NZ712373B2

NZ712373B2 - Modified t lymphocytes

Info

Publication number: NZ712373B2
Application number: NZ712373A
Authority: NZ
Inventors: Stewart Abbot; Tianjian Li; Bitao Liang
Original assignee: Celgene Corporation
Priority date: 2013-03-15
Filing date: 2014-03-14
Publication date: 2021-11-30

Abstract

Provided herein are T lymphocytes expressing cell death polypeptides having an extracellular domain with a CD20 epitope and a intracellular domain with an apoptosis-inducing domain of caspase 3, caspase 8, or caspase 9, wherein an apoptosis-inducing signal is generated when the polypeptide is dimerized using a CD20 antibody. Also provided herein is use of such cells, e.g., T lymphocytes, to treat diseases such as cancer. zed using a CD20 antibody. Also provided herein is use of such cells, e.g., T lymphocytes, to treat diseases such as cancer.

Description

(12) Granted patent speciﬁcaon (19) NZ (11) 712373 (13) B2 (47) aon date: 2021.12.24 (54) MODIFIED T LYMPHOCYTES (51) Internaonal Patent Classiﬁcaon(s): C07K 1/00 (22) Filing date: (73) Owner(s): 2014.03.14 E CORPORATION (23) Complete speciﬁcaon ﬁling date: (74) Contact: 2014.03.14 HENRY HUGHES IP LIMITED (30) Internaonal Priority Data: (72) or(s): US 61/794,294 3.15 ABBOT, Stewart LIANG, Bitao (86) Internaonal Applicaon No.: LI, Tianjian (87) Internaonal Publicaon number: WO/2014/152177 (57) ct: Provided herein are T lymphocytes expressing cell death polypepdes having an extracellular domain with a CD20 epitope and a intracellular domain with an apoptosis-inducing domain of caspase 3, caspase 8, or caspase 9, wherein an apoptosis-inducing signal is generated when the polypepde is dimerized using a CD20 anbody. Also provided herein is use of such cells, e.g., T lymphocytes, to treat diseases such as cancer. 712373 B2 MODIFIED T LYMPHOCYTES This application claims priority to U.S. Provisional Patent Application No. 61/794,294, filed March 15, 2013, the disclosure of which is incorporated herein by reference in its entirety. 1. FIELD The disclosure herein relates to the field of immunology, and more specifically, to the modification of T lymphocytes or other immune cells. 2. BACKGROUND T lymphocytes recognize and interact with specific antigens, ing tumor- associated or specific antigens. Because T lymphocytes are able to kill tumor cells, the last 25 years has seen a great deal of interest in targeting tumor cells with T lymphocytes, either antigen-specific T lymphocytes, or T lymphocytes genetically modified to express one or more chimeric antigen ors (CARs; see, e.g., Eshhar, U.S. Patent No. 7,741,465; Eshhar, U.S. Patent Application Publication No. 2012/0093842). However, given the ability of T lymphocytes to kill not only tumor cells displaying a certain antigen but normal cells displaying the same antigen, it is ble to incorporate into the T lymphocytes a safety mechanism that enables rapid killing of the cells after administration to a patient should offtarget s prove deleterious to the patient.

While a system to kill T cells has described (Straathof et al. (2005) Blood 105(11):4247-4254), this system was dependent upon specific and difficult-to-make protein modifications, ing the system undesirable for practical use. As such, there exists a need in the art for a safety system to rapidly kill therapeutic T lymphocytes that are vely simple and straightforward to construct. T lymphocytes comprising such a safety system are provided herein. 3. SUMMARY The present invention particularly provides aspects and embodiments as set out in the clauses below: 1. An isolated T lymphocyte comprising an artificial cell death polypeptide comprising an apoptosis-inducing domain, wherein said artificial cell death polypeptide is a embrane n comprising an extracellular domain that ses a CD20 epitope, a transmembrane , and an intracellular domain comprising said apoptosis-inducing domain, wherein said apoptosis-inducing domain is caspase 3, caspase 8 or caspase 9, wherein said (followed by page 1A) polypeptide is dimerizable using an anti-CD20 antibody that binds to said CD20 epitope, and wherein when said antibody dimerizes said polypeptide, an apoptosis-inducing signal is generated in said T lymphocyte. 2. The T lymphocyte of 1, wherein said antibody is rituximab and said extracellular domain comprises a CD20 epitope to which said rituximab binds. 3. The T lymphocyte of 1, wherein said antibody is tositumumab and said extracellular domain ses a CD20 epitope to which said tositumumab binds. 4. The T lymphocyte of 1, wherein said antibody is ibritumomab and said extracellular domain comprises a CD20 epitope to which said ibritumomab binds.

. The T lymphocyte of 1, wherein said dy is ofatumumab and said ellular domain comprises a CD20 epitope to which said ofatumumab binds. 6. The T lymphocyte of any one of 1-5, which additionally comprises a chimeric antigen receptor (CAR) that recognizes an antigen on a tumor cell. 7. The T lymphocyte of 6, n said tumor cell is a cell of a solid tumor. 8. The T lymphocyte of 6, wherein said tumor cell is a cell of a blood cancer. 9. The T lymphocyte of 6, wherein said antigen is Her2, prostate stem cell antigen , alpha-fetoprotein (AFP), oembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A, myo-D1, muscle-specific actin (MSA), ilament, neuron-specific enolase (NSE), placental alkaline phosphatase, ophysis, thyroglobulin, thyroid transcription factor-1, or the dimeric form of the pyruvate kinase isoenzyme type M2.

. The use of a T lymphocyte according to any one of 1-9 in the manufacture of a medicament for treating cancer in an individual, wherein said T lymphocyte additionally comprises a chimeric antigen receptor (CAR) that recognizes an n on a tumor cell, and wherein said treatment comprises stration of an anti-CD20 antibody that binds to a CD20 epitope in the event that the administration of said T cyte causes any unwanted or deleterious effects in said individual receiving said T lymphocyte or in the event that the presence of the T cyte in said individual is no longer ary. (followed by page 1B) 11. The use of 10, wherein the unwanted or deleterious effects comprise signs of ss sing one or more of difficulty breathing, fever, abnormal serum cytokine levels, or rash. 12. Use of an anti-CD20 antibody that binds to a CD20 epitope in the manufacture of a medicament for eutic killing of a T lymphocyte in an individual to whom the T lymphocyte has been administered, wherein said T lymphocyte comprises a plurality of cial cell death polypeptides each comprising an apoptosis-inducing domain, wherein said artificial cell death polypeptide is a transmembrane protein comprising an extracellular domain that comprises the CD20 epitope, a transmembrane domain, and an intracellular domain comprising said apoptosis-inducing domain, wherein said apoptosis-inducing domain is caspase 3, e 8 or caspase 9, wherein upon administration of the D20 antibody to the individual, said artificial cell death ptides dimerize and an apoptosis-inducing signal is generated in said T lymphocyte, wherein the therapeutic killing of said T lymphocyte comprises administering to the individual the medicament comprising an amount of said anti-CD20 antibody sufficient for said plurality of artificial cell death polypeptides to dimerize and generate an aggregate apoptosis-inducing signal sufficient to kill said T cyte in the event that the administration of said T lymphocyte causes any unwanted or deleterious effects in said individual to whom the T cyte has been administered or in the event that the presence of said T lymphocyte in said individual is no longer necessary. 13. The use of any one of 10, 11, and 12, wherein said antibody is rituximab and said extracellular domain comprises a CD20 epitope to which said rituximab binds. 14. The use of any one of 10, 11, and 12, wherein said antibody is mumab and said extracellular domain comprises a CD20 e to which said tositumumab binds.

. The use of any one of 10, 11, and 12, wherein said antibody is ibritumomab and said extracellular domain comprises a CD20 epitope to which said ibritumomab binds. 16. The use of any one of 10, 11, and 12, wherein said antibody is ofatumumab and said extracellular domain comprises a CD20 e to which said ofatumumab binds. (followed by page 1C) 17. The use of 12, n said T lymphocyte onally comprises a chimeric antigen receptor (CAR) that recognizes an n on a tumor cell. 18. The use of any one of 10, 11, and 17, wherein said tumor cell is a cell in a solid tumor. 19. The use of any one of 10, 11, and 17, wherein said tumor cell is a cell of a blood cancer.

. The use of any one of 10, 11, and 17, n said antigen is Her2, prostate stem cell antigen, alpha-fetoprotein, carcinoembryonic antigen, cancer antigen-125, CA19-9, calretinin, MUC-1, epithelial membrane protein, epithelial tumor antigen, tyrosinase, melanoma-associated antigen, CD34, CD45, CD99, CD 117, chromogranin, ratin, desmin, glial fibrillary acidic protein, gross cystic disease fluid protein, HMB-45 antigen, protein melan-A, myo-D1, muscle-specific actin, neurofilament, neuron-specific enolase, placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, or the c form of the pyruvate kinase isoenzyme type M2. [0005a] However, the invention is also described herein in more general terms for completeness. Certain embodiments which are described herein form the subject matter of the divisional application, NZ 751539.

] Thus generally provided herein are cally modified cells, for example immune cells, such as T lymphocytes, e.g., human T cytes, that comprise an artificial multimerizable, e.g., dimerizable, polypeptide (referred to herein as a "cell death polypeptide") that, when multimerized, e.g., dimerized, by a multimerizing agent, e.g., dimerizing agent, generates an apoptosis-inducing signal in a cell, e.g., a T lymphocyte, that expresses the polypeptide, resulting (followed by page 2) 2014/027039 in cell death, e.g., via apoptosis. Without wishing to be bound by any particular mechanism or theory, it is thought that when a sufﬁcient number of a plurality of cell death polypeptides of the cell are multimerized, e.g., dimerized, that the aggregate apoptosis-inducing signal thereby generated is sufﬁcient to kill the cell, e.g., cause the cell to undergo apoptosis.

The cell death polypeptides provided herein may be used in conjunction with any cells, in particular, any mammalian cells, for example, any human cells. For example, such cell death polypeptides provide, for example, a useful safety feature for cell therapeutics. As such, the cell death polypeptides can, for example, be important for a drug product comprising a cell therapeutic, e.g., a chimeric antigen receptor-expressing CAR T lymphocytes, because the cell death polypeptides enable rapid killing of the cell therapeutic, e. g., the T lymphocytes, should such rapid killing become desirable, e.g., in the event administration of the cells causes any unwanted or deleterious effects in a patient receiving them, or if the presence of the cell therapeutic, e. g., the T lymphocytes, in a subject is no longer necessary. Thus, in n embodiments, the cell death polypeptides provided herein can be used in conjunction with any administrable cells, for example cell therapeutics, such as ian cell therapeutics, e. g., human cell therapeutics. Non-limiting es of cells in which the cell death polypeptides and multimerizing or dimerizing agents may be used include, but are not limited to, natural killer (NK) cells, tic cells (DC), placental stem cells (e.g., the placental stem cells sed in US. Patent Nos. 7,468,276; 8,057,788 and 8,202,703, the disclosures of which are hereby incorporated by reference in their entireties), mesenchymal-like stem cells from umbilical cord blood, placental blood, peripheral blood, bone , dental pulp, adipose tissue, hondral tissue, and the like; embryonic stem cells, embryonic germ cells, neural crest stem cells, neural stem cells, and differentiated cells (e. g., ﬁbroblasts, etc.). The cell death polypeptides, and multimerizing or dimerizing agents, may also be used in tumor cell lines, e.g., for animal model experimental purposes.

Typically, the cell death polypeptide is erizable or dimerizable using an administrable multimerizing or dimerizing agent, e. g., a small molecule, polypeptide (other than the cell death ptide) such as an dy, an oligonucleotide, or a polysaccharide. The cell death polypeptides do not comprise a FK506 binding protein (FKBP), functional portion f, ofmodiﬁed form thereof, and the multimerizing agent or dimerizing agent is not an FKBP ligand.

In a ﬁrst aspect, provided herein is a cell, e.g., a T lymphocyte, comprising a cell death polypeptide sing an sis-inducing domain, wherein said cell death polypeptide is erizable using a multimerizing agent, wherein when said multimerizing agent multimerizes said ptide, an apoptosis-inducing signal is generated in said cell. In a speciﬁc embodiment, said multimerizing agent is a dimerizing agent; that is, the multimerizing agent causes the cell death ptide to dimerize. In another speciﬁc embodiment, when said dimerizing agent dimerizes said polypeptide, an apoptosis-inducing signal is generated in said cell.

In certain ments, said cell death polypeptide is a transmembrane polypeptide comprising an extracellular domain, a embrane domain, and an intracellular domain comprising said apoptosis-inducing domain. In particular embodiments, the apoptosis-inducing domain of the cell death polypeptide is or comprises a caspase, e.g., caspase 9, caspase 8, or caspase 3, for example a human caspase 9, caspase 8, or caspase 3.

In certain ments, the dimerizing agent is a polypeptide comprising at least two sites that speciﬁcally bind to a cell death polypeptide, e. g., an extracellular domain of a cell death polypeptide. In particular embodiments, the polypeptide is an antibody, e.g., an antibody comprising at least two epitope or mimotope binding sites. In certain embodiments, only the antigen binding domain of an antibody is used as a multimerizing or dimerizing agent. In certain embodiments, an extracellular domain of a cell death polypeptide ses at least one epitope or mimotope to which the antibody speciﬁcally binds. In particular embodiments, the antibody is a iﬁc dy comprising two different epitope or mimotope binding sites that bind two different epitopes or mimotopes present on an extracellular domain of a cell death polypeptide.

In certain embodiments, the antibody is an IgG or an IgM antibody. In a particular embodiment, an antibody useful as a multimerizing or dimerizing agent is one that has been approved by the United States Food and Drug Administration for any use.

In one ment, an antibody useful as a multimerizing or dimerizing agent is one that speciﬁcally binds to a CD20 epitope or mimotope, e. g., a human CD20 epitope or mimotope, and an extracellular domain of a cell death polypeptide comprises a CD20 epitope or mimotope to which the antibody speciﬁcally binds. In certain speciﬁc embodiments, the antibody is rituximab and an ellular domain of a cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that speciﬁcally binds to said rituximab. In another speciﬁc embodiment, the antibody is tositumumab and an extracellular domain of a cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that speciﬁcally binds to said tositumumab. In yet another embodiment, the antibody is ibritumomab and an extracellular domain of a cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that speciﬁcally binds to said ibritumomab. In still another embodiment, the dy is ofatumumab and an extracellular domain of a cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that speciﬁcally binds to said ofatumumab.

