper-catalyzed azide-alkyne cycloaddition) using, for example, a fluorescent probe (e.g., Click-iT® EdU Alexa Fluor® 488), facilitating detection of newly synthesized DNA with either image-based techniques or flow cytometry (see, e.g., Salic and Mitchison, Proc Natl Acad Sci USA 1 05(7) :241 5-20, 2008).

In other examples, 5-bromo-2'-deoxyuridine (BrdU) can be used to detect cell proliferation. When cells are cultured with labeling medium containing BrdU, this pyrimidine analog is incorporated in place of thymidine in newly synthesized DNA. Detection of incorporated BrdU can be accomplished using an anti- BrdU antibody (see, e.g., Porstmann et al., J Immunol Methods 82(1 ) :169-79, 1 985).

Another example for detecting DNA synthesis, as a measure of CART cell proliferation, utilizes a radioactive nucleotide, 3H-thymidine, and involves detection of 3H-thymidine incorporation into new strands of chromosomal DNA during cell division (see, e.g. , Denton , Methods Mol Biol 79:1 69-77, 1998). A scintillation counter can be used to measure the radioactivity in DNA recovered from the 3H-thymidine- treated cells.

As noted above, assays in which proliferation-specific cellular markers are detected can also be utilized in the context of the invention. One exemplary assay involves the detection of the nuclear- specific proliferation antigen, Ki-67. Detection of Ki-67 protein expression in proliferating cells is accomplished through the use of an anti-Ki-67 antibody, followed by either imaging techniques or flow cytometry (see, e.g., Soares et al ., J Immunol Methods 362(1 -2) :43-50, 2010). Ki-67 protein is present during all active phases of the cell cycle (Gi , S, G2, and mitosis), but is absent from resting cells.

In other examples, cell membrane binding dyes can be utilized in the context of the invention for assessment of cellular proliferation. Some exemplary dyes that can be used include carboxyfluorescein succinimidyl ester (CFSE) and CellTrace™ Far Red. Such dyes cross cellular plasma membranes and covalently bind to all free amines on the surface and inside of cells, and can be retained for long periods of time. The fluorescent signal of the dyes following incorporation into cells can be detected by flow cytometry. The probe signal can subsequently be used to monitor proliferation, due to the progressive halving of the fluorescence within daughter cells following each cell division (see, e.g. , Tario et al ., J Vis Exp (70) :e4287, 2012; and Filby et al., Methods 82:29-37, 2015).

Cellular DNA content can also be measured to determine the extent of cellular proliferation. One exemplary method that can be used is the CyQUANT® cell proliferation assay, which employs a green fluorescent nucleic acid stain and a background suppression dye that is impermeable in live cells and suppresses the nucleic acid stain, blocking staining of dead cells and cells with compromised cell membranes. Detection of stained DNA is accomplished by measuring fluorescence with a plate reader (see, e.g. , Jones et al., J Immunol Methods 254(1 -2):85-98, 2001 ).

In other examples, cellular metabolism can be assessed in the context of the invention to determine cellular proliferation. Tetrazolium salts, such as MTT and MTS, can be utilized to assess cellular metabolic activity, reflecting the number of viable cell present in a sample. NAD(P)H-dependent cellular oxidoreductase enzymes, under defined conditions, are capable of reducing such tetrazolium salts into colored, insoluble formazan dyes. The amount of colored product formed can be quantified by measuring light absorption at a specific wavelength through the solution (see, e.g., Mosmann, J Immunol Methods 65(1 -2):55-63, 1 983; and Cory et al., Cancer Commun 3(7) :207-12, 1991 ). A related compound for measuring cell metabolic activity, alamarBlue®, has a fluorescence-based readout that is proportional to the cell number in a given sample (see, e.g. , Ahmed et al., J Immunol Methods 170(2):21 1 -24, 1 994). Additionally, ATP bioluminescence can be used as a measure of cell proliferation, correlating the concentration of ATP in a given sample with the number of viable cells (see, e.g., Crouch et al. , J Immunol Methods 160(1 ):81 -8, 1993).

Determining Proliferation Index and Potency

The potency of a CART cell therapy product can be expressed in terms reflecting the level of antigen-specific proliferation of the product, according to the methods of the invention . This level of proliferation can be compared, for example, to the level of proliferation of a control sample of the CART cell therapy product that is not exposed to antigen-specific stimulation (e.g., unstimulated or stimulated, but in a non-antigen specific manner). Furthermore, the calculations can be normalized based on, for example, the number of cells in the test samples that express the CAR. Based on this information, a Proliferation Index (PI) according to the following expression can be used as a measure of CART cell therapy product potency:

PI = [(proliferation in stimulated group) - (proliferation in unstimulated group)]/

% cells expressing CAR

In the case of the use of nucleotide analog (e.g ., EdU) incorporation into DNA, as a measure of proliferation, and also the use of retrovirally-transduced T cells in the production of a CART cell therapy product, the PI calculation can be expressed as follows: PI = [(%nucleotid8 analog+ in stimulated group)-(%nucleotide analog+ in unstimulated group)]/ % transduction

In either of the examples set forth above, the "unstimulated group" can be replaced with "non- specifically stimulated group" as described elsewhere herein.

Methods known in the art can be used to determine the percentage of cells expressing the CAR (e.g., the level of transduction), for the normalization. For example, in tests utilizing flow cytometry, antibodies against the CAR can be included in the assay and used to quantify the level of CAR expressing cells, relative to the total number of T cells.

The level of antigen-specific in vitro proliferation of CART T cell therapy products correlates with in vivo pharmacokinetic (PK) and pharmacodynamics (PD) properties of the products, as is described further below. PK/PD features of a CART cell preparation that can be considered, according to the invention, include, for example, the Cmax, Tmax, and Area Under the Curve (AUC), which can be determined in clinical samples using standard methods in the art. The relationship between in vitro proliferation of a CART cell therapy product, as reflected in, e.g. , a proliferation index as described above, and the in vivo PK/PD characteristics of the product, can be shown using standard methods such as, for example, the Spearman correlation coefficient method, which can be used to assess linear associations between these features. The methods of the invention thus provide a basis for predicting PK/PD parameters, based on antigen-specific in vitro proliferation as shown, e.g. , by determination of a PI . Chimeric Antigen Receptor Technology

In general, the invention can involve the use of isolated nucleic acid molecules encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain (e.g., antibody or antibody fragment, TCR or TCR fragment) that binds to a tumor antigen as described herein, a transmembrane domain (e.g. , a transmembrane domain described herein), and an intracellular signaling domain (e.g. , an intracellular signaling domain described herein) (e.g. , an intracellular signaling domain comprising a costimulatory domain (e.g. , a costimulatory domain described herein) and/or a primary signaling domain (e.g., a primary signaling domain described herein). In other aspects, the invention involves: host cells containing the above nucleic acids and isolated proteins encoded by such nucleic acid molecules. CAR nucleic acid constructs, encoded proteins, containing vectors, host cells, pharmaceutical compositions, and methods of administration and treatment related to the present invention are disclosed in detail in International Patent Application Publication No. WO201 5142675, which is incorporated by reference in its entirety.

In one aspect, the invention involves the use of isolated nucleic acid molecules encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain (e.g., antibody or antibody fragment, TCR or TCR fragment) that binds to a tumor-supporting antigen (e.g. , a tumor- supporting antigen as described herein), a transmembrane domain (e.g., a transmembrane domain described herein), and an intracellular signaling domain (e.g. , an intracellular signaling domain described herein) (e.g., an intracellular signaling domain comprising a costimulatory domain (e.g. , a costimulatory domain described herein) and/or a primary signaling domain (e.g., a primary signaling domain described herein). In some embodiments, the tumor-supporting antigen is an antigen present on a stromal cell or a myeloid-derived suppressor cell (MDSC). In other aspects, the invention involves polypeptides encoded by such nucleic acids and host cells containing such nucleic acids and/or polypeptides.

Targets

The present invention involves immune effector cells (e.g., T cells, NK cells) that are engineered to contain one or more CARs that direct the immune effector cells to undesired cells (e.g. , cancer cells). This is achieved through an antigen binding domain on the CAR that is specific for a cancer associated antigen. There are two classes of cancer associated antigens (tumor antigens) that can be targeted by the CARs of the instant invention: (1 ) cancer associated antigens that are expressed on the surface of cancer cells; and (2) cancer associated antigens that itself is intracellular, however, a fragment of such antigen (peptide) is presented on the surface of the cancer cells by HC (major histocompatibility complex).

In some embodiments, the tumor antigen is chosen from one or more of: CD19; CD123; CD22; CD30; CD171 ; CS-1 (also referred to as CD2 subset 1 , CRACC, SLA F7, CD31 9, and 1 9A24) ; C-type lectin-like molecule-1 (CLL-1 or CLECL1 ) ; CD33; epidermal growth factor receptor variant I II (EGFRvll l); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGaip(1 -4)bDGicp(1 -1 )Cer) ; TNF receptor family member B cell maturation (BCMA) ; Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)) ; prostate- specific membrane antigen (PSMA) ; Receptor tyrosine kinase-like orphan receptor 1 (ROR1 ) ; Fms-Like Tyrosine Kinase 3 (FLT3) ; Tumor-associated glycoprotein 72 (TAG 72) ; CD38; CD44v6;

Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276) ; KIT (CD1 1 7) ; lnterleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin ; Interleukin 1 1 receptor alpha (IL-1 1 Ra) ; prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21 ); vascular endothelial growth factor receptor 2 (VEG FR2); Lewis(Y) antigen ; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2/neu); Mucin 1 , cell surface associated (MUC1 ) ; epidermal growth factor receptor (EGFR) ; neural cell adhesion molecule (NCAM) ; Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2 ; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX) ; Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2) ; glycoprotein 1 00 (gp1 00) ; oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl) ; tyrosinase; ephrin type-A receptor 2 (EphA2); Fucosyl GM1 ; sialyl Lewis adhesion molecule (sLe) ; ganglioside GM3 (aNeu5Ac(2-3)bDGaip(1 -4)bDG!cp(1 -1 )Cer); transglutaminase 5 (TGS5) ; high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-G D2 ganglioside (OAcGD2) ; Folate receptor beta; tumor endothelial marker 1 (TEM1 /CD248); tumor endothelial marker 7- related (TEM7R); claudin 6 (CLDN6) ; thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (G PRC5D); chromosome X open reading frame 61 (CXORF61 ) ; CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1 ) ;

hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY- BR-1 ); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1 ) ; adrenoceptor beta 3 (ADRB3) ; pannexin 3 (PANX3) ; G protein-coupled receptor 20 (GPR20) ; lymphocyte antigen 6 complex, locus K 9 (LY6K) ; Olfactory receptor 51 E2 (OR51 E2) ; TCR Gamma Alternate Reading Frame Protein (TARP) ; Wilms tumor protein (WT1 ) ; Cancer/testis antigen 1 (NY-ESO-1 ); Cancer/testis antigen 2 (LAGE-1 a); Melanoma-associated antigen 1 (MAG E-A1 ) ; ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML) ; sperm protein 1 7 (SPA1 7) ; X Antigen Family, Member 1 A (XAGE1 ) ; angiopoietin- binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen-1 (MAD-CT-1 ) ; melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1 ; tumor protein p53 (p53) ; p53 mutant; prostein ; surviving; telomerase; prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1 ); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT) ; sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP) ; ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl- transferase V (NA17) ; paired box protein Pax -3 (PAX3) ; Androgen receptor; Cyclin B1 ; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC) ; Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1 B1 (CYP1 B1 ); CCCTC- Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3) ; Paired box protein Pax-5 (PAX5) ; proacrosin binding protein sp32 (OY-TES1 ) ; lymphocyte-specific protein tyrosine kinase (LCK) ; A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2) ; Receptor for Advanced Glycation Endproducts (RAGE-1 ); renal ubiquitous 1 (RU1 ); renal ubiquitous 2 (RU2) ; legumain ; human papilloma virus E6 (HPV E6) ; human papilloma virus E7 (H PV E7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2) ; CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAI R1 ) ; Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2) ; CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A) ; bone marrow stromal cell antigen 2 (BST2) ; EG F-like module-containing mucin-like hormone receptor-like 2 (EMR2) ; lymphocyte antigen 75 (LY75) ; Glypican-3 (G PC3); Fc receptor-like 5 (FCRL5) ; and immunoglobulin lambda-like polypeptide 1 (IGLL1 ).

A CAR described herein can comprise an antigen binding domain (e.g. , antibody or antibody fragment, TCR or TCR fragment) that binds to a tumor-supporting antigen (e.g. , a tumor-supporting antigen as described herein). In some embodiments, the tumor-supporting antigen is an antigen present on a stromal cell or a myeloid-derived suppressor cell (MDSC). Stromal cells can secrete growth factors to promote cell division in the microenvironment. MDSC cells can inhibit T cell proliferation and activation. Without wishing to be bound by theory, in some embodiments, the CAR-expressing cells destroy the tumor-supporting cells, thereby indirectly inhibiting tumor growth or survival.

In embodiments, the stromal cell antigen is chosen from one or more of: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) and tenascin. In an embodiment, the FAP- specific antibody is, competes for binding with, or has the same CDRs as, sibrotuzumab. In

embodiments, the MDSC antigen is chosen from one or more of: CD33, CD1 1 b, C14, CD15, and CD66b. Accordingly, in some embodiments, the tumor-supporting antigen is chosen from one or more of: bone marrow stromal cell antigen 2 (BST2), fibroblast activation protein (FAP) or tenascin, CD33, CD1 1 b, C1 4, CD1 5, and CD66b. Antigen Binding Domain Structures

In some embodiments, the antigen binding domain of the encoded CAR molecule comprises an antibody, an antibody fragment, an scFv, a Fv, a Fab, a (Fab')2, a single domain antibody (SDAB), a VH or VL domain, a camelid VHH domain or a bi-functional (e.g. bi-specific) hybrid antibody (e.g. ,

Lanzavecchia et al. , Eur. J. Immunol. 1 7, 105 (1 987)).

In some instances, scFvs can be prepared according to method known in the art (see, for example, Bird et al. , (1 988) Science 242:423-426 and Huston et al. , (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). ScFv molecules can be produced by linking VH and VL regions together using flexible polypeptide linkers. The scFv molecules comprise a linker (e.g., a Ser-Gly linker) with an optimized length and/or amino acid composition. The linker length can greatly affect how the variable regions of a scFv fold and interact. In fact, if a short polypeptide linker is employed (e.g., between 5-1 0 amino acids) intrachain folding is prevented. Interchain folding is also required to bring the two variable regions together to form a functional epitope binding site. For examples of linker orientation and size see, e.g., Hollinger et al . 1 993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448, U.S. Patent Application Publication Nos. 2005/01 00543, 2005/0175606, 2007/0014794, and PCT publication Nos. WO2006/020258 and WO2007/02471 5, is incorporated herein by reference.

An scFv can comprise a linker of at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 1 5, 16, 1 7, 1 8, 1 9, 20, 25, 30, 35, 40, 45, 50, or more amino acid residues between its VL and VH regions. The linker sequence may comprise any naturally occurring amino acid. In some embodiments, the linker sequence comprises amino acids glycine and serine. In another embodiment, the linker sequence comprises sets of glycine and serine repeats such as (Gly4Ser)n, where n is a positive integer equal to or greater than 1 (SEQ ID NO:22). In one embodiment, the linker can be (Gly4Ser)4 (SEQ ID NO:29) or (Gly4Ser)3(SEQ ID NO:30). Variation in the linker length may retain or enhance activity, giving rise to superior efficacy in activity studies.

In another aspect, the antigen binding domain is a T cell receptor ("TCR"), or a fragment thereof, for example, a single chain TCR (scTCR). Methods to make such TCRs are known in the art. See, e.g. , Willemsen RA et al, Gene Therapy 7: 1369-1377 (2000); Zhang T et al, Cancer Gene Ther 1 1 : 487-496 (2004) ; Aggen et al, Gene Ther. 1 9(4) :365-74 (2012) (references are incorporated herein by its entirety). For example, scTCR can be engineered that contains the Va and νβ genes from a T cell clone linked by a linker (e.g., a flexible peptide). This approach is very useful to cancer associated target that itself is intracellular, however, a fragment of such antigen (peptide) is presented on the surface of the cancer cells by MHC.

In certain embodiments, the encoded antigen binding domain has a binding affinity KD of 1 0-4 M to 10"8 M.

In one embodiment, the encoded CAR molecule comprises an antigen binding domain that has a binding affinity KD of 1 0 4 M to 10 8 M, e.g., 1 0 5 M to 1 0 7 M, e.g., 10 6 M or 10 7 M, for the target antigen. In one embodiment, the antigen binding domain has a binding affinity that is at least five-fold, 1 0-fold, 20- fold, 30-fold, 50-fold, 100-fold or 1 , 000-fold less than a reference antibody, e.g ., an antibody described herein. In one embodiment, the encoded antigen binding domain has a binding affinity at least 5-fold less than a reference antibody (e.g., an antibody from which the antigen binding domain is derived). In one aspect such antibody fragments are functional in that they provide a biological response that can include, but is not limited to, activation of an immune response, inhibition of signal-transduction origination from its target antigen, inhibition of kinase activity, and the like, as will be understood by a skilled artisan.

In one aspect, the antigen binding domain of the CAR is a scFv antibody fragment that is humanized compared to the murine sequence of the scFv from which it is derived.

In one aspect, the antigen binding domain of a CAR of the invention (e.g. , a scFv) is encoded by a nucleic acid molecule whose sequence has been codon optimized for expression in a mammalian cell. In one aspect, entire CAR construct of the invention is encoded by a nucleic acid molecule whose entire sequence has been codon optimized for expression in a mammalian cell. Codon optimization refers to the discovery that the frequency of occurrence of synonymous codons (i.e. , codons that code for the same amino acid) in coding DNA is biased in different species. Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. A variety of codon optimization methods is known in the art, and include, e.g., methods disclosed in at least US Patent Numbers 5,786,464 and 6,1 1 4,1 48.

Specific Antigen Antibody Pairs

In one embodiment, an antigen binding domain against CD22 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Haso et al ., Blood, 121 (7): 1 1 65-1 1 74 (2013) ; Wayne et al., Clin Cancer Res 16(6): 1 894-1 903 (2010); Kato et al., Leuk Res 37(1 ):83-88 (201 3) ; Creative BioMart (creativebiomart.net): MOM-18047-S(P).

In one embodiment, an antigen binding domain against CS-1 is an antigen binding portion, e.g. , CDRs, of Elotuzumab (BMS), see e.g. , Tai et al., 2008, Blood 1 12(4):1329-37; Tai et al. , 2007, Blood. 1 10(5) :1 656-63.

In one embodiment, an antigen binding domain against CLL-1 is an antigen binding portion, e.g., CDRs, of an antibody available from R&D, ebiosciences, Abeam, for example, PE-CLL1 -hu Cat# 353604 (BioLegend); and PE-CLL1 (CLEC12A) Cat# 562566 (BD) .

In one embodiment, an antigen binding domain against CD33 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Bross et al. , Clin Cancer Res 7(6) :1490-1496 (2001 )

(Gemtuzumab Ozogamicin, hP67.6), Caron et al., Cancer Res 52(24) :6761 -6767 (1 992) (Lintuzumab, HuM1 95), Lapusan et al., Invest New Drugs 30(3):1 121 -1 131 (2012) (AVE9633), Aigner et al. , Leukemia 27(5) : 1 107-1 1 1 5 (2013) (AMG330, CD33 BiTE), Dutour et al. , Adv hematol 2012:683065 (2012), and Pizzitola et al., Leukemia doi:1 0.1 038/Lue.2014.62 (2014).

In one embodiment, an antigen binding domain against GD2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Mujoo et al., Cancer Res. 47(4):1 098-1 104 (1987) ; Cheung et al., Cancer Res 45(6) :2642-2649 (1 985), Cheung et al. , J Clin Oncol 5(9):1 430-1440 (1 987), Cheung et al., J Clin Oncol 16(9) :3053-3060 (1 998), Handgretinger et al., Cancer Immunol Immunother 35(3) :199- 204 (1 992). In some embodiments, an antigen binding domain against GD2 is an antigen binding portion of an antibody selected from mAb 14.18, 1 4G2a, ch14.18, hu14.18, 3F8, hu3F8, 3G6, 8B6, 60C3, 1 0B8, ME36.1 , and 8H9, see e.g. , WO2012033885, WO2013040371 , WO20131 92294, WO2013061273, WO2013123061 , WO2013074916, and WO201385552. In some embodiments, an antigen binding domain against GD2 is an antigen binding portion of an antibody described in US Publication No. :

201001 50910 or PCT Publication No. : WO 201 1 1601 1 9.

In one embodiment, an antigen binding domain against BC A is an antigen binding portion, e.g ., CDRs, of an antibody described in, e.g., WO20121 63805, WO2001 12812, and WO2003062401 .

In one embodiment, an antigen binding domain against Tn antigen is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US8, 440,798, Brooks et al., PNAS 1 07(22) :1 0056-1 0061 (201 0), and Stone et al. , Oncolmmunology 1 (6) :863-873(2012).

In one embodiment, an antigen binding domain against PSMA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Parker et al ., Protein Expr Purif 89(2) :136-145 (2013), US 201 10268656 (J591 ScFv) ; Frigerio et al, European J Cancer 49(9) :2223-2232 (2013) (scFvD2B); WO 2006125481 (mAbs 3/A12, 3/E7 and 3/F1 1 ) and single chain antibody fragments (scFv A5 and D7).

In one embodiment, an antigen binding domain against ROR1 is an antigen binding portion, e.g. , CDRs, of an antibody described in, e.g., Hudecek et al. , Clin Cancer Res 1 9(12) :3153-31 64 (201 3) ; WO 201 1 1 59847; and US20130101 607.

In one embodiment, an antigen binding domain against FLT3 is an antigen binding portion, e.g. ,

CDRs, of an antibody described in, e.g., WO201 1076922, US5777084, EP0754230, US20090297529, and several commercial catalog antibodies (R&D, ebiosciences, Abeam).

In one embodiment, an antigen binding domain against TAG72 is an antigen binding portion, e.g. , CDRs, of an antibody described in, e.g., Hombach et al ., Gastroenterology 1 13(4):1 1 63-1 170 (1 997); and Abeam ab691 .

In one embodiment, an antigen binding domain against FAP is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Ostermann et al., Clinical Cancer Research 14:4584-4592 (2008) (FAP5), US Pat. Publication No. 2009/030471 8; sibrotuzumab (see e.g., Hofheinz et al., Oncology Research and Treatment 26(1 ), 2003) ; and Tran et al. , J Exp Med 210(6):1 125-1 135 (2013).

In one embodiment, an antigen binding domain against CD38 is an antigen binding portion, e.g.,

CDRs, of daratumumab (see, e.g., Groen et al., Blood 1 16(21 ):1261 -1262 (201 0) ; MOR202 (see, e.g., US8263746) ; or antibodies described in US836221 1 .

In one embodiment, an antigen binding domain against CD44v6 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Casucci et al. , Blood 122(20) :3461 -3472 (201 3).

In one embodiment, an antigen binding domain against CEA is an antigen binding portion, e.g.,

CDRs, of an antibody described in, e.g., Chmielewski et al., Gastroenterology 1 43(4) :1095-1 1 07 (2012).

In one embodiment, an antigen binding domain against EPCAM is an antigen binding portion, e.g., CDRS, of an antibody selected from MT1 1 0, EpCAM-CD3 bispecific Ab (see, e.g. ,

clinicaltrials.gov/ct2/show/NCT00635596); Edrecolomab; 3622W94; ING-1 ; and adecatumumab (MT201 ).

