AU2017204617B2

AU2017204617B2 - Chimeric antigen receptors

Info

Publication number: AU2017204617B2
Application number: AU2017204617A
Authority: AU
Inventors: Stewart Abbot; Tianjian Li; Bitao Liang
Original assignee: Celgene Corp
Current assignee: Celgene Corp
Priority date: 2012-12-20
Filing date: 2017-07-06
Publication date: 2019-08-22
Anticipated expiration: 2033-04-12
Also published as: AU2015210373B2; AU2015210373A1; AU2017204617A1

Abstract

Provided herein are therapeutic polypeptides, e.g., chimeric antigen receptors, able to direct an immune cell, e.g., a T lymphocyte to a target antigen, and able to cause the T cell to proliferate or to kill cells displaying the antigen when the antigen binds to the polypeptide, wherein the polypeptides comprise a transmembrane domain from a T cell co-inhibitory protein such as CTLA4 or PD-1. Also provided herein are T lymphocytes expressing the polypeptides, and use of such T lymphocytes to treat diseases such as cancer.

Description

[01] The disclosure herein relates to the field of immunology, and more specifically, to the modification of T lymphocytes or other immune cells.

2. BACKGROUND [02] Cells of the immune system such as T lymphocytes (also referred to as T cells) recognize and interact with specific antigens through receptors or receptor complexes which, upon recognition or an interaction with such antigens, cause activation of the cell. An example of such a receptor is the antigen-specific T lymphocyte receptor complex (TCR/CD3), a complex of eight proteins. The T cell receptor (TCR) is expressed on the surface of T lymphocytes. One component, CD3, which has an invariant structure, is responsible for intracellular signaling following occupancy of the TCR by ligand. The T lymphocyte receptor for antigen-CD3 complex (TCR/CD3) recognizes antigenic peptides that are presented to it by the proteins of the major histocompatibility complex (MHC).

Complexes of MHC and peptide are expressed on the surface of antigen presenting cells and other T lymphocyte targets. Stimulation of the TCR/CD3 complex results in activation of the T lymphocyte and a consequent antigen-specific immune response. The TCR/CD3 complex plays a central role in the effector function and regulation of the immune system.

[03] T lymphocytes require a second, co-stimulatory signal to become fully active. Without such a signal, T lymphocytes are either non-responsive to antigen binding to the TCR, or become anergic. Such a co-stimulatory signal, for example, is provided by CD28, a T lymphocyte protein, which interacts with CD80 and CD86 on antigen-producing cells. ICOS (Inducible CO Stimulator), another T lymphocyte protein, provides a co-stimulatory signal when bound to ICOS ligand. CTLA4 (cytotoxic T-Lymphocyte Antigen 4), also known as

CD 152, is a receptor expressed on the surface of helper T cells and CD4+ T cells, that downregulates T cell activity. Binding of CTLA4 to its cognate ligands, CD80 and CD86, results in reduced T cell activation and proliferation. PD-1 (Programmed Cell Death-1), also

2017204617 31 Jul 2019 known as CD279, is currently understood to negatively regulate T Cell Receptor (TCR) signals, and to broadly negatively regulate immune responses.

[04] The essential antigen-binding, signaling, and stimulatory functions of the TCR complex have been reduced by genetic recombination methods to a single polypeptide chain, generally referred to as a Chimeric Antigen Receptor (CAR). See, e.g., Eshhar, U.S. Patent No. 7,741,465; Eshhar, U.S. Patent Application Publication No. 2012/0093842. T lymphocytes bearing such CARs are generally referred to as CAR-T lymphocytes. CARs are constructed specifically to stimulate T cell activation and proliferation in response to a specific antigen to which the CAR binds.

[04a] Throughout the description and claims of the specification, the word “comprise” and variations of the word, such as “comprising” and “comprises”, is not intended to exclude other additives, components, integers or steps.

[04b] A reference herein to a patent document or other matter which is given as prior art is not to be taken as admission that the document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

3. SUMMARY [05] In one aspect, provided herein is a polynucleotide encoding a polypeptide comprising (i) a transmembrane domain from CTLA4 or PD-1, (ii) an intracellular domain of an

Ό endogenous protein expressed on the surface of lymphocytes that triggers the activation and/or proliferation of said lymphocytes, (iii) one or more co-stimulatory domains, and (iv) an extracellular domain that binds to an antigen, wherein if the transmembrane domain is from CTLA4, the intracellular domain and extracellular domain of said polypeptide are not from CTLA4; and if the transmembrane domain is from PD-1, the intracellular domain and 25 extracellular domain of said polypeptide are not from PD-1.

[05a] In a further aspect, provided herein are polypeptides, e.g., chimeric antigen receptors (see, e.g., Eshhar, U.S. Patent No. 7,741,465), that can be expressed by immune system cells, e.g., T lymphocytes (T cells), are membrane-bound in such immune system cells, and which comprise a transmembrane domain from an immune system protein that normally transmits 30 an inhibitory signal to such immune system cells, e.g., a transmembrane domain from

2017204617 31 Jul 2019

CTLA4 (Cytotoxic T-Lymphocyte Antigen 4 or Cytotoxic T-Lymphocyte Associated protein 4) or PD-1 (Programmed Cell Death-1).

[06] In one embodiment, provided herein is a polypeptide comprising (i) a transmembrane domain comprising the transmembrane domain from CTLA4 (e.g., GenBank Accession No.

NM_005214.4 (CTLA4 cytotoxic T-lymphocyte-associated protein 4 (Homo sapiens)', Gene

ID: 1493)) or PD-1 (e.g., GenBank Accession No. NM 005018.2 (programmed cell death 1 (Homo sapiens ); Gene ID: 5133)), or a portion thereof, (ii) an intracellular domain (e.g., cytoplasmic domain) of an endogenous protein expressed on the surface of lymphocytes that triggers the activation and/or proliferation of said lymphocytes, and (iii) an extracellular domain that binds to an antigen of interest, wherein if the transmembrane domain is from CTLA4, the intracellular domain and extracellular domain of said polypeptide are not from CTLA4; and if the transmembrane domain is from PD-1, the intracellular domain and extracellular domain of said polypeptide are not from PD-1. In a specific embodiment, the polypeptide is a chimeric antigen receptor (CAR). 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 polypeptide binds to said antigen. In a specific

2a

2017204617 06 Jul 2017 embodiment, the polypeptide, when expressed on the surface of a T lymphocyte, directs the T lymphocyte to kill a cell expressing said antigen.

[07] In a specific embodiment, provided herein is a polypeptide comprising a transmembrane domain from CTLA4, wherein the CTLA4 transmembrane domain is the polypeptide sequence encoded by exon 3 of a human ctla4 gene (e.g., GenBank Accession No.