In another speciﬁc embodiment, the antibody is alemtuzumab and an extracellular domain of a cell death ptide ses a CD52 epitope or a CD52 mimotope that speciﬁcally binds to said alemtuzumab. In yet another embodiment, the antibody is basiliximab and an extracellular domain of the cell death polypeptide comprises a CD25 epitope or a CD25 mimotope that speciﬁcally binds to said basiliximab. In another embodiment, the antibody is daclizumab and an extracellular domain of a cell death polypeptide comprises a CD25 epitope or a CD25 mimotope that speciﬁcally binds to said daclizumab. In still r ment, the antibody is brentuximab and an extracellular domain of a cell death polypeptide comprises a CD30 epitope or a CD30 mimotope that speciﬁcally binds to said brentuximab. In another embodiment, the antibody is belimumab and an extracellular domain of a cell death polypeptide comprises a B-cell activating factor (BAFF) epitope or a BAFF mimotope that speciﬁcally binds to said belimumab. In another embodiment, the antibody is cetuximab and an extracellular domain of a cell death polypeptide comprises an epidermal growth factor receptor (EGFR) e or an EGFR mimotope that speciﬁcally binds to said cetuximab. In yet another ment, the antibody is mumab and an extracellular domain of a cell death ptide comprises an epidermal growth factor receptor (EGFR) epitope or an EGFR mimotope that cally binds to said panitumumab. In r embodiment, the antibody is efalizumab and an extracellular domain of a cell death polypeptide ses an epitope of CD1 la or a mimotope of CD1 la that speciﬁcally binds to said efalizumab. In still another embodiment, the antibody is ipilimumab and an extracellular domain of a cell death polypeptide ses a CD152 epitope or CDl52 mimotope that speciﬁcally binds to said ipilimumab. In still another embodiment, the antibody is natalizumab and an extracellular domain of a cell death polypeptide comprises an epitope of alpha-4 integrin or a mimotope of alpha 4 integrin that speciﬁcally binds to said natalizumab. In another embodiment, the antibody is basiliximab and an extracellular domain of a cell death ptide comprises a CD25 e or CD25 mimotope that speciﬁcally binds to said basiliximab.

In certain embodiments, when a multimerizing agent or a dimerizing agent binds to at least two cell death polypeptides, dimerization or multimerization of the cell death polypeptides occurs, e.g., zation or multimerization of the cell death polypeptides occurs. In certain embodiments, an extracellular domain of a cell death polypeptide is or comprises a or or a ligand-binding portion thereof. In a speciﬁc embodiment, a erizing agent or dimerizing agent is or comprises at least two ligands for said or or ligand binding portion thereof. In another speciﬁc embodiment, said multimerizing agent or dimerizing agent binds to said receptor or said ligand binding n thereof on two of the cell death polypeptides, and said polypeptides are multimerized or dimerized, e.g., the intracellular s of said polypeptides are multimerized or dimerized. In particular embodiments the cell death polypeptides comprise intracellular domains comprising a caspase domain, and multimerization or dimerization of the caspase domains occurs. In speciﬁc embodiments, said multimerization or dimerization, for example, multimerization or dimerization of intracellular domains, e.g., multimerization or dimerization of caspase domains, initiates an apoptosis-inducing signal in said cell, e.g., T lymphocyte.

In speciﬁc embodiments, when an antibody speciﬁcally binds to an epitope or mimotope of at least two cell death polypeptides, dimerization of the cell death polypeptides occurs, e.g., dimerization of the ellular domains of the cell death polypeptides occurs. In particular embodiments the cell death polypeptides se intracellular domains comprising a caspase , and dimerization of the caspase domains occurs. In speciﬁc embodiments, said dimerization, for example, dimerization of intracellular domains, e.g., dimerization of e domains, initiates an apoptosis-inducing signal in said cell, e. g., T lymphocyte.

In certain other embodiments of the cell, e.g., T lymphocyte, said extracellular domain of the cell death polypeptide comprises a ligand for a receptor. In a c embodiment, said multimerizing agent or dimerizing agent comprises at least two receptors or -binding portions f that bind to said ligand. In a speciﬁc embodiment, when said multimerizing agent or dimerizing agent binds to said receptor or said ligand binding portion thereof on at least two of the cell death ptides, said polypeptides are multimerized or dimerized. In a speciﬁc embodiment, when the cell death polypeptides are multimerized or dimerized, an apoptosis-inducing signal is generated in said cell.

In certain other embodiments, an extracellular domain of a cell death ptide comprises an artiﬁcial oligonucleotide sequence. For example, in particular embodiments, a modiﬁed cell, e. g., T lymphocyte, comprises a cell death polypeptide comprising an extracellular domain that comprises an artiﬁcial oligonucleotide sequence. In a speciﬁc embodiment, a multimerizing or zing agent is or comprises at least one multimerizing or zing oligonucleotide comprising a ﬁrst oligonucleotide and a second oligonucleotide, optionally joined by a linker, wherein said ﬁrst oligonucleotide and said second oligonucleotide are complementary to said artiﬁcial oligonucleotide sequence. In certain speciﬁc embodiments, said ﬁrst oligonucleotide and said second oligonucleotide have the same sequence. In speciﬁc embodiments, said ﬁrst oligonucleotide and said second oligonucleotide are joined in a head-to- head or tail-to-tail conformation. In speciﬁc embodiments, when said erizing or dimerizing oligonucleotide of said multimerizing agent or dimerizing agent hybridizes to the artiﬁcial oligonucleotide ce of two of said cell death polypeptides, the cell death polypeptides are erized or dimerized. In another speciﬁc ment, when the cell death polypeptides are multimerized or dimerized, an apoptosis-inducing signal is generated in said cell. In particular embodiments, the cell death polypeptides se ellular caspase domains, and when the intracellular caspase domains are multimerized or dimerized, an apoptosis-inducing signal is generated in said cell.

In certain other embodiments of the cell, e.g., T lymphocyte, said multimerizing or dimerizing agent is an artiﬁcial polypeptide comprising two or more g domains joined by one or more s.

In a speciﬁc embodiment, provided herein is a cell, e. g., a T lymphocyte, comprising a cell death polypeptide comprising an extracellular domain sing an epitope, a transmembrane domain, and an intracellular domain comprising a caspase 9, e. g., a human caspase 9, or a functional portion f. In another speciﬁc embodiment, provided herein is a cell, e. g., a T lymphocyte, comprising an artiﬁcial ptide comprising an extracellular domain comprising a receptor or ligand-binding portion thereof, and an intracellular domain comprising a caspase 9, e. g., a human caspase 9, or a functional portion thereof. In another speciﬁc embodiment, provided herein is a cell, e.g., a T lymphocyte, comprising an artiﬁcial polypeptide comprising an extracellular domain comprising a ligand or a receptor-binding portion thereof, wherein said ligand binds a receptor or ligand-binding portion thereof, and an intracellular domain comprising a caspase 9, e. g., a human caspase 9, or ﬁanctional n f. In a specific embodiment, said cell is a T cyte.

In another aspect, provided herein is cell, e. g., a T lymphocyte, safety system comprising a cell sing (a) a cell death polypeptide comprising an extracellular domain comprising an epitope, a embrane domain, and an intracellular domain comprising a caspase or a functional portion thereof; and (b) a dimerizing agent comprising two epitope- binding or mimotope-binding domains that when contacted with two of said cell death polypeptides dimerizes said ptides, wherein said caspase is e 3, caspase 8 or caspase 9, e. g., human caspase 3, caspase 8, or caspase 9, and wherein said dimerization generates an apoptosis-inducing signal in said cell. In a specific embodiment, said cell is a T lymphocyte.

In another embodiment, ed herein is a cell, e.g., a T lymphocyte, safety system sing (a) a cell comprising an cial polypeptide comprising an extracellular domain comprising a receptor or ligand-binding portion thereof, and an intracellular domain comprising a caspase or a ﬁanctional portion thereof; and (b) a dimerizing agent sing two ligands that bind to said receptor or ligand-binding portion thereof, wherein when said dimerizing agent is contacted with two of said polypeptides said dimerizing agent dimerizes said polypeptides, wherein said e is caspase 3, caspase 8 or caspase 9, e. g., human caspase 3, caspase 8, or caspase 9, and wherein said dimerization generates an apoptosis-inducing signal in said cell. In a specific embodiment, said cell is a T lymphocyte.

In another ment, provided herein is a cell, e.g., a T lymphocyte, safety system comprising (a) a cell comprising an artificial cell death ptide sing an extracellular domain comprising a ligand or a receptor-binding portion thereof, and an intracellular domain comprising a caspase or functional portion thereof; and (b) a dimerizing agent comprising two receptors or ligand-binding portions thereof that bind to said ligand or receptor-binding portion thereof, wherein when said zing agent is contacted with two of said polypeptides said zing agent dimerizes said polypeptides, wherein said caspase is caspase 3, caspase 8 or caspase 9, e. g., human caspase 3, caspase 8, or e 9, and wherein said dimerization generates an apoptosis-inducing signal in said cell. In a speciﬁc embodiment, said cell is a T lymphocyte.

In a speciﬁc embodiment of any of the embodiments herein, when a plurality of said apoptosis-inducing signals are generated in said cell, e. g., T lymphocyte, said signal is sufﬁcient to kill said cell. In a c embodiment, said cell is a T lymphocyte.

In another aspect, r ed herein is a method of killing a cell, e.g., a T cyte, wherein said cell comprises a plurality of artiﬁcial cell death ptides each comprising an apoptosis-inducing domain, wherein the cell death polypeptides are multimerizable or dimerizable using a erizing agent or dimerizing agent that is not an FK506 binding protein (FKBP) ligand, and wherein when said multimerizing agent or dimerizing agent multimerizes or dimerizes said polypeptide, an apoptosis-inducing signal is generated in said T lymphocyte, comprising contacting said cell with an amount of said multimerizing agent or dimerizing agent sufﬁcient for said plurality of the cell death polypeptides to multimerize or dimerize and generate an aggregate apoptosis-inducing signal sufﬁcient to kill said cell. In certain embodiments, the cell death polypeptide is a embrane polypeptide sing an extracellular domain, a transmembrane domain, and an intracellular domain comprising said apoptosis-inducing domain. In speciﬁc embodiments, said apoptosis- inducing domain of said polypeptide is or comprises a e, e.g., caspase 3, caspase 8 or caspase 9, for example a human caspase 9, caspase 8, or caspase 3. In speciﬁc embodiments, the multimerizing agent or dimerizing agent is a protein, an oligonucleotide or a polysaccharide. In a speciﬁc embodiment, said cell is a T lymphocyte.

In certain embodiments, the multimerizing agent or dimerizing agent is a protein, an oligonucleotide, or a polysaccharide. In c ments, the multimerizing agent or dimerizing agent is a polypeptide comprising at least two sites that speciﬁcally bind to a cell death polypeptide, e. g., an extracellular domain of a cell death polypeptide. In particular embodiments, the ptide is an antibody, e.g., an antibody that comprises at least two epitope-binding sites or at least two mimotope-binding sites. In certain embodiments, an extracellular domain of a cell death polypeptide comprises at least one epitope or mimotope to WO 52177 which the antibody speciﬁcally binds. In particular embodiments, the antibody is a bispecific antibody comprising two ent epitope or mimotope binding sites that bind two different epitopes or mimotopes present on an extracellular domain of a cell death polypeptide. In certain embodiments, the antibody is an IgG or an IgM antibody. In a specific ment, an antibody useful as a erizing or dimerizing agent is one that has been approved by the United States Food and Drug Administration for any use.

In a specific embodiment, when the multimerizing agent or dimerizing agent is an antibody, said antibody is one that cally binds to a CD20 epitope or mimotope, e. g., a human CD20 epitope or mimotope, and said extracellular domain of a cell death polypeptide comprises a CD20 epitope or mimotope to which said antibody speciﬁcally binds. In n specific embodiments, when the multimerizing agent or zing agent is an antibody, said antibody is mab and said extracellular domain of the cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that binds to said rituximab; said antibody is tositumumab and said extracellular domain of the cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that binds to said tositumumab; said antibody is ibritumomab and said extracellular domain of the cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that binds said ibritumomab; said antibody is ofatumumab and said extracellular domain of the cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that binds said ofatumumab; said antibody is alemtuzumab and said extracellular domain of the cell death polypeptide comprises a CD52 epitope or a CD52 mimotope that binds to said alemtuzumab; said antibody is basiliximab and said extracellular domain of the cell death polypeptide comprises a CD25 epitope or a CD25 mimotope that binds said basiliximab; said antibody is daclizumab and said extracellular domain of the cell death polypeptide comprises a CD25 e or a CD25 mimotope that binds said daclizumab; said antibody is brentuximab and said extracellular domain of the cell death polypeptide comprises a CD30 epitope or a CD30 mimotope that binds said ximab; said dy is belimumab and said extracellular domain of the cell death ptide comprises a B-cell activating factor (BAFF) epitope or a BAFF mimotope that binds said belimumab; said antibody is cetuximab and said ellular domain of the cell death polypeptide comprises an mal growth factor receptor (EGFR) epitope or an EGFR mimotope that binds said cetuximab; said antibody is panitumumab and said extracellular domain of the cell death polypeptide comprises an epidermal growth factor receptor (EGFR) epitope or an EGFR mimotope that binds said panitumumab; said antibody is efalizumab and said extracellular domain of the cell death polypeptide comprises an e of CD1 la or a mimotope of CD1 la that binds to said efalizumab; said antibody is ipilimumab and said extracellular domain of the cell death polypeptide comprises a CDl52 epitope or CDl52 mimotope that binds said ipilimumab; said antibody is natalizumab and said extracellular domain of the cell death polypeptide comprises an epitope of alpha-4 integrin or a mimotope of alpha 4 integrin that binds said natalizumab; or said antibody is basiliximab and said extracellular domain of the cell death ptide comprises a CD25 epitope or CD25 mimotope that binds said basiliximab. In a c ment of any of the above embodiments, when said dy binds to said epitope or mimotope on at least two of said cell death polypeptides, the intracellular domains of said polypeptides, and/or the respective caspases in said intracellular domains multimerize or dimerize. In speciﬁc embodiments, when said antibody speciﬁcally binds to an epitope or mimotope of at least two cell death polypeptides, dimerization of the cell death polypeptides , e.g., dimerization of the intracellular domains of the cell death polypeptides occurs. In particular embodiments the cell death polypeptides comprise intracellular domains comprising a caspase domain, and dimerization of the caspase domains occurs. In speciﬁc embodiments, said dimerization, for example, dimerization of intracellular domains, e.g., dimerization of caspase domains, initiates an apoptosis-inducing signal in said cell, e.g., T lymphocyte.

In certain c embodiments, said extracellular domain of the cell death ptide is or comprises a receptor or a ligand-binding portion f. In such ments, said multimerizing agent or dimerizing agent comprises at least two ligands for said receptor or ligand binding portion f. In speciﬁc embodiments, when said erizing agent or dimerizing agent binds to said receptor or said ligand g portion thereof on at least two of said cell death polypeptides, said polypeptides are multimerized or dimerized. In speciﬁc embodiments, said multimerization or dimerization of said polypeptides initiates an apoptosis- inducing signal in said cell, e. g., T lymphocyte.

In certain speciﬁc embodiments, said extracellular domain of the cell death polypeptide comprises a ligand for a receptor. In such embodiments, said multimerizing agent or dimerizing agent comprises at least two receptors or ligand-binding portions thereof that bind to said ligand.

In speciﬁc ments, when said multimerizing agent or dimerizing agent binds to said receptor or said ligand g portion thereof on two or more of said polypeptides, the intracellular domains in said polypeptides and/or the caspase domains of said polypeptides are multimerized or dimerized. In speciﬁc ments, said multimerization or dimerization of said intracellular domains and/or e domains initiates an apoptosis-inducing signal is generated in said cell, e.g., T cyte.