In one embodiment, an antigen binding domain against PRSS21 is an antigen binding portion, e.g., CDRs, of an antibody described in US Patent No. : 8,080,650.

In one embodiment, an antigen binding domain against B7H3 is an antigen binding portion, e.g., CDRs, of an antibody MGA271 (Macrogenics). In one embodiment, an antigen binding domain against KIT is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US7915391 , US20120288506, and several commercial catalog antibodies.

In one embodiment, an antigen binding domain against IL-13Ra2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., WO2008/14691 1 , WO2004087758, several commercial catalog antibodies, and WO2004087758.

In one embodiment, an antigen binding domain against CD30 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US7090843 B1 , and EP0805871 .

In one embodiment, an antigen binding domain against GD3 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US7253263; US 8,207,308; US 20120276046; EP1013761 ; WO2005035577; and US6437098.

In one embodiment, an antigen binding domain against CD171 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Hong et al., J Immunother 37(2):93-104 (2014).

In one embodiment, an antigen binding domain against IL-1 1 Ra is an antigen binding portion, e.g., CDRs, of an antibody available from Abeam (cat# ab55262) or Novus Biologicals (cat# EPR5446). In another embodiment, an antigen binding domain again IL-1 1 Ra is a peptide, see, e.g., Huang et al., Cancer Res 72(1 ):271 -281 (2012).

In one embodiment, an antigen binding domain against PSCA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Morgenroth et al., Prostate 67(10):1 121 -1 131 (2007) (scFv 7F5); Nejatollahi et al., J of Oncology 2013(2013), article ID 839831 (scFv C5-II); and US Pat Publication No. 2009031 1 181 .

In one embodiment, an antigen binding domain against VEGFR2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Chinnasamy et al., J Clin Invest 120(1 1 ):3953-3968 (2010).

In one embodiment, an antigen binding domain against LewisY is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Kelly et al., Cancer Biother Radiopharm 23(4) :41 1 -423 (2008) (hu3S193 Ab (scFvs)); Dolezal et al., Protein Engineering 16(1 ):47-56 (2003) (NC10 scFv).

In one embodiment, an antigen binding domain against CD24 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Maliar et al., Gastroenterology 143(5):1375-1384 (2012).

In one embodiment, an antigen binding domain against PDGFR-beta is an antigen binding portion, e.g., CDRs, of an antibody Abeam ab32570.

In one embodiment, an antigen binding domain against SSEA-4 is an antigen binding portion, e.g., CDRs, of antibody C813 (Cell Signaling), or other commercially available antibodies.

In one embodiment, an antigen binding domain against CD20 is an antigen binding portion, e.g., CDRs, of the antibody Rituximab, Ofatumumab, Ocrelizumab, Veltuzumab, or GA101 .

In one embodiment, an antigen binding domain against Folate receptor alpha is an antigen binding portion, e.g., CDRs, of the antibody I GN853, or an antibody described in US20120009181 ; US4851332, LK26: US5952484.

In one embodiment, an antigen binding domain against ERBB2 (Her2/neu) is an antigen binding portion, e.g., CDRs, of the antibody trastuzumab, or pertuzumab. In one embodiment, an antigen binding domain against MUC1 is an antigen binding portion, e.g., CDRs, of the antibody SAR566658.

In one embodiment, the antigen binding domain against EGFR is antigen binding portion, e.g., CDRs, of the antibody cetuximab, panitumumab, zalutumumab, nimotuzumab, or matuzumab.

In one embodiment, an antigen binding domain against NCAM is an antigen binding portion, e.g.,

CDRs, of the antibody clone 2-2B: MAB5324 (EMD Millipore)

In one embodiment, an antigen binding domain against Ephrin B2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Abengozar et al., Blood 1 19(19):4565-4576 (2012).

In one embodiment, an antigen binding domain against IGF-I receptor is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US83441 12 B2; EP2322550 A1 ; WO 2006/138315, or PCT/US2006/022995.

In one embodiment, an antigen binding domain against CAIX is an antigen binding portion, e.g., CDRs, of the antibody clone 303123 (R&D Systems).

In one embodiment, an antigen binding domain against LMP2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US7410640, or US20050129701 .

In one embodiment, an antigen binding domain against gp100 is an antigen binding portion, e.g., CDRs, of the antibody HMB45, NKIbetaB, or an antibody described in WO2013165940, or

US20130295007

In one embodiment, an antigen binding domain against tyrosinase is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US5843674; or US19950504048.

In one embodiment, an antigen binding domain against EphA2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Yu et al., Mol Ther 22(1 ):102-1 1 1 (2014).

In one embodiment, an antigen binding domain against GD3 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US7253263; US 8,207,308; US 20120276046; EP1013761 A3; 20120276046; WO2005035577; or US6437098.

In one embodiment, an antigen binding domain against fucosyl GM1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US20100297138; or WO2007/067992.

In one embodiment, an antigen binding domain against sLe is an antigen binding portion, e.g., CDRs, of the antibody G193 (for lewis Y), see Scott AM et al, Cancer Res 60: 3254-61 (2000), also as described in Neeson et al, J Immunol May 2013 190 (Meeting Abstract Supplement) 177.10.

In one embodiment, an antigen binding domain against GM3 is an antigen binding portion, e.g., CDRs, of the antibody CA 2523449 (mAb 14F7).

In one embodiment, an antigen binding domain against HMWMAA is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Kmiecik et al., Oncoimmunology 3(1 ):e27185 (2014) (PMID: 24575382) (mAb9.2.27); US6528481 ; WO2010033866; or US 20140004124.

In one embodiment, an antigen binding domain against o-acetyl-GD2 is an antigen binding portion, e.g., CDRs, of the antibody 8B6.

In one embodiment, an antigen binding domain against TEM1/CD248 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Marty et al., Cancer Lett 235(2) :298-308 (2006); Zhao et al., J Immunol Methods 363(2):221 -232 (201 1 ). In one embodiment, an antigen binding domain against CLDN6 is an antigen binding portion, e.g., CDRs, of the antibody IMAB027 (Ganymed Pharmaceuticals), see e.g.,

clinicaltrial.gov/show/NCT02054351 .

In one embodiment, an antigen binding domain against TSHR is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US8603466; US8501415; or US8309693.

In one embodiment, an antigen binding domain against GPRC5D is an antigen binding portion, e.g., CDRs, of the antibody FAB6300A (R&D Systems); or LS-A4180 (Lifespan Biosciences).

In one embodiment, an antigen binding domain against CD97 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., US6, 846,91 1 ;de Groot et al., J Immunol 183(6) :4127-4134 (2009); or an antibody from R&D:MAB3734.

In one embodiment, an antigen binding domain against ALK is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Mino-Kenudson et al., Clin Cancer Res 16(5):1561 -1571 (2010).

In one embodiment, an antigen binding domain against polysialic acid is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Nagae et al., J Biol Chem 288(47) :33784-33796 (2013).

In one embodiment, an antigen binding domain against PLAC1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Ghods et al., Biotechnol AppI Biochem 2013

doi:10.1002/bab.1 177.

In one embodiment, an antigen binding domain against GloboH is an antigen binding portion of the antibody VK9; or an antibody described in, e.g., Kudryashov V et al, Glycoconj J.15(3):243-9 ( 1998), Lou et al., Proc Natl Acad Sci USA 1 1 1 (7):2482-2487 (2014) ; MBM : Bremer E-G et al. J Biol Chem 259:14773-14777 (1984).

In one embodiment, an antigen binding domain against NY-BR-1 is an antigen binding portion, e.g., CDRs of an antibody described in, e.g., Jager et al., AppI Immunohistochem Mol Morphol 15(1 ):77- 83 (2007).

In one embodiment, an antigen binding domain against WT-1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Dao et al., Sci Transl Med 5(1 76):176ra33 (2013); or

WO2012/135854.

In one embodiment, an antigen binding domain against MAGE-A1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Willemsen et al., J Immunol 174(12):7853-7858 (2005) (TCR-like scFv).

In one embodiment, an antigen binding domain against sperm protein 17 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Song et al., Target Oncol 2013 Aug 14 (PMID: 23943313); Song et al., Med Oncol 29(4):2923-2931 (2012).

In one embodiment, an antigen binding domain against Tie 2 is an antigen binding portion, e.g.,

CDRs, of the antibody AB33 (Cell Signaling Technology).

In one embodiment, an antigen binding domain against MAD-CT-2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., PMID: 2450952; US7635753.

In one embodiment, an antigen binding domain against Fos-related antigen 1 is an antigen binding portion, e.g., CDRs, of the antibody 12F9 (Novus Biologicals). In one embodiment, an antigen binding domain against MelanA/MART1 is an antigen binding portion, e.g., CDRs, of an antibody described in, EP2514766 A2; or US 7,749,719.

In one embodiment, an antigen binding domain against sarcoma translocation breakpoints is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Luo et al, EMBO Mol. Med.

4(6):453-461 (2012).

In one embodiment, an antigen binding domain against TRP-2 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Wang et al, J Exp Med. 184(6):2207-16 (1996).

In one embodiment, an antigen binding domain against CYP1 B1 is an antigen binding portion, e.g., CDRs, of an antibody described in, e.g., Maecker et al, Blood 102 (9): 3287-3294 (2003).

In one embodiment, an antigen binding domain against RAGE-1 is an antigen binding portion, e.g., CDRs, of the antibody MAB5328 (EMD Millipore).

In one embodiment, an antigen binding domain against human telomerase reverse transcriptase is an antigen binding portion, e.g., CDRs, of the antibody cat no: LS-B95-100 (Lifespan Biosciences)

In one embodiment, an antigen binding domain against intestinal carboxyl esterase is an antigen binding portion, e.g., CDRs, of the antibody 4F12: cat no: LS-B6190-50 (Lifespan Biosciences).

In one embodiment, an antigen binding domain against mut hsp70-2 is an antigen binding portion, e.g., CDRs, of the antibody Lifespan Biosciences: monoclonal: cat no: LS-C133261 -100

(Lifespan Biosciences).

In one embodiment, an antigen binding domain against CD79a is an antigen binding portion, e.g., CDRs, of the antibody Anti-CD79a antibody [HM47/A9] (ab3121 ), available from Abeam; antibody CD79A Antibody #3351 available from Cell Signalling Technology; or antibody HPA01 7748 - Anti-CD79A antibody produced in rabbit, available from Sigma Aldrich.

In one embodiment, an antigen binding domain against CD79b is an antigen binding portion, e.g., CDRs, of the antibody polatuzumab vedotin, anti-CD79b described in Dornan et al., "Therapeutic potential of an anti-CD79b antibody-drug conjugate, anti-CD79b-vc-MMAE, for the treatment of non- Hodgkin lymphoma" Blood. 2009 Sep 24;1 14(13):2721 -9. doi: 10.1 182/blood-2009-02-205500. Epub 2009 Jul 24, or the bispecific antibody Anti-CD79b/CD3 described in "4507 Pre-Clinical Characterization of T Cell-Dependent Bispecific Antibody Anti-CD79b/CD3 As a Potential Therapy for B Cell Malignancies" Abstracts of 56th ASH Annual Meeting and Exposition, San Francisco, CA December 6-9 2014.

In one embodiment, an antigen binding domain against CD72 is an antigen binding portion, e.g.,

CDRs, of the antibody J3-109 described in Myers, and Uckun, "An anti-CD72 immunotoxin against therapy-refractory B-lineage acute lymphoblastic leukemia." Leuk Lymphoma. 1995 Jun;18(1 -2):1 19-22, or anti-CD72 (10D6.8.1 , mlgG1 ) described in Poison et al., "Antibody-Drug Conjugates for the Treatment of Non-Hodgkin's Lymphoma: Target and Linker-Drug Selection" Cancer Res March 15, 2009 69; 2358.

In one embodiment, an antigen binding domain against LAIR1 is an antigen binding portion, e.g.,

CDRs, of the antibody ANT-301 LAIR1 antibody, available from ProSpec; or anti-human CD305 (LAIR1 ) Antibody, available from BioLegend.

In one embodiment, an antigen binding domain against FCAR is an antigen binding portion, e.g., CDRs, of the antibody CD89/FCARAntibody (Catalog#10414-H08H), available from Sino Biological Inc. In one embodiment, an antigen binding domain against LILRA2 is an antigen binding portion, e.g., CDRs, of the antibody LILRA2 monoclonal antibody (M17), clone 3C7, available from Abnova, or Mouse Anti-LILRA2 antibody, Monoclonal (2D7), available from Lifespan Biosciences.

In one embodiment, an antigen binding domain against CD300LF is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-CMRF35-like molecule 1 antibody, Monoclonal[UP-D2], available from BioLegend, or Rat Anti-CMRF35-like molecule 1 antibody, Monoclonal[234903], available from R&D Systems.

In one embodiment, an antigen binding domain against CLEC12A is an antigen binding portion, e.g., CDRs, of the antibody Bispecific T cell Engager (BiTE) scFv-antibody and ADC described in Noordhuis et al., "Targeting of CLEC1 2A In Acute Myeloid Leukemia by Antibody-Drug-Conjugates and Bispecific CLL-1 xCD3 BiTE Antibody" 53rd ASH Annual Meeting and Exposition, December 10-13, 201 1 , and MCLA-1 1 7 (Merus).

In one embodiment, an antigen binding domain against BST2 (also called CD31 7) is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-CD31 7 antibody, Monoclonal[3H4], available from Antibodies-Online or Mouse Anti-CD31 7 antibody, Monoclonal[696739], available from R&D Systems.

In one embodiment, an antigen binding domain against EMR2 (also called CD312) is an antigen binding portion, e.g., CDRs, of the antibody Mouse Anti-CD312 antibody, Monoclonal [LS-B8033] available from Lifespan Biosciences, or Mouse Anti-CD312 antibody, Monoclonal [494025] available from R&D Systems.

In one embodiment, an antigen binding domain against LY75 is an antigen binding portion, e.g. ,

CDRs, of the antibody Mouse Anti-Lymphocyte antigen 75 antibody, Monoclonal [HD30] available from EMD Millipore or Mouse Anti-Lymphocyte antigen 75 antibody, Monoclonal [A15797] available from Life Technologies.

In one embodiment, an antigen binding domain against GPC3 is an antigen binding portion, e.g., CDRs, of the antibody hGC33 described in Nakano K, Ishiguro T, Konishi H, et al . Generation of a humanized anti-glypican 3 antibody by CDR grafting and stability optimization. Anticancer Drugs. 201 0 Nov;21 (1 0):907-91 6, or MDX-1414, HN3, or YP7, all three of which are described in Feng et al., "Glypican-3 antibodies: a new therapeutic target for liver cancer." FEBS Lett. 2014 Jan 21 ;588(2):377-82.

In one embodiment, an antigen binding domain against FCRL5 is an antigen binding portion, e.g. , CDRs, of the anti-FcRL5 antibody described in Elkins et al., "FcRL5 as a target of antibody-drug conjugates for the treatment of multiple myeloma" Mol Cancer Ther. 2012 Oct;1 1 (10):2222-32. .

In one embodiment, an antigen binding domain against IGLL1 is an antigen binding portion, e.g. , CDRs, of the antibody Mouse Anti-lmmunoglobulin lambda-like polypeptide 1 antibody, Monoclonal [AT1 G4] available from Lifespan Biosciences, Mouse Anti-lmmunoglobulin lambda-like polypeptide 1 antibody, Monoclonal [HSL1 1 ] available from BioLegend.

In one embodiment, the antigen binding domain comprises one, two three (e.g., all three) heavy chain CDRs, HC CDR1 , HC CDR2 and HC CDR3, from an antibody listed above, and/or one, two, three (e.g., all three) light chain CDRs, LC CDR1 , LC CDR2 and LC CDR3, from an antibody listed above. In one embodiment, the antigen binding domain comprises a heavy chain variable region and/or a variable light chain region of an antibody listed above. In another aspect, the antigen binding domain comprises a humanized antibody or an antibody fragment. In some aspects, 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 or fragment thereof. In one aspect, the antigen binding domain is humanized.

Bispecific CARs

In certain embodiments, the antigen binding domain is a bi- or multi- specific molecule (e.g., a multispecific antibody molecule). 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. In an embodiment the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In an embodiment the first and second epitopes overlap. In an embodiment the first and second epitopes do not overlap. In an embodiment the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In an embodiment a bispecific antibody molecule comprises a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a first epitope and a heavy chain variable domain sequence and a light chain variable domain sequence which have binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a half antibody having binding specificity for a first epitope and a half antibody having binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a half antibody, or fragment thereof, having binding specificity for a first epitope and a half antibody, or fragment thereof, having binding specificity for a second epitope. In an embodiment a bispecific antibody molecule comprises a scFv, or fragment thereof, have binding specificity for a first epitope and a scFv, or fragment thereof, have binding specificity for a second epitope.

In certain embodiments, the antibody molecule is a multi-specific {e.g., a bispecific or a trispecific) antibody molecule. Such molecules include bispecific fusion proteins, e.g., an expression construct containing two scFvs with a hydrophilic helical peptide linker between them and a full constant region , as described in, e.g., US5637481 ; minibody constructs with linked VL and VH chains further connected with peptide spacers to an antibody hinge region and CH3 region, which can be dimerized to form

bispecific/multivalent molecules, as described in, e.g., US5837821 ; String of VH domains (or VL domains in family members) connected by peptide linkages with crosslinkable groups at the C-terminus further associated with VL domains to form a series of FVs (or scFvs), as described in, e.g., US586401 9; and single chain binding polypeptides with both a VH and a VL domain linked through a peptide linker are combined into multivalent structures through non-covalent or chemical crosslinking to form, e.g., homobivalent, heterobivalent, trivalent, and tetravalent structures using both scFV or diabody type format, as described in, e.g., US5869620. The contents of the above-referenced applications are incorporated herein by reference in their entireties.

Within each antibody or antibody fragment (e.g., scFv) of a bispecific antibody molecule, the VH can be upstream or downstream of the VL. In some embodiments, the upstream antibody or antibody fragment (e.g. , scFv) is arranged with its VH (VHi) upstream of its VL (VLi) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL2) upstream of its VH (VH2), such that the overall bispecific antibody molecule has the arrangement VH1 -VL1 -VL2-VH2. In other embodiments, the upstream antibody or antibody fragment (e.g. , scFv) is arranged with its VL (VLi) upstream of its VH (VHi) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH2) upstream of its VL (VL2), such that the overall bispecific antibody molecule has the arrangement VL1 -VH1- VH2-VL2. Optionally, a linker is disposed between the two antibodies or antibody fragments (e.g. , scFvs), e.g., between VLi and VL2 if the construct is arranged as VH1-VL1-VL2-VH2, or between VHi and VH2 if the construct is arranged as VL1-VH1 -VH2-VL2. The linker may be a linker as described herein, e.g., a (Gly4-Ser)n linker, wherein n is 1 , 2, 3, 4, 5, or 6, preferably 4. In general, the linker between the two scFvs should be long enough to avoid mispairing between the domains of the two scFvs. Optionally, a linker is disposed between the VL and VH of the first scFv. Optionally, a linker is disposed between the VL and VH of the second scFv. In constructs that have multiple linkers, any two or more of the linkers can be the same or different. Accordingly, in some embodiments, a bispecific CAR comprises VLs, VHs, and optionally one or more linkers in an arrangement as described herein.

Transmembrane domains

With respect to the transmembrane domain, in various embodiments, a CAR can be designed to comprise a transmembrane domain that is attached to the extracellular domain of the CAR. A transmembrane domain can include one or more additional amino acids adjacent to the transmembrane region, e.g., one or more amino acid associated with the extracellular region of the protein from which the transmembrane was derived (e.g. , 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 0 up to 15 amino acids of the extracellular region) and/or one or more additional amino acids associated with the intracellular region of the protein from which the transmembrane protein is derived (e.g. , 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 0 up to 15 amino acids of the intracellular region). In one aspect, the transmembrane domain is one that is associated with one of the other domains of the CAR e.g., in one embodiment, the transmembrane domain may be from the same protein that the signaling domain, costimulatory domain or the hinge domain is derived from . In another aspect, the transmembrane domain is not derived from the same protein that any other domain of the CAR is derived from. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, e.g., to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerization with another CAR on the cell surface of a CAR-expressing cell. In a different aspect, the amino acid sequence of the transmembrane domain may be modified or substituted so as to minimize interactions with the binding domains of the native binding partner present in the same CAR-expressing cell.

The transmembrane domain may be derived either from a natural or from a recombinant source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. In one aspect the transmembrane domain is capable of signaling to the intracellular domain(s) whenever the CAR has bound to a target. A transmembrane domain of particular use in this invention may include at least the transmembrane region(s) of e.g., the alpha, beta or zeta chain of the T-cell receptor, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. In some embodiments, a transmembrane domain may include at least the transmembrane region(s) of, e.g., KIRDS2, OX40, CD2, CD27, LFA-1 (CD1 1 a, CD18), ICOS (CD278), 4-1 BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLA F7, NKp80 (KLRF1 ), NKp44, NKp30, NKp46, CD160, CD19, IL2R beta, IL2R gamma, IL7R a, ITGA1 , VLA1 , CD49a, ITGA4, IA4,

CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1 d, ITGAE, CD103, ITGAL, CD1 1 a, LFA-1 , ITGAM, CD1 1 b, ITGAX, CD1 1 c, ITGB1 , CD29, ITGB2, CD18, LFA-1 , ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACA 1 , CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKG2D, NKG2C.

In some instances, the transmembrane domain can be attached to the extracellular region of the CAR, e.g., the antigen binding domain of the CAR, via a hinge, e.g., a hinge from a human protein. For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge (e.g., an lgG4 hinge, an IgD hinge), a GS linker (e.g., a GS linker described herein), a KIR2DS2 hinge or a CD8a hinge. In one embodiment, the hinge or spacer comprises (e.g., consists of) the amino acid sequence of SEQ ID NO:4. In one aspect, the transmembrane domain comprises (e.g., consists of) a transmembrane domain of SEQ ID NO: 12.

In certain embodiments, the encoded transmembrane domain comprises an amino acid sequence of a CD8 transmembrane domain having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO: 12, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO: 12. In one embodiment, the encoded transmembrane domain comprises the sequence of SEQ ID NO: 12.

In other embodiments, the nucleic acid molecule encoding the CAR comprises a nucleotide sequence of a CD8 transmembrane domain, e.g., comprising the sequence of SEQ ID NO: 13, or a sequence with 95-99% identity thereof.

In certain embodiments, the encoded antigen binding domain is connected to the transmembrane domain by a hinge region. In one embodiment, the encoded hinge region comprises the amino acid sequence of a CD8 hinge, e.g., SEQ ID NO: 4; or the amino acid sequence of an lgG4 hinge, e.g., SEQ ID NO: 6, or a sequence with 95-99% identity to SEQ ID NO:4 or 6. In other embodiments, the nucleic acid sequence encoding the hinge region comprises a sequence of SEQ ID NO: 5 or SEQ ID NO: 7, corresponding to a CD8 hinge or an lgG4 hinge, respectively, or a sequence with 95-99% identity to SEQ ID NO:5 or 7.