NM_005214.4 (CTLA4 cytotoxic T-lymphocyte-associated protein 4 (Homo sapiens)', Gene ID: 1493)).

[08] In another specific embodiment, the CTLA4 transmembrane domain of a polypeptide provided herein is or comprises the amino acid sequence

PEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKM (in three-letter code, Pro-Glu-ProCys-Pro-Asp-Ser-Asp-Phe-Leu-Leu-Trp-Ile-Leu-Ala-Ala-Val-Ser-Ser-Gly-Leu-Phe-Phe-TyrSer-Phe-Leu-Leu-Thr-Ala-Val-Ser-Leu-Ser-Lys-Met) (SEQ ID NO:1).

[09] In another specific embodiment, the CTLA4 transmembrane domain of a polypeptide provided herein is or comprises the polypeptide sequence encoded by nucleotides 610-722 of 15 GenBank Accession No. NM_005214.4 (CTLA4 cytotoxic T-lymphocyte-associated protein 4 (Homo sapiens)', Gene ID: 1493).

[010] In another specific embodiment, the CTLA4 transmembrane domain of a polypeptide provided herein is or comprises the amino acid sequence

PDSDFLLWILAAVSSGLFFYSFLLTAVSL (in three-letter code, Pro-Asp-Ser-Asp-Phe-Leu20 Leu-Trp-Ile-Leu-Ala-Ala-Val-Ser-Ser-Gly-Leu-Phe-Phe-Tyr-Ser-Phe-Leu-Leu-Thr-Ala-ValSer-Leu) (SEQ ID NO:2).

[Oil] In another specific embodiment, the CTLA4 transmembrane domain of a polypeptide provided herein is or comprises the polypeptide sequence encoded by nucleotides 636-699 of GenBank Accession No. NM_005214.4 (CTLA4 cytotoxic T-lymphocyte-associated protein 4 25 (Homo sapiens)', Gene ID: 1493).

[012] In another specific embodiment, the CTLA4 transmembrane domain of a polypeptide provided herein 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-TyrSer-Phe-Leu-Leu-Thr-Ala-Val) (SEQ ID NO:3).

[013] In another specific embodiment, the CTLA4 transmembrane domain of a polypeptide provided herein 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-SerPhe-Leu-Leu-Thr) (SEQ ID NO:4).

2017204617 06 Jul 2017 [014] In another specific embodiment, the CTLA4 transmembrane domain of a polypeptide provided herein is or comprises the polypeptide sequence FLLWILVAVSLGLFFYSFLVSAVSLS (in three-letter code, Phe-Leu-Leu-Trp-Ile-Leu-ValAla-Val-Ser-Leu-Gly-Leu-Phe-Phe-Tyr-Ser-Phe-Leu-Val-Ser-Ala-Val-Ser-Leu-Ser) (SEQ ID 5 NO:5).

[015] In another specific embodiment, the PD-1 transmembrane domain of a polypeptide provided herein is or comprises the amino acid sequence TLVVGVVGGLLGSLVLLVWVLAVICSRAA (in three-letter code, Thr-Leu-Val-Val-GlyV al-V al-Gly-Gly-Leu-Leu-Gly-S er-Leu-V al-Leu-Leu-V al-Trp-V al-Leu-Ala-V al-Ile-Cys-Ser10 Arg-Ala-Ala) (SEQ ID NO:6).

[016] In another specific embodiment, the PD-1 transmembrane domain of a polypeptide provided herein 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-TrpVal-Leu-Ala-Val-Ile) (SEQ ID NO:7).

[017] In another specific embodiment, the PD-1 transmembrane domain of a polypeptide provided herein is or comprises the amino acid sequence FQTLVVGWGGLLGSLVLLVWVLAVI (in three-letter code, Phe-Glu-Thr-Leu-Val-ValGly-V al-V al-Gly-Gly-Leu-Leu-Gly-Ser-Leu-V al-Leu-Leu-V al-Trp-V al-Leu-Ala-V al-Ile) (SEQ ID NO:8).

[018] In certain embodiments, a nucleotide sequence that encodes one or the polypeptides disclosed 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.

[019] In a specific embodiment, provided herein is a polypeptide that comprises a transmembrane domain, wherein the transmembrane domain is or comprises at least 10, 11,

12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutive amino acids disclosed in SEQIDNO: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, provided herein is a nucleic acid that encodes a polypeptide described herein, wherein the nucleic acid comprises a nucleotide sequence that 30 encodes at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutive amino acids 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.

2017204617 06 Jul 2017 [020] In certain embodiments, the extracellular domain of any of the polypeptides described herein comprises a receptor, or a portion of a receptor, that binds to an antigen. The extracellular domain may be, e.g., a receptor, or a portion of a receptor, that binds to said antigen. In certain embodiments, the extracellular domain comprises, or is, an antibody or an antigen-binding portion thereof. In a specific embodiment, the extracellular domain comprises, or is, a single-chain Fv domain. The single-chain Fv domain can comprise, for example, a V_L linked to Νh by a flexible linker, wherein said N_L and Nh are from an antibody that binds said antigen.

[021] The antigen to which the extracellular domain of the polypeptide binds can be any antigen of interest, e.g., an antigen on a tumor cell. The tumor cell may be, e.g., a cell in a solid tumor, or a cell of non-solid tumor, e.g., a cell of a blood cancer. In certain embodiments, the antigen is a tumor-associated antigen or a tumor-specific antigen. In a specific embodiment, the tumor-associated antigen or tumor-specific antigen is, without limitation, Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanomaassociated 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 (melanoma antigen recognized by T lymphocytes; MART-1), myo20 DI, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2-PK), CD 19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein (Spl7), mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate 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 ανβ3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), or Ral-B.

[022] In certain embodiments, the extracellular domain of the polypeptides described herein is joined to the transmembrane domain of the polypeptide by a linker, spacer or hinge polypeptide/peptide sequence, e.g., a sequence from CD8, CD28, CTLA4 or PD-1. [023] In certain embodiments, the intracellular domain of the polypeptides described herein is or comprises an intracellular domain of a protein that is expressed on the surface of T cells

2017204617 06 Jul 2017 and triggers activation and/or proliferation of said T cells. In a specific embodiment, the intracellular domain is a CD3q intracellular signaling domain. In another specific embodiment, the intracellular domain is from a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit, or an IL-2 receptor subunit.