In certain embodiments, said extracellular domain of said cell death polypeptide comprises an artiﬁcial oligonucleotide sequence. For example, in particular ments, said cell death polypeptide comprises an extracellular domain that comprises an artiﬁcial oligonucleotide sequence. In a speciﬁc embodiment, a erizing or dimerizing agent is or comprises at least one multimerizing or dimerizing oligonucleotide comprising a ﬁrst oligonucleotide and a second oligonucleotide, optionally joined by a linker, wherein said ﬁrst oligonucleotide and said second oligonucleotide are complementary to said artiﬁcial oligonucleotide sequence. In certain speciﬁc ments, said ﬁrst oligonucleotide and said second oligonucleotide have the same sequence. In speciﬁc embodiments, said ﬁrst oligonucleotide and said second oligonucleotide are joined in a head-to-head or tail-to-tail conformation. In c embodiments, when said multimerizing or dimerizing oligonucleotide of said multimerizing agent or dimerizing agent izes to the ial oligonucleotide sequence of two of said cell death ptides, the cell death polypeptides are multimerized or dimerized. In another speciﬁc embodiment, when the cell death polypeptides are multimerized or dimerized, an apoptosis-inducing signal is generated in said cell. In particular embodiments, the cell death polypeptides comprise intracellular caspase domains, and when the intracellular caspase s are multimerized or dimerized, an apoptosis-inducing signal is generated in said cell. In a speciﬁc embodiment, said cell is a T lymphocyte.

In a speciﬁc embodiment of any of the embodiments herein, when a plurality of said apoptosis-inducing signals are ted in said T lymphocyte, said signal is ent to kill said cell, e.g., T lymphocyte.

In other embodiments, said multimerizing agent or dimerizing agent is an artiﬁcial polypeptide comprising two or more binding domains joined by one or more linkers.

In a speciﬁc embodiment, provided herein is a method of killing a cell, e. g., a T lymphocyte, wherein said cell comprises a ity of artiﬁcial cell death polypeptides each comprising a caspase or functional portion f, wherein said caspase is caspase 3, caspase 8 or caspase 9 (e.g., human caspase 3, caspase 8, or caspase 9), and, wherein said cell death polypeptide is dimerizable using an antibody, and n when said antibody dimerizes said polypeptide, an apoptosis-inducing signal is generated in said cell, comprising contacting said cell with an amount of said antibody sufﬁcient to dimerize a sufﬁcient number of said plurality of artiﬁcial polypeptides to dimerize and generate an aggregate apoptosis-inducing signal sufﬁcient to kill said cell. In a speciﬁc embodiment, said cell is a T cyte.

In another speciﬁc embodiment, provided herein is a method of killing a cell, e.g., a T lymphocyte, wherein said cell ses a plurality of artiﬁcial cell death polypeptides each comprising a caspase or functional portion thereof, wherein said caspase is caspase 3, caspase 8 or caspase 9 (e.g., human caspase 3, caspase 8, or caspase 9), and, wherein said plurality of artiﬁcial polypeptides each comprises an extracellular domain comprising a receptor or ligand- binding portion thereof that bind a ligand, n said polypeptide is dimerizable using a dimerizing agent comprising two said ligands, and wherein when said dimerizing agent zes two of said ptides, an apoptosis-inducing signal is generated in cell, sing contacting said cell with an amount of said dimerizing agent sufﬁcient to dimerize a sufﬁcient number of said plurality of artiﬁcial cell death polypeptides to dimerize and generate an aggregate sis-inducing signal sufﬁcient to kill said cell. In a speciﬁc embodiment, said cell is a T lymphocyte.

In another speciﬁc embodiment, ed herein is a method of killing a cell, e.g., a T lymphocyte, wherein said cell comprises a plurality of artiﬁcial cell death polypeptides each comprising a caspase or functional portion thereof, wherein said caspase is caspase 3, caspase 8 or caspase 9 (e.g., human e 3, caspase 8, or caspase 9), and, wherein said plurality of artiﬁcial polypeptides each comprises an extracellular domain comprising a ligand or receptor- binding portion thereof that bind a receptor or -binding portion thereof, wherein said polypeptides are zable using a dimerizing agent comprising two said receptors or ligand- g portion f, and n when said dimerizing agent dimerizes two of said polypeptides, an apoptosis-inducing signal is generated in said cell, comprising contacting said cell with an amount of said dimerizing agent sufﬁcient to dimerize a sufﬁcient number of said plurality of artiﬁcial polypeptides to dimerize and generate an aggregate apoptosis-inducing signal sufﬁcient to kill said cell. In a speciﬁc embodiment, said cell is a T lymphocyte.

In r speciﬁc embodiment, provided herein is a method of killing a cell, e.g., a T lymphocyte, wherein said cell comprises a plurality of artiﬁcial cell death polypeptides each comprising a caspase or functional portion thereof, wherein said caspase is caspase 3, e 8 or caspase 9 (e.g., human caspase 3, caspase 8, or caspase 9), and, wherein said plurality of artiﬁcial polypeptides each comprising an extracellular domain comprising an artiﬁcial oligonucleotide, wherein said plurality of polypeptides are dimerizable using a dimerizing agent comprising an oligonucleotide comprising a ﬁrst oligonucleotide and a second oligonucleotide, wherein said ﬁrst oligonucleotide and said second ucleotide have the same nucleotide sequence, and wherein said ﬁrst oligonucleotide and second oligonucleotide optionally are joined by a linker, and wherein said ﬁrst oligonucleotide and said second oligonucleotide are complementary to said ial oligonucleotide in said extracellular domain of said polypeptide, and wherein when said dimerizing agent dimerizes two of said cell death polypeptides, an apoptosis-inducing signal is ted in said cell, comprising contacting said cell with an amount of said dimerizing agent sufﬁcient to dimerize a ent number of said plurality of artiﬁcial polypeptides to dimerize and generate an aggregate apoptosis-inducing signal sufﬁcient to kill said cell. In a c embodiment, said cell is a T lymphocyte.

In certain embodiments, the cells (e. g., T lymphocytes) killed in accordance with the methods described herein se a polypeptide that acts to target the cell to a particular n, e.g., a tumor-associated n or tumor-speciﬁc antigen, wherein said polypeptide, when bound to said antigen, causes the cell to kill a cell ying said antigen, for example, a chimeric antigen receptor (CAR). T lymphocytes comprising CARs are referred to herein as CAR-T cytes. The chimeric antigen receptors typically comprise (i) an intracellular domain (e.g., asmic domain) of an endogenous protein expressed on the surface of lymphocytes and that rs the activation and/or proliferation of said lymphocytes, (ii) a transmembrane domain, and (iii) an extracellular domain that binds to an antigen of interest, e.g., a tumor-associated antigen or tumor-speciﬁc antigen. The CAR-T lymphocytes also typically comprise one or more co-stimulatory domains. In certain ments, a CAR-T lymphocyte comprises at least two CAR polypeptides, at least one of which provides a y stimulatory signal to the CAR-T lymphocyte, and at least one that provides a costimulatory signal to the CAR-T lymphocyte. CAR-T lymphocytes comprising a cell death polypeptide and comprising speciﬁc embodiments of CARs are provided below.

In another , provided herein are methods of treating an individual having a disease or disorder, wherein the disease or disorder is characterized, or is characterizable, by cells sing an antigen, comprising administering to the individual cells, e.g., T lymphocytes, expressing a polypeptide, as described herein. In certain embodiments, when the modiﬁed cells, e.g., d T lymphocytes described herein are administered to a subject in need thereof, the combination of multimerizing agent and cell death polypeptide selected are chosen such that they are compatible with the patient population (or ulation) in which the cells, e.g., T lymphocytes, have been administered. By way of example only, if the multimerizing agent selected is the antibody rituximab, then in certain embodiments the t population is individuals having a cancer of the B cells, e.g., B cell lymphoma. 4. DETAILED DESCRIPTION 4.1. Cells sing Cell Death Polypeptides Provided herein are genetically modiﬁed cells, for example immune cells, such as T cytes, e. g., human T lymphocytes, that comprise an artiﬁcial multimerizable, e. g., dimerizable, polypeptide (referred to herein as a "cell death ptide") that, when multimerized, e.g., dimerized, by a erizing agent, e. g., zing agent, generates an apoptosis-inducing signal in a cell, e. g., a T lymphocyte, that expresses the polypeptide, resulting in cell death, e. g., via apoptosis. Without wishing to be bound by any particular mechanism or theory, it is thought that when a sufﬁcient number of a plurality of cell death polypeptides of the cell are multimerized, e.g., dimerized, that the ate apoptosis-inducing signal thereby generated is sufﬁcient to kill the cell, e.g., cause the cell to undergo apoptosis. In a speciﬁc embodiment, the genetically modiﬁed cells provided herein are T lymphocytes.

In certain embodiments, the cell death polypeptide can be multimerized or dimerized by an administrable multimerizing agent or dimerizing agent, e.g., a protein (e.g., antibody, receptor or ligand-binding portion thereof, a ligand or receptor-binding n f), oligonucleotide, or the like. In certain embodiments, the multimerizing agent is not a small molecule. The multimerizing or zing agent can be used to kill T lymphocytes comprising the cell death polypeptide either in vitro or in viva.

Thus, in a ﬁrst aspect, provided herein is a T lymphocyte comprising an artiﬁcial polypeptide (cell death polypeptide) comprising an apoptosis-inducing domain, wherein said cell death ptide is multimerizable using a multimerizing agent, n said multimerizing agent is not an FK506 g protein (FKBP) , and wherein when said multimerizing agent multimerizes said polypeptide, an apoptosis-inducing signal is generated in said T lymphocyte. In a speciﬁc embodiment, said multimerizing agent is a dimerizing agent; that is, the multimerizing agent causes the cell death polypeptide to dimerize. In another speciﬁc embodiment, when said dimerizing agent dimerizes said cell death polypeptide, an apoptosis- inducing signal is generated in said T lymphocyte. The cell death polypeptide does not se an FK506 binding protein, functional portion f, or modiﬁed form f.

In certain embodiments, said cell death ptide is a transmembrane polypeptide sing an extracellular domain, a transmembrane domain, and an ellular domain comprising said apoptosis-inducing domain. In certain embodiments, the apoptosis-inducing domain of the cell death polypeptide can be, for example, any protein or portion thereof that when dimerized initiates an apoptosis-inducing signal in the cell. In certain embodiments, the apoptosis-inducing domain is any caspase that homodimerizes, and preferably is or comprises a caspase, e.g., caspase 9, caspase 8, or caspase 3 (e.g., human caspase 9, e 8, or caspase 3).

The amino acid sequences of human caspases, including human caspase 9, human caspase 8, and human caspase 3 are well known in the art. For example, human e 3 has been assigned NCBI Gene ID: 836; human caspase 8 has been assigned NCBI Gene ID: 841; and human caspase 9 has been assigned NCBI Gene ID: 842. In certain embodiments, the intracellular domain that is, or comprises, a caspase domain, and the extracellular domain, which comprises the epitope or pe, are joined by a CD80L stalk or CD80 stalk, at least part of which can function as a transmembrane domain.

In certain embodiments, the dimerizing agent is a polypeptide comprising at least two sites that speciﬁcally bind to a cell death polypeptide, e. g., an extracellular domain of a cell death polypeptide. In particular embodiments, the polypeptide is an antibody, e.g., an antibody sing at least two epitope or mimotope binding sites. In certain embodiments, only the antigen binding domain of an antibody is used as a multimerizing or dimerizing agent. In certain embodiments, an extracellular domain of a cell death polypeptide comprises at least one epitope or mimotope to which the antibody speciﬁcally binds. In particular embodiments, the antibody is a bispeciﬁc antibody comprising two different epitope or mimotope binding sites that bind two ent epitopes or mimotopes present on an extracellular domain of a cell death polypeptide. 2014/027039 In certain embodiments, the antibody is an IgG or an IgM antibody. Artiﬁcial antibody constructs sing epitope-binding or mimotope-binding domains from antibodies, optionally joined by one or more linkers, may also be used.

In a speciﬁc embodiment, said dy, useful as a multimerizing or dimerizing agent, has been approved by a mental regulatory authority, e.g., the United States Food and Drug Administration for any use. This ensures, e.g., that the antibody, when used as a dimerizing or multimerizing agent, has a known toxicity and patient safety proﬁle. Any combination of antibody and associated target may be used in the T lymphocytes provided herein. In one embodiment, an antibody useful as a multimerizing or dimerizing agent is one that speciﬁcally binds to a CD20 epitope or mimotope, e.g., a human CD20 epitope or pe, and an extracellular domain of a cell death polypeptide comprises a CD20 epitope or mimotope to which the antibody speciﬁcally binds. In certain speciﬁc ments, the antibody is mab and an extracellular domain of a cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that speciﬁcally binds to said rituximab. In another speciﬁc embodiment, the antibody is tositumumab and an extracellular domain of a cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that speciﬁcally binds to said tositumumab. In yet r embodiment, the antibody is ibritumomab and an extracellular domain of a cell death polypeptide comprises a CD20 epitope or a CD20 pe that speciﬁcally binds to said ibritumomab. In still another embodiment, the antibody is ofatumumab and an extracellular domain of a cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that speciﬁcally binds to said atumumab.

In r speciﬁc embodiment, the antibody is alemtuzumab and an extracellular domain of a cell death polypeptide comprises a CD52 epitope or a CD52 mimotope that speciﬁcally binds to said alemtuzumab. In yet another ment, the antibody is ximab and an extracellular domain of the cell death polypeptide comprises a CD25 epitope or a CD25 mimotope that speciﬁcally binds to said ximab. In another embodiment, the antibody is daclizumab and an extracellular domain of a cell death polypeptide comprises a CD25 epitope or a CD25 mimotope that speciﬁcally binds to said daclizumab. In still another embodiment, the antibody is brentuximab and an ellular domain of a cell death polypeptide comprises a CD30 epitope or a CD30 mimotope that speciﬁcally binds to said brentuximab. In another embodiment, the antibody is belimumab and an ellular domain of a cell death polypeptide comprises a B-cell activating factor (BAFF) epitope or a BAFF mimotope that speciﬁcally binds to said belimumab. In another embodiment, the antibody is cetuximab and an extracellular domain of a cell death ptide comprises an epidermal growth factor receptor (EGFR) epitope or an EGFR mimotope that speciﬁcally binds to said mab. In yet another embodiment, the antibody is panitumumab and an ellular domain of a cell death polypeptide comprises an mal growth factor receptor (EGFR) epitope or an EGFR mimotope that speciﬁcally binds to said panitumumab. In another embodiment, the antibody is efalizumab and an extracellular domain of a cell death polypeptide comprises an epitope of CD11a or a mimotope of CD1 1a that speciﬁcally binds to said efalizumab. In still r embodiment, the antibody is ipilimumab and an extracellular domain of a cell death ptide comprises a CD152 epitope or CD152 mimotope that cally binds to said ipilimumab. In still another embodiment, the antibody is natalizumab and an extracellular domain of a cell death polypeptide comprises an epitope of alpha-4 integrin or a mimotope of alpha 4 integrin that speciﬁcally binds to said natalizumab. In another embodiment, the antibody is basiliximab and an extracellular domain of a cell death polypeptide ses a CD25 epitope or CD25 mimotope that speciﬁcally binds to said basiliximab.