In one aspect, the hinge or spacer comprises an lgG4 hinge. For example, in one embodiment, the hinge or spacer comprises a hinge of the amino acid sequence

ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNA KTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCS VMHEALHNHYTQKSLSLSLGKM (SEQ ID NO:6). In some embodiments, the hinge or spacer comprises a hinge encoded by a nucleotide sequence of

GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTTCCTGGGCGGACCCAGCGT GTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGT GGTGGTGGACGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGG TGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAGTTCAATAGCACCTACCGGGTGGTGTCCGTG CTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAGGGC CTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCTCGGGAGCCCCAGGTGTA CACCCTGCCCCCTAGCCAAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGG GCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAG ACCACCCCCCCTGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCCGGCTGACCGTGGACAAG AGCCGGTGGCAGGAGGGCAACGTCTTTAGCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTAC ACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG (SEQ ID N0:7).

In one aspect, the hinge or spacer comprises an IgD hinge. For example, in one embodiment, the hinge or spacer comprises a hinge of the amino acid sequence

RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEKEKEEQEERETKTPECPSHTQ PLGVYLLTPAVQDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLT LPRSLWNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEVSGFSPPNIL LMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLE VSYVTDH (SEQ ID NO:8). In some embodiments, the hinge or spacer comprises a hinge encoded by a nucleotide sequence of

AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGCACAGCCCCAGGCAGAAGG CAGCCTAGCCAAAGCTACTACTGCACCTGCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAA GAAAAAGGAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCTGAATGTCCATCCCA TACCCAGCCGCTGGGCGTCTATCTCTTGACTCCCGCAGTACAGGACTTGTGGCTTAGAGATAAGGC CACCTTTACATGTTTCGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTTGCCGGA AAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAGCGCCATTCCAATGGCTCTCAGAGCCA GCACTCAAGACTCACCCTTCCGAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAAT CATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCCGCCCAGGCACCAGTTAAG CTTAGCCTGAATCTGCTCGCCAGTAGTGATCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTG TCCGGCTTTAGCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGTGAACACCAGC GGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTTCTACCACATTCTGGGCCTGGAGTGTCTTA AGGGTCCCAGCACCACCTAGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAGC AGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGACTGACCATT (SEQ ID NO:9).

In one aspect, the transmembrane domain may be recombinant, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. In one aspect a triplet of phenylalanine, tryptophan and valine can be found at each end of a recombinant transmembrane domain.

Optionally, a short oligo- or polypeptide linker, between 2 and 1 0 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic region of the CAR. A glycine-serine doublet provides a particularly suitable linker. For example, in one aspect, the linker comprises the amino acid sequence of GGGGSGGGGS (SEQ ID NO:10). In some embodiments, the linker is encoded by a nucleotide sequence of GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC (SEQ ID NO:1 1 ).

In one aspect, the hinge or spacer comprises a KIR2DS2 hinge. Signaling domains

In embodiments of the invention having an intracellular signaling domain, such a domain can contain, e.g., one or more of a primary signaling domain and/or a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a sequence encoding a primary signaling domain. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a primary signaling domain and a costimulatory signaling domain .

The intracellular signaling sequences within the cytoplasmic portion of the CAR of the invention may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, for example, between 2 and 10 amino acids (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids) in length may form the linkage between intracellular signaling sequences. In one embodiment, a glycine-serine doublet can be used as a suitable linker. In one embodiment, a single amino acid, e.g., an alanine, a glycine, can be used as a suitable linker.

In one aspect, the intracellular signaling domain is designed to comprise two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains. In an embodiment, the two or more, e.g., 2, 3, 4, 5, or more, costimulatory signaling domains, are separated by a linker molecule, e.g., a linker molecule described herein. In one embodiment, the intracellular signaling domain comprises two costimulatory signaling domains. In some embodiments, the linker molecule is a glycine residue. In some

embodiments, the linker is an alanine residue.

Primary Signaling domains

A primary signaling domain regulates primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary intracellular signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITA s.

Examples of ITA containing primary intracellular signaling domains that are of particular use in the invention include those of CD3 zeta, common FcR gamma (FCER1 G), Fc gamma Rlla, FcR beta (Fc Epsilon R1 b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP10, and DAP12. In one embodiment, a CAR of the invention comprises an intracellular signaling domain, e.g., a primary signaling domain of CD3-zeta.

In one embodiment, the encoded primary signaling domain comprises a functional signaling domain of CD3 zeta. The encoded CD3 zeta primary signaling domain can comprise an amino acid sequence having at least one, two or three modifications but not more than 20, 1 0 or 5 modifications of an amino acid sequence of SEQ ID NO: 18 or SEQ ID NO: 20, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO:1 8 or SEQ I D NO: 20. In some embodiments, the encoded primary signaling domain comprises a sequence of SEQ I D NO:18 or SEQ ID NO: 20. In other embodiments, the nucleic acid sequence encoding the primary signaling domain comprises a sequence of SEQ I D NO:19 or SEQ ID NO: 21 , or a sequence with 95-99% identity thereof. Cost imulatory Signaling Domains

In some embodiments, the encoded intracellular signaling domain comprises a costimulatory signaling domain. For example, the intracellular signaling domain can comprise a primary signaling domain and a costimulatory signaling domain. In some embodiments, the encoded costimulatory signaling domain comprises a functional signaling domain of a protein chosen from one or more of CD27, CD28, 4-1 BB (CD137), OX40, CD30, CD40, PD-1 , ICOS, lymphocyte function-associated antigen-1 (LFA-1 ), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1 , GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1 ), CD160, CD1 9, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1 , CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1 d, ITGAE, CD103, ITGAL, CD1 1 a, LFA-1 , ITGA , CD1 1 b, ITGAX, CD1 1 c, ITGB1 , CD29, ITGB2, CD18, LFA-1 , ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1 , CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1 , CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, or NKG2D.

In certain embodiments, the encoded costimulatory signaling domain comprises an amino acid sequence having at least one, two or three modifications but not more than 20, 10 or 5 modifications of an amino acid sequence of SEQ ID NO:14 or SEQ ID NO: 16, or a sequence with 95-99% identity to an amino acid sequence of SEQ ID NO:14 or SEQ ID NO: 16. In one embodiment, the encoded costimulatory signaling domain comprises a sequence of SEQ ID NO: 14 or SEQ ID NO: 16. In other embodiments, the nucleic acid sequence encoding the costimulatory signaling domain comprises a sequence of SEQ ID NO:15 or SEQ ID NO: 17, or a sequence with 95-99% identity thereof.

In other embodiments, the encoded intracellular domain comprises the sequence of SEQ ID NO: 14 or SEQ ID NO: 16, and the sequence of SEQ ID NO: 18 or SEQ ID NO: 20, wherein the sequences comprising the intracellular signaling domain are expressed in the same frame and as a single polypeptide chain.

In certain embodiments, the nucleic acid sequence encoding the intracellular signaling domain comprises a sequence of SEQ ID NO:15 or SEQ ID NO: 17, or a sequence with 95-99% identity thereof, and a sequence of SEQ ID NO:19 or SEQ ID NO:21 , or a sequence with 95-99% identity thereof.

In some embodiments, the nucleic acid molecule further encodes a leader sequence. In one embodiment, the leader sequence comprises the sequence of SEQ ID NO: 2.

In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1 BB. In one aspect, the signaling domain of 4-1 BB is a signaling domain of SEQ ID NO: 14. In one aspect, the signaling domain of CD3-zeta is a signaling domain of SEQ ID NO: 18.

In one aspect, the intracellular signaling domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD27. In one aspect, the signaling domain of CD27 comprises an amino acid sequence of QRRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP (SEQ ID NO:16). In one aspect, the signaling domain of CD27 is encoded by a nucleic acid sequence of AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCC CACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ I D NO:1 7).

Vectors

In another aspect, the invention includes the use of vectors comprising a nucleic acid sequence encoding a CAR described herein. In one embodiment, the vector is chosen from a DNA vector, an RNA vector, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector. In one embodiment, the vector is a lentivirus vector.

The present invention also provides for the use of vectors in which a DNA used in the present invention is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco- retroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity. A retroviral vector may also be, e.g., a gammaretroviral vector. A gammaretroviral vector may include, e.g. , a promoter, a packaging signal (ψ), a primer binding site (PBS), one or more (e.g., two) long terminal repeats (LTR), and a transgene of interest, e.g. , a gene encoding a CAR. A gammaretroviral vector may lack viral structural gens such as gag, pol, and env. Exemplary gammaretroviral vectors include Murine Leukemia Virus (MLV), Spleen-Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Virus (MPSV), and vectors derived therefrom. Other gammaretroviral vectors are described, e.g., in Tobias Maetzig et al. , "Gammaretroviral Vectors: Biology, Technology and Application" Viruses. 201 1 Jun ; 3(6): 677-713.

In another embodiment, the vector comprising the nucleic acid encoding the desired CAR of the invention is an adenoviral vector (A5/35). In another embodiment, the expression of nucleic acids encoding CARs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See below June et al. 2009 Nature Reviews Immunology 9.1 0: 704-71 6, is incorporated herein by reference.

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

Disclosed herein are methods for producing an in vitro transcribed RNA CAR. The present invention also includes a CAR encoding RNA construct that can be directly transfected into a cell. A method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3' and 5' untranslated sequence ("UTR"), a 5' cap and/or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length. RNA so produced can efficiently transfect different kinds of cells. In one aspect, the template includes sequences for the CAR.

Non-viral delivery methods

In some aspects, non-viral methods can be used to deliver a nucleic acid encoding a CAR described herein into a cell or tissue or a subject. In some embodiments, the non-viral method includes the use of a transposon (also called a transposable element). In some embodiments, a transposon is a piece of DNA that can insert itself at a location in a genome, for example, a piece of DNA that is capable of self-replicating and inserting its copy into a genome, or a piece of DNA that can be spliced out of a longer nucleic acid and inserted into another place in a genome. For example, a transposon comprises a DNA sequence made up of inverted repeats flanking genes for transposition.

In some embodiments, cells, e.g., T or NK cells, are generated that express a CAR described herein by using a combination of gene insertion using the SBTS and genetic editing using a nuclease (e.g., Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR/Cas system, or engineered meganuclease re-engineered homing endonucleases).

In some embodiments, use of a non-viral method of delivery permits reprogramming of cells, e.g. , T or NK cells, and direct infusion of the cells into a subject. Advantages of non-viral vectors include but are not limited to the ease and relatively low cost of producing sufficient amounts required to meet a patient population, stability during storage, and lack of immunogenicity.

Inhibitory domains

In an embodiment, the vector comprises a nucleic acid sequence that encodes a CAR, e.g., a CAR described herein, and a nucleic acid sequence that encodes an inhibitory molecule comprising : an inhKIR cytoplasmic domain; a transmembrane domain, e.g., a KI R transmembrane domain ; and an inhibitor cytoplasmic domain, e.g., an ITIM domain, e.g. , an inhKIR ITIM domain. In an embodiment the inhibitory molecule is a naturally occurring inhKIR, or a sequence sharing at least 50, 60, 70, 80, 85, 90, 95, or 99% homology with, or that differs by no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 1 5, or 20 residues from, a naturally occurring inhKIR.

In an embodiment, the nucleic acid sequence that encodes an inhibitory molecule comprises: a SLAM family cytoplasmic domain ; a transmembrane domain, e.g., a SLAM family transmembrane domain ; and an inhibitor cytoplasmic domain, e.g., a SLAM family domain, e.g., an SLAM family ITIM domain. In an embodiment the inhibitory molecule is a naturally occurring SLAM family member, or a sequence sharing at least 50, 60, 70, 80, 85, 90, 95, or 99% homology with, or that differs by no more than 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 5, or 20 residues from, a naturally occurring SLAM family member.

In one embodiment, the vector is an in vitro transcribed vector, e.g., a vector that transcribes RNA of a nucleic acid molecule described herein. In one embodiment, the nucleic acid sequence in the vector further comprises a poly(A) tail, e.g. , a poly A tail. In one embodiment, the nucleic acid sequence in the vector further comprises a 3'UTR, e.g., a 3' UTR described herein, e.g., comprising at least one repeat of a 3'UTR derived from human beta-globulin. In one embodiment, the nucleic acid sequence in the vector further comprises promoter, e.g. , a T2A promoter.

Promoters

In one embodiment, the vector further comprises a promoter. In some embodiments, the promoter is chosen from an EF-1 promoter, a CMV IE gene promoter, an EF-1 a promoter, an ubiquitin C promoter, or a phosphoglycerate kinase (PGK) promoter. In one embodiment, the promoter is an EF-1 promoter. In one embodiment, the EF-1 promoter comprises a sequence of SEQ I D NO: 1 .

Host cells for CAR expression

As noted above, in some aspects the invention pertains to a cell, e.g. , an immune effector cell, (e.g., a population of cells, e.g., a population of immune effector cells) comprising a nucleic acid molecule, a CAR polypeptide molecule, or a vector as described herein.

In certain aspects of the present disclosure, immune effector cells, e.g. , T cells, can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In one preferred aspect, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one aspect, the cells collected by apheresis may be washed to remove the plasma fraction and, optionally, to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations.

Initial activation steps in the absence of calcium can lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated "flow-through" centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

It is recognized that the methods of the application can utilize culture media conditions comprising 5% or less, for example 2%, human AB serum, and employ known culture media conditions and compositions, for example those described in Smith et al. , "Ex vivo expansion of human T cells for adoptive immunotherapy using the novel Xeno-free CTS Immune Cell Serum Replacement" Clinical & Translational Immunology (2015) 4, e31 ; doi :10.1038/cti.2014.31 .

In one aspect, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation.

The methods described herein can include, e.g., selection of a specific subpopulation of immune effector cells, e.g., T cells, that are a T regulatory cell-depleted population, CD25+ depleted cells, using, e.g., a negative selection technique, e.g., described herein. Preferably, the population of T regulatory depleted cells contains less than 30%, 25%, 20%, 15%, 1 0%, 5%, 4%, 3%, 2%, 1 % of CD25+ cells.

In one embodiment, T regulatory cells, e.g. , CD25+ T cells, are removed from the population using an anti-CD25 antibody, or fragment thereof, or a CD25-binding ligand, IL-2. In one embodiment, the anti-CD25 antibody, or fragment thereof, or CD25-binding ligand is conjugated to a substrate, e.g., a bead, or is otherwise coated on a substrate, e.g., a bead. In one embodiment, the anti-CD25 antibody, or fragment thereof, is conjugated to a substrate as described herein. In one embodiment, the T regulatory cells, e.g., CD25+ T cells, are removed from the population using CD25 depletion reagent from Miltenyi™. In one embodiment, the ratio of cells to CD25 depletion reagent is 1 e7 cells to 20 uL, or 1 e7 cells to15 uL, or 1 e7 cells to 10 uL, or 1 e7 cells to 5 uL, or 1 e7 cells to 2.5 uL, or 1 e7 cells to 1 .25 uL. In one embodiment, e.g., for T regulatory cells, e.g., CD25+ depletion , greater than 500 million cells/ml is used. In a further aspect, a concentration of cells of 600, 700, 800, or 900 million cells/ml is used.

In one embodiment, the population of immune effector cells to be depleted includes about 6 x 1 09 CD25+ T cells. In other aspects, the population of immune effector cells to be depleted include about 1 x 109 to 1 x 1 010 CD25+ T cell, and any integer value in between. In one embodiment, the resulting population T regulatory depleted cells has 2 x 109 T regulatory cells, e.g., CD25+ cells, or less (e.g., 1 x 1 09, 5 x 108 , 1 x 1 08, 5 x 1 07, 1 x 1 07, or less CD25+ cells).

In one embodiment, the T regulatory cells, e.g., CD25+ cells, are removed from the population using the Clini AC system with a depletion tubing set, such as, e.g ., tubing 1 62-01 . In one embodiment, the CliniMAC system is run on a depletion setting such as, e.g. , DEPLETION2.1 .

Without wishing to be bound by a particular theory, decreasing the level of negative regulators of immune cells (e.g., decreasing the number of unwanted immune cells, e.g. , TREG cells), in a subject prior to apheresis or during manufacturing of a CAR-expressing cell product can reduce the risk of subject relapse. For example, methods of depleting TREG cells are known in the art. Methods of decreasing TREG cells include, but are not limited to, cyclophosphamide, anti-GITR antibody (an anti-GITR antibody described herein), CD25-depletion, and combinations thereof.

In some embodiments, the manufacturing methods comprise reducing the number of (e.g., depleting) TREG cells prior to manufacturing of the CAR-expressing cell. For example, manufacturing methods comprise contacting the sample, e.g., the apheresis sample, with an anti-GITR antibody and/or an anti-CD25 antibody (or fragment thereof, or a CD25-binding ligand), e.g., to deplete TREG cells prior to manufacturing of the CAR-expressing cell (e.g., T cell, NK cell) product.

In an embodiment, a subject is pre-treated with one or more therapies that reduce TREG cells prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of subject relapse to CAR-expressing cell treatment. In an embodiment, methods of decreasing TREG cells include, but are not limited to, administration to the subject of one or more of cyclophosphamide, anti-GITR antibody, CD25-depletion, or a combination thereof. Administration of one or more of cyclophosphamide, anti-G ITR antibody, CD25-depletion, or a combination thereof, can occur before, during or after an infusion of the CAR-expressing cell product.

In an embodiment, a subject is pre-treated with cyclophosphamide prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of subject relapse to CAR- expressing cell treatment. In an embodiment, a subject is pre-treated with an anti-GITR antibody prior to collection of cells for CAR-expressing cell product manufacturing, thereby reducing the risk of subject relapse to CAR-expressing cell treatment.

In one embodiment, the population of cells to be removed are neither the regulatory T cells or tumor cells, but cells that otherwise negatively affect the expansion and/or function of CART cells, e.g. cells expressing CD14, CD1 1 b, CD33, CD1 5, or other markers expressed by potentially immune suppressive cells. In one embodiment, such cells are envisioned to be removed concurrently with regulatory T cells and/or tumor cells, or following said depletion, or in another order.

The methods described herein can include more than one selection step, e.g., more than one depletion step. Enrichment of a T cell population by negative selection can be accomplished, e.g. , with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail can include antibodies to CD14, CD20, CD1 1 b, CD16, HLA-DR, and CD8.

The methods described herein can further include removing cells from the population which express a tumor antigen, e.g., a tumor antigen that does not comprise CD25, e.g. , CD1 9, CD30, CD38, CD123, CD20, CD14 or CD1 1 b, to thereby provide a population of T regulatory depleted, e.g. , CD25+ depleted, and tumor antigen depleted cells that are suitable for expression of a CAR, e.g. , a CAR described herein. In one embodiment, tumor antigen expressing cells are removed simultaneously with the T regulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody, or fragment thereof, and an anti-tumor antigen antibody, or fragment thereof, can be attached to the same substrate, e.g., bead, which can be used to remove the cells or an anti-CD25 antibody, or fragment thereof, or the anti-tumor antigen antibody, or fragment thereof, can be attached to separate beads, a mixture of which can be used to remove the cells. In other embodiments, the removal of T regulatory cells, e.g. , CD25+ cells, and the removal of the tumor antigen expressing cells is sequential, and can occur, e.g., in either order.

Also provided are methods that include removing cells from the population which express a check point inhibitor, e.g. , a check point inhibitor described herein, e.g., one or more of PD1 + cells, LAG3+ cells, and TIM3+ cells, to thereby provide a population of T regulatory depleted, e.g., CD25+ depleted cells, and check point inhibitor depleted cells, e.g., PD1 +, LAG3+ and/or TIM3+ depleted cells. Exemplary check point inhibitors include B7-H1 , B7-1 , CD1 60, P1 H, 2B4, PD1 , TIM3, CEACAM (e.g., CEACAM-1 ,

CEACAM-3 and/or CEACAM-5), LAG 3, TIGIT, CTLA-4, BTLA and LAIR1 . In one embodiment, check point inhibitor expressing cells are removed simultaneously with the T regulatory, e.g. , CD25+ cells. For example, an anti-CD25 antibody, or fragment thereof, and an anti-check point inhibitor antibody, or fragment thereof, can be attached to the same bead which can be used to remove the cells, or an anti- CD25 antibody, or fragment thereof, and the anti-check point inhibitor antibody, or fragment there, can be attached to separate beads, a mixture of which can be used to remove the cells. In other embodiments, the removal of T regulatory cells, e.g., CD25+ cells, and the removal of the check point inhibitor expressing cells is sequential, and can occur, e.g. , in either order.

Methods described herein can include a positive selection step. For example, T cells can isolated by incubation with anti-CD3/anti-CD28 (e.g., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3/CD28 T, for a time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1 , 2, 3, 4, 5, or 6 hours. In yet another embodiment, the time period is 10 to 24 hours, e.g. , 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points.

In one embodiment, a T cell population can be selected that expresses one or more of I FN-Y, TNFa, IL-1 7A, IL-2, IL-3, IL-4, GM-CSF, IL-1 0, IL-13, granzyme B, and perforin, or other appropriate molecules, e.g. , other cytokines. Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No. : WO 2013/126712.

For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain aspects, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (e.g. , increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one aspect, a concentration of 10 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6 billion/ml, or 5 billion/ml is used. In one aspect, a concentration of 1 billion cells/ml is used. In yet one aspect, a concentration of cells from 75, 80, 85, 90, 95, or 1 00 million cells/ml is used. In further aspects, concentrations of 125 or 1 50 million cells/ml can be used.

Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

In a related aspect, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In one aspect, the concentration of cells used is 5 x 1 06/ml. In other aspects, the concentration used can be from about 1 x 1 05/ml to 1 x 106/ml, and any integer value in between.

In other aspects, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10°C or at room temperature.

T cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 1 0% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31 .25% Plasmalyte-A, 31 .25% Dextrose 5%, 0.45% NaCI, 1 0% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80°C at a rate of 1 ° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20 ° C or in liquid nitrogen.

In certain aspects, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using methods relating to the present invention.

Also contemplated in the context of the invention is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in immune effector cell therapy for any number of diseases or conditions that would benefit from immune effector cell therapy, such as those described herein. In one aspect a blood sample or an apheresis is taken from a generally healthy subject. In certain aspects, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain aspects, the T cells may be expanded, frozen, and used at a later time. In certain aspects, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further aspect, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, Cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.

In a further aspect of the present invention, T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present invention to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, in certain aspects, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system . In one embodiment, the immune effector cells expressing a CAR molecule, e.g. , a CAR molecule described herein, are obtained from a subject that has received a low, immune enhancing dose of an mTOR inhibitor. In an embodiment, the population of immune effector cells, e.g. , T cells, to be engineered to express a CAR, are harvested after a sufficient time, or after sufficient dosing of the low, immune enhancing, dose of an mTOR inhibitor, such that the level of PD1 negative immune effector cells, e.g., T cells, or the ratio of PD1 negative immune effector cells, e.g., T cells/ PD1 positive immune effector cells, e.g., T cells, in the subject or harvested from the subject has been, at least transiently, increased.

In other embodiments, population of immune effector cells, e.g. , T cells, which have, or will be engineered to express a CAR, can be treated ex vivo by contact with an amount of an mTOR inhibitor that increases the number of PD1 negative immune effector cells, e.g ., T cells or increases the ratio of PD1 negative immune effector cells, e.g., T cells/ PD1 positive immune effector cells, e.g., T cells.

In one embodiment, a T cell population is diaglycerol kinase (DGK)-deficient. DGK-deficient cells include cells that do not express DGK RNA or protein, or have reduced or inhibited DGK activity. DGK- deficient cells can be generated by genetic approaches, e.g., administering RNA-interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or prevent DGK expression. Alternatively, DGK-deficient cells can be generated by treatment with DGK inhibitors described herein.