[024] In certain embodiments, the polypeptides provided herein additionally comprise one or more co-stimulatory domains, e.g., as part of the intracellular domain of the polypeptide. The one or more co-stimulatory domains can be, or can comprise, without limitation, 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 costimulatory (ICOS) polypeptide sequence.

[025] In a specific embodiment, a polypeptide provided herein comprises, in order, from Nterminus to C-terminus: (i) an antigen-binding domain (e.g., an antigen binding domain that binds an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CD28 15 or CTLA4 hinge polypeptide sequence; (iii) a CTLA4 or PD-1 transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain. In a specific embodiment, the antigen-binding domain of the polypeptide binds to CD 19.

[026] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a V/, linked to V//by 20 a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CD28 hinge polypeptide sequence; (iii) a CTLA4 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a CD3ζ intracellular signaling domain.

[027] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a Nl linked to V// by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CTLA4 hinge polypeptide sequence; (iii) a CTLA4 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a CD3ζ intracellular signaling domain.

[028] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a Nl linked to V//by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CD28 hinge

2017204617 06 Jul 2017 polypeptide sequence; (iii) a PD-1 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a 0Ο3ζ intracellular signaling domain.

[029] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a Nl linked to V//by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CTLA4 hinge polypeptide sequence; (iii) a PD-1 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a 6Ο3ζ intracellular signaling domain.

[030] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a Nl linked to Nh by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CTLA4 hinge polypeptide sequence; (iii) a PD-1 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a CD3C intracellular signaling domain.

[031] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) an antigen-binding domain (e.g., an antigen binding domain that binds an antigen on a tumor cell, e.g., an antigen on a tumor cell described above);

(ii) a PD-1 hinge polypeptide sequence; (iii) a CTLA4 or PD-1 transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain.

[032] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a Nl linked to V//by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a PD-1 hinge polypeptide sequence; (iii) a CTLA4 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a CD3ζ intracellular signaling domain.

[033] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a Nl linked to V//by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a PD-1 hinge polypeptide sequence; (iii) a PD-1 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a CD3ζ intracellular signaling domain.

[034] In another aspect, provided herein are T lymphocytes, e.g., T cells, that comprise, e.g., express on their cell surface, a membrane-bound polypeptide, wherein said polypeptide

2017204617 06 Jul 2017 comprises (i) a transmembrane domain comprising the transmembrane domain from CTLA4 or PD-1, or a portion thereof, (ii) an intracellular domain of an endogenous protein expressed on the surface of lymphocytes and that triggers the activation and/or proliferation of said lymphocytes, and (iii) an extracellular domain that binds to an antigen of interest, wherein if the transmembrane domain is from CTLA4, the intracellular domain and extracellular domain (optionally excluding a CTLA4 linker) of said polypeptide are not from CTLA4; and if the transmembrane domain is from PD-1, the intracellular domain and extracellular domain of said polypeptide are not from PD-1. In a specific embodiment, the polypeptide is a chimeric antigen receptor (CAR).

[035] In a specific embodiment, provided herein is a T lymphocyte comprising a polypeptide that comprises a transmembrane domain from CTLA4, wherein the CTLA4 transmembrane domain is the polypeptide sequence encoded by exon 3 of a human CTLA4 gene (e.g., GenBank Accession No. NM_005214.4 (CTLA4 cytotoxic T-lymphocyte-associated protein 4 (Homo sapiens); Gene ID: 1493)).

[036] In another specific embodiment, provided herein is a T lymphocyte comprising a polypeptide that comprises a transmembrane domain from CTLA4, wherein the CTLA4 transmembrane domain is or comprises the amino acid sequence PEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKM (in three-letter code, Pro-Glu-ProCys-Pro-Asp-Ser-Asp-Phe-Leu-Leu-Trp-Ile-Leu-Ala-Ala-Val-Ser-Ser-Gly-Leu-Phe-Phe-Tyr20 Ser-Phe-Leu-Leu-Thr-Ala-Val-Ser-Leu-Ser-Lys-Met) (SEQ ID NO:1).

[037] In another specific embodiment, provided herein is a T lymphocyte comprising a polypeptide that comprises a transmembrane domain from CTLA4, wherein the CTLA4 transmembrane domain is or comprises the polypeptide sequence encoded by nucleotides 610722 of GenBank Accession No. NM_005214.4 (CTLA4 cytotoxic T-lymphocyte-associated 25 protein 4 (Homo sapiens); Gene ID: 1493).

[038] In another specific embodiment, provided herein is a T lymphocyte comprising a polypeptide that comprises a transmembrane domain from CTLA4, wherein the CTLA4 transmembrane domain is or comprises the amino acid sequence PDSDFLLWILAAVSSGLFFYSFLLTAVSL (in three-letter code, Pro-Asp-Ser-Asp-Phe-Eeu30 Eeu-Trp-Ile-Eeu-Ala-Ala-Val-Ser-Ser-Gly-Eeu-Phe-Phe-Tyr-Ser-Phe-Eeu-Eeu-Thr-Ala-ValSer-Eeu) (SEQ ID NO:2).

[039] In another specific embodiment, provided herein is a T lymphocyte comprising a polypeptide that comprises a transmembrane domain from CTEA4, wherein the CTEA4

2017204617 06 Jul 2017 transmembrane domain is or comprises the polypeptide sequence encoded by nucleotides 636699 of GenBank Accession No. NM 005214.4 (CTLA4 cytotoxic T-lymphocyte-associated protein 4 (Homo sapiens); Gene ID: 1493).

[040] In another specific embodiment, provided herein is a T lymphocyte comprising a polypeptide that comprises a transmembrane domain from CTLA4, wherein the CTLA4 transmembrane domain is or comprises the amino acid sequence FLLWILAAVSSGLFFYSFLLTAV (in three-letter code, Phe-Leu-Leu-Trp-Ile-Leu-Ala-AlaVal-Ser-Ser-Gly-Leu-Phe-Phe-Tyr-Ser-Phe-Leu-Leu-Thr-Ala-Val) (SEQ ID NO:3).

[041] In another specific embodiment, provided herein is a T lymphocyte comprising a polypeptide that comprises a transmembrane domain from CTLA4, wherein the CTLA4 transmembrane domain is or comprises the polypeptide sequence FLLWILAAVSSGLFFYSFLLT (in three-letter code, Phe-Leu-Leu-Trp-Ile-Leu-Ala-Ala-ValSer-Ser-Gly-Leu-Phe-Phe-Tyr-Ser-Phe-Leu-Leu-Thr) (SEQ ID NO:4).