Ligands and receptors can be utilized in the construction of the cell death polypeptides provided herein, and multimerizing agents or dimerizing agents comprising the receptors’ respective ligands can be used to multimerize or dimerize the polypeptides. In n embodiments, when a multimerizing agent or a dimerizing agent binds to at least two cell death polypeptides, dimerization or multimerization of the cell death polypeptides occurs, e.g., dimerization or multimerization of the cell death polypeptides occurs. In certain embodiments, an extracellular domain of a cell death polypeptide is or comprises a receptor or a ligand-binding portion thereof. In a speciﬁc embodiment, a multimerizing agent or dimerizing agent is or ses at least two ligands for said receptor or ligand binding portion f In another speciﬁc embodiment, said multimerizing agent or dimerizing agent binds to said receptor or said ligand binding n thereof on two of the cell death polypeptides, and said polypeptides are multimerized or zed, e.g., the intracellular s of said polypeptides are multimerized or dimerized. In particular embodiments the cell death polypeptides comprise intracellular domains comprising a caspase , and multimerization or dimerization of the caspase domains occurs. In speciﬁc embodiments, said multimerization or dimerization, for example, multimerization or dimerization of intracellular domains, e.g., multimerization or dimerization of caspase domains, tes an apoptosis-inducing signal in said cell, e.g., T lymphocyte.

In speciﬁc ments, when an antibody speciﬁcally binds to an epitope or mimotope of at least two cell death polypeptides, dimerization of the cell death polypeptides occurs, e.g., dimerization of the ellular domains of the cell death polypeptides occurs. In particular embodiments the cell death polypeptides comprise intracellular domains comprising a caspase domain, and dimerization of the caspase domains occurs. In speciﬁc embodiments, said dimerization, for example, dimerization of intracellular domains, e.g., dimerization of caspase domains, initiates an apoptosis-inducing signal in said cell, e. g., T lymphocyte.

In certain other ments of the cell, e.g., T lymphocyte, said extracellular domain of the cell death polypeptide comprises a ligand for a receptor. In a speciﬁc ment, said multimerizing agent or dimerizing agent comprises at least two receptors or ligand-binding portions thereof that bind to said ligand. In a speciﬁc embodiment, when said multimerizing agent or dimerizing agent binds to said receptor or said ligand binding portion thereof on at least two of the cell death polypeptides, said ptides are multimerized or dimerized. In a speciﬁc embodiment, when the cell death polypeptides are multimerized or dimerized, an sis-inducing signal is generated in said cell. In a speciﬁc embodiment, said cell is a T lymphocyte.

In certain other embodiments, an extracellular domain of a cell death polypeptide comprises an artiﬁcial oligonucleotide sequence. For example, in particular embodiments, a modiﬁed cell, e. g., T lymphocyte, comprises a cell death polypeptide comprising an ellular domain that comprises an artiﬁcial oligonucleotide sequence. In a speciﬁc ment, a multimerizing or dimerizing agent is or comprises at least one multimerizing or dimerizing oligonucleotide comprising a ﬁrst oligonucleotide and a second oligonucleotide, optionally joined by a linker, wherein said ﬁrst oligonucleotide and said second oligonucleotide are complementary to said artiﬁcial ucleotide sequence. In certain c embodiments, said ﬁrst oligonucleotide and said second oligonucleotide have the same ce. In speciﬁc ments, said ﬁrst oligonucleotide and said second oligonucleotide are joined in a head-to- head or o-tail conformation. In c embodiments, when said multimerizing or dimerizing oligonucleotide of said multimerizing agent or dimerizing agent hybridizes to the artiﬁcial oligonucleotide sequence of two of said cell death polypeptides, the cell death polypeptides are multimerized or dimerized. In another speciﬁc embodiment, when the cell death polypeptides are multimerized or dimerized, an apoptosis-inducing signal is generated in said cell. In particular embodiments, the cell death polypeptides comprise intracellular caspase domains, and when the intracellular caspase domains are multimerized or dimerized, an apoptosis-inducing signal is generated in said cell. In a speciﬁc embodiment, said cell is a T lymphocyte.

In certain other ments of the T lymphocyte, the multimerizing or dimerizing agent is an artiﬁcial polypeptide comprising two or more g domains joined by one or more linkers.

In a speciﬁc embodiment, provided herein is a cell, e. g., a T lymphocyte, comprising a cell death polypeptide comprising an extracellular domain comprising an e, a embrane domain, and an ellular domain sing a caspase 9, e. g., a human caspase 9, or a onal portion thereof. In another speciﬁc embodiment, provided herein is a cell, e. g., a T lymphocyte, comprising an artiﬁcial polypeptide comprising an extracellular domain comprising a receptor or -binding portion thereof, and an ellular domain comprising a caspase 9, e. g., a human caspase 9, or a functional portion thereof. In r speciﬁc embodiment, provided herein is a cell, e.g., a T lymphocyte, comprising an artiﬁcial polypeptide comprising an extracellular domain comprising a ligand or a receptor-binding portion thereof, wherein said ligand binds a receptor or ligand-binding portion thereof, and an intracellular domain comprising a caspase 9, e. g., a human caspase 9, or ﬁlnctional portion thereof. In a speciﬁc embodiment, said cell is a T lymphocyte.

In any of the embodiments , wherein the modiﬁed cells are T lymphocytes, the T lymphocytes may be CD4+ T lymphocytes or CD8+ T cytes. The T lymphocytes may be, without genetic modiﬁcation, speciﬁc for a particular antigen (e.g., a tumor-associated antigen, tumor-speciﬁc antigen, viral antigen, or the like). The T lymphocytes may be genetically modiﬁed to express one or more polypeptides, e.g., chimeric antigen receptors, that target the T lymphocyte to a speciﬁc antigen. 4.2. s of Killing Cells that Comprise Cell Death Polypeptides The cell death polypeptides provided herein can be used in methods of killing cells, e. g., T lymphocytes, that comprise the cell death polypeptides. The cell death polypeptides provided herein may be used in conjunction with any cells, in particular, any mammalian cells, for example, any human cells. Such cell death polypeptides provide, for example, a useful safety feature for cell therapeutics. As such, the cell death polypeptides can, for example, be ant for a drug product sing a cell therapeutic, e. g., a chimeric n receptor-expressing CAR T lymphocytes, because the cell death polypeptides enable rapid killing of the cell therapeutic, e. g., the T cytes, should such rapid killing become desirable, e. g., in the event administration of the cells causes any unwanted or deleterious s in a patient receiving them, or if the presence of the cell therapeutic, e.g., the T lymphocytes, in a subject is no longer necessary. Thus, in certain embodiments, the cell death polypeptides provided herein can be used in conjunction with any administrable cells, for example cell therapeutics, such as mammalian cell therapeutics, e.g., human cell therapeutics. Non-limiting examples of cells in which the cell death polypeptides and multimerizing or dimerizing agents may be used include, but are not limited to, natural killer (NK) cells, dendritic cells (DC), placental stem cells (e.g., the placental stem cells disclosed in US. Patent Nos. 7,468,276; 8,057,788 and 8,202,703, the disclosures of which are hereby orated by reference in their entireties), mesenchymal-like stem cells from umbilical cord blood, placental blood, peripheral blood, bone marrow, dental pulp, adipose tissue, osteochondral , and the like; embryonic stem cells, embryonic germ cells, neural crest stem cells, neural stem cells, and differentiated cells (e.g., ﬁbroblasts, etc.). The cell death polypeptides, and erizing or dimerizing agents, may also be used in tumor cell lines, e. g., for animal model experimental purposes.

Cell killing by the cell death ptides bed herein can take place either in viva, e. g., in an individual to whom the cells, e. g., T lymphocytes, have been administered, or in vitro, e. g., in a laboratory, e.g., as part of quality control experiments. In one embodiment, provided herein is a method of killing a cell, e.g., a T lymphocyte, wherein said cell comprises a plurality of artiﬁcial cell death polypeptides each sing an apoptosis-inducing domain, wherein said polypeptides are multimerizable or dimerizable using a multimerizing agent or dimerizing agent that is not an FK506 binding n (FKBP) ligand, and wherein when said multimerizing agent or dimerizing agent multimerizes or dimerizes said polypeptide, an apoptosis-inducing signal is generated in said cell, comprising contacting said cell with an amount of said multimerizing agent or dimerizing agent sufﬁcient for said plurality of artiﬁcial polypeptides to dimerize and generate an aggregate apoptosis-inducing signal sufﬁcient to kill said cell. In certain embodiments, the cell death ptide is a transmembrane polypeptide comprising an extracellular domain, a transmembrane domain, and an intracellular domain comprising said apoptosis-inducing . In certain ments, the intracellular domain that is, or comprises, a caspase domain, and the extracellular , which comprises the epitope or mimotope, are joined by a CD80L stalk or CD80 stalk, at least part of which can ﬁlnction as a transmembrane domain. In certain c embodiments of the method, the apoptosis-inducing domain of said polypeptide is or comprises a caspase, e. g., caspase 3, caspase 8, or caspase 9 (e.g., human caspase 9, e 8, or caspase 3).

The multimerizing agent or dimerizing agent used in the method can be any compound, other than a small molecule, that can dimerize or multimerizes a cell death polypeptide, e.g., a protein, an oligonucleotide or a polysaccharide. In certain embodiments, the multimerizing agent or dimerizing agent is an antibody, e.g., an dy that comprises at least two epitope- binding sites or at least two mimotope-binding sites. In certain embodiments, only the antigen g domain of an dy is used as a erizing or dimerizing agent. In such embodiments, the extracellular domain of the cell death polypeptide comprises an epitope or pe to which the antibody binds. The antibody can be an antibody of any e, but is preferably an IgG or an IgM antibody.

In a speciﬁc embodiment, said antibody, useful as a multimerizing or dimerizing agent, has been approved by a governmental regulatory authority, e.g., the United States Food and Drug Administration for any use. Any combination of antibody and associated target may be used in the methods of killing T lymphocytes provided herein.

In a speciﬁc embodiment, when the multimerizing agent or dimerizing agent is an antibody, said antibody is one that speciﬁcally binds to a CD20 epitope or pe, e. g., a human CD20 epitope or mimotope, and said extracellular domain of a cell death polypeptide comprises a CD20 epitope or mimotope to which said antibody speciﬁcally binds. In certain speciﬁc embodiments, when the multimerizing agent or dimerizing agent is an antibody, said antibody is rituximab and said extracellular domain of the cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that binds to said mab; said antibody is mumab and said extracellular domain of the cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that binds to said tositumumab; said antibody is ibritumomab and said extracellular domain of the cell death polypeptide comprises a CD20 epitope or a CD20 mimotope that binds said ibritumomab; said antibody is ofatumumab and said ellular domain of the cell death polypeptide comprises a CD20 e or a CD20 mimotope that binds said ofatumumab; or said antibody is alemtuzumab and said extracellular domain of the cell death polypeptide comprises a CD52 epitope or a CD52 mimotope that binds to said alemtuzumab.

In certain speciﬁc embodiments, when the multimerizing agent or dimerizing agent is an antibody, said antibody is said antibody is basiliximab and said extracellular domain of the cell death polypeptide comprises a CD25 epitope or a CD25 mimotope that binds said basiliximab; said antibody is umab and said extracellular domain of the cell death polypeptide comprises a CD25 epitope or a CD25 pe that binds said daclizumab; said antibody is brentuximab and said extracellular domain of the cell death polypeptide comprises a CD30 epitope or a CD30 mimotope that binds said brentuximab; said antibody is belimumab and said extracellular domain of the cell death polypeptide comprises a B-cell activating factor (BAFF) epitope or a BAFF pe that binds said belimumab; said antibody is cetuximab and said extracellular domain of the cell death ptide comprises an epidermal growth factor receptor (EGFR) epitope or an EGFR mimotope that binds said cetuximab; said antibody is panitumumab and said extracellular domain of the cell death polypeptide comprises an epidermal growth factor receptor (EGFR) epitope or an EGFR mimotope that binds said panitumumab; said antibody is efalizumab and said extracellular domain of the cell death polypeptide comprises an epitope of CD1 la or a mimotope of CD1 la that binds to said efalizumab; said antibody is ipilimumab and said extracellular domain of the cell death ptide comprises a CD152 e or CDl52 pe that binds said ipilimumab; said antibody is natalizumab and said extracellular domain of the cell death polypeptide comprises an epitope of 4 integrin or a mimotope of alpha 4 integrin that binds said natalizumab; or said antibody is basiliximab and said extracellular domain of the cell death polypeptide ses a CD25 epitope or CD25 mimotope that binds said basiliximab.

In a speciﬁc embodiment of any of the above embodiments, when said antibody binds to said epitope or pe on at least two of said cell death polypeptides, the intracellular domains of said polypeptides, and/or the respective caspases in said intracellular domains multimerize or dimerize. In speciﬁc embodiments, when said antibody speciﬁcally binds to an epitope or mimotope of at least two cell death polypeptides, dimerization of the cell death polypeptides occurs, e.g., dimerization of the intracellular domains of the cell death polypeptides occurs. In particular embodiments the cell death polypeptides comprise intracellular s comprising a caspase domain, and dimerization of the caspase domains occurs. In speciﬁc embodiments, said dimerization, for example, dimerization of ellular domains, e. g., dimerization of caspase domains, initiates an apoptosis-inducing signal in said cell, e.g., T lymphocyte. t ing to be limited by , when the antibody binds to the respective epitopes or mimotopes on at least two of said polypeptides, the intracellular domains of said polypeptides multimerizes, e.g., dimerize, at which time the respective caspases in said intracellular domains ze. Dimerization of said polypeptides tes an apoptosis- inducing signal in said T lymphocyte.

As above, receptors and their respective ligands may be used to multimerize or dimerize cell death ptides, and thereby effect killing of a cell, e. g., a T lymphocyte, comprising the polypeptide. For example, the extracellular domain of said cell death polypeptide is or comprises a receptor or a ligand-binding portion thereof. In such embodiments, the multimerizing agent or dimerizing agent comprises at least two ligands for said receptor or ligand binding n thereof, ng multimerization or dimerization of the cell death ptide when the multimerizing agent or zing agent binds to said or or said ligand binding portion thereof on at least two of said polypeptides, said polypeptides are dimerized. In preferred ments, dimerization of said polypeptides initiates an apoptosis- inducing signal in said cell.

In other embodiments of the method of killing cells, e. g., T lymphocytes, the extracellular domain of the cell death polypeptide comprises a ligand for a receptor. In such embodiments of the method, the multimerizing agent or dimerizing agent comprises at least two receptors or ligand-binding portions thereof that bind to said ligand. When the multimerizing dimerizing agent binds to said receptor or said ligand binding portion thereof on two of said cell death polypeptides, the intracellular domains, and thus preferably the caspase domains, in said polypeptides are zed. Dimerization of said intracellular domains, and the e domains, preferably initiates an apoptosis-inducing signal is generated in said cell.

In certain other embodiments of the method of killing cells, e.g., T lymphocytes, said extracellular domain of said cell death polypeptide ses an artiﬁcial oligonucleotide sequence. For example, in particular embodiments, said cell death polypeptide comprises an extracellular domain that comprises an artiﬁcial oligonucleotide sequence. In a speciﬁc embodiment, a multimerizing or dimerizing agent is or comprises at least one multimerizing or dimerizing oligonucleotide comprising a ﬁrst oligonucleotide and a second oligonucleotide, optionally joined by a linker, wherein said ﬁrst oligonucleotide and said second oligonucleotide are complementary to said artiﬁcial oligonucleotide sequence. In certain speciﬁc ments, said ﬁrst oligonucleotide and said second oligonucleotide have the same ce. In speciﬁc embodiments, said ﬁrst ucleotide and said second oligonucleotide are joined in a head-to- head or tail-to-tail conformation. In speciﬁc embodiments, when said multimerizing or dimerizing oligonucleotide of said multimerizing agent or dimerizing agent hybridizes to the artiﬁcial oligonucleotide sequence of two of said cell death ptides, the cell death ptides are multimerized or dimerized. In another speciﬁc embodiment, when the cell death polypeptides are multimerized or dimerized, an apoptosis-inducing signal is generated in said cell. In particular embodiments, the cell death polypeptides comprise intracellular caspase domains, and when the intracellular caspase domains are multimerized or zed, an apoptosis-inducing signal is ted in said cell. In a speciﬁc embodiment, said cell is a T lymphocyte.