In one embodiment, a T cell population is Ikaros-deficient. Ikaros-deficient cells include cells that do not express Ikaros RNA or protein, or have reduced or inhibited Ikaros activity, Ikaros-deficient cells can be generated by genetic approaches, e.g., administering RNA-interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or prevent Ikaros expression. Alternatively, Ikaros-deficient cells can be generated by treatment with Ikaros inhibitors, e.g. , lenalidomide.

In embodiments, a T cell population is DGK-deficient and Ikaros-deficient, e.g., does not express DGK and Ikaros, or has reduced or inhibited DGK and Ikaros activity. Such DGK and Ikaros-deficient cells can be generated by any of the methods described herein.

In an embodiment, the NK cells are obtained from the subject. In another embodiment, the NK cells are an NK cell line, e.g., NK-92 cell line (Conkwest).

Additional Expressed Agents

In another embodiment, a CAR-expressing immune effector cell described herein can further express another agent, e.g., an agent which enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Examples of inhibitory molecules include PD-1 , PD-L1 , CTLA-4, TIM-3, CEACAM (e.g. , CEACAM-1 , CEACAM-3 and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1 , CD160, 2B4 and TGFR beta, e.g., as described herein. In one embodiment, the agent that inhibits an inhibitory molecule comprises a first polypeptide, e.g., an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g. , an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD-1 , PD-L1 , CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1 , CEACAM-3 and/or CEACAM-5), LAG-3, VISTA, BTLA, TIG IT, LAIR1 , CD160, 2B4 or TGFR beta, or a fragment of any of these, and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g., 41 BB, CD27 or CD28, e.g. , as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of PD-1 or a fragment thereof, and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28, CD27, OX40 or 4-IBB signaling domain described herein and/or a CD3 zeta signaling domain described herein).

In one embodiment, the CAR-expressing immune effector cell described herein can further comprise a second CAR, e.g., a second CAR that includes a different antigen binding domain, e.g., to the same target (e.g., a target described above) or a different target. In one embodiment, the second CAR includes an antigen binding domain to a target expressed on the same cancer cell type as the target of the first CAR. In one embodiment, the CAR-expressing immune effector cell comprises a first CAR that targets a first antigen and includes an intracellular signaling domain having a costimulatory signaling domain but not a primary signaling domain, and a second CAR that targets a second, different, antigen and includes an intracellular signaling domain having a primary signaling domain but not a costimulatory signaling domain.

While not wishing to be bound by theory, placement of a costimulatory signaling domain, e.g., 4-

1 BB, CD28, CD27 or OX-40, onto the first CAR, and the primary signaling domain, e.g. , CD3 zeta, on the second CAR can limit the CAR activity to cells where both targets are expressed. In one embodiment, the CAR expressing immune effector cell comprises a first CAR that includes an antigen binding domain that targets, e.g., a target described above, a transmembrane domain and a costimulatory domain and a second CAR that targets an antigen other than antigen targeted by the first CAR (e.g. , an antigen expressed on the same cancer cell type as the first target) and includes an antigen binding domain, a transmembrane domain and a primary signaling domain . In another embodiment, the CAR expressing immune effector cell comprises a first CAR that includes an antigen binding domain that targets, e.g., a target described above, a transmembrane domain and a primary signaling domain and a second CAR that targets an antigen other than antigen targeted by the first CAR (e.g., an antigen expressed on the same cancer cell type as the first target) and includes an antigen binding domain to the antigen, a transmembrane domain and a costimulatory signaling domain.

In one embodiment, the CAR-expressing immune effector cell comprises a CAR described herein, e.g., a CAR to a target described above, and an inhibitory CAR. In one embodiment, the inhibitory CAR comprises an antigen binding domain that binds an antigen found on normal cells but not cancer cells, e.g. , normal cells that also express the target. In one embodiment, the inhibitory CAR comprises the antigen binding domain, a transmembrane domain and an intracellular domain of an inhibitory molecule. For example, the intracellular domain of the inhibitory CAR can be an intracellular domain of PD1 , PD-L1 , CTLA-4, TIM-3, CEACA (e.g. , CEACAM-1 , CEACAM-3 and/or CEACA -5), LAG -3, VISTA, BTLA, TIGIT, LAIR1 , CD160, 2B4 or TGFR beta.

In one embodiment, an immune effector cell (e.g., T cell, NK cell) comprises a first CAR comprising an antigen binding domain that binds to a tumor antigen as described herein, and a second CAR comprising a PD1 extracellular domain or a fragment thereof.

In one embodiment, the cell further comprises an inhibitory molecule as described above. In one embodiment, the second CAR in the cell is an inhibitory CAR, wherein the inhibitory CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular domain of an inhibitory molecule. The inhibitory molecule can be chosen from one or more of: PD1 , PD-L1 , CTLA-4, TI -3, LAG -3, VISTA, BTLA, TIG IT, LAIR1 , CD1 60, 2B4, TGFR beta, CEACAM-1 , CEACA -3, and CEACAM-5. In one embodiment, the second CAR molecule comprises the extracellular domain of PD1 or a fragment thereof.

In embodiments, the second CAR molecule in the cell further comprises an intracellular signaling domain comprising a primary signaling domain and/or an intracellular signaling domain.

In other embodiments, the intracellular signaling domain in the cell comprises a primary signaling domain comprising the functional domain of CD3 zeta and a costimulatory signaling domain comprising the functional domain of 4-1 BB.

In one embodiment, the second CAR molecule in the cell comprises the amino acid sequence of SEQ ID NO: 26.

In certain embodiments, the antigen binding domain of the first CAR molecule comprises a scFv and the antigen binding domain of the second CAR molecule does not comprise a scFv. For example, the antigen binding domain of the first CAR molecule comprises a scFv and the antigen binding domain of the second CAR molecule comprises a camelid VHH domain.

Split CAR

In some embodiments, the CAR-expressing cell uses a split CAR. The split CAR approach is described in more detail in publications WO2014/055442 and WO2014/055657. Briefly, a split CAR system comprises a cell expressing a first CAR having a first antigen binding domain and a costimulatory domain (e.g. , 41 BB), and the cell also expresses a second CAR having a second antigen binding domain and an intracellular signaling domain (e.g. , CD3 zeta). When the cell encounters the first antigen, the costimulatory domain is activated, and the cell proliferates. When the cell encounters the second antigen, the intracellular signaling domain is activated and cell-killing activity begins. Thus, the CAR-expressing cell is only fully activated in the presence of both antigens.

Multiple CAR expression

In one aspect, the CAR-expressing cell described herein can further comprise a second CAR, e.g., a second CAR that includes a different antigen binding domain, e.g. , to the same target or a different target (e.g. , a target other than a cancer associated antigen described herein or a different cancer associated antigen described herein). In one embodiment, the second CAR includes an antigen binding domain to a target expressed the same cancer cell type as the cancer associated antigen. In one embodiment, the CAR-expressing cell comprises a first CAR that targets a first antigen and includes an intracellular signaling domain having a costimulatory signaling domain but not a primary signaling domain, and a second CAR that targets a second, different, antigen and includes an intracellular signaling domain having a primary signaling domain but not a costimulatory signaling domain. While not wishing to be bound by theory, placement of a costimulatory signaling domain, e.g., 4-1 BB, CD28, CD27 or OX-40, onto the first CAR, and the primary signaling domain, e.g. , CD3 zeta, on the second CAR can limit the CAR activity to cells where both targets are expressed. In one embodiment, the CAR expressing cell comprises a first cancer associated antigen CAR that includes an antigen binding domain that binds a target antigen described herein, a transmembrane domain and a costimulatory domain and a second CAR that targets a different target antigen (e.g., an antigen expressed on that same cancer cell type as the first target antigen) and includes an antigen binding domain, a transmembrane domain and a primary signaling domain. In another embodiment, the CAR expressing cell comprises a first CAR that includes an antigen binding domain that binds a target antigen described herein, a transmembrane domain and a primary signaling domain and a second CAR that targets an antigen other than the first target antigen (e.g., an antigen expressed on the same cancer cell type as the first target antigen) and includes an antigen binding domain to the antigen, a transmembrane domain and a costimulatory signaling domain.

In some embodiments, the invention includes the use of a first and second CAR, wherein the antigen binding domain of one of said first CAR said second CAR does not comprise a variable light domain and a variable heavy domain. In some embodiments, the antigen binding domain of one of said first CAR said second CAR is an scFv, and the other is not an scFv. In some embodiments, the antigen binding domain of one of said first CAR said second CAR comprises a single VH domain, e.g., a camelid, shark, or lamprey single VH domain, or a single VH domain derived from a human or mouse sequence. In some embodiments, the antigen binding domain of one of said first CAR said second CAR comprises a nanobody. In some embodiments, the antigen binding domain of one of said first CAR said second CAR comprises a camelid VHH domain.

Telomerase expression

While not wishing to be bound by any particular theory, in some embodiments, a therapeutic T cell has short term persistence in a patient, due to shortened telomeres in the T cell ; accordingly, transfection with a telomerase gene can lengthen the telomeres of the T cell and improve persistence of the T cell in the patient. See Carl June, "Adoptive T cell therapy for cancer in the clinic", Journal of Clinical Investigation, 1 17:1466-1476 (2007). Thus, in an embodiment, an immune effector cell, e.g., a T cell, ectopically expresses a telomerase subunit, e.g., the catalytic subunit of telomerase, e.g., TERT, e.g., hTERT. In some aspects, this disclosure provides a method of producing a CAR-expressing cell, comprising contacting a cell with a nucleic acid encoding a telomerase subunit, e.g. , the catalytic subunit of telomerase, e.g., TERT, e.g., hTERT. The cell may be contacted with the nucleic acid before, simultaneous with , or after being contacted with a construct encoding a CAR. Expansion and Activation

Immune effector cells such as T cells may be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681 ; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843 ; 5,883,223; 6,905,874; 6,797,51 4; 6,867,041 ; and U.S. Patent Application Publication No. 20060121 005, each of which is incorporated by reference in its entirety.

Generally, a population of immune effector cells e.g., T regulatory cell depleted cells, may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g. , bryostatin) in conjunction with a calcium ionophore. For co- stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody can be used. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besangon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1 998; Haanen et al., J. Exp. Med. 1 90(9) :13191328, 1999; Garland et al., J. Immunol Meth. 227(1 -2):53-63, 1999).

In certain aspects, the primary stimulatory signal and the costimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e. , in "cis" formation) or to separate surfaces (i.e. , in "trans" formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In one aspect, the agent providing the costimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain aspects, both agents can be in solution. In one aspect, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. 20040101 519 and 20060034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present invention.

In one aspect, the two agents are immobilized on beads, either on the same bead, i.e. , "cis," or to separate beads, i.e., "trans." By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the costimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts. In one aspect, a 1 :1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell growth is used. In certain aspects of the present invention, a ratio of anti CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1 :1 . In one particular aspect an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1 :1 . In one aspect, the ratio of CD3:CD28 antibody bound to the beads ranges from 1 00:1 to 1 :100 and all integer values there between. In one aspect, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e. , the ratio of CD3:CD28 is less than one. In certain aspects, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2:1 . In one particular aspect, a 1 :1 00 CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1 :75 CD3:CD28 ratio of antibody bound to beads is used. In a further aspect, a 1 :50 CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1 :30 CD3:CD28 ratio of antibody bound to beads is used. In one preferred aspect, a 1 :1 0 CD3:CD28 ratio of antibody bound to beads is used. In one aspect, a 1 :3 CD3 :CD28 ratio of antibody bound to the beads is used. In yet one aspect, a 3: 1 CD3:CD28 ratio of antibody bound to the beads is used. Ratios of particles to cells from 1 :500 to 500:1 and any integer values in between may be used to stimulate T cells or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many. In certain aspects the ratio of cells to particles ranges from 1 :1 00 to 1 00:1 and any integer values in-between and in further aspects the ratio comprises 1 :9 to 9:1 and any integer values in between, can also be used to stimulate T cells. The ratio of anti-CD3- and anti-CD28-coupled particles to T cells that result in T cell stimulation can vary as noted above, however certain preferred values include 1 :1 00, 1 :50, 1 :40, 1 :30, 1 :20, 1 :1 0, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 :1 , 2:1 , 3:1 , 4:1 , 5:1 , 6:1 , 7:1 , 8 :1 , 9:1 , 1 0 :1 , and 1 5:1 with one preferred ratio being at least 1 :1 particles per T cell. In one aspect, a ratio of particles to cells of 1 :1 or less is used. In one particular aspect, a preferred particle: cell ratio is 1 :5. In further aspects, the ratio of particles to cells can be varied depending on the day of stimulation. For example, in one aspect, the ratio of particles to cells is from 1 :1 to 10:1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 1 0 days, at final ratios of from 1 :1 to 1 :10 (based on cell counts on the day of addition). In one particular aspect, the ratio of particles to cells is 1 :1 on the first day of stimulation and adjusted to 1 :5 on the third and fifth days of stimulation . In one aspect, particles are added on a daily or every other day basis to a final ratio of 1 :1 on the first day, and 1 :5 on the third and fifth days of stimulation . In one aspect, the ratio of particles to cells is 2:1 on the first day of stimulation and adjusted to 1 :10 on the third and fifth days of stimulation. In one aspect, particles are added on a daily or every other day basis to a final ratio of 1 :1 on the first day, and 1 :10 on the third and fifth days of stimulation. One of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present invention. In particular, ratios will vary depending on particle size and on cell size and type. In one aspect, the most typical ratios for use are in the neighborhood of 1 :1 , 2:1 and 3:1 on the first day.

In further aspects, the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative aspect, prior to culture, the agent-coated beads and cells are not separated but are cultured together. In a further aspect, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

By way of example, cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3x28 beads) to contact the T cells. In one aspect the cells (for example, 1 04 to 1 09 T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 T paramagnetic beads at a ratio of 1 :1 ) are combined in a buffer, for example PBS (without divalent cations such as, calcium and magnesium). Again, those of ordinary skill in the art can readily appreciate any cell concentration may be used. For example, the target cell may be very rare in the sample and comprise only 0.01 % of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest.

Accordingly, any cell number is within the context of the present invention. In certain aspects, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and particles. For example, in one aspect, a concentration of about 1 0 billion cells/ml, 9 billion/ml, 8 billion/ml, 7 billion/ml, 6 billion/ml, 5 billion/ml, or 2 billion cells/ml is used. In one aspect, greater than 100 million cells/ml is used. In a further aspect, a concentration of cells of 10, 1 5, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used. In yet one aspect, a concentration of cells from 75, 80, 85, 90, 95, or 1 00 million cells/ml is used. In further aspects, concentrations of 125 or 1 50 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such populations of cells may have therapeutic value and would be desirable to obtain in certain aspects. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

In one embodiment, cells transduced with a nucleic acid encoding a CAR, e.g., a CAR described herein, are expanded, e.g., by a method described herein. In one embodiment, the cells are expanded in culture for a period of several hours (e.g. , about 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 15, 1 8, 21 hours) to about 14 days (e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13 or 14 days). In one embodiment, the cells are expanded for a period of 4 to 9 days. In one embodiment, the cells are expanded for a period of 8 days or less, e.g. , 7, 6 or 5 days. In one embodiment, the cells are expanded in culture for 5 days, and the resulting cells are more potent than the same cells expanded in culture for 9 days under the same culture conditions. Potency can be defined, e.g., by various T cell functions, e.g. proliferation, target cell killing, cytokine production, activation, migration, or combinations thereof. In one embodiment, the cells are expanded for 5 days show at least a one, two, three or four fold increase in cells doublings upon antigen stimulation as compared to the same cells expanded in culture for 9 days under the same culture conditions. In one embodiment, the cells are expanded in culture for 5 days, and the resulting cells exhibit higher proinflammatory cytokine production, e.g., IFN-γ and/or GM-CSF levels, as compared to the same cells expanded in culture for 9 days under the same culture conditions. In one embodiment, the cells expanded for 5 days show at least a one, two, three, four, five, ten-fold or more increase in pg/ml of proinflammatory cytokine production, e.g., IFN-γ and/or GM-CSF levels, as compared to the same cells expanded in culture for 9 days under the same culture conditions.

Several cycles of stimulation may also be desired such that culture time of T cells can be 60 days or more. Conditions appropriate for T cell culture include an appropriate media (e.g. , Minimal Essential Media or RPMI Media 1 640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin , IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFp, and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1 640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g. , penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% CO2). In one embodiment, the cells are expanded in an appropriate media (e.g. , media described herein) that includes one or more interleukin that result in at least a 200-fold (e.g. , 200-fold, 250-fold, 300- fold, 350-fold) increase in cells over a 14 day expansion period, e.g., as measured by a method described herein such as flow cytometry. In one embodiment, the cells are expanded in the presence of IL-15 and/or IL-7 (e.g. , IL-15 and IL-7).

In embodiments, methods described herein, e.g. , CAR-expressing cell manufacturing methods, comprise removing T regulatory cells, e.g. , CD25+ T cells, from a cell population, e.g. , using an anti- CD25 antibody, or fragment thereof, or a CD25-binding ligand, IL-2. Methods of removing T regulatory cells, e.g., CD25+ T cells, from a cell population are described herein. In embodiments, the methods, e.g., manufacturing methods, further comprise contacting a cell population (e.g., a cell population in which T regulatory cells, such as CD25+ T cells, have been depleted; or a cell population that has previously contacted an anti-CD25 antibody, fragment thereof, or CD25-binding ligand) with IL-15 and/or IL-7. For example, the cell population (e.g. , that has previously contacted an anti-CD25 antibody, fragment thereof, or CD25-binding ligand) is expanded in the presence of IL-1 5 and/or IL-7.

In some embodiments a CAR-expressing cell described herein is contacted with a composition comprising a interleukin-15 (IL-1 5) polypeptide, a interleukin-1 5 receptor alpha (IL-1 5Ra) polypeptide, or a combination of both a IL-1 5 polypeptide and a IL-15Ra polypeptide e.g., hetlL-1 5, during the manufacturing of the CAR-expressing cell, e.g ., ex vivo. In embodiments, a CAR-expressing cell described herein is contacted with a composition comprising an IL-15 polypeptide during the

manufacturing of the CAR-expressing cell, e.g ., ex vivo. In embodiments, a CAR-expressing cell described herein is contacted with a composition comprising a combination of both an IL-1 5 polypeptide and a IL-15 Ra polypeptide during the manufacturing of the CAR-expressing cell, e.g., ex vivo. In embodiments, a CAR-expressing cell described herein is contacted with a composition comprising hetlL- 1 5 during the manufacturing of the CAR-expressing cell, e.g. , ex vivo.

In one embodiment the CAR-expressing cell described herein is contacted with a composition comprising hetlL-1 5 during ex vivo expansion . In an embodiment, the CAR-expressing cell described herein is contacted with a composition comprising an IL-1 5 polypeptide during ex vivo expansion. In an embodiment, the CAR-expressing cell described herein is contacted with a composition comprising both an IL-15 polypeptide and an IL-1 5Ra polypeptide during ex vivo expansion. In one embodiment the contacting results in the survival and proliferation of a lymphocyte subpopulation, e.g., CD8+ T cells.

T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree. Further, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes.

Once a CAR described herein is constructed, various assays can be used to evaluate the activity of the molecule, such as but not limited to, the ability to expand T cells following antigen stimulation, sustain T cell expansion in the absence of re-stimulation, and anti-cancer activities in appropriate in vitro and animal models. Assays to evaluate the effects of a CAR of the present invention are described in further detail below

Western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers and dimers. See, e.g., Milone er a/., Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, T cells (1 :1 mixture of CD4+ and CD8+ T cells) expressing the CARs are expanded in vitro for more than 1 0 days followed by lysis and SDS-PAGE under reducing conditions. CARs containing the full length TCR-ζ cytoplasmic domain and the endogenous TCR-ζ chain are detected by western blotting using an antibody to the TCR-ζ chain. The same T cell subsets are used for SDS-PAGE analysis under non-reducing conditions to permit evaluation of covalent dimer formation.

In vitro expansion of CAR+ T cells following antigen stimulation can be measured by flow cytometry. For example, a mixture of CD4+ and CD84 T cells are stimulated with aCD3/aCD28 aAPCs followed by transduction with lentiviral vectors expressing GFP under the control of the promoters to be analyzed. Exemplary promoters include the CMV I E gene, EF-1 a, ubiquitin C, or phosphoglycerokinase (PGK) promoters. GFP fluorescence is evaluated on day 6 of culture in the CD4+ and/or CD8+ T cell subsets by flow cytometry. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).

Alternatively, a mixture of CD4+ and CD84 T cells are stimulated with aCD3/aCD28 coated magnetic beads on day 0, and transduced with CAR on day 1 using a bicistronic lentiviral vector expressing CAR along with eGFP using a 2A ribosomal skipping sequence. Cultures are re-stimulated with either a cancer associated antigen as described herein K562 cells (K562 expressing a cancer associated antigen as described herein), wild-type K562 cells (K562 wild type) or K562 cells expressing hCD32 and 4-1 BBL in the presence of antiCD3 and anti-CD28 antibody (K562-BBL-3/28) following washing. Exogenous IL-2 is added to the cultures every other day at 100 lU/ml. G FP+ T cells are enumerated by flow cytometry using bead-based counting. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).

Sustained CAR4 T cell expansion in the absence of re-stimulation can also be measured. See, e.g., Milone et al., Molecular Therapy 1 7(8) : 1453-1464 (2009). Briefly, mean T cell volume (fl) is measured on day 8 of culture using a Coulter Multisizer III particle counter, a Nexcelom Cellometer Vision or Millipore Scepter, following stimulation with aCD3/aCD28 coated magnetic beads on day 0, and transduction with the indicated CAR on day 1 .

Animal models can also be used to measure a CART activity. For example, xenograft model using human a cancer associated antigen described herein-specific CAR4 T cells to treat a primary human pre-B ALL in immunodeficient mice can be used. See, e.g., Milone et al., Molecular Therapy 1 7(8) : 1453-1464 (2009). Very briefly, after establishment of ALL, mice are randomized as to treatment groups. Different numbers of a cancer associated antigen -specific CAR engineered T cells are coinjected at a 1 :1 ratio into NOD-SCID-v-'- mice bearing B-ALL. The number of copies of a cancer associated antigen -specific CAR vector in spleen DNA from mice is evaluated at various times following T cell injection. Animals are assessed for leukemia at weekly intervals. Peripheral blood a cancer associate antigen as described herein+ B-ALL blast cell counts are measured in mice that are injected with a cancer associated antigen described herein-ζ CAR4 T cells or mock-transduced T cells. Survival curves for the groups are compared using the log-rank test. In addition, absolute peripheral blood CD4+ and CD8+ T cell counts 4 weeks following T cell injection in NOD-SCID-γ-'- mice can also be analyzed. Mice are injected with leukemic cells and 3 weeks later are injected with T cells engineered to express CAR by a bicistronic lentiviral vector that encodes the CAR linked to eGFP. T cells are normalized to 45- 50% input GFP+ T cells by mixing with mock-transduced cells prior to injection, and confirmed by flow cytometry. Animals are assessed for leukemia at 1 -week intervals. Survival curves for the CAR+ T cell groups are compared using the log-rank test.

Dose dependent CAR treatment response can be evaluated. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). For example, peripheral blood is obtained 35-70 days after establishing leukemia in mice injected on day 21 with CAR T cells, an equivalent number of mock- transduced T cells, or no T cells. Mice from each group are randomly bled for determination of peripheral blood a cancer associate antigen as described herein* ALL blast counts and then killed on days 35 and 49. The remaining animals are evaluated on days 57 and 70.