[042] In another specific embodiment, provided herein is a T lymphocyte comprising a polypeptide that comprises a transmembrane domain from CTLA4, wherein the CTLA4 transmembrane domain is or comprises the polypeptide sequence FLLWILVAVSLGLFFYSFLVSAVSLS (in three-letter code, Phe-Leu-Leu-Trp-Ile-Leu-ValAla-Val-Ser-Leu-Gly-Leu-Phe-Phe-Tyr-Ser-Phe-Leu-Val-Ser-Ala-Val-Ser-Leu-Ser) (SEQ ID NO:5).

[043] In another specific embodiment, provided herein is a T lymphocyte comprising a polypeptide that comprises a transmembrane domain from PD-1, wherein the PD-1 transmembrane domain is or comprises the amino acid sequence TLVVGVVGGLLGSLVLLVWVLAVICSRAA (in three-letter code, Thr-Leu-Val-Val-GlyV al-V al-Gly-Gly-Leu-Leu-Gly-S er-Leu-V al-Leu-Leu-V al-Trp-V al-Leu-Ala-V al-Ile-Cys-Ser25 Arg-Ala-Ala) (SEQ ID NO:6).

[044] In another specific embodiment, provided herein is a T lymphocyte comprising a polypeptide that comprises a transmembrane domain from PD-1, wherein the PD-1 transmembrane domain is or comprises the amino acid sequence VGVVGGLLGSLVLLVWVLAVI (in three-letter code, Val-Gly-Val-Val-Gly-Gly-Leu-Leu30 Gly-Ser-Leu-Val-Leu-Leu-Val-Trp-Val-Leu-Ala-Val-Ile) (SEQ ID NO:7).

[045] In another specific embodiment, provided herein is a T lymphocyte comprising a polypeptide that comprises a transmembrane domain from PD-1, wherein the PD-1 transmembrane domain is or comprises the amino acid sequence

2017204617 06 Jul 2017

FQTLVVGWGGLLGSLVLLVWVLAVI (in three-letter code, Phe-Glu-Thr-Leu-Val-ValGly-V al-V al-Gly-Gly-Leu-Leu-Gly-Ser-Leu-V al-Leu-Leu-V al-Trp-V al-Leu-Ala-V al-Ile) (SEQ ID NO:8).

[046] In certain embodiments, a nucleotide sequence expressed or encoded by a T lymphocyte provided herein (i.e., a T lymphocyte comprising a polypeptide described 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.

[047] In another specific embodiment, provided herein is a T lymphocyte comprising a polypeptide that comprises a transmembrane domain, wherein the transmembrane domain is or comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutive amino acids 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 certain embodiments, provided herein is a T lymphocyte comprising a nucleic acid that encodes a polypeptide described herein, wherein the nucleic acid comprises a nucleotide sequence that encodes at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutive amino acids 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. [048] In certain embodiments, the extracellular domain of a polypeptide expressed by the T lymphocytes provided herein comprises a receptor, or a portion of a receptor, that binds to an antigen of interest. The extracellular domain may be, e.g., a receptor, or a portion of a receptor, that binds to said antigen. In certain embodiments, the extracellular domain comprises, or is, an antibody or an antigen-binding portion thereof. In specific embodiments, the extracellular domain comprises, or is, a single-chain Fv domain. The single-chain Fv domain can comprise, for example, a Nl linked to Nh by a flexible linker, wherein said Nl and

Nh are from an antibody that binds said antigen.

[049] The antigen to which the extracellular domain of the polypeptide expressed by a T lymphocyte provided herein binds, and therefore to which the T cell is directed by the polypeptide, can be any antigen of interest, e.g., an antigen on a tumor cell. The tumor cell may be, e.g., a cell in a solid tumor, or a cell of a non-solid tumor, e.g., a cell of a blood cancer. In certain embodiments, the antigen is a tumor-associated antigen or a tumor-specific antigen. In certain embodiments, the antigen is one or more of Kappa, Lambda, CD 19, CD22, CD27, CD30, CD70, GD2, HER2, CEA, EGFRvIII, Sperm Proteinl7, PSCA, mesothelin, PAP (prostatic acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading

2017204617 06 Jul 2017 frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), and/or MUC-1. In various specific embodiments, without 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 (CA-125), CA19-9, calretinin,

MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD34, CD45, CD99, CDI 17, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin (MSA), neurofilament, neuron10 specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2PK), an abnormal ras protein, or an abnormal p53 protein. In another specific embodiment, said tumor-associated antigen or tumor-specific antigen is integrin ανβ3 (CD61), galactin, KRas (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), or Ral-B.

[050] In certain embodiments, the extracellular domain of a polypeptide expressed by a T lymphocyte described herein is joined to said transmembrane domain of the polypeptide by a linker, spacer or hinge polypeptide sequence, e.g., a sequence from CD8, CD28, CTLA4 or PD-1.

[051] In certain embodiments, the intracellular domain of a polypeptide expressed by a T lymphocyte described herein is or comprises an intracellular domain of a protein that is normally expressed on the surface of T cells and which triggers activation and/or proliferation of said T cells. In a specific embodiment, the intracellular domain is a Οϋ3ζ intracellular signaling domain. In another embodiment, the intracellular domain is from a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit or an IL-2 receptor subunit.

[052] In certain embodiments, a polypeptide expressed by a T lymphocyte described herein additionally comprises one or more co-stimulatory domains, e.g., as part of the intracellular domain of the polypeptide. The one or more co-stimulatory domains can be, or comprise, one or more of a co-stimulatory CD27 polypeptide sequence, a co-stimulatory CD28 polypeptide sequence, a co-stimulatory 0X40 (CD 134) polypeptide sequence, a co-stimulatory 4-IBB (CD137) polypeptide sequence, or a co-stimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence.

2017204617 06 Jul 2017 [053] In a specific embodiment, the T lymphocytes provided herein express or comprise a polypeptide that comprises, in order, fromN-terminus to C-terminus: (i) an antigen-binding domain (e.g., an antigen binding domain that binds an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CD28 or CTLA4 hinge polypeptide sequence; (iii) a

CTLA4 or PD-1 transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain. In a specific embodiment, the antigen-binding domain of the polypeptide binds to CD 19.

[054] In another specific embodiment, the T lymphocytes provided herein express or comprise a polypeptide that comprises, in order, from N-terminus to C-terminus: (i) a single10 chain Fv domain comprising a Nl linked to Nh by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CD28 hinge polypeptide sequence; (iii) a CTLA4 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a €Ό3ζ intracellular signaling domain.

[055] In another specific embodiment, the T lymphocytes provided herein express or comprise a polypeptide that comprises, in order, fromN-terminus to C-terminus: (i) a singlechain Fv domain comprising a Nl linked to Nh by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CTLA4 hinge polypeptide sequence; (iii) a CTLA4 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a ϋΟ3ζ intracellular signaling domain.