In certain other embodiments of the method of killing T lymphocytes, the multimerizing or dimerizing agent is an artiﬁcial polypeptide comprising two or more binding s joined by one or more linkers.

In a speciﬁc embodiment, provided herein is a method of killing a cell, e. g., a T lymphocyte, wherein said cell comprises a plurality of artiﬁcial cell death polypeptides each sing a caspase or functional n thereof, wherein said caspase is caspase 3, e 8 or caspase 9 (e.g., human caspase 3, caspase 8, or caspase 9), and, wherein said cell death polypeptide is dimerizable using an antibody, and wherein when said antibody zes said polypeptide, an apoptosis-inducing signal is generated in said cell, comprising contacting said cell with an amount of said antibody sufﬁcient to dimerize a sufﬁcient number of said plurality of artiﬁcial polypeptides to dimerize and generate an ate apoptosis-inducing signal sufﬁcient to kill said cell. In a speciﬁc embodiment, said cell is a T cyte.

In another speciﬁc embodiment, provided herein is a method of killing a cell, e.g., a T lymphocyte, wherein said cell comprises a plurality of artiﬁcial cell death polypeptides each comprising a caspase or functional portion thereof, wherein said caspase is caspase 3, caspase 8 or caspase 9 (e.g., human caspase 3, caspase 8, or caspase 9), and, wherein said ity of artiﬁcial polypeptides each ses an extracellular domain comprising a receptor or ligand- binding portion f that bind a ligand, wherein said polypeptide is dimerizable using a zing agent comprising two said ligands, and wherein when said dimerizing agent dimerizes two of said polypeptides, an apoptosis-inducing signal is generated in cell, comprising contacting said cell with an amount of said dimerizing agent sufﬁcient to dimerize a ent number of said plurality of artiﬁcial cell death polypeptides to dimerize and generate an aggregate apoptosis-inducing signal sufﬁcient to kill said cell. In a speciﬁc embodiment, said cell is a T lymphocyte.

In another speciﬁc embodiment, provided herein is a method of killing a cell, e.g., a T lymphocyte, wherein said cell comprises a plurality of artiﬁcial cell death polypeptides each comprising a caspase or functional portion thereof, wherein said caspase is caspase 3, caspase 8 or caspase 9 (e.g., human caspase 3, caspase 8, or caspase 9), and, wherein said plurality of artiﬁcial polypeptides each comprises an extracellular domain comprising a ligand or receptor- binding portion f that bind a receptor or ligand-binding portion thereof, wherein said polypeptides are zable using a dimerizing agent sing two said receptors or ligandbinding portion thereof, and wherein when said dimerizing agent zes two of said polypeptides, an apoptosis-inducing signal is generated in said cell, sing ting said cell with an amount of said dimerizing agent sufﬁcient to dimerize a sufﬁcient number of said plurality of artiﬁcial polypeptides to dimerize and te an aggregate apoptosis-inducing signal ent to kill said cell. In a speciﬁc embodiment, said cell is a T lymphocyte.

In another speciﬁc embodiment, provided herein is method of killing a cell, e.g., a T lymphocyte, wherein said cell comprises a plurality of artiﬁcial cell death ptides each comprising a caspase or functional portion f, wherein said caspase is caspase 3, caspase 8 or caspase 9 (e.g., human caspase 3, caspase 8, or caspase 9), and, wherein said plurality of artiﬁcial polypeptides each comprising an extracellular domain comprising an artiﬁcial oligonucleotide, wherein said plurality of polypeptides are dimerizable using a dimerizing agent comprising an ucleotide comprising a ﬁrst oligonucleotide and a second oligonucleotide, n said ﬁrst oligonucleotide and said second oligonucleotide have the same nucleotide sequence, and n said ﬁrst oligonucleotide and second oligonucleotide optionally are joined by a linker, and wherein said ﬁrst oligonucleotide and said second oligonucleotide are mentary to said artiﬁcial oligonucleotide in said extracellular domain of said polypeptide, and wherein when said dimerizing agent dimerizes two of said cell death polypeptides, an apoptosis-inducing signal is generated in said cell, comprising contacting said cell with an amount of said dimerizing agent sufﬁcient to dimerize a sufﬁcient number of said ity of ial polypeptides to dimerize and generate an ate apoptosis-inducing signal sufﬁcient to kill said cell. In a speciﬁc embodiment, said cell is a T cyte.

In a speciﬁc embodiment, the T cytes killed in accordance with the methods described herein are CAR-T lymphocytes. 4.3. Chimeric Antigen Receptors When the cells provided herein are T lymphocytes which comprise the cell death polypeptides described above, such T lymphocytes can, in certain embodiments, comprise chimeric antigen receptors (CARs), which are artiﬁcial membrane-bound proteins that direct a T lymphocyte to an antigen, and stimulate the T lymphocyte to kill a cell displaying the antigen.

See, e.g., Eshhar, US. Patent No. 7,741,465. At a m, the CAR ses an extracellular domain that binds to an antigen, e.g., an antigen on a cell, a transmembrane domain, and an intracellular (cytoplasmic) signaling domain that transmits a primary activation signal to an immune cell. All other conditions being satisﬁed, when the CAR is expressed on the surface of, e.g., a T cyte, and the extracellular domain of the CAR binds to an antigen, the intracellular signaling domain transmits a signal to the T lymphocyte to activate and/or proliferate, and, if the antigen is present on a cell e, to kill the cell expressing the antigen.

Because T lymphocytes require two signals, a primary activation signal and a costimulatory signal, in order to activate, typically CARs also comprise a costimulatory domain such that 2014/027039 binding of the antigen to the extracellular domain results in transmission of both a primary activation signal and a costimulatory signal. 4.3.1. General CAR Structure Intracellular Domain In certain embodiments, the intracellular domain of the CAR is or comprises an ellular domain or motif of a protein that is expressed on the surface of T lymphocytes and triggers activation and/or eration of said T cytes. Such a domain or motif is able to transmit a y antigen-binding signal that is necessary for the activation of a T lymphocyte in response to the n’s g to the CAR’s ellular portion. Typically, this domain or motif comprises, or is, an ITAM (immunoreceptor tyrosine-based activation motif). ITAM- containing polypeptides suitable for CARs include, for example, the zeta CD3 chain (CD3<‘;) or ITAM-containing portions thereof. In a specific embodiment, the intracellular domain is a CD35; intracellular signaling domain. In other specific embodiments, the intracellular domain is from a lymphocyte receptor chain, a TCIVCD3 complex protein, an Fc receptor subunit or an IL-2 receptor subunit.

In certain embodiments, the CAR additionally comprises one or more co-stimulatory domains or motifs, e.g., as part of the intracellular domain of the polypeptide. The one or more co-stimulatory domains or motifs can be, or comprise, one or more of a co-stimulatory CD27 polypeptide sequence, a co-stimulatory CD28 polypeptide sequence, a co-stimulatory 0X40 (CD134) polypeptide sequence, a co-stimulatory 4-lBB (CD137) polypeptide sequence, or a costimulatory inducible T-cell costimulatory (ICOS) ptide ce, or other costimulatory domain or motif.

The transmembrane region can be any transmembrane region that can be incorporated into a functional CAR, typically a transmembrane region from a CD4 or a CD8 molecule. 4.3.2. CAR embrane Domains From CTLA4 0r PD-l In certain embodiments, the transmembrane domain of the CAR is from an immune system protein that normally transmits an inhibitory signal to such immune system cells, e.g., a transmembrane domain from CTLA4 (Cytotoxic T-Lymphocyte Antigen 4 or Cytotoxic T- Lymphocyte Associated protein 4) or PD-l ammed Death-l).

In certain embodiments, any of the T lymphocytes provided herein, which comprise a plurality of cell death polypeptides, comprise a transmembrane domain from CTLA4 or PD-l (Programmed Cell Death 1) In a speciﬁc embodiment, a T lymphocyte expressing said polypeptide, or any of such polypeptides described herein, is activated or stimulated to proliferate when said ptide binds to said antigen. In a speciﬁc embodiment, the polypeptide, when expressed on the surface of a T lymphocyte, directs the T lymphocyte to kill a cell expressing said antigen.

In speciﬁc embodiments of any of the polypeptides herein, in which the transmembrane domain of the polypeptide is from CTLA4, the CTLA4 embrane domain is from a mammalian CTLA4, e. g., human, primate, or rodent, e. g., murine CTLA4. Preferably, the transmembrane domain does not se amino acids from the intracellular domain, extracellular domain, or either intracellular or extracellular domain of CTLA4 or PD-l. Speciﬁc, non-limiting examples of CTLA4 or PD-l embrane domain sequences are provided below.

In a speciﬁc embodiment, the CTLA4 transmembrane domain is the polypeptide sequence encoded by exon 3 of a human CTLA4 gene. In another speciﬁc embodiment, the CTLA4 transmembrane domain is or comprises the amino acid ce PEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKM (in three-letter code, u-Pro-Cys- Pro-Asp-Ser-Asp-Phe-Leu-Leu-Trp-Ile-Leu-Ala-Ala-Val-Ser-Ser-Gly-Leu-Phe-Phe-Tyr-Ser- u-Leu-Thr-Ala-Val-Ser-Leu-Ser-Lys-Met) (SEQ ID NO: 1). In another speciﬁc embodiment, the CTLA4 transmembrane domain is or comprises the polypeptide sequence encoded by nucleotides 610-722 of GenBank Accession No. 214.4. In r speciﬁc embodiment, the CTLA4 transmembrane domain is or comprises the amino acid sequence PDSDFLLWILAAVSSGLFFYSFLLTAVSL (in three-letter code, Pro-Asp-Ser-Asp-Phe-Leu- p-Ile-Leu-Ala-Ala-Val-Ser-Ser-Gly-Leu-Phe-Phe-Tyr-Ser—Phe-Leu-Leu-Thr-Ala-Val- Ser-Leu) (SEQ ID NO:2). In another speciﬁc embodiment, the CTLA4 transmembrane domain is or comprises the polypeptide sequence encoded by nucleotides 636-699 of GenBank Accession No. NM_005214.4. In another speciﬁc embodiment, the CTLA4 transmembrane domain is or comprises the amino acid sequence FLLWILAAVSSGLFFYSFLLTAV (in three- letter code, Phe-Leu-Leu-Trp-Ile-Leu-Ala-Ala-Val-Ser-Ser—Gly-Leu-Phe-Phe-Tyr—Ser-Phe-Leu- r-Ala-Val) (SEQ ID NO:3). See, e.g., Ensembl n reference no.

ENSP00000303939.3. In another speciﬁc embodiment, the CTLA4 transmembrane domain is or comprises the polypeptide sequence FLLWILAAVSSGLFFYSFLLT (in three-letter code, Phe- Leu-Leu-Trp-Ile-Leu-Ala-Ala-Val-Ser-Ser-Gly-Leu-Phe-Phe-Tyr—Ser-Phe-Leu-Leu-Thr) (SEQ ID NO:4), see, e.g., UNIPROT Accession No. P16410. In another speciﬁc embodiment, the CTLA4 transmembrane domain is or comprises the polypeptide sequence VAVSLGLFFYSFLVSAVSLS (in three-letter code, Phe-Leu-Leu-Trp-Ile-Leu-Val- l-Ser-Leu-Gly-Leu-Phe-Phe-Tyr-Ser-Phe-Leu-Val-Ser-Ala-Val-Ser-Leu-Ser) (SEQ ID NO:5). See, e.g., Shin et al., Blood 119:5678-5687 . In another speciﬁc embodiment, the PD-1 transmembrane domain is or comprises the amino acid sequence TLVVGVVGGLLGSLVLLVWVLAVICSRAA (in three-letter code, Thr-Leu-Val-Val-Gly-Val- Val-Gly-Gly-Leu-Leu-Gly-Ser-Leu-Val-Leu-Leu-Val-Trp-Val-Leu-Ala-Val-Ile-Cys-Ser-Arg- Ala-Ala) (SEQ ID NO:6). See Finger et a1., Gene 197(1-2):177-187 (1997). In another speciﬁc embodiment, the PD-1 embrane domain is or comprises the amino acid sequence VGVVGGLLGSLVLLVWVLAVI (in three-letter code, Val-Gly-Val-Val-Gly-Gly-Leu-Leu- Gly-Ser-Leu-Val-Leu-Leu-Val-Trp-Val-Leu-Ala-Val-Ile) (SEQ ID NO:7). See, e.g., UNIPROT ion No. Q15116. In another speciﬁc embodiment, the PD-1 transmembrane domain is or comprises the amino acid sequence FQTLVVGVVGGLLGSLVLLVWVLAVI (in three-letter code, Phe-Glu-Thr-Leu-Val-Val-Gly-Val-Val-Gly-Gly-Leu-Leu-Gly-Ser-Leu-Val-Leu-Leu-Val- Trp-Val-Leu—Ala-Val-Ile) (SEQ ID NO:8). See, e.g., GenBank Accession No. NM_005018.2.

In certain embodiments, a nucleotide sequence that encodes one of the transmembrane polypeptides sed herein comprises a nucleotide sequence that encodes any of the amino acid sequences disclosed in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. In another speciﬁc embodiment, the PD-l transmembrane domain is or comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutive amino acids sed in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. In certain embodiments, a tide sequence that encodes one of the polypeptides disclosed herein comprises a nucleotide sequence that encodes at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20 or 21 consecutive amino acids sed in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. In constructing the polypeptide, e.g. CAR, in certain embodiments, human sequences may be combined with non- human ces. For example, a polypeptide, e.g. CAR comprising human extracellular and intracellular domain amino acid sequences may comprise a transmembrane domain from a non- human species; e.g., may comprise a murine CTLA4 transmembrane domain or a murine PD-l transmembrane domain. In a more c embodiment, the polypeptide, e. g. CAR, comprises human amino acid ces for the extracellular and intracellular domains, and comprises a embrane domain having, or consisting of, the amino acid sequence of SEQ ID NO:5. 4.3.3. CAR Intracellular Domain The extracellular domain of the polypeptide binds to an antigen of interest. In certain embodiments of any of the polypeptides described herein, the ellular domain comprises a receptor, or a portion of a receptor, that binds to said antigen. The extracellular domain may be, e.g., a receptor, or a portion of a receptor, that binds to said antigen. In certain ments, the extracellular domain comprises, or is, an antibody or an antigen-binding portion f. In speciﬁc embodiments, the extracellular domain comprises, or is, a single-chain FV domain. The single-chain FV domain can comprise, for example, a VL linked to VH by a ﬂexible linker, wherein said VL and VH are from an antibody that binds said antigen.