Assessment of cell proliferation and cytokine production has been previously described, e.g., at Milone ef al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, assessment of CAR-mediated proliferation is performed in microtiter plates by mixing washed T cells with K562 cells expressing a cancer associated antigen described herein (K1 9) or CD32 and CD137 (KT32-BBL) for a final T-cell :K562 ratio of 2:1 . K562 cells are irradiated with gamma-radiation prior to use. Anti-CD3 (clone OKT3) and anti- CD28 (clone 9.3) monoclonal antibodies are added to cultures with KT32-BBL cells to serve as a positive control for stimulating T-cell proliferation since these signals support long-term CD8+ T cell expansion ex vivo. T cells are enumerated in cultures using CountBright™ fluorescent beads (Invitrogen, Carlsbad,

CA) and flow cytometry as described by the manufacturer. CAR+ T cells are identified by G FP expression using T cells that are engineered with eG FP-2A linked CAR-expressing lentiviral vectors. For CAR+ T cells not expressing GFP, the CAR+ T cells are detected with biotinylated recombinant a cancer associate antigen as described herein protein and a secondary avidin-PE conjugate. CD4+ and CD8+ expression on T cells are also simultaneously detected with specific monoclonal antibodies (BD

Biosciences). Cytokine measurements are performed on supernatants collected 24 hours following re- stimulation using the human TH1 TH2 cytokine cytometric bead array kit (BD Biosciences, San Diego, CA) according the manufacturer's instructions. Fluorescence is assessed using a FACScalibur flow cytometer, and data is analyzed according to the manufacturer's instructions.

Cytotoxicity can be assessed by a standard 51 Cr-release assay. See, e.g., Milone ef al.,

Molecular Therapy 1 7(8) : 1453-1464 (2009). Briefly, target cells (K562 lines and primary pro-B-ALL cells) are loaded with 51 Cr (as NaCr04, New England Nuclear, Boston, MA) at 37°C for 2 hours with frequent agitation, washed twice in complete RPMI and plated into microtiter plates. Effector T cells are mixed with target cells in the wells in complete RPMI at varying ratios of effector cell:target cell (E:T). Additional wells containing media only (spontaneous release, SR) or a 1 % solution of triton-X 100 detergent (total release, TR) are also prepared. After 4 hours of incubation at 37°C, supernatant from each well is harvested. Released 51 Cr is then measured using a gamma particle counter (Packard Instrument Co., Waltham, MA). Each condition is performed in at least triplicate, and the percentage of lysis is calculated using the formula: % Lysis = (ER- SR) / (TR - SR), where ER represents the average 51 Cr released for each experimental condition.

Imaging technologies can be used to evaluate specific trafficking and proliferation of CARs in tumor-bearing animal models. Such assays have been described, for example, in Barrett et al., Human Gene Therapy 22:1 575-1586 (201 1 ). Briefly, NOD/SCI D/ cr'- (NSG) mice are injected IV with Nalm-6 cells followed 7 days later with T cells 4 hour after electroporation with the CAR constructs. The T cells are stably transfected with a lentiviral construct to express firefly luciferase, and mice are imaged for bioluminescence. Alternatively, therapeutic efficacy and specificity of a single injection of CAR+ T cells in Nalm-6 xenograft model can be measured as the following : NSG mice are injected with Nalm-6 transduced to stably express firefly luciferase, followed by a single tail-vein injection of T cells electroporated with CARs of the present invention 7 days later. Animals are imaged at various time points post injection. For example, photon-density heat maps of firefly luciferase positive leukemia in representative mice at day 5 (2 days before treatment) and day 8 (24 hr post CAR+ PBLs) can be generated.

Other assays, including those described in the Example section herein as well as those that are known in the art can also be used to evaluate the CARs described herein.

Methods of treatment/Combination therapies

In another aspect, the present invention includes methods comprising administering a CAR molecule, e.g. , a CAR molecule described herein, or a cell comprising a nucleic acid encoding a CAR molecule, e.g. , a CAR molecule described herein. In one embodiment, the subject has a disorder described herein, e.g. , the subject has cancer, e.g., the subject has a cancer which expresses a target antigen described herein. In one embodiment, the subject is a human.

In another aspect, the invention pertains to a method of treating a subject having a disease associated with expression of a cancer associated antigen as described herein comprising administering to the subject an effective amount of a cell comprising a CAR molecule, e.g. , a CAR molecule described herein.

In yet another aspect, the invention features a method of treating a subject having a disease associated with expression of a tumor antigen (e.g. , an antigen described herein), comprising administering to the subject an effective amount of a cell, e.g. , an immune effector cell (e.g., a population of immune effector cells) comprising a CAR molecule, wherein the CAR molecule comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, said intracellular domain comprises a costimulatory domain and/or a primary signaling domain, wherein said antigen binding domain binds to the tumor antigen associated with the disease, e.g. a tumor antigen as described herein.

In a related aspect, the invention features a method of treating a subject having a disease associated with expression of a tumor antigen. The method comprises administering to the subject an effective amount of a cell, e.g., an immune effector cell (e.g., a population of immune effector cells) comprising a CAR molecule, in combination with an agent that increases the efficacy of the immune cell, wherein:

the agent that increases the efficacy of the immune cell is chosen from one or more of: (i) a protein phosphatase inhibitor;

(ii) a kinase inhibitor;

(iii) a cytokine;

(iv) an inhibitor of an immune inhibitory molecule ; or

(v) an agent that decreases the level or activity of a TREG cell.

In another aspect, the invention features a composition comprising an immune effector cell (e.g., a population of immune effector cells) comprising a CAR molecule (e.g., a CAR molecule as described herein) for use in the treatment of a subject having a disease associated with expression of a tumor antigen, e.g., a disorder as described herein .

In certain embodiments of any of the aforesaid methods or uses, the disease associated with a tumor antigen, e.g., a tumor antigen described herein, is selected from a proliferative disease such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia, or is a non-cancer related indication associated with expression of a tumor antigen described herein. In one embodiment, the disease is a cancer described herein, e.g. , a cancer described herein as being associated with a target described herein. In one embodiment, the disease is a hematologic cancer. In one embodiment, the hematologic cancer is leukemia. In one embodiment, the cancer is selected from the group consisting of one or more acute leukemias including but not limited to B-cell acute lymphoid leukemia ("BALL"), T-cell acute lymphoid leukemia ("TALL"), acute lymphoid leukemia (ALL) ; one or more chronic leukemias including but not limited to chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL) ; additional hematologic cancers or hematologic conditions including, but not limited to B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm , Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm , Waldenstrom macroglobulinemia, and "preleukemia" which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and to disease associated with expression of a tumor antigen described herein include, but not limited to, atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing a tumor antigen as described herein ; and any combination thereof. In another embodiment, the disease associated with a tumor antigen described herein is a solid tumor.

In certain embodiments, the methods or uses are carried out in combination with an agent that increases the efficacy of the immune effector cell, e.g. , an agent as described herein.

In any of the aforesaid methods or uses, the disease associated with expression of the tumor antigen is selected from the group consisting of a proliferative disease, a precancerous condition, a cancer, and a non-cancer related indication associated with expression of the tumor antigen. The cancer can be a hematologic cancer, e.g. , a cancer chosen from one or more of chronic lymphocytic leukemia (CLL), acute leukemias, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (C L), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-fol licular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, or pre-leukemia.

The cancer can also be chosen from colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's

Disease, non-Hodgkin's lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma, environmentally induced cancers, combinations of said cancers, and metastatic lesions of said cancers.

In certain embodiments of the methods or uses described herein, the CAR molecule is administered in combination with an agent that increases the efficacy of the immune effector cell, e.g., one or more of a protein phosphatase inhibitor, a kinase inhibitor, a cytokine, an inhibitor of an immune inhibitory molecule; or an agent that decreases the level or activity of a TREG cell.

In certain embodiments of the methods or uses described herein, the protein phosphatase inhibitor is a SHP-1 inhibitor and/or an SHP-2 inhibitor.

In other embodiments of the methods or uses described herein, kinase inhibitor is chosen from one or more of a CDK4 inhibitor, a CDK4/6 inhibitor (e.g., palbociclib), a BTK inhibitor (e.g., ibrutinib or RN-486), an mTOR inhibitor (e.g., rapamycin or everolimus (RAD001 )), an MNK inhibitor, or a dual P13K/mTOR inhibitor. In one embodiment, the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin-2-inducible kinase (ITK).

In other embodiments of the methods or uses described herein, the agent that inhibits the immune inhibitory molecule comprises an antibody or antibody fragment, an inhibitory nucleic acid, a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription-activator like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN) that inhibits the expression of the inhibitory molecule. In other embodiments of the methods or uses described herein, the agent that decreases the level or activity of the TREG cells is chosen from cyclophosphamide, anti-GITR antibody, CD25-depletion, or a combination thereof.

In certain embodiments of the methods or uses described herein, the immune inhibitory molecule is selected from the group consisting of PD1 , PD-L1 , CTLA-4, TIM-3, LAG -3, VISTA, BTLA, TIGIT, LAIR1 , CD1 60, 2B4, TGFR beta, CEACAM-1 , CEACAM-3, and CEACAM-5.

In other embodiments, the agent that inhibits the inhibitory molecule comprises a first polypeptide comprising an inhibitory molecule or a fragment thereof and a second polypeptide that provides a positive signal to the cell, and wherein the first and second polypeptides are expressed on the CAR-containing immune cells, wherein (i) the first polypeptide comprises PD1 , PD-L1 , CTLA-4, TIM-3, LAG3, VISTA,

BTLA, TIG IT, LAIR1 , CD1 60, 2B4, TGFR beta, CEACAM-1 , CEACAM-3, and CEACAM-5 or a fragment thereof; and/or (ii) the second polypeptide comprises an intracellular signaling domain comprising a primary signaling domain and/or a costimulatory signaling domain. In one embodiment, the primary signaling domain comprises a functional domain of CD3 zeta; and/or the costimulatory signaling domain comprises a functional domain of a protein selected from 41 BB, CD27 and CD28.

In other embodiments, cytokine is chosen from IL-7, IL-15 or IL-21 , or both.

In other embodiments, the immune effector cell comprising the CAR molecule and a second, e.g. , any of the combination therapies disclosed herein (e.g . , the agent that that increases the efficacy of the immune effector cell) are administered substantially simultaneously or sequentially.

In other embodiments, the immune cell comprising the CAR molecule is administered in combination with a molecule that targets GITR and/or modulates G ITR function. In certain embodiments, the molecule targeting GITR and/or modulating G ITR function is administered prior to the CAR- expressing cell or population of cells, or prior to apheresis.

In one embodiment, lymphocyte infusion, for example allogeneic lymphocyte infusion, is used in the treatment of the cancer, wherein the lymphocyte infusion comprises at least one CAR-expressing cell of the present invention . In one embodiment, autologous lymphocyte infusion is used in the treatment of the cancer, wherein the autologous lymphocyte infusion comprises at least one CAR-expressing cell described herein.

In one embodiment, the cell is a T cell and the T cell is diaglycerol kinase (DGK) deficient. In one embodiment, the cell is a T cell and the T cell is Ikaros deficient. In one embodiment, the cell is a T cell and the T cell is both DGK and Ikaros deficient.

In one embodiment, the method includes administering a cell expressing the CAR molecule, as described herein, in combination with an agent which enhances the activity of a CAR-expressing cell, wherein the agent is a cytokine, e.g., IL-7, IL-15, IL-18, IL-21 , or a combination thereof. The cytokine can be delivered in combination with , e.g., simultaneously or shortly after, administration of the CAR- expressing cell. Alternatively, the cytokine can be delivered after a prolonged period of time after administration of the CAR-expressing cell, e.g., after assessment of the subject's response to the CAR- expressing cell. In one embodiment the cytokine is administered to the subject simultaneously (e.g. , administered on the same day) with or shortly after administration (e.g., administered 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration) of the cell or population of cells described herein. In other embodiments, the cytokine is administered to the subject after a prolonged period of time (e.g., e.g., at least 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, or more) after administration of the cell or population of cells described herein , or after assessment of the subject's response to the cell.

In other embodiments, the cells expressing a CAR molecule are administered in combination with an agent that ameliorates one or more side effects associated with administration of a cell expressing a CAR molecule. Side effects associated with the CAR-expressing cell can be chosen from cytokine release syndrome (CRS) or hemophagocytic lymphohistiocytosis (HLH).

In embodiments of any of the aforesaid methods or uses, the cells expressing the CAR molecule are administered in combination with an agent that treats the disease associated with expression of the tumor antigen, e.g., any of the second or third therapies disclosed herein. Additional exemplary combinations include one or more of the following.

In another embodiment, the cell expressing the CAR molecule, e.g., as described herein, can be administered in combination with another agent, e.g ., a kinase inhibitor and/or checkpoint inhibitor described herein. In an embodiment, a cell expressing the CAR molecule can further express another agent, e.g., an agent which enhances the activity of a CAR-expressing cell.

For example, in one embodiment, the agent that enhances the activity of a CAR-expressing cell can be an agent which inhibits an inhibitory molecule (e.g., an immune inhibitor molecule). Examples of inhibitory molecules include PD1 , PD-L1 , CTLA-4, TIM-3, CEACAM (e.g. , CEACAM-1 , CEACAM-3 and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAI R1 , CD1 60, 2B4 and TG FR beta.

In one embodiment, the agent that inhibits the inhibitory molecule is an inhibitory nucleic acid is a dsRNA, a siRNA, or a shRNA. In embodiments, the inhibitory nucleic acid is linked to the nucleic acid that encodes a component of the CAR molecule. For example, the inhibitory molecule can be expressed on the CAR-expressing cell.

In another embodiment, the agent which inhibits an inhibitory molecule, e.g., is a molecule described herein, e.g. , an agent that comprises a first polypeptide, e.g. , an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD-1 , PD-L1 , CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1 , CEACAM-3 and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAI R1 , CD1 60, 2B4 or TGFR beta, or a fragment of any of these (e.g., at least a portion of the extracellular domain of any of these), and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g. , 41 BB, CD27 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of PD1 or a fragment thereof (e.g. , at least a portion of the extracellular domain of PD1 ), and a second polypeptide of an intracellular signaling domain described herein (e.g. , a CD28 signaling domain described herein and/or a CD3 zeta signaling domain described herein).

In one embodiment, the CAR-expressing immune effector cell of the present invention , e.g., T cell or NK cell, is administered to a subject that has received a previous stem cell transplantation, e.g., autologous stem cell transplantation. In one embodiment, the CAR-expressing immune effector cell of the present invention, e.g., T cell or NK cells, is administered to a subject that has received a previous dose of melphalan.

In one embodiment, the cell expressing a CAR molecule, e.g., a CAR molecule described herein, is administered in combination with an agent that increases the efficacy of a cell expressing a CAR molecule, e.g. , an agent described herein.

In one embodiment, the cells expressing a CAR molecule, e.g ., a CAR molecule described herein, are administered in combination with a low, immune enhancing dose of an mTOR inhibitor. While not wishing to be bound by theory, it is believed that treatment with a low, immune enhancing, dose (e.g ., a dose that is insufficient to completely suppress the immune system but sufficient to improve immune function) is accompanied by a decrease in PD-1 positive T cells or an increase in PD-1 negative cells. PD-1 positive T cells, but not PD-1 negative T cells, can be exhausted by engagement with cells which express a PD-1 ligand, e.g., PD-L1 or PD-L2.

In an embodiment this approach can be used to optimize the performance of CAR cells described herein in the subject. While not wishing to be bound by theory, it is believed that, in an embodiment, the performance of endogenous, non-modified immune effector cells, e.g., T cells or NK cells, is improved. While not wishing to be bound by theory, it is believed that, in an embodiment, the performance of a target antigen CAR- expressing cell is improved. In other embodiments, cells, e.g. , T cells or NK cells, which have, or will be engineered to express a CAR, can be treated ex vivo by contact with an amount of an mTOR inhibitor that increases the number of PD1 negative immune effector cells, e.g., T cells or increases the ratio of PD1 negative immune effector cells, e.g., T cells/ PD1 positive immune effector cells, e.g., T cells.

In an embodiment, administration of a low, immune enhancing, dose of an mTOR inhibitor, e.g., an allosteric inhibitor, e.g. , RAD001 , or a catalytic inhibitor, is initiated prior to administration of an CAR expressing cell described herein, e.g ., T cells or NK cells. In an embodiment, the CAR cells are administered after a sufficient time, or sufficient dosing, of an mTOR inhibitor, such that the level of PD1 negative immune effector cells, e.g. , T cells or NK cells, or the ratio of PD1 negative immune effector cells, e.g., T cells/ PD1 positive immune effector cells, e.g. , T cells, has been, at least transiently, increased.

In an embodiment, the cell, e.g. , T cell or NK cell, to be engineered to express a CAR, is harvested after a sufficient time, or after sufficient dosing of the low, immune enhancing, dose of an mTOR inhibitor, such that the level of PD1 negative immune effector cells, e.g., T cells, or the ratio of PD1 negative immune effector cells, e.g., T cells/ PD1 positive immune effector cells, e.g., T cells, in the subject or harvested from the subject has been, at least transiently, increased.

In one embodiment, the cell expressing a CAR molecule, e.g., a CAR molecule described herein, is administered in combination with an agent that ameliorates one or more side effect associated with administration of a cell expressing a CAR molecule, e.g., an agent described herein.

In one embodiment, the cell expressing a CAR molecule, e.g., a CAR molecule described herein, is administered in combination with an agent that treats the disease associated with a cancer associated antigen as described herein, e.g., an agent described herein. In one embodiment, a cell expressing two or more CAR molecules, e.g., as described herein, is administered to a subject in need thereof to treat cancer. In one embodiment, a population of cells including a CAR expressing cell, e.g. , as described herein, is administered to a subject in need thereof to treat cancer.

In one embodiment, the cell expressing a CAR molecule, e.g., a CAR molecule described herein, is administered at a dose and/or dosing schedule described herein.

In one embodiment, the CAR molecule is introduced into immune effector cells (e.g., T cells, NK cells), e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of cells comprising a CAR molecule, and one or more subsequent administrations of cells comprising a CAR molecule, wherein the one or more subsequent administrations are administered less than 1 5 days, e.g. , 14, 13, 1 2, 1 1 , 1 0, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration. In one embodiment, more than one administration of cells comprising a CAR molecule are administered to the subject (e.g., human) per week, e.g. , 2, 3, or 4 administrations of cells comprising a CAR molecule are administered per week. In one embodiment, the subject (e.g., human subject) receives more than one administration of cells comprising a CAR molecule per week (e.g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no administration of cells comprising a CAR molecule, and then one or more additional administration of cells comprising a CAR molecule (e.g ., more than one administration of the cells comprising a CAR molecule per week) is administered to the subject. In another embodiment, the subject (e.g. , human subject) receives more than one cycle of cells comprising a CAR molecule, and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the cells comprising a CAR molecule are administered every other day for 3 administrations per week. In one embodiment, the cells comprising a CAR molecule are administered for at least two, three, four, five, six, seven, eight or more weeks.

In one embodiment, the cells expressing a CAR molecule, e.g ., a CAR molecule described herein, are administered as a first line treatment for the disease, e.g., the cancer, e.g., the cancer described herein. In another embodiment, the cells expressing a CAR molecule, e.g., a CAR molecule described herein, are administered as a second, third, fourth line treatment for the disease, e.g. , the cancer, e.g. , the cancer described herein .

In one embodiment, a population of cells described herein is administered.

In another aspect, the invention pertains to the isolated nucleic acid molecule encoding a CAR of the invention, the isolated polypeptide molecule of a CAR of the invention, the vector comphsing a CAR of the invention, and the cell comprising a CAR of the invention for use as a medicament.

In another aspect, the invention pertains to a the isolated nucleic acid molecule encoding a CAR of the invention, the isolated polypeptide molecule of a CAR of the invention, the vector comprising a CAR of the invention, and the cell comprising a CAR of the invention for use in the treatment of a disease expressing a cancer associated antigen as described herein.

In another aspect, the invention pertains to a cell expressing a CAR molecule described herein for use as a medicament in combination with a cytokine, e.g. , IL-7, IL-1 5 and/or IL-21 as described herein. In another aspect, the invention pertains to a cytokine described herein for use as a medicament in combination with a cell expressing a CAR molecule described herein. In another aspect, the invention pertains to a cell expressing a CAR molecule described herein for use as a medicament in combination with a kinase inhibitor and/or a checkpoint inhibitor as described herein. In another aspect, the invention pertains to a kinase inhibitor and/or a checkpoint inhibitor described herein for use as a medicament in combination with a cell expressing a CAR molecule described herein.

In another aspect, the invention pertains to a cell expressing a CAR molecule described herein for use in combination with a cytokine, e.g., IL-7, IL-1 5 and/or IL-21 as described herein, in the treatment of a disease expressing a tumor antigen targeted by the CAR. In another aspect, the invention pertains to a cytokine described herein for use in combination with a cell expressing a CAR molecule described herein, in the treatment of a disease expressing a tumor antigen targeted by the CAR.

In another aspect, the invention pertains to a cell expressing a CAR molecule described herein for use in combination with a kinase inhibitor and/or a checkpoint inhibitor as described herein, in the treatment of a disease expressing a tumor antigen targeted by the CAR. In another aspect, the invention pertains to a kinase inhibitor and/or a checkpoint inhibitor described herein for use in combination with a cell expressing a CAR molecule described herein, in the treatment of a disease expressing a tumor antigen targeted by the CAR.

In another aspect, the present invention provides a method comprising administering a CAR molecule, e.g. , a CAR molecule described herein, or a cell comprising a nucleic acid encoding a CAR molecule, e.g. , a CAR molecule described herein. In one embodiment, the subject has a disorder described herein, e.g. , the subject has cancer, e.g., the subject has a cancer and has tumor-supporting cells which express a tumor-supporting antigen described herein. In one embodiment, the subject is a human.

In another aspect, the invention pertains to a method of treating a subject having a disease associated with expression of a tumor-supporting antigen as described herein comprising administering to the subject an effective amount of a cell comprising a CAR molecule, e.g., a CAR molecule described herein.

In yet another aspect, the invention features a method of treating a subject having a disease associated with expression of a tumor-supporting antigen, comprising administering to the subject an effective amount of a cell, e.g., an immune effector cell {e.g., a population of immune effector cells) comprising a CAR molecule, wherein the CAR molecule comprises an antigen binding domain, a transmembrane domain, and an intracellular domain, said intracellular domain comprises a costimulatory domain and/or a primary signaling domain, wherein said antigen binding domain binds to the tumor- supporting antigen associated with the disease, e.g. a tumor-supporting antigen as described herein.

In another aspect, the invention features a composition comprising an immune effector cell (e.g., a population of immune effector cells) comprising a CAR molecule (e.g. , a CAR molecule as described herein) for use in the treatment of a subject having a disease associated with expression of a tumor- supporting antigen, e.g. , a disorder as described herein.

In any of the aforesaid methods or uses, the disease associated with expression of the tumor- supporting antigen is selected from the group consisting of a proliferative disease, a precancerous condition, a cancer, and a non-cancer related indication associated with expression of the tumor- supporting antigen. In an embodiment, the disease associated with a tumor-supporting antigen described herein is a solid tumor.