[056] In another specific embodiment, the T lymphocytes provided herein express or comprise a polypeptide that comprises, in order, fromN-terminus to C-terminus: (i) a singlechain Fv domain comprising a Nl linked to Nh by a linker, wherein said Nl and Nh are from 25 an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CD28 hinge polypeptide sequence; (iii) a PD-1 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a ϋΟ3ζ intracellular signaling domain.

[057] In another specific embodiment, the T lymphocytes provided herein express or comprise a polypeptide that comprises, in order, from N-terminus to C-terminus: (i) a singlechain Fv domain comprising a Nl linked to Nh by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CTLA4 hinge polypeptide sequence; (iii) a PD-1

2017204617 06 Jul 2017 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a 6Ο3ζ intracellular signaling domain.

[058] In another specific embodiment, the T lymphocytes provided herein express or comprise a polypeptide that comprises, in order, fromN-terminus to C-terminus: (i) an antigen-binding domain (e.g., an antigen binding domain that binds an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a PD-1 hinge polypeptide sequence; (iii) a CTLA4 or PD-1 transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain.

[059] In another specific embodiment, the T lymphocytes provided herein express or comprise a polypeptide that comprises, in order, from N-terminus to C-terminus: (i) a singlechain Fv domain comprising a Nl linked to Nh by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a PD-1 hinge polypeptide sequence; (iii) a CTLA4 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a CO3Q intracellular signaling domain.

[060] In another specific embodiment, the T lymphocytes provided herein express or comprise a polypeptide that comprises, in order, fromN-terminus to C-terminus: (i) a singlechain Fv domain comprising a V/. linked to Nh by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a PD-1 hinge polypeptide sequence; (iii) a PD-1 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a 6Ό3ζ intracellular signaling domain.

[061] In a specific embodiment, the T lymphocytes provided herein that express or comprise one or more of the polypeptides provided herein, become activated or stimulated to proliferate 25 when said polypeptide binds to the antigen to which the antigen binding domain or singlechain Fv domain of the polypeptide is specific. In another specific embodiment, the T lymphocytes provided herein that express or comprise one or more of the polypeptides provided herein, kill cells that express or comprise the antigen to which the antigen binding domain or single-chain Fv domain of the polypeptide is specific when the T lymphocytes come in contact with said antigen-expressing cells.

[062] In another aspect, 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 expressing an antigen, comprising administering to the individual one or more of the T

2017204617 06 Jul 2017 lymphocytes provided herein, i.e., T lymphocytes that comprise or express a polypeptide described herein.

4. DETAILED DESCRIPTION [063] In one aspect, provided herein are polypeptides, e.g., chimeric antigen receptors (see,

e.g., Eshhar, U.S. Patent No. 7,741,465), that can be expressed by immune system cells, e.g., T lymphocytes (T cells), are membrane-bound in such immune system cells, and which comprise a transmembrane domain 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 PD10 1 (Programmed Cell Death-1). Further provided herein are nucleic acid sequences encoding the polypeptides described herein. Also provided herein are immune system cells, e.g., T lymphocytes (e.g., T cells), expressing such polypeptides.

[064] The polypeptides provided herein comprise an extracellular domain that binds to an antigen, e.g., an antigen on a cell, a transmembrane domain, and an intracellular (cytoplasmic) 15 signaling domain that transmits a primary activation signal to an immune cell. When the polypeptides provided herein are expressed on the surface of, e.g., a T lymphocyte, and when 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 surface, to kill the cell expressing the antigen. Because T lymphocytes 20 require two signals in order to fully activate, a primary activation signal and a costimulatory signal, in certain embodiments, the polypeptides described herein can comprise a costimulatory domain such that binding of the antigen to the extracellular domain results in transmission of both a primary activation signal and a costimulatory signal.

[065] The polypeptides, e.g., CARs, provided herein are functional, immune stimulatory 25 polypeptides that comprise a transmembrane domain from a T cell co-inhibitory protein, e.g.,

CTLA4 or PD-1. In one aspect, provided herein is a polypeptide comprising (i) a transmembrane domain from CTLA4 or PD-1, (ii) an intracellular domain (e.g., cytoplasmic domain) of an endogenous protein expressed on the surface of lymphocytes and that triggers the activation and/or proliferation of said lymphocytes, and (iii) an extracellular domain that 30 binds to an antigen, wherein if the transmembrane domain is from CTLA4, the intracellular domain and extracellular domain of said polypeptide are not from CTLA4; and if the transmembrane domain is from PD-1, the intracellular domain and extracellular domain of

2017204617 06 Jul 2017 said polypeptide are not from PD-1. In a specific embodiment, a T lymphocyte expressing a polypeptide described herein is activated or stimulated to proliferate when said polypeptide binds to an antigen to which the polypeptide is specific (i.e., an antigen that is bound by the extracellular domain of the polypeptide). 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.

[066] In certain embodiments the polypeptides provided herein comprise a transmembrane domain from CTLA4 or PD-1, or a portion thereof, wherein the CTLA4 or PD-1 transmembrane domain is from a mammalian CTLA4 or PD-1, e.g., human, primate, or rodent, e.g., murine CTLA4 or PD-1. In a specific embodiment, the transmembrane domain does not comprise amino acids from the intracellular domain, extracellular domain, or either the intracellular or extracellular domain of CTLA4 or PD-1. Specific, non-limiting examples of CTLA4 or PD-1 transmembrane domain sequences are provided below.

[067] In a specific embodiment, provided herein is a polypeptide comprising a transmembrane domain from CTLA4, wherein the CTLA4 transmembrane domain is the polypeptide sequence encoded by exon 3 of a human ctla4 gene (e.g., GenBank Accession No. NM_005214.4 (CTLA4 cytotoxic T-lymphocyte-associated protein 4 (Homo sapiens)', Gene ID: 1493)).

[068] In another specific embodiment, the CTLA4 transmembrane domain of a polypeptide 20 provided herein is or comprises the amino acid sequence

[069] In another specific embodiment, the CTLA4 transmembrane domain of a polypeptide 25 provided herein is or comprises the polypeptide sequence encoded by nucleotides 610-722 of

GenBank Accession No. NM_005214.4 (CTLA4 cytotoxic T-lymphocyte-associated protein 4 (Homo sapiens)', Gene ID: 1493).