The antigen to which the extracellular domain of the polypeptide binds can be any antigen of interest, e. g., can be an antigen on a tumor cell. The tumor cell may be, e.g., a cell in a solid tumor, or a cell of a blood cancer. The antigen can be any antigen that is expressed on a cell of any tumor or cancer type, e.g., cells of a lymphoma, a lung , a breast cancer, a prostate cancer, an adrenocortical carcinoma, a thyroid carcinoma, a nasopharyngeal carcinoma, a melanoma, e.g., a malignant melanoma, a skin carcinoma, a colorectal carcinoma, a desmoid tumor, a desmoplastic small round cell tumor, an endocrine tumor, an Ewing sarcoma, a peripheral primitive ctodermal tumor, a solid germ cell tumor, a hepatoblastoma, a neuroblastoma, a abdomyosarcoma soft tissue a, an osteosarcoma, a blastoma, a rhabdomyosarcoma, a Wilms tumor, a glioblastoma, a myxoma, a ﬁbroma, a lipoma, or the like. In more speciﬁc embodiments, said lymphoma can be chronic cytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic ia, lymphoplasmacytic lymphoma, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal marginal zone B cell lymphoma, MALT lymphoma, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary efﬁlsion lymphoma, Burkitt’s lymphoma, T cyte phocytic leukemia, T lymphocyte large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T lymphocyte leukemia/lymphoma, extranodal NK/T lymphocyte lymphoma, nasal type, pathy-type T cyte ma, hepatosplenic T lymphocyte lymphoma, blastic NK cell lymphoma, mycosis ﬁlngoides, Sezary me, y cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T lymphocyte lymphoma, eral T cyte lymphoma (unspeciﬁed), stic large cell lymphoma, Hodgkin lymphoma, or a non-Hodgkin lymphoma.

In a speciﬁc embodiment, in which the cancer is chronic lymphocytic leukemia (CLL), the B cells of the CLL have a normal karyotype. In other speciﬁc embodiments, in which the cancer is chronic lymphocytic leukemia (CLL), the B cells of the CLL carry a l7p deletion, an llq deletion, a 12q trisomy, a l3q deletion or a p53 deletion.

In n embodiments, the antigen is a tumor-associated antigen or a tumor-speciﬁc antigen. In various speciﬁc embodiments, t limitation, the tumor-associated antigen or tumor-speciﬁc antigen is Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 5), CAl9-9, inin, MUC-l, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma- associated antigen (MAGE), CD19, CD34, CD45, CD99, CD1 17, chromogranin, cytokeratin, desmin, glial ﬁbrillary acidic protein (GFAP), gross cystic disease ﬂuid protein (GCDFP-lS), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-l), myo-Dl, muscle-speciﬁc actin (MSA), neuroﬁlament, neuron-speciﬁc enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription -l, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2-PK), an abnormal ras protein, or an abnormal p53 protein.

In certain embodiments, the TAA or TSA is a cancer/testis (CT) antigen, e.g., BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-l, NY-SAR—35, OY-TES-l, SPANXBl, SPAl7, SSX, SYCPl, or TPTE.

In certain other embodiments, the TAA or TSA is a carbohydrate or ganglioside, e.g., fuc-GMl, GM2 (oncofetal antigen-immunogenic-l; OFA-I-l); GD2 (OFA-I-2), GM3, GD3, and the like.

WO 52177 2014/027039 In n other embodiments, the TAA or TSA is alpha-actinin-4, Bage-l, BCR-ABL, Bcr—Abl fusion protein, beta-catenin, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1, dek-can fusion protein, EBNA, EF2, Epstein Barr virus antigens, ML1 fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, ROL fusion protein, PTPRK, K-ras, N—ras, phosphate isomerase, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-l, NA-88, NY-Eso-l/Lage-2, SP17, SSX-2, TRP2-Int2, , gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-l, MAGE-3, RAGE, GAGE-1, GAGE-2, p15(58), RAGE, , SCP-l, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, E2A-PRL, H4-RET, IGH-IGK, R, human omavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, 13-Catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, Ga733 (EpCAM), HTgp-175, M344, , P1, CO-029, FGF-5, G250, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-l, RCASl, 16, TA-90, TAAL6, TAG72, TLP, TPS, CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma a1temate reading frame protein), Trp-p8, STEAP1 (six- transmembrane epithelial antigen of the prostate 1), an abnormal ras protein, or an abnormal p53 protein. In another specific embodiment, said tumor-associated antigen or tumor-specific antigen is integrin (va3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), or Ral-B. Other tumor-associated and tumor-specific antigens are known to those in the art.

Antibodies, and scFvs, that bind to TSAs and TAAs are known in the art, as are nucleotide sequences that encode them.

In certain specific embodiments, the antigen is an antigen not considered to be a TSA or a TAA, but which is nevertheless associated with tumor cells, or damage caused by a tumor. In certain ments, for example, the antigen is, e.g., a growth factor, cytokine or interleukin, e.g., a growth factor, ne, or interleukin ated with angiogenesis or vasculogenesis.

Such growth factors, cytokines, or interleukins can include, e. g., vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), n-like growth factor (IGF), or interleukin-8 (IL-8).

Tumors can also create a hypoxic environment local to the tumor. As such, in other specific embodiments, the antigen is a hypoxia-associated factor, e.g., HIF-lu, HIF-l B, HIP-20L, HIF-ZB, L, or HIF-3B. Tumors can also cause localized damage to normal tissue, causing the e of molecules known as damage associated molecular n molecules (DAMPs; also known as alarmins). In certain other speciﬁc embodiments, therefore, the antigen is a DAMP, e. g., a heat shock protein, chromatin-associated protein high mobility group box 1 (HMGBl), SlOOA8 (MRP8, calgranulin A), SlOOA9 (MRPl4, calgranulin B), serum amyloid A (SAA), or can be a deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.

In certain ments of the polypeptides described herein, the extracellular domain is joined to said transmembrane domain by a linker, spacer or hinge polypeptide sequence, e.g., a ce from CD28 or a sequence from CTLA4. 4.3.4. Bispeciﬁc CARS In certain embodiments of the T lymphocytes or methods bed herein, the T lymphocytes, in addition to comprising a cell death ptide, comprise two or more CARs in which the primary signaling mechanism and ulatory mechanism are split into two or more polypeptides.

In certain embodiments, for example, the T cytes comprise a cell death polypeptide, and at least two different other polypeptides, e.g., chimeric receptors, in which the immune signal derived from g of a primary ing polypeptide, e.g., chimeric receptor, to a first antigen is separated from a costimulatory signal produced by a costimulatory polypeptide, e.g., chimeric receptor, wherein the costimulatory signal is dependent on antigen binding of a second antigen by the second chimeric receptor.

In one embodiment, the T lymphocyte comprises a y signaling polypeptide comprising a first extracellular n binding domain that binds a first n, and a first intracellular signaling domain, wherein said primary signaling polypeptide does not comprise a co-stimulatory domain; and a co-stimulatory comprising a second extracellular antigen binding domain binding a second antigen, or a receptor that binds said second antigen; and a second intracellular signaling domain; wherein said T lymphocyte becomes maximally cytotoxic only when said first signaling domain and said second signaling domain are both activated by said first antigen and said second antigen, respectively. In a specific embodiment, binding of said first antigen to said first antigen binding domain without binding of said second antigen to said second binding domain, or binding of said second antigen to said second antigen binding domain Without binding of ﬁrst second antigen to said ﬁrst binding domain, induces anergy of said T lymphocyte, or non-responsiveness of said T-lymphocyte to said ﬁrst antigen or said second antigen.

In another speciﬁc embodiment, said ﬁrst antigen binding domain and said second antigen binding domain are ndently an antigen-binding n of a receptor, an antigen- binding portion of an antibody, or other peptide-based macromolecular antigen binding agent. In certain speciﬁc embodiments, either or both of said ﬁrst antigen binding domain or said second antigen binding domain are scFv antibody fragments. In speciﬁc embodiments, either or both of said primary signaling ptide or said co-stimulatory polypeptide additionally comprise a transmembrane domain. In other speciﬁc embodiments, said primary signaling polypeptide or said co-stimulatory ptide comprises a T lymphocyte survival motif. In a speciﬁc embodiment, the T cyte survival motif is a CD28 T lymphocyte survival motif. In other speciﬁc embodiments, said T lymphocyte survival motif is an intracellular signaling domain of IL-7 receptor (IL-7R), an ellular signaling domain of IL-l2 receptor, an intracellular signaling domain of IL-lS receptor, an intracellular signaling domain of IL-2l or, or an intracellular signaling domain of transforming growth factor B (TGFB) receptor. In another more speciﬁc ment, said primary signaling polypeptide or said mulatory polypeptide comprise a portion of a CD28 molecule that ses a T lymphocyte survival motif. In a more speciﬁc embodiment, said y signaling polypeptide or said co-stimulatory polypeptide comprise a CD28 molecule that comprises a T lymphocyte survival motif. In certain speciﬁc embodiments, said ﬁrst intracellular signaling domain comprises a polypeptide sequence comprising an immunoreceptor tyrosine-based activation motif (ITAM). In a more speciﬁc embodiment, said polypeptide sequence is a CD35; signaling domain.

In certain speciﬁc embodiments, said ﬁrst antigen is an antigen on a tumor cell. In a more speciﬁc embodiment, said tumor cell is a cell in a solid tumor. In another more speciﬁc embodiment, said tumor cell is a blood cancer cell. In another speciﬁc embodiment, said n is a associated antigen or a speciﬁc n. In more speciﬁc embodiments, said tumor-associated antigen or tumor-speciﬁc n is Her2, prostate stem cell antigen (PSCA), fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CAl9- 9, calretinin, MUC-l, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD1 17, chromogranin, cytokeratin, desmin, glial ﬁbrillary acidic protein (GFAP), gross cystic disease ﬂuid protein (GCDFP-l5), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-l), myo-Dl, muscle-speciﬁc actin (MSA), neuroﬁlament, neuron-speciﬁc enolase (NSE), placental ne phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-l, the dimeric form of the te kinase isoenzyme type M2 (tumor M2- PK), an abnormal ras protein, or an abnormal p53 protein.

In another speciﬁc embodiment, said second antigen is a growth factor, cytokine, or interleukin. The second antigen is a growth factor, ne, or interleukin associated with angiogenesis or vasculogenesis. In more speciﬁc embodiments, said second antigen is vascular endothelial growth factor (VEGF), basic ast growth factor (bFGF), platelet-derived growth factor , hepatocyte growth factor (HGF), n-like growth factor (IGF), or interleukin-8 .

In r speciﬁc embodiment, signal transduction activation provided by said second antigen is non-antigenic, but is associated with hypoxia. In more c ments, said stimulus is induced by activation of hypoxia-inducible factor-10L (HIF-lu), HIF-lB, HIP-20L, HIF- 2B, HIF-30L, or HIF-3B.

In another c embodiment, said second antigen is an interleukin.

In another speciﬁc ment, said second antigen is a damage associated molecular pattern molecule (DAMP; also known as an alarmin). In more speciﬁc embodiments, said DAMP is a heat shock protein, chromatin-associated protein high mobility group box 1 (HMGBl), SlOOA8 (also known as MRP8, or calgranulin A), SlOOA9 (also known as MRPl4, or calgranulin B), serum amyloid A (SAA), deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.

In certain speciﬁc embodiments, said second n is an antigen on an antibody that binds to an antigen presented by a tumor cell.

In a speciﬁc embodiment of any of the embodiments herein, said co-stimulatory polypeptide comprises one or more co-stimulatory domains. In speciﬁc embodiments, said one or more co-stimulatory s comprises one or more of a co-stimulatory CD27 polypeptide sequence, a co-stimulatory CD28 polypeptide ce, a co-stimulatory 0X40 (CD134) polypeptide sequence, a co-stimulatory 4-lBB (CD137) polypeptide sequence, or a costimulatory inducible T-cell co-stimulatory (ICOS) polypeptide sequence.

In a c embodiment, said primary signaling polypeptide comprises an extracellular tumor antigen-binding domain and a CD35; signaling , and wherein said co-stimulatory polypeptide comprises an antigen-binding domain wherein said antigen is an angiogenic or vasculogenic factor, and one or more co-stimulatory molecule signaling domains. Said angiogenic factor can be, e. g., VEGF. Said one or more co-stimulatory molecule signaling motifs can comprise, e.g., mulatory signaling domains from each of CD28, 0X40, and 4- lBB. In a more specific embodiment, said primary signaling polypeptide comprises an extracellular tumor antigen-binding domain and a CD35; signaling domain, and wherein said co- stimulatory polypeptide comprises an n-binding domain wherein said antigen is VEGF, and co-stimulatory signaling domains from each of CD28, 0X40, and 4-lBB.

In a more specific ment, said primary signaling polypeptide or said co- stimulatory polypeptide comprises a T cyte survival motif. In more specific embodiments, said T lymphocyte survival motif is, or is derived from, an intracellular ing domain of IL-7 receptor (IL-7R), an intracellular signaling domain of IL-l2 receptor, an intracellular signaling domain of IL-lS receptor, an intracellular signaling domain of IL-2l receptor, or an intracellular ing domain of transforming growth factor B (TGFB) receptor.

In a more specific embodiment of said T cyte, therefore, said primary ing polypeptide comprises an extracellular tumor antigen-binding domain and a CD35; signaling , and wherein said co-stimulatory polypeptide comprises an antigen-binding domain wherein said antigen is VEGF, an IL-7 receptor intracellular T cyte survival motif, and co-stimulatory signaling s from each of CD28, 0X40, and 4-lBB.

In another specific embodiment of the T lymphocyte, said first antigen is a tumor- speciﬁc antigen or a tumor-associated antigen, and said first intracellular signaling domain comprises a CD35; signaling domain; and wherein said co-stimulatory polypeptide comprises an antigen-binding domain that binds said second antigen, and co-stimulatory signaling domains from each of CD28, 0X40, and 4-lBB. In a more specific embodiment, said co-stimulatory polypeptide further ses an intracellular T lymphocyte survival motif, e.g., a T lymphocyte survival motif that is, or is derived ﬁom, an intracellular ing domain of IL-7 receptor (IL- 7R), an intracellular signaling domain of IL-12 receptor, an intracellular ing domain of IL- receptor, an intracellular signaling domain of IL-21 receptor, or an intracellular signaling domain of transforming growth factor B (TGFB) receptor.

In a speciﬁc embodiment of any of the T lymphocytes provided herein, said second antigen is VEGF or IL-4.