In one embodiment of the methods or uses described herein, the CAR molecule is administered in combination with another agent. In one embodiment, the agent can be a kinase inhibitor, e.g., a CDK4/6 inhibitor, a BTK inhibitor, an mTOR inhibitor, a MNK inhibitor, or a dual PI3K/mTOR inhibitor, and combinations thereof. In one embodiment, the kinase inhibitor is a CDK4 inhibitor, e.g. , a CDK4 inhibitor described herein, e.g. , a CD4/6 inhibitor, such as, e.g. , 6-Acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1 - yl-pyridin-2-ylamino)-8H-pyrido[2,3-Gflpyrimidin-7-one, hydrochloride (also referred to as palbociclib or PD0332991 ). In one embodiment, the kinase inhibitor is a BTK inhibitor, e.g. , a BTK inhibitor described herein, such as, e.g. , ibrutinib. In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g., an mTOR inhibitor described herein, such as, e.g., rapamycin, a rapamycin analog, OSI-027. The mTOR inhibitor can be, e.g. , an mTORCI inhibitor and/or an mTORC2 inhibitor, e.g., an mTORCI inhibitor and/or mTORC2 inhibitor described herein. In one embodiment, the kinase inhibitor is a MNK inhibitor, e.g., a MNK inhibitor described herein, such as, e.g. , 4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d| pyrimidine. The MNK inhibitor can be, e.g., a MNK1 a, MNK1 b, MNK2a and/or MNK2b inhibitor. The dual PI3K/mTOR inhibitor can be, e.g. , PF-046951 02.

In one embodiment of the methods or uses described herein, the kinase inhibitor is a CDK4 inhibitor selected from aloisine A; flavopiridol or HMR-1275, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)- 3-hydroxy-1 -methyl-4-piperidinyl]-4-chromenone; crizotinib (PF-02341066; 2-(2-Chlorophenyl)-5,7- dihydroxy-8-[(2R,3S)-2-(hydroxymethyl)-1 -methyl-3-pyrrolidinyl]- 4H-1 -benzopyran-4-one, hydrochloride (P276-00); 1 -methyl-5-[[2-[5-(trifluoromethyl)-1 H-imidazol-2-yl]-4-pyridinyl]oxy]-/V-[4- (trifluoromethyl)phenyl]-1 H-benzimidazol-2-amine (RAF265); indisulam (E7070) ; roscovitine (CYC202); palbociclib (PD0332991 ) ; dinaciclib (SCH727965); N-[5-[[(5-ferf-butyloxazol-2-yl)methyl]thio]thiazol-2- yl]piperidine-4-carboxamide (BMS 387032) ; 4-[[9-chloro-7-(2,6-difluorophenyl)-5H-pyrimido[5,4- aq[2]benzazepin-2-yl]amino]-benzoic acid (MLN8054) ; 5-[3-(4,6-difluoro-1 H-benzimidazol-2-yl)-1 H- indazol-5-yl]-N-ethyl-4-methyl-3-pyridinemethanamine (AG-024322); 4-(2,6-dichlorobenzoylamino)-1 H- pyrazole-3-carboxylic acid N-(piperidin-4-yl)amide (AT7519); 4-[2-methyl-1 -(1 -methylethyl)-1 H-imidazol-5- yl]-A/-[4-(methylsulfonyl)phenyl]- 2-pyrimidinamine (AZD5438) ; and XL281 (BMS908662).

In one embodiment of the methods or uses described herein, the kinase inhibitor is a CDK4 inhibitor, e.g. , palbociclib (PD0332991 ), and the palbociclib is administered at a dose of about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 1 00 mg, 1 05 mg, 1 10 mg, 1 15 mg, 120 mg, 125 mg, 130 mg, 135 mg (e.g., 75 mg, 100 mg or 125 mg) daily for a period of time, e.g., daily for 14-21 days of a 28 day cycle, or daily for 7-12 days of a 21 day cycle. In one embodiment, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 1 0, 1 1 , 1 2, or more cycles of palbociclib are administered.

In one embodiment of the methods or uses described herein, the kinase inhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765); G DC-0834; RN-486; CG I-560; CG I-1 764; HM-71224; CC- 292; ONO-4059; CNX-774; and LFM-A13. In one embodiment, the BTK inhibitor does not reduce or inhibit the kinase activity of interleukin-2-inducible kinase (ITK), and is selected from G DC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13. In one embodiment of the methods or uses described herein, the kinase inhibitor is a BTK inhibitor, e.g. , ibrutinib (PCI-32765), and the ibrutinib is administered at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (e.g., 250 mg, 420 mg or 560 mg) daily for a period of time, e.g., daily for 21 day cycle, or daily for 28 day cycle. In one embodiment, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or more cycles of ibrutinib are administered.

In one embodiment of the methods or uses described herein, the kinase inhibitor is a BTK inhibitor that does not inhibit the kinase activity of ITK, e.g., RN-486, and RN-486 is administered at a dose of about 100 mg, 1 10 mg, 120 mg, 130 mg, 140 mg, 1 50 mg, 1 60 mg, 1 70 mg, 180 mg, 190 mg, 200 mg, 210 mg, 220 mg, 230 mg, 240 mg, 250 mg (e.g., 1 50 mg, 200 mg or 250 mg) daily for a period of time, e.g. , daily a 28 day cycle. In one embodiment, 1 , 2, 3, 4, 5, 6, 7, or more cycles of RN-486 are administered.

In one embodiment of the methods or uses described herein, the kinase inhibitor is an mTOR inhibitor selected from temsirolimus; ridaforolimus (1 R,2R,AS)-A-[{2R)-2

[(1 fl,9S,12S,1 5fl,16E,1 8fl,1 9fl,21 R, 23S,24E,26E,28Z,30S,32S,35fl)-1 ,1 8-dihydroxy-1 9,30-dimethoxy- 1 5,1 7,21 ,23, 29,35-hexamethyl-2,3, 1 0, 14,20-pentaoxo-1 1 ,36-dioxa-4-azatricyclo[30.3.1 .049]

hexatriaconta-1 6,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl dimethylphosphinate, also known as AP23573 and MK8669; everolimus (RAD001 ); rapamycin (AY22989); simapimod; (5-{2,4-bis[(3S)-3- methylmorpholin-4-yl]pyrido[2,3-c|pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8- [irans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-d|pyrimidin-7(8 - )- one (PF04691 502); and Λ -[1 ,4-dioxo-4-[[4-(4-oxo-8-phenyl-4 - -1 -benzopyran-2-yl)morpholinium-4- yl]methoxy]butyl]-L-arginylglycyl-L-a-aspartylL-serine-, inner salt (SF1 126); and XL765.

In one embodiment of the methods or uses described herein, the kinase inhibitor is an mTOR inhibitor, e.g. , rapamycin, and the rapamycin is administered at a dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g., 6 mg) daily for a period of time, e.g. , daily for 21 day cycle, or daily for 28 day cycle. In one embodiment, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or more cycles of rapamycin are administered. In one embodiment, the kinase inhibitor is an mTOR inhibitor, e.g., everolimus and the everolimus is administered at a dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 1 0 mg, 1 1 mg, 12 mg, 13 mg, 14 mg, 1 5 mg (e.g., 10 mg) daily for a period of time, e.g., daily for 28 day cycle. In one embodiment, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12 or more cycles of everolimus are administered.

In one embodiment of the methods or uses described herein, the kinase inhibitor is an MNK inhibitor selected from CGP052088; 4-amino-3-(p-fluorophenylamino)-pyrazolo [3,4-cfl pyrimidine (CGP57380) ; cercosporamide; ETC-1 780445-2; and 4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-cfl pyrimidine.

In one embodiment of the methods or uses described herein, the kinase inhibitor is a dual phosphatidylinositol 3-kinase (PI3K) and mTOR inhibitor selected from 2-Amino-8-[frans-4-(2- hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methyl-pyrido[2,3-G'|pyrimidin-7(8 - )-one (PF- 04691 502) ; /V-[4-[[4-(Dimethylamino)-1 -piperidinyl]carbonyl]phenyl]-/V-[4-(4,6-di-4-morpholinyl-1 ,3,5- triazin-2-yl)phenyl]urea (PF-05212384, PKI-587) ; 2-Methyl-2-{4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3- dihydro-1 /-imidazo[4,5-c]quinolin-1 -yl]phenyl}propanenitrile (BEZ-235); apitolisib (G DC-0980, RG7422); 2,4-Difluoro-N-{2-(methyloxy)-5-[4-(4-pyn^

(GSK2126458) ; 8-(6-methoxypyridin-3-yl)-3-methyl-1 -(4-(piperazin-1 -yl)-3-(trifluoromethyl)^

imidazo[4,5-c]quinolin-2(3H)-one aleic acid (NVP-BGT226) ; 3-[4-(4-Morpholinylpyrido[3',2':4,5]furo[3,2- d]pyrimidin-2-yl]phenol (PI- 1 03); 5-(9-isopropyl-8-methyl-2-morpholino-9H-purin-6-yl)pyrimidin-2-amine (VS-5584, SB2343); and N-[2-[(3,5-Dimethoxyphenyl)amino]quinoxalin-3-yl]-4-[(4-methyl-3- methoxyph6nyl)carbonyl]aminoph6nylsulfonamide (XL765).

In one embodiment of the methods or uses described herein, a CAR expressing immune effector cell described herein is administered to a subject in combination with a protein tyrosine phosphatase inhibitor, e.g. , a protein tyrosine phosphatase inhibitor described herein. In one embodiment, the protein tyrosine phosphatase inhibitor is an SHP-1 inhibitor, e.g., an SHP-1 inhibitor described herein, such as, e.g., sodium stibogluconate. In one embodiment, the protein tyrosine phosphatase inhibitor is an SHP-2 inhibitor.

In one embodiment of the methods or uses described herein, the CAR molecule is administered in combination with another agent, and the agent is a cytokine. The cytokine can be, e.g., IL-7, IL-15, IL- 21 , or a combination thereof. In another embodiment, the CAR molecule is administered in combination with a checkpoint inhibitor, e.g., a checkpoint inhibitor described herein. For example, in one

embodiment, the check point inhibitor inhibits an inhibitory molecule selected from PD-1 , PD-L1 , CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1 , CEACAM-3 and/or CEACAM-5), LAG-3, VISTA, BTLA, TIGIT, LAIR1 , CD1 60, 2B4 and TGFR beta.

In one aspect, the CAR of the invention can be used to eradicate a normal cell that express a tumor antigen as described herein, thereby applicable for use as a cellular conditioning therapy prior to cell transplantation. In one aspect, the normal cell that expresses a tumor antigen as described herein is a normal stem cell and the cell transplantation is a stem cell transplantation. Therapeutic Application

In another aspect, a method of treating a subject, e.g. , reducing or ameliorating, a

hyperproliferative condition or disorder (e.g., a cancer), e.g., solid tumor, a soft tissue tumor, or a metastatic lesion, in a subject is provided. As used herein, the term "cancer" is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. Examples of solid tumors include malignancies, e.g. , sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting liver, lung, breast, lymphoid, gastrointestinal (e.g., colon), genitourinary tract (e.g., renal, urothelial cells), prostate and pharynx. Adenocarcinomas include malignancies such as most colon cancers, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus. In one embodiment, the cancer is a melanoma, e.g., an advanced stage melanoma. Metastatic lesions of the aforementioned cancers can also be treated or prevented using the methods and compositions of the invention. Examples of other cancers that can be treated include bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin Disease, non-Hodgkin lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, chronic or acute leukemias including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder, cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasm of the central nervous system (CNS), primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer, T-cell lymphoma,

environmentally induced cancers including those induced by asbestos, and combinations of said cancers. Treatment of metastatic cancers, e.g., metastatic cancers that express PD-L1 (Iwai et al. (2005) Int. Immunol. 1 7:133-144) can be effected using the antibody molecules described herein.

Exemplary cancers whose growth can be inhibited include cancers typically responsive to immunotherapy. Non-limiting examples of cancers for treatment include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), breast cancer, colon cancer and lung cancer (e.g. non-small cell lung cancer). Additionally, refractory or recurrent malignancies can be treated using the molecules described herein .

In one aspect, the invention pertains to a vector comprising a CAR operably linked to promoter for expression in mammalian immune effector cells (e.g. , T cells, NK cells). In one aspect, the invention provides a recombinant immune effector cell expressing a CAR of the present invention for use in treating cancer expressing a cancer associate antigen as described herein. In one aspect, CAR-expressing cells of the invention is capable of contacting a tumor cell with at least one cancer associated antigen expressed on its surface such that the CAR-expressing cell targets the cancer cell and growth of the cancer is inhibited.

In one aspect, the invention pertains to a method of inhibiting growth of a cancer, comprising contacting the cancer cell with a CAR-expressing cell of the present invention such that the CART is activated in response to the antigen and targets the cancer cell, wherein the growth of the tumor is inhibited.

In one aspect, the invention pertains to a method of treating cancer in a subject. The method comprises administering to the subject CAR-expressing cell of the present invention such that the cancer is treated in the subject. In one aspect, the cancer associated with expression of a cancer associate antigen as described herein is a hematological cancer. In one aspect, the hematological cancer is a leukemia or a lymphoma. In one aspect, a cancer associated with expression of a cancer associate antigen as described herein includes cancers and malignancies including, but not limited to, e.g., one or more acute leukemias including but not limited to, e.g., B-cell acute Lymphoid Leukemia ("BALL"), T-cell acute Lymphoid Leukemia ("TALL"), acute lymphoid leukemia (ALL) ; one or more chronic leukemias including but not limited to, e.g. , chronic myelogenous leukemia (CML), Chronic Lymphoid Leukemia (CLL). Additional cancers or hematologic conditions associated with expression of a cancer associate antigen as described herein include, but are not limited to, e.g., B cell prolymphocyte leukemia, blastic plasmacytoid dendritic cell neoplasm , Burkitt's lymphoma, diffuse large B cell lymphoma, Follicular lymphoma, Hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplasia syndrome, non-Hodgkin lymphoma,

plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and "preleukemia" which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and the like. Further a disease associated with a cancer associate antigen as described herein expression include, but not limited to, e.g. , atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases associated with expression of a cancer associate antigen as described herein.

In some embodiments, a cancer that can be treated with CAR-expressing cell of the present invention is multiple myeloma. Generally, myeloma cells are thought to be negative for a cancer associate antigen as described herein expression by flow cytometry. Thus, in some embodiments, a CD1 9 CAR, e.g. , as described herein, may be used to target myeloma cells. In some embodiments, cars of the present invention therapy can be used in combination with one or more additional therapies, e.g ., lenalidomide treatment.

The invention includes a type of cellular therapy where immune effector cells (e.g. , T cells, NK cells) are genetically modified to express a chimeric antigen receptor (CAR) and the CAR-expressing T cell or NK cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Unlike antibody therapies, CAR-modified immune effector cells (e.g. , T cells, NK cells) are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control. In various aspects, the immune effector cells (e.g., T cells, NK cells) administered to the patient, or their progeny, persist in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after administration of the T cell or NK cell to the patient.

The invention also includes a type of cellular therapy where immune effector cells (e.g., T cells, NK cells) are modified, e.g. , by in vitro transcribed RNA, to transiently express a chimeric antigen receptor (CAR) and the CAR T cell or NK cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Thus, in various aspects, the immune effector cells (e.g. , T cells, NK cells) administered to the patient, is present for less than one month, e.g., three weeks, two weeks, one week, after administration of the T cell or NK cell to the patient.

Without wishing to be bound by any particular theory, the anti-tumor immunity response elicited by the CAR-modified immune effector cells (e.g ., T cells, NK cells) may be an active or a passive immune response, or alternatively may be due to a direct vs indirect immune response. In one aspect, the CAR transduced immune effector cells (e.g., T cells, NK cells) exhibit specific proinflammatory cytokine secretion and potent cytolytic activity in response to human cancer cells expressing the a cancer associate antigen as described herein, resist soluble a cancer associate antigen as described herein inhibition, mediate bystander killing and mediate regression of an established human tumor. For example, antigen-less tumor cells within a heterogeneous field of a cancer associate antigen as described herein- expressing tumor may be susceptible to indirect destruction by a cancer associate antigen as described herein-redirected immune effector cells (e.g. , T cells, NK cells) that has previously reacted against adjacent antigen-positive cancer cells.

In one aspect, the fully-human CAR-modified immune effector cells (e.g., T cells, NK cells) of the invention may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal. In one aspect, the mammal is a human.

With respect to ex vivo immunization, at least one of the following occurs in vitro prior to administering the cell into a mammal : i) expansion of the cells, ii) introducing a nucleic acid encoding a CAR to the cells or iii) cryopreservation of the cells.

Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e. , transduced or transfected in vitro) with a vector expressing a CAR disclosed herein . The CAR-modified cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the CAR-modified cell can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.

The procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described in U.S. Pat. No. 5,199,942, incorporated herein by reference, can be applied to the cells of the present invention. Other suitable methods are known in the art, therefore the present invention is not limited to any particular method of ex vivo expansion of the cells. Briefly, ex vivo culture and expansion of immune effector cells (e.g., T cells, NK 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 U.S. Pat. No. 5, 199,942, other factors such as flt3-L, IL-1 , IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.

In addition to using a cell-based vaccine in terms of ex vivo immunization , the present invention also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.

Generally, the cells activated and expanded as described herein may be utilized in the treatment and prevention of diseases that arise in individuals who are immunocompromised. In particular, the CAR- modified immune effector cells (e.g. , T cells, NK cells) of the invention are used in the treatment of diseases, disorders and conditions associated with expression of a cancer associate antigen as described herein. In certain aspects, the cells of the invention are used in the treatment of patients at risk for developing diseases, disorders and conditions associated with expression of a cancer associate antigen as described herein. Thus, the present invention provides methods for the treatment or prevention of diseases, disorders and conditions associated with expression of a cancer associate antigen as described herein comprising administering to a subject in need thereof, a therapeutically effective amount of the CAR-modified immune effector cells (e.g ., T cells, NK cells) of the invention.

In one aspect the CAR-expressing cells of the inventions may be used to treat a proliferative disease such as a cancer or malignancy or is a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia. Further a disease associated with a cancer associate antigen as described herein expression include, but not limited to, e.g. , atypical and/or non-classical cancers, malignancies, precancerous conditions or proliferative diseases expressing a cancer associated antigen as described herein. Non-cancer related indications associated with expression of a cancer associate antigen as described herein include, but are not limited to, e.g. , autoimmune disease, (e.g. , lupus), inflammatory disorders (allergy and asthma) and transplantation.

The CAR-modified immune effector cells (e.g., T cells, NK cells) of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations.

Hematologic Cancer

Hematological cancer conditions are the types of cancer such as leukemia, lymphoma, and malignant lymphoproliferative conditions that affect blood, bone marrow and the lymphatic system.

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.

Lymphoma is a group of blood cell tumors that develop from lymphocytes. Exemplary lymphomas include non-Hodgkin lymphoma and Hodgkin lymphoma.

The present invention also provides methods for inhibiting the proliferation or reducing a cancer associated antigen as described herein-expressing cell population, the methods comprising contacting a population of cells comprising a cancer associated antigen as described herein-expressing cell with a CAR-expressing T cell or NK cell of the invention that binds to the a cancer associate antigen as described herein-expressing cell. In a specific aspect, the present invention provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing a cancer associated antigen as described herein, the methods comprising contacting a cancer associate antigen as described herein-expressing cancer cell population with a CAR-expressing T cell or NK cell of the invention that binds to a cancer associated antigen as described herein-expressing cell. In one aspect, the present invention provides methods for inhibiting the proliferation or reducing the population of cancer cells expressing a cancer associated antigen as described herein, the methods comprising contacting a cancer associated antigen as described herein-expressing cancer cell population with a CAR-expressing T cell or NK cell of the invention that binds to a cancer associated antigen as described herein-expressing cell. In certain aspects, a CAR-expressing T cell or NK cell of the invention reduces the quantity, number, amount or percentage of cells and/or cancer cells by at least 25%, at least 30%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 95%, or at least 99% in a subject with or animal model for myeloid leukemia or another cancer associated with a cancer associated antigen as described herein-expressing cells relative to a negative control. In one aspect, the subject is a human.

The present invention also provides methods for preventing, treating and/or managing a disease associated with a cancer associated antigen as described herein-expressing cells (e.g., a hematologic cancer or atypical cancer expressing a cancer associated antigen as described herein), the methods comprising administering to a subject in need a CAR T cell or NK cell of the invention that binds to a cancer associated antigen as described herein-expressing cell. In one aspect, the subject is a human. Non-limiting examples of disorders associated with a cancer associated antigen as described herein- expressing cells include autoimmune disorders (such as lupus), inflammatory disorders (such as allergies and asthma) and cancers (such as hematological cancers or atypical cancers expressing a cancer associated antigen as described herein).

The present invention also provides methods for preventing, treating and/or managing a disease associated with a cancer associated antigen as described herein-expressing cells, the methods comprising administering to a subject in need a CAR T cell or NK cell of the invention that binds to a cancer associated antigen as described herein-expressing cell. In one aspect, the subject is a human.

The present invention provides methods for preventing relapse of cancer associated with a cancer associated antigen as described herein-expressing cells, the methods comprising administering to a subject in need thereof a CAR T cell or NK cell of the invention that binds to a cancer associated antigen as described herein-expressing cell. In one aspect, the methods comprise administering to the subject in need thereof an effective amount of a CAR-expressing T cell or NK cell described herein that binds to a cancer associated antigen as described herein-expressing cell in combination with an effective amount of another therapy.

Pharmaceutical compositions and treatments

Pharmaceutical compositions of the present invention may comprise a CAR-expressing cell, e.g., a plurality of CAR-expressing cells, as described herein, in combination with one or more

pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g. , aluminum hydroxide) ; and preservatives. Compositions of the present invention are in one aspect formulated for intravenous administration.

Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages may be determined by clinical trials.

In one embodiment, the pharmaceutical composition is substantially free of, e.g. , there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin , mycoplasma, replication competent lentivirus (RCL), p24, VSV-G nucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum , culture media components, vector packaging cell or plasmid components, a bacterium and a fungus. In one embodiment, the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli , Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A. When "an immunologically effective amount," "an anti-tumor effective amount," "a tumor-inhibiting effective amount," or "therapeutic amount" is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the immune effector cells (e.g. , T cells, NK cells) described herein may be administered at a dosage of 1 04 to 1 09 cells/kg body weight, in some instances 105 to 1 06 cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al ., New Eng. J. of Med. 31 9:1 676, 1 988).

In certain aspects, it may be desired to administer activated immune effector cells (e.g., T cells, NK cells) to a subject and then subsequently redraw blood (or have an apheresis performed), activate immune effector cells (e.g., T cells, NK cells) therefrom according to the present invention, and reinfuse the patient with these activated and expanded immune effector cells (e.g., T cells, NK cells). This process can be carried out multiple times every few weeks. In certain aspects, immune effector cells (e.g., T cells, NK cells) can be activated from blood draws of from 10cc to 400cc. In certain aspects, immune effector cells (e.g. , T cells, NK cells) are activated from blood draws of 20cc, 30cc, 40cc, 50cc, 60cc, 70cc, 80cc, 90cc, or 1 0Occ.

The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one aspect, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. I n one aspect, the T cell compositions of the present invention are administered by i.v. injection. The compositions of immune effector cells (e.g., T cells, NK cells) may be injected directly into a tumor, lymph node, or site of infection.

In a particular exemplary aspect, subjects may undergo leukapheresis, wherein leukocytes are collected, enriched, or depleted ex vivo to select and/or isolate the cells of interest, e.g. , T cells. These T cell isolates may be expanded by methods known in the art and treated such that one or more CAR constructs of the invention may be introduced, thereby creating a CAR T cell of the invention. Subjects in need thereof may subsequently undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain aspects, following or concurrent with the transplant, subjects receive an infusion of the expanded CAR T cells of the present invention. In an additional aspect, expanded cells are administered before or following surgery.