[070] In another specific embodiment, the CTLA4 transmembrane domain of a polypeptide provided herein is or comprises the amino acid sequence

PDSDFLLWILAAVSSGLFFYSFLLTAVSL (in three-letter code, Pro-Asp-Ser-Asp-Phe-LeuLeu-Trp-Ile-Leu-Ala-Ala-Val-Ser-Ser-Gly-Leu-Phe-Phe-Tyr-Ser-Phe-Leu-Leu-Thr-Ala-ValSer-Leu) (SEQ ID NO:2).

2017204617 06 Jul 2017 [071] In another specific embodiment, the CTLA4 transmembrane domain of a polypeptide provided herein is or comprises the polypeptide sequence encoded by nucleotides 636-699 of GenBank Accession No. NM_005214.4 (CTLA4 cytotoxic T-lymphocyte-associated protein 4 (Homo sapiens)', Gene ID: 1493).

[072] In another specific embodiment, the CTLA4 transmembrane domain of a polypeptide provided herein 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-TyrSer-Phe-Leu-Leu-Thr-Ala-Val) (SEQ ID NO:3).

[073] In another specific embodiment, the CTLA4 transmembrane domain of a polypeptide provided herein 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-SerPhe-Leu-Leu-Thr) (SEQ ID NO:4).

[074] In another specific embodiment, the CTLA4 transmembrane domain of a polypeptide provided herein is or comprises the polypeptide sequence

FLLWILVAVSLGLFFYSFLVSAVSLS (in three-letter code, Phe-Leu-Leu-Trp-Ile-Leu-ValAla-Val-Ser-Leu-Gly-Leu-Phe-Phe-Tyr-Ser-Phe-Leu-Val-Ser-Ala-Val-Ser-Leu-Ser) (SEQ ID NO:5).

[075] In another specific embodiment, the PD-1 transmembrane domain of a polypeptide provided herein is or comprises the amino acid sequence

TLVVGVVGGLLGSLVLLVWVLAVICSRAA (in three-letter code, Thr-Leu-Val-Val-GlyV al-V al-Gly-Gly-Leu-Leu-Gly-S er-Leu-V al-Leu-Leu-V al-Trp-V al-Leu-Ala-V al-Ile-Cys-SerArg-Ala-Ala) (SEQ ID NO:6).

[076] In another specific embodiment, the PD-1 transmembrane domain of a polypeptide provided herein 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-TrpVal-Leu-Ala-Val-Ile) (SEQ ID NO:7).

[077] In another specific embodiment, the PD-1 transmembrane domain of a polypeptide provided herein is or comprises the amino acid sequence FQTLVVGWGGLLGSLVLLVWVLAVI (in three-letter code, Phe-Glu-Thr-Leu-Val-Val30 Gly-V al-V al-Gly-Gly-Leu-Leu-Gly-Ser-Leu-V al-Leu-Leu-V al-Trp-V al-Leu-Ala-V al-Ile) (SEQ ID NO:8).

[078] In certain embodiments, a nucleotide sequence that encodes one or the polypeptides disclosed herein comprises a nucleotide sequence that encodes any of the amino acid

2017204617 06 Jul 2017 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.

[079] In a specific embodiment, provided herein is a polypeptide that comprises a transmembrane domain, wherein the transmembrane domain is or comprises at least 10, 11, 5 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutive amino acids 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, provided herein is a nucleic acid that encodes a polypeptide described herein, wherein the nucleic acid comprises a nucleotide sequence that encodes at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutive amino acids disclosed in SEQ ID NO: 1, SEQIDNO:2, SEQIDNO:3, SEQIDNO:4, SEQIDNO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[080] In constructing the polypeptides provided herein, in certain embodiments, human sequences may be combined with non-human sequences. For example, a polypeptide comprising human extracellular and intracellular domain amino acid sequences may comprise 15 a transmembrane domain from a non-human species; e.g., may comprise a murine CTLA4 transmembrane domain or a murine PD-1 transmembrane domain. In a more specific embodiment, the polypeptide comprises human amino acid sequences for the extracellular and intracellular domains, and comprises a transmembrane domain having, or consisting of, the amino acid sequence of SEQ ID NO:5.

[081] The extracellular domains of the polypeptides provided herein bind to an antigen of interest. In certain embodiments, the extracellular domain of a polypeptide provided herein 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 embodiments, the extracellular domain comprises, or is, an antibody or an antigen25 binding portion thereof. In specific embodiments, the extracellular domain comprises, or is, a single-chain Fv domain. The single-chain Fv domain can comprise, for example, a \_L linked to Nh by a flexible linker, wherein said Nl and Nh are from an antibody that binds said antigen.

[082] The antigen to which the extracellular domain of the polypeptides provided herein binds/recognizes 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 cell of a non-solid tumor, e.g., 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 cancer, a breast cancer, a prostate cancer, an

2017204617 06 Jul 2017 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 neuroectodermal tumor, a solid germ cell tumor, a hepatoblastoma, a neuroblastoma, a non-rhabdomyosarcoma soft tissue sarcoma, an osteosarcoma, a retinoblastoma, 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 lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, 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 effusion 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 lymphoma, blastic NK cell lymphoma, mycosis fungoides, Sezary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T lymphocyte lymphoma, peripheral T lymphocyte lymphoma (unspecified), anaplastic large cell lymphoma, Hodgkin lymphoma, or a non-Hodgkin lymphoma. Antigens specific to certain cancers, as well as methods for identifying such antigens, are known in the art.

[083] 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 17p deletion, an 25 1 lq deletion, a 12q trisomy, a 13q deletion or a p53 deletion.

[084] In certain embodiments, the antigen recognized by the extracellular domain of a polypeptide described herein is a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA). In various specific embodiments, the tumor-associated antigen or tumor-specific antigen is, without limitation, Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic 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), CD19, CD22, CD27, CD30, CD34, CD45, CD70, CD99, CD117, EGFRvIII (epidermal growth factor variant III), mesothelin, PAP (prostatic

2017204617 06 Jul 2017 acid phosphatase), prostein, TARP (T cell receptor gamma alternate reading frame protein), Trp-p8, STEAP1 (six-transmembrane epithelial antigen of the prostate 1), chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, protein melan-A (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin (MSA), neurofilament, neuronspecific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2PK), an abnormal ras protein, or an abnormal p53 protein.

[085] In certain embodiments, the TAA or TSA recognized by the extracellular domain of a polypeptide described herein is integrin ανβ3 (CD61), galactin, or Ral-B.

[086] In certain embodiments, the TAA or TSA recognized by the extracellular domain of a polypeptide described herein is a cancer/testis (CT) antigen, e.g., BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-1, NY-SAR-35, OY-TES-1, SPANXB1, SPA17, SSX, SYCP1, or TPTE.