In another aspect, provided herein is a T lymphocyte comprising a cell death polypeptide, a co-stimulatory polypeptide comprising a ﬁrst extracellular antigen binding domain that binds a ﬁrst antigen, and a ﬁrst intracellular ing domain; and a primary signaling polypeptide comprising a second extracellular n binding domain binding a second antigen, or a receptor that binds said second antigen; and a second intracellular signaling domain, wherein said primary signaling polypeptide does not comprise a co-stimulatory ; wherein said modiﬁed lymphocyte becomes maximally cytotoxic only when said ﬁrst ing domain and said second signaling domain are both activated by said ﬁrst antigen and said second antigen, respectively. In a speciﬁc ment, binding of said ﬁrst antigen to said ﬁrst antigen binding domain without g of said second antigen to said second binding domain, or binding of said second antigen to said second antigen binding domain without binding of ﬁrst second antigen to said ﬁrst binding domain induces anergy of said T lymphocyte, or non- responsiveness of said T lymphocyte to said ﬁrst antigen. In a speciﬁc embodiment, said ﬁrst antigen-binding domain and said antigen-binding domain are independently an antigen-binding portion of a receptor or an antigen-binding portion of an dy. In another speciﬁc embodiment, either or both of said ﬁrst antigen binding domain or said second antigen binding domain are scFv antibody fragments. In speciﬁc embodiments, said co-stimulatory polypeptide and/or said primary signaling polypeptide additionally comprise a embrane domain. In a more speciﬁc embodiment, said co-stimulatory polypeptide or said primary ing polypeptide comprises a T lymphocyte al motif, e.g. ., any of the T lymphocyte survival motifs bed herein. In another c embodiment, said ﬁrst antigen is an antigen on a tumor cell, e. g., a cell in a solid tumor or a blood cancer cell. In a speciﬁc embodiment, said ﬁrst antigen is a tumor-associated antigen or a tumor-speciﬁc antigen, e. g., Her2, prostate stem cell antigen (PSCA), fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), , calretinin, MUC-l, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, granin, cytokeratin, desmin, glial ﬁbrillary acidic protein (GFAP), gross cystic disease ﬂuid protein (GCDFP-lS), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-D1, muscle-speciﬁc actin (MSA), neuroﬁlament, - speciﬁc e (NSE), placental alkaline phosphatase, synaptophysin, thyroglobulin, thyroid transcription factor-1, the c form of the pyruvate kinase yme type M2 (tumor M2- PK), an abnormal ras protein, an al p53 protein, CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, or STEAPl (six-transmembrane epithelial antigen of the te 1). In another speciﬁc embodiment, said tumor-associated antigen or tumor-speciﬁc antigen is integrin (va3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), or Ral-B.

In certain speciﬁc embodiments, said second intracellular signaling domain comprises a polypeptide sequence sing an immunoreceptor tyrosine-based activation motif (ITAM), e. g., a CD35; signaling domain. In a c embodiment, said second antigen is a growth factor, ne, or interleukin. In another c embodiment, said second antigen is a growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis, e.g., VEGF, bFGF, PDGF, HGF, IGF, or IL-8. In other more speciﬁc embodiments, signal transduction by said second chimeric receptor is induced by activation of a hypoxia-associated factor, e. g., HIP-10L, HIF-l B, HIP-20L, HIF-2B, HIP-30L, or . In other speciﬁc ments, said second antigen is an interleukin. In other speciﬁc embodiments, said second antigen is a DAMP, e. g., a heat shock protein, HMGBl, SlOOA8, SlOOA9, SAA, DNA, ATP, uric acid, or heparin sulfate.

In other speciﬁc embodiments, said second n is an administered peptide, e. g., an antibody or a synthetic polypeptide. In other speciﬁc embodiments, said second antigen is an antigen on an dy that binds to an antigen presented by a tumor cell. In certain speciﬁc embodiments, said co-stimulatory polypeptide ses one or more co-stimulatory domains, e.g., one or more of a co-stimulatory CD27 polypeptide sequence, a co-stimulatory CD28 polypeptide sequence, a co-stimulatory 0X40 (CD134) polypeptide sequence, a co-stimulatory 4-1BB (CD137) polypeptide sequence, or a co-stimulatory inducible T-cell co-stimulatory (ICOS) polypeptide sequence. In any of the above embodiments, in a speciﬁc embodiment, said co- atory polypeptide or said primary signaling polypeptide comprises a T lymphocyte survival motif, e.g., said T lymphocyte survival motif is, or is derived from, an intracellular ing domain of lL-7 receptor (IL-7R), an intracellular signaling domain of IL-12 receptor, an intracellular signaling domain of IL-15 receptor, an intracellular signaling domain of IL-21 receptor, or an ellular signaling domain of transforming growth factor B (TGFB) receptor. 4.4. Isolated Polypeptides Any of the polypeptides, comprising a CTLA4 or PD-l transmembrane domain, provided herein, may be modiﬁed by, e.g., acylation, amidation, glycosylation, methylation, phosphorylation, sulfation, sumoylation, ubiquitylation, or the like. The polypeptides may be labeled with a label capable of providing a able , e.g., with radioisotopes and ﬂuorescent compounds. One or more side chains of the ﬁrst or second polypeptides may be derivatized, e.g., derivatization of l and amino terminal residues with succinic or other carboxylic acid anhydrides, or derivatization with, e.g., imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 edione; and transaminase-catalyzed reaction with glyoxylate.

Carboxyl side groups, aspartyl or glutamyl, may be selectively modiﬁed by reaction with carbodiimides =N—R') such as ohexyl(2-morpholinyl-(4-ethyl)carbodiimide or 1-ethyl(4-azonia-4,4-dimethylpentyl)carbodiimide. 4.5. Isolated Nucleic Acids The polypeptides provided herein (e.g., chimeric receptors) can be encoded by cleotide sequences ing to well-known methods in the art. The polynucleotides may be contained within any polynucleotide vector suitable for the transformation of immune cells, e. g., T lymphocytes. For example, T lymphocytes may be transformed using synthetic vectors, lentiviral or retroviral vectors, mously replicating plasmids, a virus (e.g., a retrovirus, lentivirus, adenovirus, or herpes virus), or the like, containing polynucleotides encoding the ﬁrst and second polypeptides (e. g., chimeric receptors). LentiViral vectors suitable for transformation of T lymphocytes include, but are not limited to, e.g., the iral vectors described in US.

Patent Nos. 5,994,136; 6,165,782; 953; 7,083,981; and 7,250,299, the disclosures ofwhich are hereby incorporated by reference in their entireties. HIV vectors le for transformation of T lymphocytes include, but are not d to, e. g., the vectors described in US. Patent No. ,665,577, the disclosure of which is hereby incorporated by reference in its entirety.

Nucleic acids useful in the tion of the ﬁrst and second polypeptides, e. g., within a T cyte, include DNA, RNA, or nucleic acid analogs. Nucleic acid analogs can be modiﬁed at the base moiety, sugar moiety, or phosphate backbone, and can include deoxyuridine substitution for hymidine, 5-methyl-2'-deoxycytidine or 5-bromo-2'-deoxycytidine substitution for deoxycytidine. Modiﬁcations of the sugar moiety can include modiﬁcation of the 2' hydroxyl of the ribose sugar to form 2'-O-methyl or 2'-O-allyl . The deoxyribose phosphate backbone can be modiﬁed to produce lino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide c acids, in which the deoxyphosphate backbone is replaced by a pseudopeptide backbone and the four bases are retained. See, for example, Summerton and Weller (1997) Antisense Nucleic Acid Drug Dev. 7:l87-l95; and Hyrup et al. (1996) Bioorgan. Med. Chain. 4:5-23. In addition, the deoxyphosphate backbone can be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a oroamidite, or an alkyl phosphotriester backbone. 4.6. Cells Non-limiting examples of cells in which the cell death polypeptides and multimerizing or dimerizing agents may be used include, but are not limited to, natural killer (NK) cells, dendritic cells (DC), placental stem cells (e.g., the placental stem cells disclosed in US. Patent Nos. 7,468,276; 8,057,788 and 703, the sures of which are hereby incorporated by reference in their ties), mesenchymal-like stem cells from umbilical cord blood, placental blood, peripheral blood, bone marrow, dental pulp, adipose , osteochondral tissue, and the like; embryonic stem cells, embryonic germ cells, neural crest stem cells, neural stem cells, and differentiated cells (e. g., ﬁbroblasts, etc.). The cell death polypeptides, and erizing or dimerizing agents, may also be used in tumor cell lines, e.g., for animal model experimental purposes.

In a speciﬁc embodiment, the cells comprising the ptides provided herein are T lymphocytes. The T lymphocytes comprising the polypeptides provided herein may be naive T lymphocytes or MHC-restricted T lymphocytes. In certain embodiments, the T lymphocytes are tumor inﬁltrating lymphocytes (TILs). In certain embodiments, the T lymphocytes have been isolated from a tumor biopsy, or have been expanded from T lymphocytes isolated from a tumor . In certain other embodiments, the T lymphocytes have been isolated from, or are ed from T lymphocytes expanded from, peripheral blood, cord blood, or lymph.

The immune cells, e. g., T lymphocytes, used in the present methods are preferably autologous to an individual to Whom the T lymphocytes are to be administered. In certain other embodiments, the T lymphocytes are allogeneic to an indiVidual to Whom the T lymphocytes are to be administered. Where allogeneic T cytes are used to prepare T lymphocytes, it is preferable to select T lymphocytes that will reduce the possibility of graft-versus-host e (GVHD) in the individual. For example, in certain embodiments, virus-speciﬁc T lymphocytes are selected for preparation of T lymphocytes; such lymphocytes will be expected to have a greatly d native capacity to bind to, and thus become activated by, any recipient antigens.

In n embodiments, recipient-mediated rejection of allogeneic T lymphocytes can be reduced by co-administration to the host of one or more suppressive , e.g., cyclosporine, tacrolimus, sirolimus, cyclophosphamide, or the like.

In one embodiment, T lymphocytes are obtained from an indiVidual, optionally then expanded, and then transformed with a polynucleotide ng a cell death polypeptide, and optionally one or more polynucleotides encoding one or more CAR(s), and optionally then expanded. In another embodiment, T lymphocytes are obtained from an indiVidual, ally then expanded, and then transformed with a polynucleotide encoding a cell death polypeptide, and optionally one or more polynucleotides encoding one or more CAR(s), and optionally then expanding. Cells containing any of the polynucleotide may be selected using one or more selectable markers.

In certain embodiments, any of the T lymphocytes provided herein express or comprise native TCR proteins, e.g., TCR-0L and TCR-B that are capable of forming native TCR complexes, in addition to the CTLA4 or PD-l transmembrane -containing polypeptide.

In certain other embodiments, either or both of the native genes encoding TCR-0L and TCR-B in the T lymphocytes are modified to be non-functional, e.g., a portion or all are deleted, a mutation is inserted, etc.

In certain ments, any of the T lymphocytes provided herein are isolated from a tumor lesion, e.g., are tumor-infiltrating lymphocytes; such T cytes are expected to be speciﬁc for a TSA or TAA.

T lymphocytes, and T lymphocytes comprising a polypeptide comprising a CD35; signaling domain and a CD28 co-stimulatory domain can be expanded using antibodies to CD3 and CD28, e. g., antibodies attached to beads, or to the surface of a cell culture plate; see, e. g., US. Patent Nos. 5,948,893; 6,534,055; 6,352,694; 6,692,964; 6,887,466; and 6,905,681.

In any of the above embodiments, the n and/or antibody can exist free in the medium in which the T lymphocytes are cultures, or either or both can be attached to a solid support, e.g., tissue culture plastic surface, beads, or the like.

The T lymphocytes provided herein can optionally comprise a second type of "suicide gene" or "safety switch", in addition to the cell death ptide. For example, the T lymphocytes, in certain embodiments, can comprise an HSV thymidine kinase gene (HSV-TK), which causes death of the T lymphocytes upon contact with gancyclovir. In another embodiment, the T lymphocytes express or comprise an inducible caspase, e.g., an inducible e 9 ase9), e.g., a fusion protein between caspase 9 and human FK506 binding protein allowing for dimerization using a specific small molecule pharmaceutical. See Straathof et al., Blood 105(11);4247—4254 (2005). 4.7. Methods of Using Cells Comprising Cell Death ptides The cells, e.g., T lymphocytes, provided herein that comprise cell death polypeptides and optionally one or more CARs, as described elsewhere herein, can be used to treat an individual having one or more types of cells desired to be targeted by the cells described herein, e. g., one or more types of cells to be killed. In certain embodiments, the cells to be killed are cancer cells, e.g., tumor cells. In c embodiments, the cancer cells are cells of a solid tumor. In specific embodiments, the cells are cells of a lymphoma, a lung cancer, a breast cancer, a prostate cancer, an adrenocortical carcinoma, a d carcinoma, a nasopharyngeal carcinoma, a melanoma, e.g., a malignant melanoma, a skin carcinoma, a colorectal carcinoma, a desmoid tumor, a desmoplastic small round cell tumor, an ine tumor, an Ewing sarcoma, a peripheral primitive neuroectodermal tumor, a solid germ cell tumor, a hepatoblastoma, a neuroblastoma, a non-rhabdomyosarcoma soft tissue sarcoma, an osteosarcoma, a retinoblastoma, a myosarcoma, a Wilms tumor, a astoma, a , a f1broma, a lipoma, or the like. In more specific embodiments, said lymphoma can be chronic cytic ia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma, extranodal al zone B cell lymphoma, MALT lymphoma, nodal marginal zone B cell lymphoma, follicular lymphoma, mantle cell ma, diffuse large B cell lymphoma, mediastinal (thymic) large B cell lymphoma, intravascular large B cell lymphoma, primary efﬁlsion lymphoma, Burkitt’s lymphoma, T lymphocyte prolymphocytic leukemia, T lymphocyte large granular lymphocytic leukemia, aggressive NK cell leukemia, adult T lymphocyte leukemia/lymphoma, extranodal NK/T lymphocyte lymphoma, nasal type, enteropathy-type T lymphocyte lymphoma, hepatosplenic T lymphocyte ma, blastic NK cell lymphoma, mycosis ﬁangoides, Sezary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T cyte lymphoma, peripheral T lymphocyte lymphoma (unspecified), stic large cell lymphoma, Hodgkin lymphoma, or a non-Hodgkin lymphoma.

In certain embodiments, when the modiﬁed cells, e. g., modif1ed T lymphocytes described herein are administered to a subject in need thereof, the combination of erizing agent and cell death polypeptide selected are chosen such that they are compatible with the patient population (or ulation) in which the cells, e.g., T lymphocytes, have been administered. By way of example only, if the erizing agent selected is the antibody mab, then in certain embodiments the patient population is individuals having a cancer of the B cells, e.g., B cell lymphoma.

Efficacy of the cells, e.g., T lymphocytes, after administration to an individual having a disease or disorder remediable by such cells, e. g., T lymphocytes, e.g., an individual having cancer, can be assessed by one or more criteria, speciﬁc to the particular e or disorder, known to those of ordinary skill in the art, to be indicative of progress of the e or disorder.

Generally, administration of the cells to such an dual is effective when one or more of said criteria detectably, e.g., significantly, moves from a disease state value or range to, or s, a normal value or range.

The cells, e.g., T lymphocytes, may be formulated in any pharmaceutically-acceptable solution, ably a solution suitable for the ry of living cells, e. g., saline solution (such as Ringer's solution), gelatins, carbohydrates (e.g., lactose, amylose, starch, or the like), fatty acid esters, hydroxymethylcellulose, polyvinyl pyrolidine, etc. Such preparations are preferably sterilized prior to addition of the cells, and may be mixed with auxiliary agents such as lubricants, preservatives, stabilizers, ﬁers, salts for inﬂuencing osmotic pressure, buffers, and coloring. Pharmaceutical carriers suitable for use in formulating the cells are known in the art and are described, for example, in WO 96/05309.

In certain embodiments, the cells, e. g., T lymphocytes, are formulated into individual doses, wherein said individual doses comprise at least, at most, or about 1x104, 5x104, 1x105, 5x105,1x106,5x106,1x107,5x107,1x108,5x108,1x109,5x109,1x101°,5><101°,or1><10"T lymphocytes. In certain embodiments, the cells are formulated for intravenous, rterial, parenteral, intramuscular, subcutaneous, intrathecal, or intraocular administration, or administration within a particular organ or tissue.

. EXAMPLES .1. e 1: Treatment of B Cell Lymphoma ] An individual presents with B-cell chronic lymphocytic leukemia, a B cell lymphoma.