The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices. The dose for CAMPATH, for example, will generally be in the range 1 to about 1 00 mg for an adult patient, usually administered daily for a period between 1 and 30 days. The preferred daily dose is 1 to 10 mg per day although in some instances larger doses of up to 40 mg per day may be used (described in U.S. Patent No. 6, 120,766). In one embodiment, the CAR is introduced into immune effector cells (e.g. , T cells, NK cells), e.g., using in vitro transcription, and the subject (e.g., human) receives an initial administration of CAR immune effector cells (e.g., T cells, NK cells) of the invention, and one or more subsequent

administrations of the CAR immune effector cells (e.g. , T cells, NK cells) of the invention, wherein the one or more subsequent administrations are administered less than 1 5 days, e.g. , 14, 13, 12, 1 1 , 1 0, 9, 8, 7, 6, 5, 4, 3, or 2 days after the previous administration . In one embodiment, more than one administration of the CAR immune effector cells (e.g., T cells, NK cells) of the invention are administered to the subject (e.g., human) per week, e.g. , 2, 3, or 4 administrations of the CAR immune effector cells (e.g. , T cells, NK cells) of the invention are administered per week. In one embodiment, the subject (e.g., human subject) receives more than one administration of the CAR immune effector cells (e.g., T cells, NK cells) per week (e.g., 2, 3 or 4 administrations per week) (also referred to herein as a cycle), followed by a week of no CAR immune effector cells (e.g., T cells, NK cells) administrations, and then one or more additional administration of the CAR immune effector cells (e.g., T cells, NK cells) (e.g., more than one

administration of the CAR immune effector cells (e.g., T cells, NK cells) per week) is administered to the subject. In another embodiment, the subject (e.g., human subject) receives more than one cycle of CAR immune effector cells (e.g., T cells, NK cells), and the time between each cycle is less than 10, 9, 8, 7, 6, 5, 4, or 3 days. In one embodiment, the CAR immune effector cells (e.g. , T cells, NK cells) are administered every other day for 3 administrations per week. In one embodiment, the CAR immune effector cells (e.g., T cells, NK cells) of the invention are administered for at least two, three, four, five, six, seven , eight or more weeks.

In one aspect, CAR-expressing cells of the present inventions are generated using lentiviral viral vectors, such as lentivirus. Cells, e.g., CARTs, generated that way will have stable CAR expression.

In one aspect, CAR-expressing cells, e.g., CARTs, are generated using a viral vector such as a gammaretroviral vector, e.g., a gammaretroviral vector described herein. CARTs generated using these vectors can have stable CAR expression .

In one aspect, CARTs transiently express CAR vectors for 4, 5, 6, 7, 8, 9, 1 0, 1 1 , 12, 1 3, 14, 1 5 days after transduction. Transient expression of CARs can be effected by RNA CAR vector delivery. In one aspect, the CAR RNA is transduced into the T cell by electroporation.

A potential issue that can arise in patients being treated using transiently expressing CAR immune effector cells (e.g., T cells, NK cells) (particularly with murine scFv bearing CARTs) is anaphylaxis after multiple treatments.

Without being bound by this theory, it is believed that such an anaphylactic response might be caused by a patient developing humoral anti-CAR response, i.e. , anti-CAR antibodies having an anti-lgE isotype. It is thought that a patient's antibody producing cells undergo a class switch from IgG isotype (that does not cause anaphylaxis) to IgE isotype when there is a ten to fourteen day break in exposure to antigen.

If a patient is at high risk of generating an anti-CAR antibody response during the course of transient CAR therapy (such as those generated by RNA transductions), CART infusion breaks should not last more than ten to fourteen days. Methods of making CAR-expressing cells

In another aspect, the invention pertains to a method of making a cell (e.g., an immune effector cell or population thereof) comprising introducing into (e.g. , transducing) a cell, e.g., a T cell or a NK cell described herein, with a vector of comprising a nucleic acid encoding a CAR, e.g. , a CAR described herein ; or a nucleic acid encoding a CAR molecule e.g., a CAR described herein.

The cell in the methods is an immune effector cell (e.g. , a T cell or a NK cell, or a combination thereof). In some embodiments, the cell in the methods is diaglycerol kinase (DGK) and/or Ikaros deficient.

In some embodiment, the introducing the nucleic acid molecule encoding a CAR comprises transducing a vector comprising the nucleic acid molecule encoding a CAR, or transfecting the nucleic acid molecule encoding a CAR, wherein the nucleic acid molecule is an in vitro transcribed RNA.

In some embodiments, the method further comprises:

a. providing a population of immune effector cells (e.g. , T cells or NK cells); and

b. removing T regulatory cells from the population, thereby providing a population of T regulatory-depleted cells;

wherein steps a) and b) are performed prior to introducing the nucleic acid encoding the CAR to the population.

In embodiments of the methods, the T regulatory cells comprise CD25+ T cells, and are removed from the cell population using an anti-CD25 antibody, or fragment thereof. The anti-CD25 antibody, or fragment thereof, can be conjugated to a substrate, e.g., a bead.

In other embodiments, the population of T regulatory-depleted cells provided from step (b) contains less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1 % of CD25+ cells.

In yet other embodiments, the method further comprises removing cells from the population which express a tumor antigen that does not comprise CD25 to provide a population of T regulatory- depleted and tumor antigen depleted cells prior to introducing the nucleic acid encoding a CAR to the population. The tumor antigen can be selected from CD1 9, CD30, CD38, CD123, CD20, CD14 or CD1 1 b, or a combination thereof.

In other embodiments, the method further comprises removing cells from the population which express a checkpoint inhibitor, to provide a population of T regulatory-depleted and inhibitory molecule depleted cells prior to introducing the nucleic acid encoding a CAR to the population. The checkpoint inhibitor can be chosen from PD-1 , LAG-3, TIM3, B7-H1 , CD1 60, P1 H, 2B4, CEACAM (e.g., CEACAM-1 , CEACAM-3, and/or CEACAM-5), TIG IT, CTLA-4, BTLA, and LAIR1 .

Further embodiments disclosed herein encompass providing a population of immune effector cells. The population of immune effector cells provided can be selected based upon the expression of one or more of CD3, CD28, CD4, CD8, CD45RA, and/or CD45RO. In certain embodiments, the population of immune effector cells provided are CD3+ and/or CD28+.

In certain embodiments of the method, the method further comprises expanding the population of cells after the nucleic acid molecule encoding a CAR has been introduced.

In embodiments, the population of cells is expanded for a period of 8 days or less. In certain embodiments, the population of cells is expanded in culture for 5 days, and the resulting cells are more potent than the same cells expanded in culture for 9 days under the same culture conditions.

In other embodiments, the population of cells is expanded in culture for 5 days show at least a one, two, three or four fold increase in cell doublings upon antigen stimulation as compared to the same cells expanded in culture for 9 days under the same culture conditions.

In yet other embodiments, the population of cells is expanded in culture for 5 days, and the resulting cells exhibit higher proinflammatory IFN-γ and/or GM-CSF levels, as compared to the same cells expanded in culture for 9 days under the same culture conditions.

In other embodiments, the population of cells is expanded by culturing the cells in the presence of an agent that stimulates a CD3/TCR complex associated signal and/or a ligand that stimulates a costimulatory molecule on the surface of the cells. The agent can be a bead conjugated with anti-CD3 antibody, or a fragment thereof, and/or anti-CD28 antibody, or a fragment thereof.

In other embodiments, the population of cells is expanded in an appropriate media that includes one or more interleukin that result in at least a 200-fold, 250-fold, 300-fold, or 350-fold increase in cells over a 14 day expansion period, as measured by flow cytometry.

In other embodiments, the population of cells is expanded in the presence IL-1 5 and/or IL-7. In certain embodiments, the method further includes cryopreserving the population of the cells after the appropriate expansion period.

In yet other embodiments, the method of making disclosed herein further comprises contacting the population of immune effector cells with a nucleic acid encoding a telomerase subunit, e.g. , hTERT. The nucleic acid encoding the telomerase subunit can be DNA.

The present invention also provides a method of generating a population of RNA-engineered cells, e.g., cells described herein, e.g., immune effector cells (e.g., T cells, NK cells), transiently expressing exogenous RNA. The method comprises introducing an in vitro transcribed RNA or synthetic RNA into a cell, where the RNA comprises a nucleic acid encoding a CAR molecule described herein.

In another aspect, the invention pertains to a method of providing an anti-tumor immunity in a subject comprising administering to the subject an effective amount of a cell comprising a CAR molecule, e.g., a cell expressing a CAR molecule described herein. In one embodiment, the cell is an autologous T cell or NK cell. In one embodiment, the cell is an allogeneic T cell or NK cell. In one embodiment, the subject is a human.

In one aspect, the invention includes a population of autologous cells that are transfected or transduced with a vector comprising a nucleic acid molecule encoding a CAR molecule, e.g., as described herein. In one embodiment, the vector is a retroviral vector. In one embodiment, the vector is a self-inactivating lentiviral vector as described elsewhere herein. In one embodiment, the vector is delivered (e.g., by transfecting or electroporating) to a cell, e.g. , a T cell or a NK cell , wherein the vector comprises a nucleic acid molecule encoding a CAR of the present invention as described herein, which is transcribed as an mRNA molecule, and the CARs of the present invention is translated from the RNA molecule and expressed on the surface of the cell. In another aspect, the present invention provides a population of CAR-expressing cells, e.g., CAR-expressing immune effector cells (e.g., T cells or NK cells). In some embodiments, the population of CAR-expressing cells comprises a mixture of cells expressing different CARs. For example, in one embodiment, the population of CAR-expressing immune effector cells (e.g., T cells or NK cells) can include a first cell expressing a CAR having an antigen binding domain that binds to a first tumor antigen as described herein, and a second cell expressing a CAR having a different antigen binding domain that binds to a second tumor antigen as described herein. As another example, the population of CAR- expressing cells can include a first cell expressing a CAR that includes an antigen binding domain that binds to a tumor antigen as described herein, and a second cell expressing a CAR that includes an antigen binding domain to a target other than a tumor antigen as described herein. In one embodiment, the population of CAR-expressing cells includes, e.g., a first cell expressing a CAR that includes a primary intracellular signaling domain, and a second cell expressing a CAR that includes a secondary signaling domain, e.g., a costimulatory signaling domain.

In another aspect, the present invention provides a population of cells wherein at least one cell in the population expresses a CAR having an antigen binding domain that binds to a tumor antigen as described herein, and a second cell expressing another agent, e.g., an agent which enhances the activity of a CAR-expressing cell. For example, in one embodiment, the agent can be an agent which inhibits an inhibitory molecule. Examples of inhibitory molecules include PD-1 , PD-L1 , CTLA-4, TIM-3, CEACAM (e.g., CEACAM-1 , CEACAM-3 and/or CEACAM-5), LAG-3, VISTA, BTLA, TIG IT, LAI R1 , CD1 60, 2B4 and TGFR beta. In one embodiment, the agent which inhibits an inhibitory molecule, e.g. , is a molecule described herein, e.g. , an agent that comprises a first polypeptide, e.g. , an inhibitory molecule, associated with a second polypeptide that provides a positive signal to the cell, e.g., an intracellular signaling domain described herein. In one embodiment, the agent comprises a first polypeptide, e.g., of an inhibitory molecule such as PD-1 , LAG-3, CTLA-4, CD1 60, BTLA, LAIR1 , TIM-3, CEACAM (e.g., CEACAM-1 , CEACAM-3 and/or CEACAM-5), 2B4 and TIGIT, or a fragment of any of these, and a second polypeptide which is an intracellular signaling domain described herein (e.g., comprising a costimulatory domain (e.g ., 41 BB, CD27 or CD28, e.g., as described herein) and/or a primary signaling domain (e.g., a CD3 zeta signaling domain described herein). In one embodiment, the agent comprises a first polypeptide of PD-1 or a fragment thereof, and a second polypeptide of an intracellular signaling domain described herein (e.g., a CD28, CD27, OX40 or 4-IBB signaling domain described herein and/or a CD3 zeta signaling domain described herein).

In one embodiment, the nucleic acid molecule encoding a CAR of the present invention molecule, e.g., as described herein, is expressed as an mRNA molecule. In one embodiment, the genetically modified CAR of the present invention-expressing cells, e.g., immune effector cells (e.g. , T cells, NK cells), can be generated by transfecting or electroporating an RNA molecule encoding the desired CARs (e.g., without a vector sequence) into the cell. In one embodiment, a CAR of the present invention molecule is translated from the RNA molecule once it is incorporated and expressed on the surface of the recombinant cell.

A method for generating mRNA for use in transfection involves in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3' and 5' untranslated sequence ("UTR") (e.g. , a 3' and/or 5' UTR described herein), a 5' cap (e.g., a 5' cap described herein) and/or Internal Ribosome Entry Site (IRES) (e.g., an I RES described herein), the nucleic acid to be expressed, and a polyA tail, typically 50-2000 bases in length. RNA so produced can efficiently transfect different kinds of cells. In one embodiment, the template includes sequences for the CAR. In an embodiment, an RNA CAR vector is transduced into a cell, e.g. , a T cell or a NK cell, by electroporation.

Kits

The invention also provides kits for carrying out the methods described herein. The kits can include one or more reagents or assay components for determining the proliferation of a CART cell, as described herein, and optionally instructions for carrying out the methods. For example, the kits may include modified nucleotides, which are incorporated into DNA that is synthesized in proliferating cells. Further, the kits can optionally include controls such as, for example, anti-idiotypic antibodies, beads including antibodies against CD3 and/or CD28, or beads including control IgG antibodies. The kits may further an antigen preparation for stimulating the CART cells (e.g., antigen immobilized on a bead or present on cells, such as killed cells). The components of the kits can be present in unit forms, for use in carrying out a single or multiple assays, as desired.

Sequence Tables

Sequences of some examples of various components of CARs and vectors for making them are listed in Table 1 , where aa stands for amino acids, and na stands for nucleic acids that encode the corresponding peptide.

Table 1. Sequences of various components of CARs and vectors for making them (aa - amino acids, na - nucleic acids that encodes the corresponding protein)

SEQ. description Sequence Corresp. ID NO to

huCD19

1 EF-1 CGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCAC 100

promoter AGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGC

CTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA

CTGGCTCCGCC 1 1 1 1 1 CCCGAGGGTGGGGGAGAACCGTATATAAGT

GCAGTAGTCGCCGTGAACG 1 1 L I 1 1 1 1 CGCAACGGGTTTGCCGCCAG

AACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTA

CGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAG

TACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGA

GTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTT

GAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGG

CACCTTCG CG CCTGTCTCG CTGCTTTCG ATAAGTCTCTAG CCATTT AA

AA I 1 1 1 I A I GACC I C I CGACGC I 1 1 1 1 1 1 C 1 GGCAAGATAGTCTT

GTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGG 1 1 1 1 1 GGGGC

CGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGC

GAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTA GTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGT

GTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGT

TGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGC

TCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCA

CCCACACAAAGGAAAAGGGCC 1 1 1 CCGTCCTCAGCCGTCGCTTCATG

TGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCT

CGAGC 1 1 1 1 GGAG 1 AC 1 1 C 1 1 1 AGG 1 1 GGGGGAGGGG 1 1 1 1 AT

GCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGC

CAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCC 1 1 1 1 1 GAGT

TTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAG 1 1 1 1 1

TTCTTCCATTTCAGGTGTCGTGA

Leader (aa) MALPVTALLLPLALLLHAARP 13

Leader (na) ATGGCCCTGCCTGTGACAGCCCTGCTGCTGCCTCTGGCTCTGCTGCT 54

GCATGCCGCTAGACCC

CD 8 hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 14 (aa)

CD8 hinge ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCG 55 (na) CGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGC

GGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT

Ig4 hinge ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSQ 102 (aa) EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN

GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVM HEALHNHYTQ.KSLSLSLGKM

Ig4 hinge GAGAGCAAGTACGGCCCTCCCTGCCCCCCTTGCCCTGCCCCCGAGTT 103 (na) CCTGGGCGGACCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACA

CCCTGATGATCAGCCGGACCCCCGAGGTGACCTGTGTGGTGGTGGA

CGTGTCCCAGGAGGACCCCGAGGTCCAGTTCAACTGGTACGTGGAC

GGCGTGGAGGTGCACAACGCCAAGACCAAGCCCCGGGAGGAGCAG

TTCAATAGCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCA

GGACTGGCTGAACGGCAAGGAATACAAGTGTAAGGTGTCCAACAAG

GGCCTGCCCAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCC

AGCCTCGGGAGCCCCAGGTGTACACCCTGCCCCCTAGCCAAGAGGA

GATGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAAGGGCTTCT

ACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCG

AGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAG

CTTCTTCCTGTACAGCCGGCTGACCGTGGACAAGAGCCGGTGGCAG

G AG G G CAACGTCTTTAG CTG CTCCGTG ATG C ACG AG G CCCTGC AC A

ACCACTACACCCAGAAGAGCCTGAGCCTGTCCCTGGGCAAGATG

IgD hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKEK 47 (aa) EKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVVGS

DLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSLWN

AGTSVTCTLNH PSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASW

LLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTTFWAWSV

LRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH

IgD hinge AGGTGGCCCGAAAGTCCCAAGGCCCAGGCATCTAGTGTTCCTACTGC 48 (na) ACAGCCCCAGGCAGAAGGCAGCCTAGCCAAAGCTACTACTGCACCT

GCCACTACGCGCAATACTGGCCGTGGCGGGGAGGAGAAGAAAAAG

GAGAAAGAGAAAGAAGAACAGGAAGAGAGGGAGACCAAGACCCCT

GAATGTCCATCCCATACCCAGCCGCTGGGCGTCTATCTCTTGACTCCC

GCAGTACAG G ACTTGTG G CTTAG AG ATAAG G CC ACCTTTACATGTTT CGTCGTGGGCTCTGACCTGAAGGATGCCCATTTGACTTGGGAGGTT

GCCGGAAAGGTACCCACAGGGGGGGTTGAGGAAGGGTTGCTGGAG

CGCCATTCCAATGGCTCTCAGAGCCAGCACTCAAGACTCACCCTTCC

GAGATCCCTGTGGAACGCCGGGACCTCTGTCACATGTACTCTAAATC

ATCCTAGCCTGCCCCCACAGCGTCTGATGGCCCTTAGAGAGCCAGCC

GCCCAGGCACCAGTTAAGCTTAGCCTGAATCTGCTCGCCAGTAGTGA

TCCCCCAGAGGCCGCCAGCTGGCTCTTATGCGAAGTGTCCGGCTTTA

GCCCGCCCAACATCTTGCTCATGTGGCTGGAGGACCAGCGAGAAGT

GAACACCAGCGGCTTCGCTCCAGCCCGGCCCCCACCCCAGCCGGGTT

CTACCACATTCTGGGCCTGGAGTGTCTTAAGGGTCCCAGCACCACCT

AGCCCCCAGCCAGCCACATACACCTGTGTTGTGTCCCATGAAGATAG

CAGGACCCTGCTAAATGCTTCTAGGAGTCTGGAGGTTTCCTACGTGA

CTGACCATT

GS hinge/ GGGGSGGGGS 49 linker (aa)

GS hinge/ GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC 50 linker (na)

CD8TM (aa) IYIWAPLAGTCGVLLLSLVITLYC 15

CD8 TM ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCT 56 (na) GTCACTG GTTATC ACCCTTTACTG C

4-1BB KRG KKLLYIFKQPFM RPVQTTQEEDGCSC FPEEEEGGCEL 16 intracellular

domain

(aa)

4-1BB AAACG G G G CAG AAAG AAACTCCTGTATATATTCAAACAACC ATTTAT 60 intraGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGA cellular TTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG

domain

(na)

CD27 (aa) QRRKYRSN KGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP 51

CD27 (na) AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGA 52

CTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCC

CCACCACGCGACTTCGCAGCCTATCGCTCC

CD3-zeta RVKFSRSADAPAYKQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGG 17 (aa) KPRRKN PQEGLYNELQKDKMAEAYSEIGM KGERRRGKGH DGLYQGLS

TATKDTYDALHMQALPPR

CD3-zeta AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACAAGCAG 101 (na) GGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGG

AGTACGATG 1 1 1 1 GGACAAGAGACGTGGCCGGGACCCTGAGATGGG GGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGA ACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGAT GAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCC 1 1 1 ACCA GGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC AGGCCCTGCCCCCTCGC

CD3-zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG 43 (aa) KPRRKN PQEGLYNELQKDKMAEAYSEIGM KGERRRGKGH DGLYQGLS

TATKDTYDALHMQALPPR

CD3-zeta AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAG 44 (na) GGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGG AGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGG

GGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGA

ACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGAT

GAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGC 1 1 1 ACCA

GGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGC

AGGCCCTGCCCCCTCGC

linker GGGGS 18 linker GGTGGCGGAGGTTCTGGAGGTGGAGGTTCC 50

PD-1 extrapgwfldspdrpwnpptfspallvvtegdnatftcsfsntsesfvlnwyrmspsnqtdkl cellular aafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgtylcgaislapkaqikeslra domain elrvterraevptahpspsprpagqfqtlv

(aa)

PD-1 extracccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcact cellular cttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacacctccgaat domain cattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtt (na) tccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaat ggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctg tgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactg agagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcct gcggggcagtttcagaccctggtc

PD-1 CAR malpvtalllplalllhaarppgwfldspdrpwnpptfspallwtegdnatftcsfsntse (aa) with sfvlnwyrmspsnqtdklaafpedrsqpgqdcrfrvtqlpngrdfhmsvvrarrndsgt signal ylcgaislapkaqikeslraelrvterraevptahpspsprpagqfqtlvtttpaprpptpa ptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkklly ifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnl grreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrg kghdglyqglstatkdtydalhmqalppr

PD-1 CAR atggccctccctgtcactgccctgcttctccccctcgcactcctgctccacgccgctagacca (na) cccggatggtttctggactctccggatcgcccgtggaatcccccaaccttctcaccggcact cttggttgtgactgagggcgataatgcgaccttcacgtgctcgttctccaacacctccgaat cattcgtgctgaactggtaccgcatgagcccgtcaaaccagaccgacaagctcgccgcgtt tccggaagatcggtcgcaaccgggacaggattgtcggttccgcgtgactcaactgccgaat ggcagagacttccacatgagcgtggtccgcgctaggcgaaacgactccgggacctacctg tgcggagccatctcgctggcgcctaaggcccaaatcaaagagagcttgagggccgaactg agagtgaccgagcgcagagctgaggtgccaactgcacatccatccccatcgcctcggcct gcggggcagtttcagaccctggtcacgaccactccggcgccgcgcccaccgactccggcc ccaactatcgcgagccagcccctgtcgctgaggccggaagcatgccgccctgccgccgga ggtgctgtgcatacccggggattggacttcgcatgcgacatctacatttgggctcctctcgc cggaacttgtggcgtgctccttctgtccctggtcatcaccctgtactgcaagcggggtcgga aaaagcttctgtacattttcaagcagcccttcatgaggcccgtgcaaaccacccaggagga ggacggttgctcctgccggttccccgaagaggaagaaggaggttgcgagctgcgcgtgaa gttctcccggagcgccgacgcccccgcctataagcagggccagaaccagctgtacaacga actgaacctgggacggcgggaagagtacgatgtgctggacaagcggcgcggccgggacc ccgaaatgggcgggaagcctagaagaaagaaccctcaggaaggcctgtataacgagctg cagaaggacaagatggccgaggcctactccgaaattgggatgaagggagagcggcgga ggggaaaggggcacgacggcctgtaccaaggactgtccaccgccaccaaggacacatac gatgccctgcacatgcaggcccttccccctcgc

linker (Gly-Gly-Gly-Ser)n, where n = 1-10 105 linker (Gly4 Ser)4 106 linker (Gly4 Ser)3 107 linker (Gly3Ser) 108