[087] In certain other embodiments, the TAA or TSA recognized by the extracellular domain of a polypeptide described herein is a carbohydrate or ganglioside, e.g., fuc-GMl, GM2 (oncofetal antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, GD3, and the like.

[088] In certain other embodiments, the TAA or TSA recognized by the extracellular domain of a polypeptide described herein is alpha-actinin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA 125, CA 15-3 (CA 27.29VBCAA), 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, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class 1, OS-9, pml-RARa fusion protein, PTPRK, K-ras, N-ras, triosephosphate isomerase, Gage 3,4,5,6,7, GnTV, Herv-K-mel,

Lage-1, NA-88, NY-Eso-l/Lage-2, SP17, SSX-2, TRP2-fnt2,, gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, pl5(58), RAGE,, SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, E2A-PRL, H4-RET, 1GH-1GK, MYL-RAR, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1,

NuMa, K-ras, 13-Catenin, Mum-1, pl6, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4,

791Tgp72, 13HCG, BCA225, BTAA,, CD68VKP1, CO-029, FGF-5, G250, Ga733 (EpCAM),

HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB\70K, NY-CO-1, RCAS1, SDCCAG16,

2017204617 06 Jul 2017

TA-90, TAAL6, TAG72, TLP, or TPS. Other tumor-associated and tumor-specific antigens are known to those in the art.

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

[090] In certain specific embodiments, the antigen recognized by the extracellular domain of a polypeptide described herein 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 embodiments, for example, the antigen is, e.g., a growth factor, cytokine or interleukin, e.g., a growth factor, cytokine, or interleukin associated with angiogenesis or vasculogenesis. Such 10 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), insulin-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-Ιβ, HlF-2a, HIF15 2β, HIF-3a, or HIF-3p. Tumors can also cause localized damage to normal tissue, causing the release of molecules known as damage associated molecular pattern 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 (HMGB1), S100A8 (MRP8, calgranulin A), S100A9 (MRP14, calgranulin B), serum amyloid A (SAA), 20 or can be a deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate.

[091] In certain embodiments, the extracellular domain of the polypeptides described herein is joined to the transmembrane domain of the polypeptide by a linker, spacer or hinge polypeptide sequence, e.g., a sequence from CD28 or a sequence from CTLA4.

[092] In certain embodiments, the intracellular domain of a polypeptide described herein is 25 or comprises an intracellular domain or motif of a protein that is expressed on the surface of T cells and triggers activation and/or proliferation of said T cells. Such a domain or motif is able to transmit a primary antigen-binding signal that is necessary for the activation of a T lymphocyte in response to the antigen’s binding to the CAR’s extracellular portion. Typically, this domain or motif comprises, or is, an ITAM (immunoreceptor tyrosine-based activation 30 motif). ITAM-containing polypeptides suitable for CARs include, for example, the zeta CD3 chain (6ϋ3ζ) or ITAM-containing portions thereof. In a specific embodiment, the intracellular domain is a €ϋ3ζ intracellular signaling domain. In other specific embodiments,

2017204617 06 Jul 2017 the intracellular domain is from a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit or an IL-2 receptor subunit.

[093] In certain embodiments, the polypeptides provided herein additionally comprise 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 (CD 134) polypeptide sequence, a co-stimulatory 4-IBB (CD137) polypeptide sequence, or a co-stimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence, or other costimulatory domain or motif.

[094] In a specific embodiment, a polypeptide provided herein comprises, in order, from Nterminus to C-terminus: (i) an antigen-binding domain (e.g., an antigen binding domain that binds an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CD28 or CTLA4 hinge polypeptide sequence; (iii) a CTLA4 or PD-1 transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain.

[095] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) an antigen-binding domain (e.g., an antigen binding domain that binds an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a PD-1 hinge polypeptide sequence; (iii) a CTLA4 or PD-1 transmembrane domain; (iv) a costimulatory domain; and (v) an intracellular signaling domain.

[096] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a \_L linked to V//by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CD28 hinge polypeptide sequence; (iii) a CTLA4 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a CD3ζ intracellular signaling domain.

[097] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a V/, linked to V//by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CTLA4 hinge polypeptide sequence; (iii) a CTLA4 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a ΰΟ3ζ intracellular signaling domain.

[098] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a Nl linked to V//by

2017204617 06 Jul 2017 a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CD28 hinge polypeptide sequence; (iii) a PD-1 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a Οϋ3ζ intracellular signaling domain.

[099] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a Nl linked to V//by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CTLA4 hinge polypeptide sequence; (iii) a PD-1 transmembrane domain; (iv) a CD28 costimulatory domain;

and (v) a ϋΟ3ζ intracellular signaling domain.

[0100] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a Nl linked to V//by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CTLA4 hinge 15 polypeptide sequence; (iii) a PD-1 transmembrane domain; (iv) a CD28 costimulatory domain;

and (v) a ΰϋ3ζ intracellular signaling domain.

[0101] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) an antigen-binding domain (e.g., an antigen binding domain that binds an antigen on a tumor cell, e.g., an antigen on a tumor cell described above);

[0102] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a Nl linked to V//by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an 25 antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a PD-1 hinge polypeptide sequence; (iii) a CTLA4 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a CD3ζ intracellular signaling domain.

[0103] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a Nl linked to V//by 30 a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a PD-1 hinge polypeptide sequence; (iii) a PD-1 transmembrane domain; (iv) a CD28 costimulatory domain; and (v) a ΰϋ3ζ intracellular signaling domain.

2017204617 06 Jul 2017 [0104] In another specific embodiment, a polypeptide provided herein comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a Nl linked to V//by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CTLA4 hinge polypeptide sequence; (iii) a CTLA4 transmembrane domain; (iv) a 4-IBB costimulatory domain; and (v) a ΟΏ3ζ intracellular signaling domain.

[0105] In another specific embodiment, the polypeptide comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a Nl linked to V// by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CD28 hinge polypeptide sequence; (iii) a PD-1 transmembrane domain; (iv) a 4-IBB costimulatory domain; and (v) a CD3C intracellular signaling domain.

[0106] In another specific embodiment, the polypeptide comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a Nl linked to Nh by a linker, wherein 15 said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a CTLA4 hinge polypeptide sequence; (iii) a PD-1 transmembrane domain; (iv) a 4-IBB costimulatory domain; and (v) a CD3ζ intracellular signaling domain.

[0107] In another specific embodiment, the polypeptide comprises, in order, from N-terminus to C-terminus: (i) a single-chain Fv domain comprising a Nl linked to Nh by a linker, wherein said Nl and Nh are from an antibody that binds an antigen of interest (e.g., an antigen on a tumor cell, e.g., an antigen on a tumor cell described above); (ii) a PD-1 hinge polypeptide sequence; (iii) a CTLA4 transmembrane domain; (iv) a 4-IBB costimulatory domain; and (v) a CD3ζ intracellular signaling domain.