Testing of B cells from the individual determines that the B cells carry a l7p deletion. T lymphocytes are obtained from the individual, transfected with a lentiviral vector comprising a tide sequence that s a ic antigen receptor (CAR), and transfected with a second lentiviral vector comprising a nucleotide ce encoding a dimerizable cell death polypeptide comprising an extracellular domain that comprises a mimotope that can be bound by the antibody rituximab, and an intracellular domain that comprises a e 9 domain. The T cytes are expanded using CD3+CD28-coated beads to sufﬁcient numbers for administration. The chimeric receptor comprises an extracellular antigen-binding region that binds to CD19; a transmembrane domain from CTLA4; intracellular co-stimulatory domain from CD28; and an intracellular CD35; domain. The individual is administered between 109 and 1010 of the T lymphocytes in 200 mL saline solution by intravenous infusion over 30 minutes. The individual is monitored for two weeks ards to establish a reduction of at least 90% of CDl9+ B cells in the dual’s blood. Where the patient, after administration of the T cytes, shows signs of distress due to the T lymphocytes (e. g., difﬁculty breathing, fever, abnormal serum cytokine levels, rash, or the like), rituximab is administered at a dosage of 200- 500 mg/m2 or until symptoms abate. .2. Example 2: Treatment of a B Cell Lymphoma ] An individual presents with B-cell chronic lymphocytic leukemia, a B cell lymphoma.

Testing of B cells from the individual determines that the B cells carry a l7p on. About 106 T lymphocytes are obtained from the individual, transfected with a lentiviral vector comprising a nucleotide ce encoding a cell death polypeptide comprising an extracellular domain that comprises a pe that can be bound by the antibody rituximab and an intracellular domain that comprises a caspase 8 domain, and transfected with a lentiviral vector comprising a nucleotide sequence that s a CAR. The CAR ses an extracellular antigen-binding region that binds to CD19; a transmembrane domain from PD-l; intracellular co-stimulatory domain from CD28; and an intracellular CD35; domain. CAR-expressing T lymphocytes are administered to the individual without prior expansion of the T lymphocytes. The individual is administered n 105 and 106 of the T lymphocytes in 200 mL saline solution by intravenous inﬁasion over 30 minutes. The individual is monitored for two weeks afterwards to establish a reduction of at least 90% of CDl9+ B cells in the individual’s blood. Where the patient, after administration of the T lymphocytes, shows signs of distress due to the T lymphocytes (e.g., difficulty breathing, fever, abnormal serum cytokine levels, rash, or the like), rituximab is administered at a dosage of 200-500 mg/m2 or until symptoms abate. .3. Example 3: Treatment of B Cell Lymphoma ] An individual presents with B-cell chronic lymphocytic leukemia, a B cell lymphoma.

Testing of B cells from the individual determines that the B cells carry a p53 deletion. T lymphocytes are obtained from the individual, transfected with a lentiviral vector comprising a nucleotide sequence encoding a cell death polypeptide comprising an extracellular domain that comprises an epitope that can be bound by the dy rituximab and an intracellular domain that comprises a e 3 domain, and transfected with a lentiviral vector comprising a nucleotide sequence that encodes a CAR. The T lymphocytes are ed using CD3+CD28- coated beads to sufficient numbers for administration. The CAR comprises an extracellular antigen-binding region that binds to CD19; a transmembrane domain from CTLA4; intracellular co-stimulatory domains from each of CD28, 4-lBB, and 0X40; and an intracellular CD35; . The individual is administered between 109 and 1010 of the T lymphocytes in 200 mL saline on by intravenous infusion over 30 minutes. The individual is red for two weeks ards to establish a reduction of at least 90% of CDl9+ B cells in the individual’s blood. Where the patient, after administration of the T lymphocytes, shows signs of distress due WO 52177 2014/027039 to the T lymphocytes (e.g., difficulty breathing, fever, abnormal serum cytokine levels, rash, or the like), rituximab is administered at a dosage of 200-500 mg/m2 or until symptoms abate. .4. Example 4: ent of a B Cell Lymphoma An individual presents with B-cell chronic lymphocytic leukemia, a B cell lymphoma.

Testing of B cells from the individual determines that the B cells carry a p53 deletion. About 106 T lymphocytes are obtained from the individual, transfected with a lentiviral vector comprising a nucleotide sequence encoding a cell death polypeptide comprising an extracellular domain that comprises an e that can be bound by the antibody rituximab and an intracellular domain that comprises a caspase 9 , and transfected with a lentiviral vector comprising a nucleotide sequence that encodes a CAR. The CAR comprises an extracellular antigen-binding region that binds to CD19; a transmembrane domain from PD-l; intracellular co-stimulatory domains from each of CD28, 4-lBB, and 0X40; and an intracellular CD35; domain. CAR- expressing T lymphocytes are administered to the individual without prior expansion of the T cytes. The individual is administered n 105 and 106 of the T lymphocytes in 200 mL saline solution by intravenous infusion over 30 minutes. The individual is monitored for two weeks afterwards to establish a reduction of at least 90% of CD19+ B cells in the individual’s blood. Where the patient, after administration of the T lymphocytes, shows signs of distress due to the T lymphocytes (e.g., difficulty breathing, fever, abnormal serum cytokine , rash, or the like), rituximab is administered at a dosage of 200-500 mg/m2 or until symptoms abate. .5. e 5: Treatment of Prostate Cancer An individual presents with stage T2 te cancer, with no spread to regional or other lymph nodes (N0, M0). Histological grade is determined to be G2. Overall, the individual is determined to have Stage II prostate cancer. The individual is stered between 109 and 1010 T lymphocytes that comprise a CAR, in 200 mL saline solution by intravenous inﬁlsion over minutes. The CAR ses an extracellular antigen-binding region that binds to PSCA, a transmembrane domain from CTLA4, intracellular co-stimulatory domain from CD28, and an intracellular CD35; domain. The T lymphocytes also comprise a cell death polypeptide comprising an extracellular domain that comprises an epitope that can be bound by the dy rituximab, and an intracellular domain that comprises a caspase 3, caspase 8, or caspase 9 domain. The individual is re-assessed for prostate cancer stage and spread to lymph nodes, and histology of biopsied prostate tissue is performed, at 30, 60 and 90 days post-administration.

Where the patient, after stration of the T lymphocytes, shows signs of distress due to the T lymphocytes (e.g., difficulty breathing, fever, abnormal serum cytokine levels, rash, or the like), rituximab is administered at a dosage of 200-500 mg/m2 or until symptoms abate. .6. Example 6: Treatment of te Cancer An individual presents with stage T2 prostate , with no spread to regional or other lymph nodes (N0, M0). Histological grade is determined to be G2. Overall, the individual is determined to have Stage II prostate cancer. The individual is administered between 109 and 1010 T lymphocytes that comprise a CAR, in 200 mL saline solution by intravenous inﬁlsion over s. The CAR comprises an extracellular antigen-binding region that binds to PSCA, a transmembrane domain from PD-l, intracellular co-stimulatory domain from CD28, and an intracellular CD35; domain. The T lymphocytes also comprise a cell death polypeptide sing an extracellular domain that ses an epitope that can be bound by the antibody rituximab, and an intracellular domain that comprises a caspase 3, caspase 8, or caspase 9 domain. The dual is re-assessed for prostate cancer stage and spread to lymph nodes, and histology of biopsied prostate tissue is performed, at 30, 60 and 90 days post-administration.

Where the patient, after administration of the T lymphocytes, shows signs of distress due to the T lymphocytes (e.g., difficulty breathing, fever, abnormal serum cytokine levels, rash, or the like), rituximab is administered at a dosage of 200-500 mg/m2 or until symptoms abate. .7. Example 7: Treatment of te Cancer An individual presents with stage T2 prostate cancer, with no spread to regional or other lymph nodes (N0, M0). Histological grade is determined to be G2. Overall, the individual is determined to have Stage II te cancer. The individual is administered between 109 and 1010 T lymphocytes that comprise a CAR, in 200 mL saline solution by intravenous inﬁlsion over s. The CAR comprises an extracellular antigen-binding region that binds to PSCA, a transmembrane domain from CTLA-4, intracellular mulatory domains from each of CD28, 4-lBB, and 0X40, and an intracellular CD35; domain. The T lymphocytes also se a cell death ptide comprising an extracellular domain that comprises an epitope that can be bound by the antibody rituximab, and an intracellular domain that ses a caspase 3, caspase 8, or caspase 9 domain. The individual is re-assessed for prostate cancer stage and spread to lymph nodes, and histology of biopsied prostate tissue is med, at 30, 60 and 90 days post- administration. Where the patient, after administration of the T lymphocytes, shows signs of distress due to the T lymphocytes (e.g., difﬁculty breathing, fever, abnormal serum cytokine levels, rash, or the like), rituximab is administered at a dosage of 200-500 mg/m2 or until symptoms abate. .8. Example 8: Treatment of Prostate Cancer An individual presents with stage T2 prostate cancer, with no spread to regional or other lymph nodes (N0, M0). Histological grade is determined to be G2. Overall, the individual is determined to have Stage II prostate cancer. The dual is stered between 109 and 1010 T lymphocytes that se a CAR, in 200 mL saline solution by intravenous inﬁlsion over minutes. The CAR comprises an extracellular antigen-binding region that binds to PSCA, a transmembrane domain from PD-l, intracellular co-stimulatory domains from each of CD28, 4- lBB, and 0X40, and an ellular CD35; domain. The T lymphocytes also comprise a cell death polypeptide comprising an extracellular domain that ses an epitope that can be bound by the antibody rituximab, and an intracellular domain that comprises a caspase 3, caspase 8, or e 9 . The individual is re-assessed for prostate cancer stage and spread to lymph nodes, and histology of biopsied prostate tissue is performed, at 30, 60 and 90 days post- administration. Where the patient, after administration of the T lymphocytes, shows signs of distress due to the T lymphocytes (e.g., difﬁculty breathing, fever, abnormal serum cytokine levels, rash, or the like), rituximab is administered at a dosage of 200-500 mg/m2 or until symptoms abate.

EQUIVALENTS The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the t matter provided herein, in on to those described, will become apparent to those skilled in the art from the foregoing description.

Such modifications are ed to fall within the scope of the appended claims.

Various ations, patents and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

What we claim is:

1. An isolated T lymphocyte comprising an artificial cell death polypeptide comprising an apoptosis-inducing domain, wherein said artificial cell death ptide is a transmembrane protein comprising an extracellular domain that comprises a CD20 epitope, a transmembrane domain, and an intracellular domain comprising said apoptosisinducing domain, wherein said apoptosis-inducing domain is caspase 3, caspase 8 or caspase 9, wherein said polypeptide is dimerizable using an D20 dy that binds to said CD20 e, and wherein when said antibody dimerizes said polypeptide, an apoptosis-inducing signal is generated in said T lymphocyte.

2. The T lymphocyte of claim 1, wherein said antibody is rituximab and said extracellular domain comprises a CD20 epitope to which said rituximab binds.

3. The T lymphocyte of claim 1, wherein said antibody is tositumumab and said extracellular domain comprises a CD20 epitope to which said tositumumab binds.

4. The T lymphocyte of claim 1, wherein said antibody is ibritumomab and said extracellular domain comprises a CD20 epitope to which said momab binds.

5. The T lymphocyte of claim 1, wherein said antibody is ofatumumab and said ellular domain comprises a CD20 epitope to which said ofatumumab binds.

6. The T lymphocyte of any one of claims 1-5, which additionally comprises a chimeric antigen receptor (CAR) that izes an antigen on a tumor cell.

7. The T cyte of claim 6, wherein said tumor cell is a cell of a solid tumor.

8. The T cyte of claim 6, wherein said tumor cell is a cell of a blood cancer.

9. The T lymphocyte of claim 6, wherein said antigen is Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), , calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid n (GCDFP-15), HMB-45 antigen, protein melan- A, myo-D1, muscle-specific actin (MSA), ilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor- 1, or the dimeric form of the pyruvate kinase isoenzyme type M2.

10. The use of a T lymphocyte according to any one of claims 1-9 in the manufacture of a medicament for treating cancer in an individual, wherein said T lymphocyte additionally comprises a chimeric antigen receptor (CAR) that recognizes an antigen on a tumor cell, and wherein said treatment comprises administration of an anti- CD20 antibody that binds to a CD20 epitope in the event that the administration of said T lymphocyte causes any unwanted or rious effects in said individual receiving said T lymphocyte or in the event that the presence of the T lymphocyte in said individual is no longer necessary.

11. The use of claim 10, wherein the unwanted or deleterious effects comprise signs of distress comprising one or more of difficulty breathing, fever, abnormal serum cytokine levels, or rash.

12. Use of an anti-CD20 antibody that binds to a CD20 epitope in the manufacture of a medicament for eutic killing of a T lymphocyte in an individual to whom the T lymphocyte has been administered, wherein said T lymphocyte comprises a plurality of artificial cell death ptides each comprising an apoptosis-inducing domain, wherein said artificial cell death ptide is a transmembrane protein comprising an extracellular domain that ses the CD20 epitope, a transmembrane domain, and an intracellular domain comprising said apoptosis-inducing domain, wherein said apoptosis-inducing domain is caspase 3, caspase 8 or caspase 9, n upon administration of the anti-CD20 antibody to the dual, said artificial cell death ptides dimerize and an apoptosis-inducing signal is generated in said T lymphocyte, wherein the eutic g of said T lymphocyte comprises stering to the individual the medicament comprising an amount of said anti-CD20 dy sufficient for said plurality of artificial cell death polypeptides to dimerize and generate an aggregate apoptosis-inducing signal sufficient to kill said T lymphocyte in the event that the administration of said T lymphocyte causes any unwanted or deleterious effects in said individual to whom the T lymphocyte has been administered or in the event that the presence of said T lymphocyte in said individual is no longer necessary.

13. The use of any one of claims 10, 11, and 12, wherein said antibody is mab and said extracellular domain comprises a CD20 epitope to which said rituximab binds.

14. The use of any one of claims 10, 11, and 12, wherein said antibody is tositumumab and said extracellular domain comprises a CD20 epitope to which said tositumumab binds.

15. The use of any one of claims 10, 11, and 12, wherein said antibody is ibritumomab and said extracellular domain comprises a CD20 epitope to which said ibritumomab binds.

16. The use of any one of claims 10, 11, and 12, wherein said antibody is ofatumumab and said extracellular domain comprises a CD20 epitope to which said ofatumumab binds.

17. The use of claim 12, wherein said T cyte additionally comprises a chimeric antigen or (CAR) that recognizes an antigen on a tumor cell.

18. The use of any one of claims 10, 11, and 17, wherein said tumor cell is a cell in a solid tumor.

19. The use of any one of claims 10, 11, and 17, wherein said tumor cell is a cell of a blood .

20. The use of any one of claims 10, 11, and 17, wherein said antigen is Her2, prostate stem cell antigen, alpha-fetoprotein, carcinoembryonic antigen, cancer antigen- 125, CA19-9, inin, MUC-1, epithelial membrane protein, epithelial tumor antigen, nase, melanoma-associated antigen, CD34, CD45, CD99, CD 117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein, gross cystic e fluid n, HMB- 45 antigen, protein melan-A, myo-D1, muscle-specific actin, neurofilament, neuron- specific enolase, placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, or the dimeric form of the te kinase isoenzyme type M2.

21. An ed T lymphocyte according to any one of claims 1 to 9, substantially as herein described or exemplified.

22. A use according to any one of claims 10-20, substantially as herein described or exemplified.