PD1 CAR DewfldsDdrDwnDDtfsDallvvtegdnatftcsfsntsesfvlnwvrmsDsnatdkl (aa) aafDedrsaDgqdcrfrvtalDngrdfhmsvvrarrndsgtvlcgaislaDkaqikeslra elrvterraevDtahDSDSDrDagafqtlvtttDaDrDDtDaDtiasaplslrDeacrDaa ggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeed gcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpe mggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdty dalhmqalppr

CD19 CAR MALPVTALLLPLALLLHAARPDIQMTQTTSSLSASLGDRVTISCRASQDIS (aa) murine KYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQ

EDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQE

SGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGS

ETTYYNSALKSRLTI IKDNSKSQVFLKM NSLQTDDTAIYYCAKHYYYGGSY

AM DYWGQGTSVTVSSTTTPAPR P PTPAPTI ASQP LS LR P EAC R PAAGG

AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPF

M RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLY

NELN LGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA

EAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD19 CAR atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggcc (na) murine ggacatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcacc atcagttgcagggcaagtcaggacattagtaaatatttaaattggtatcagcagaaacca gatggaactgttaaactcctgatctaccatacatcaagattacactcaggagtcccatcaa ggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggagcaaga agatattgccacttacttttgccaacagggtaatacgcttccgtacacgttcggagggggg accaagctggagatcacaggtggcggtggctcgggcggtggtgggtcgggtggcggcgg atctgaggtgaaactgcaggagtcaggacctggcctggtggcgccctcacagagcctgtc cgtcacatgcactgtctcaggggtctcattacccgactatggtgtaagctggattcgccagc ctccacgaaagggtctggagtggctgggagtaatatggggtagtgaaaccaca tactataattcagctctcaaatccagactgaccatcatcaaggacaactccaagagccaag ttttcttaaaaatgaacagtctgcaaactgatgacacagccatttactactgtgccaaacat tattactacggtggtagctatgctatggactactggggccaaggaacctcagtcaccgtctc ctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagc ccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagg gggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtcct tctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatat tcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgcc gatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgca gacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacg aagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaa agccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagat ggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcac gatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgc aggccctgccccctcgct

CD19 CAR MALPV ALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSCRASQDIS (aa) human KYLNWYQQKPGQAPRLLIYHTSRLHSGIPARFSGSGSGTDYTLTISSLQP

EDFAVYFCQQGNTLPYTFGQGTKLEI KGGGGSGGGGSGGGGSQVQLQ

ESGPGLVKPSETLSLTCTVSGVSLPDYGVSWI RQPPGKGLEWIGVIWGS

ETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGS

YAMDYWGQGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAG GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP

FM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGM KG E RRRG KG HDG LYQG LSTATKDTYDALH MQALPPR

36 CD19 CAR atggccctccctgtcaccgccctgctgcttccgctggctcttctgctccacgccgctcggccc

(na) human gaaattgtgatgacccagtcacccgccactcttagcctttcacccggtgagcgcgcaaccct

gtcttgcagagcctcccaagacatctcaaaataccttaattggtatcaacagaagcccgga

caggctcctcgccttctgatctaccacaccagccggctccattctggaatccctgccaggtt

cagcggtagcggatctgggaccgactacaccctcactatcagctcactgcagccagagga

cttcgctgtctatttctgtcagcaagggaacaccctgccctacacctttggacagggcacca

agctcgagattaaaggtggaggtggcagcggaggaggtgggtccggcggtggaggaagc

caggtccaactccaagaaagcggaccgggtcttgtgaagccatcagaaactctttcactga

cttgtactgtgagcggagtgtctctccccgattacggggtgtcttggatcagacagccaccg

gggaagggtctggaatggattggagtgatttggggctctgagactacttactaccaatcat

ccctcaagtcacgcgtcaccatctcaaaggacaactctaagaatcaggtgtcactgaaact

gtcatctgtgaccgcagccgacaccgccgtgtactattgcgctaagcattactattatggcg

ggagctacgcaatggattactggggacagggtactctggtcaccgtgtccagcaccactac

cccagcaccgaggccacccaccccggctcctaccatcgcctcccagcctctgtccctgcgtc

cggaggcatgtagacccgcagctggtggggccgtgcatacccggggtcttgacttcgcctg

cgatatctacatttgggcccctctggctggtacttgcggggtcctgctgctttcactcgtgatc

actctttactgtaagcgcggtcggaagaagctgctgtacatctttaagcaacccttcatgag

gcctgtgcagactactcaagaggaggacggctgttcatgccggttcccagaggaggagga

aggcggctgcgaactgcgcgtgaaattcagccgcagcgcagatgctccagcctacaagca

ggggcagaaccagctctacaacgaactcaatcttggtcggagagaggagtacgacgtgct

ggacaagcggagaggacgggacccagaaatgggcgggaagccgcgcagaaagaatcc

ccaagagggcctgtacaacgagctccaaaaggataagatggcagaagcctatagcgaga

ttggtatgaaaggggaacgcagaagaggcaaaggccacgacggactgtaccagggactc

agcaccgccaccaaggacacctatgacgctcttcacatgcaggccctgccgcctcgg

Table 2. Antigen Binding domains that bind B cell antigens

B cell SEQ ID

Name Amino Acid Sequence

antigen NO:

CD19 huscF EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYH

v1 TSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQ

GTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSG 37 VSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYSSSLKSRVTISKDNSKN QVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS

CD19 huscF EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYH

v2 TSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQ

GTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSG 38

VSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVTISKDNSK

NQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS

CD19 huscF QVQLQESG PGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIG

v3 VIWGSETTYYSSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHY

YYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPAT 39

LSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPA

RFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIK

CD19 huscF QVQLQESG PGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIG 40

v4 VIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHY YYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPAT LSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPA RFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIK

CD19 huscF EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYH

v5 TSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQ

GTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSL 41

TCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYSSSLKSRVTIS

KDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTV

SS

CD19 huscF EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYH

v6 TSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQ

GTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSL 42

TCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSb l I YYQSSLKSRVTIS

KDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYA DYWGQGTLVTV

SS

CD19 huscF QVQLQESG PGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIG

v7 VIWGSETTYYSSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHY

YYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVMT 43

QSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRL

HSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKL

EIK

CD19 huscF QVQLQESG PGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIG

v8 VIWGSETTYYQSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHY

YYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVMT 44

QSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRL

HSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKL

EIK

CD19 huscF EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYH

v9 TSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQ

GTKLEIKGGGGSGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSL 45

TCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSSLKSRVTIS

KDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYA DYWGQGTLVTV

SS

CD19 Hu QVQLQESG PGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIG

scFvl VIWGSETTYYNSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHY 0 YYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSEIVMT 46

QSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRL

HSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKL

EIK

CD19 Hu EIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYH

scFvl TSRLHSGIPARFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQ

1 GTKLEIKGGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSG 47

VSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYNSSLKSRVTISKDNSK

NQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS

CD19 Hu QVQLQESG PGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIG

scFvl VIWGSETTYYNSSLKSRVTISKDNSKNQVSLKLSSVTAADTAVYYCAKHY 2 YYGGSYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVMTQSPAT 48

LSLSPGERATLSCRASQDISKYLNWYQQKPGQAPRLLIYHTSRLHSGIPA

RFSGSGSGTDYTLTISSLQPEDFAVYFCQQGNTLPYTFGQGTKLEIK

CD19 muCT DIQ TQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYH 49

L019 TSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFG

GGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVS GVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTI IKDNSK

SQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS

Examples

The following examples describe experiments in which in vitro proliferation indices for a CART cell therapy product targeting CD19 were determined. A high concordance of in vitro proliferation and clinical cellular kinetics of CART cells in patients with pediatric acute lymphoblastic leukemia (pedALL) was identified.

Example 1

Preparation of Tumor Antigen Presenting Cells and Anti-ldiotypic Beads

Frozen CART cell product and K562 cells (parental K562 cells and K562 cells expressing tumor antigen) were thawed and washed in X-VIVO™ 15 culture medium (supplemented with 2% Human AB Serum). The viable cell concentration of each cell line was adjusted to 4 106 cells per mL. Cells were centrifuged and then re-suspended in 4% paraformaldehyde, with a final cell concentration of 4 1 06 cells per mL. The fixed K562 cells were extensively washed using sterile PBS for a total of three times after incubation with paraformaldehyde at room temperature for 1 5±5 minutes. IgG control, anti-idiotype, and anti-CD3/anti-CD28 Dynabeads™ were transferred into 1 5 mL conical tubes, washed with 10 times volume of X-VIVO 15 culture medium three times, and then resuspended in X-VIVO™ 1 5 culture medium (Table 3).

Table 3

Figure imgf000087_0001

Example 2

Stimulating the CAR of CART cells

CART cells were treated for 48 hours with beads (IgG control, anti-idiotype, or anti-CD3/anti- CD28 Dynabeads™), with a bead-to-sample cell ratio of 2:1 , or fixed K562 cells (parental K562 cells or K562 cells expressing tumor antigen), with a K562 cell-to-sample cell ratio of 1 :1 , in X-VIVO™ 15 culture medium (supplemented with 2% Human AB serum). An example of plating layout is shown in Table 4. Table 4

Figure imgf000088_0001

Example 3

Staining of CART Cells, Flow Cytometry, and Analysis

To measure antigen-specific proliferation, EdU (1 0 μ ) was added one hour before the incubation endpoint. After the incubation, beads were removed using a Dynamag™ magnet, cell pellets were washed with 200 μΙ_ PBS, and then the cell pellets were incubated at room temperature with Human Fc block for 10 minutes. For cell surface staining, 40 μΙ_ of the flow cytometry proliferation panel was added (CD3, CD4, CD8, idiotype; the volume used depends on the conjugate used) and incubated for 30+10 minutes at A °C (acceptable range 2-8 °C) in the dark. Cells were fixed with 4% paraformaldehyde for 1 5 minutes at room temperature, followed by an extensive wash with a saponin-based

permeabilization buffer. The Click-iT® EdU reaction cocktail was prepared according to the

manufacturer's instructions (a modified version is illustrated in Table 5). 1 00 μΙ_ of the permeabilization buffer and 1 00 μΙ_ of Click-iT® EdU reaction cocktail were added to the cell pellet. The mixture was pipetted to a homogenous suspension and then incubated for 30+1 0 minutes at 4°C (acceptable range 2- 8°C) in the dark. Table 5

Figure imgf000089_0001

For staining of total cellular DNA, samples were washed once in permeabilization buffer. The DNA staining solution (1 to 20 dilutions with permeabilization buffer) was added to the cells 15 minutes before flow cytometry data acquisition. After an incubation period, 200 μΙ_ of permeabilization buffer was added to the cells, and the cells were subsequently analyzed using either a Becton Dickinson (BD) FACSVerse™, BD LSRFORTESSA™, or ACEA NovoCyte™ flow cytometer utilizing either FACSuite™, FACSDiva™, or NovoExpress™ software, respectively.

Examples of the gating and analysis of the flow cytometry data is illustrated in Figures 1 -5.

Figure 1 is a plot showing FSC-A (x-axis/linear) vs. SSC-A (y-axis/linear), (WBC). The "WBC Gate" was set to gate out debris and to show the white blood cell populations. Figure 2 is a plot showing SSC-H (x- axis/linear) vs. SSC-W (y-axis/linear), (Single). The "WBC Gate" was gated off, and the "Single" gate was set to exclude doublets and encompass single white blood cells. Figure 3 is a plot showing DNA (x- axis/linear) vs. CD3 (y-axis/log), (Viable CD3). The "Single" gate was gated off, and the "Viable" gate was set to exclude the CD3 DNAlow dead or dying cells. Figure 4 is a plot showing CD8 (x-axis/log) vs. CD4 (y-axis/log) cells. The "Viable" gate was gated off, and the "CD3+/CD4+" and "CD3+/CD8+" gates were set to encompass the CD3 positive, CD4 or CD8 positive cells. Further gating on CAR+ and CAR- populations in CD3, CD4, and CD8 T cell compartments can also be done to determine, for example, the portion of T cells (total, CD4+, or CD8+) that express a particular CAR.

The design and development of an in vitro proliferation assay for CART cells is illustrated in Figure 5. The workflow for an EdU-based proliferation assay is shown and, briefly, is as follows: on day 0, CART cells are co-cultured with K562 or K562-tumor antigen at a K562-to-T cell ratio of 1 :1 . K562 cells are killed by fixation with paraformaldehyde to provide solely a stimulation on CAR receptor without influencing CART cell proliferation. On day 2, EdU is added one hour before the incubation time period endpoint. Cells are harvested for surface staining and Click-iT® reaction. Proliferating cells are identified by DNA content and EdU incorporation, as shown in the graph in Figure 5. Exemplary results obtained using an in vitro proliferation assay of the invention are set forth in Figure 6. The plots in Figure 6 show total DNA (x-axis/linear) vs. EdU (y-axis/log) FACS measurements. Cells were gated off the "CD3+/CD4+" and "CD3+/CD8+" gate (see, e.g. , Figure 4) and the "%EdU+" gate was set around the EdU+ cells. "%EdU+," "%EdU G0/G1 phase," and "%EdU G2/M phase" are denoted in both the CD4 (left) and CD8 (right) T cell subpopulations. The %EdU+ cells measured indicates the population of cells that is actively proliferating, and thus synthesizing DNA. Figure 6 shows 4.5% of CD4+ T cells and 1 5.4% of CD84 T cells were EdU+, with the remaining population of cells being EdU", and therefore not actively proliferating or synthesizing DNA. To determine the proliferation index of the antigen-stimulated CART cells, the %EdU+ cells in the antigen-stimulated cell population was subtracted from the %EdU+ cells in the unstimulated group. This difference was divided by the %transduced+ cells, which was determined by FACS analysis using an anti-idiotypic antibody, facilitating detection of the surface-expressed CAR.

CART cell product proliferation indices were plotted against in vivo PK parameters including CMAX, TMAX, and area under the curve (AUC) calculated for the indicated number of patients (Figure 7). Measurements for CD19-specific proliferation were performed using CTL019 final products in two replicates. Linear regression is shown (dark lines) along with the 95% confidence interval (shading) ; each dot or circle represents an individual patient. For each PK parameter, it was found that there was a statistically significant correlation between CART cells with a high proliferation index and improved PK parameters. The Spearman correlation coefficient method was used to assess linear association between proliferation index and PK/PD parameters, and P-values < 0.05 were considered significant.

Other embodiments are within the following claims.

Claims

Claims What is claimed is:
1 . A method for characterizing the potency of a chimeric antigen receptor (CAR)-T cell (CART cell) in vitro, the method comprising :
a. stimulating the CART cell in an antigen-specific manner, and
b. determining the level of antigen-specific proliferation of the stimulated CART cell.
2. The method of claim 1 , wherein detection of an increase in the level of proliferation of the
antigen-specific stimulated CART cell, as compared to the level of proliferation of an unstimulated CART cell or the level of proliferation of a non-specifically stimulated CART cell, indicates a stimulated CART cell for use in therapy.
3. The method of claim 1 or 2, wherein the CART cell is stimulated by an antigen for which the CAR of the CART cell is specific.
4. The method of claim 3, wherein the antigen is a tumor antigen.
5. The method of any one of claims 1 to 4, wherein the antigen is present on the surface of a cell, which optionally is fixed.
6. The method of claim 1 or 2, wherein the CART cell is stimulated by an anti-idiotypic antibody specific for the CAR of the CART cell.
7. The method of any one of claims 1 to 6, wherein the level of antigen-specific proliferation of the stimulated CART cell is determined in an assay that detects DNA synthesis, a proliferation marker, dye dilution, DNA content, or cellular metabolism.
8. The method of claim 7, wherein the assay detects DNA synthesis.
9. The method of claim 8, wherein DNA synthesis is detected by determining the level of
incorporation of a modified nucleotide into the DNA of the stimulated CART cell.
10. The method of any one of claims 1 to 9, wherein the level of antigen-specific proliferation is calculated by determination of a Proliferation Index (PI), as follows:
PI = [(level of indicator of proliferation in antigen-specific stimulated cells)
- (level of indicator of proliferation in unstimulated cells)] / %
transduction.
1 1 . The method of claim 10, wherein the indicator of proliferation is the level of incorporation of a modified nucleotide into the DNA of the antigen-specific stimulated CART cell, and the PI is determined as follows:
PI = [(% incorporation of modified nucleotide in antigen-specific
stimulated cells) - (% incorporation of modified nucleotide in
unstimulated cells)] / % transduction.
12. The method of any one of claims 7 to 1 1 , wherein the assay detects incorporation of a modified nucleotide selected from the group consisting of: 5-ethynyl-2'-deoxyuridine (EdU), 3H-thymidine, and 5-bromo-2'-deoxyuridine (BrdU) into DNA of the stimulated CART cell.
13. The method of any one of claims 1 to 12, wherein the method comprises the use of flow
cytometry.
14. The method of any one of claims 1 to 13, wherein the level of antigen-specific proliferation of the stimulated CART cell correlates with one or more in vivo clinical parameters of the CART cell.
15. The method of claim 14, wherein one or more of the in vivo clinical parameters is a
pharmacokinetic parameter of the stimulated CART cell, which optionally is selected from the group consisting of Cmax, Tmax, and Area Under the Curve (AUC).
16. The method of any one of claims 1 to 15, further comprising detecting one or more cellular
antigens of the CART cell using one or more antibodies, which optionally enables detection of a sub-population of CART cells and/or detection of cells expressing the CAR.
17. The method of claim 16, wherein CD4+ and/or CD8+ CART cells are detected by use of
antibodies specific for CD4 and/or CD8, respectively.
18. The method of any one of claims 1 to 17, further comprising determining the level of proliferation of the CART cells in the absence of an antigen-specific for the CAR of the CART cell, or in the absence of an anti-idiotypic antibody specific for the CAR of the CART cell.
19. The method of any one of claims 1 to 18, wherein the CAR of the CART cell comprises, in an N- terminal to C-terminal direction, an antigen binding domain, a transmembrane domain, and one or more signaling domains.
20. The method of claim 19, wherein the one or more signaling domains comprise one or more
primary signaling domains, and optionally one or more costimulatory signaling domains.
21 . The method of claim 20, wherein one of the primary signaling domains comprises a CD3-zeta stimulatory domain.
22. The method of claim 20 or 21 , wherein one of the costimulatory signaling domains comprises an intracellular domain selected from a costimulatory protein selected from the group consisting of CD27, CD28, 4-1 BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1 , lymphocyte function-associated antigen-1 (LFA-1 ), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLA F7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and a ligand that specifically binds with CD83.
23. The method of any one of claims 19 to 22, wherein the antigen binding domain is a scFv.
24. The method of any one of claims 1 to 23, wherein the CAR is specific for an antigen selected from the group consisting of CD19; CD123; CD22; CD30; CD171 ; CS-1 ; C-type lectin-like molecule-1 , CD33; epidermal growth factor receptor variant II I (EG FRvlll); ganglioside G2 (G D2); ganglioside GD3; TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)) ; prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1 ) ; Fms-Like Tyrosine Kinase 3 (FLT3) ; Tumor-associated glycoprotein 72 (TAG 72) ; CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD1 1 7) ; lnterleukin-13 receptor subunit alpha-2; mesothelin; Interleukin 1 1 receptor alpha (IL-1 1 Ra); prostate stem cell antigen (PSCA) ; Protease Serine 21 ; vascular endothelial growth factor receptor 2 (VEGFR2) ; Lewis(Y) antigen ; CD24; Platelet- derived growth factor receptor beta (PDG FR-beta) ; Stage-specific embryonic antigen-4 (SSEA- 4); CD20 ; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2/neu) ; Mucin 1 , cell surface associated (MUC1 ) ; epidermal growth factor receptor (EGFR) ; neural cell adhesion molecule (NCAM) ; Prostase; prostatic acid phosphatase (PAP) ; elongation factor 2 mutated (ELF2M) ; Ephrin B2; fibroblast activation protein alpha (FAP) ; insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX) ; Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2) ; glycoprotein 1 00 (gp1 00); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2) ; Fucosyl GM1 ; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; transglutaminase 5 (TGS5); high molecular weight-melanoma- associated antigen (HMWMAA) ; o-acetyl-GD2 ganglioside (OAcGD2) ; Folate receptor beta; tumor endothelial marker 1 (TEM1 /CD248) ; tumor endothelial marker 7-related (TEM7R) ; claudin 6 (CLDN6) ; thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61 ) ; CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1 ) ;
hexasaccharide portion of globoH glycoceramide (GloboH) ; mammary gland differentiation antigen (NY-BR-1 ) ; uroplakin 2 (UPK2) ; Hepatitis A virus cellular receptor 1 (HAVCR1 );
adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20) ; lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51 E2 (OR51 E2) ; TCR Gamma Alternate Reading Frame Protein (TARP) ; Wilms tumor protein (WT1 ) ; Cancer/testis antigen 1 (NY-ESO-1 ) ; Cancer/testis antigen 2 (LAG E-1 a) ; Melanoma-associated antigen 1 (MAG E-A1 ); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML) ; sperm protein 17 (SPA17); X Antigen Family, Member 1 A (XAGE1 ); angiopoietin-binding cell surface receptor 2 (Tie 2) ; melanoma cancer testis antigen-1 (MAD-CT-1 ) ; melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1 ; tumor protein p53 (p53) ; p53 mutant; prostein; surviving ; telomerase; prostate carcinoma tumor antigen-1 , melanoma antigen recognized by T cells 1 ; Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT) ; sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene) ; N-Acetyl glucosaminyl-transferase V (NA1 7) ; paired box protein Pax-3 (PAX3) ; Androgen receptor; Cyclin B1 ; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) ; Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1 B1 (CYP1 B1 ) ; CCCTC- Binding Factor (Zinc Finger Protein)-Like, Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3) ; Paired box protein Pax-5 (PAX5) ; proacrosin binding protein sp32 (OY-TES1 ); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2) ; Receptor for Advanced Glycation Endproducts (RAGE-1 ); renal ubiquitous 1 (RU1 ) ; renal ubiquitous 2 (RU2) ; legumain; human papilloma virus E6 (HPV E6) ; human papilloma virus E7 (HPV E7) ; intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2) ; CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1 ) ; Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C- type lectin domain family 12 member A (CLEC12A) ; bone marrow stromal cell antigen 2 (BST2) ; EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2) ; lymphocyte antigen 75 (LY75) ; Glypican-3 (GPC3) ; Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1 ).
25. The method of any one of claims 1 to 24, wherein the CAR is specific for CD1 9.
26. The method of any one of claims 1 to 25, further comprising :
a. determining the number of the CART cells to administer to a subject; or
b. determining the expected level of response of a subject to the CART cell.
27. The method of any one of claims 1 to 26, further comprising administering the CART cell to a subject.
28. A kit for determining the potency of a CART cell, the kit comprising :
a. an agent for stimulating CART cell in an antigen-specific manner, and
b. one or more reagents for detecting antigen-specific proliferation of the CART cell.
29. The kit of claim 28, wherein the agent for stimulating the CART cell in an antigen-specific manner is an antigen or an anti-idiotypic antibody.
30. The kit of claim 28 or 29, wherein the one or more reagents comprises a modified nucleotide for use in detecting DNA synthesis in the CART cell.
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