4.1. Isolated Polypeptides (Chimeric Antigen Receptors) [0108] The T lymphocyte-stimulatory polypeptides provided herein, which comprise a CTLA4 or PD-1 transmembrane domain, 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 detectable signal, 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 lysinyl and amino terminal residues with succinic or other carboxylic acid anhydrides, or derivatization with, e.g., imidoesters such

2017204617 06 Jul 2017 as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate. Carboxyl side groups, aspartyl or glutamyl, may be selectively modified by reaction with carbodiimides (R—N=C=N—R') such as l-cyclohexyl-3-(25 morpholinyl-(4-ethyl)carbodiimide or l-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide.

4.2. Isolated Nucleic Acids [0109] Provided herein are nucleic acid sequences (polynucleotides) that encode one or more of the polypeptides provided herein. 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, autonomously 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 lentiviral vectors described in U.S.

Patent Nos. 5,994,136; 6,165,782; 6,428,953; 7,083,981; and 7,250,299, the disclosures of which are hereby incorporated by reference in their entireties. HIV vectors suitable for transformation of T lymphocytes include, but are not limited to, e.g., the vectors described in U.S. Patent No. 5,665,577, the disclosure of which is hereby incorporated by reference in its entirety.

[0110] Nucleic acids useful in the production of the first and second polypeptides, e.g., within a modified T lymphocyte, 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 deoxythymidine, 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 sugars. The deoxyribose phosphate backbone can be modified to produce morpholino nucleic acids, in which each base moiety is linked to a six membered, morpholino ring, or peptide nucleic 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. 7:187-195; 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 phosphoroamidite, or an alkyl phosphotriester backbone.

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4.3. T Lymphocytes [0111] Provided herein are immune cells, e.g., T lymphocytes, comprising the polypeptides provided herein. The T lymphocytes provided herein may be naive T lymphocytes or MHCrestricted T lymphocytes. In certain embodiments, the T lymphocytes provided herein are tumor infiltrating lymphocytes (TILs). In certain embodiments, the T lymphocytes provided herein have been isolated from a tumor biopsy, or have been expanded from T lymphocytes isolated from a tumor biopsy. In certain other embodiments, the T lymphocytes provided herein have been isolated from, or expanded from, T lymphocytes expanded from, peripheral blood, cord blood, or lymph.

[0112] In certain embodiments, the immune cells provided herein that comprise a polypeptide provided herein, e.g., modified T lymphocytes, are autologous to an individual to whom the modified T lymphocytes are to be administered. In certain embodiments, the modified T lymphocytes provided herein are allogeneic to an individual to whom the modified T lymphocytes are to be administered. Where allogeneic T lymphocytes are used to prepare modified T lymphocytes, T lymphocytes can be selected that will reduce the possibility of graft-versus-host disease (GVHD) in the individual. For example, in certain embodiments, virus-specific T lymphocytes can be selected for preparation of modified T lymphocytes; such lymphocytes will be expected to have a greatly reduced native capacity to bind to, and thus become activated by, any recipient antigens. In certain embodiments, recipient-mediated rejection of allogeneic T lymphocytes can be reduced by co-administration to the host of one or more immunosuppressive agents, e.g., cyclosporine, tacrolimus, sirolimus, cyclophosphamide, or the like.

[0113] In one embodiment, T lymphocytes are obtained from an individual, optionally expanded, and then transformed with a polynucleotide encoding a CTLA4 or PD-1 transmembrane domain-containing polypeptide described herein, and optionally expanded. In another embodiment, T lymphocytes are obtained from an individual, optionally then expanded, and then transformed with a polynucleotide encoding a CTLA4 or PD-1 transmembrane domain-containing polypeptide described herein, and optionally then expanded at least one more time. Cells containing the polynucleotides may be selected using a selectable marker.

[0114] In certain embodiments, the modified T lymphocytes described herein express or comprise native TCR proteins, e.g., TCR-α and TCR-β that are capable of forming native TCR complexes, in addition to the CTLA4 or PD-1 transmembrane domain-containing polypeptide.

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

[0115] In certain embodiments, the T lymphocytes described herein are isolated from a tumor lesion, e.g., are tumor-infiltrating lymphocytes; such T lymphocytes are expected to be specific for a TSA or TAA.

[0116] In certain embodiments, the signaling motifs of the CTLA4 or PD-1 transmembrane domain-containing polypeptide, e.g., CAR, can be used to promote proliferation and expansion of the modified T lymphocytes described herein. For example, unmodified T lymphocytes, and T lymphocytes comprising a polypeptide comprising a 6ϋ3ζ 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., U.S. Patent Nos. 5,948,893; 6,534,055; 6,352,694; 6,692,964; 6,887,466; and 6,905,681. In certain embodiments, the antigen, to which the extracellular domain of the CTLA4 or PD-1 transmembrane domain-containing polypeptide binds, can be used to promote selective expansion of T lymphocytes expressing the polypeptide. For example, in one embodiment, in which the antigen is a TSA, T lymphocytes comprising the polypeptide cultured in the presence of the TSA, e.g., a soluble form of the TSA, resulting in increased proliferation as compared to culturing in the absence of the TSA.

[0117] In certain embodiments, T lymphocytes comprising a CTLA4 or PD-1 transmembrane domain-containing polypeptide described herein are stimulated to proliferate using an antibody that binds to a signaling domain on the polypeptide coupled with the antigen that can be bound by the extracellular antigen-binding domain of the polypeptide. For example, in embodiments in which the polypeptide’s signaling domain is CD3C and the antigen that binds to the polypeptide is a TSA, T lymphocytes comprising the polypeptide are stimulated to proliferate by culturing the cells in the presence of the TSA (e.g., a soluble form of the TSA) in combination with an antibody that binds to (2Τ)3ζ.

[0118] In any of the above embodiments, the antigen 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 30 support, e.g., tissue culture plastic surface, beads, or the like.

[0119] The T lymphocytes comprising a CTLA4 or PD-1 transmembrane domain-containing polypeptide described herein can optionally comprise a “suicide gene” or “safety switch” that enables killing of all or substantially all of the T lymphocytes when desired. For example, the

2017204617 06 Jul 2017 modified T lymphocytes described herein, in certain embodiments, can comprise an HSV thymidine kinase gene (HSV-TK), which causes death of the modified T lymphocytes upon contact with gancyclovir. In another embodiment, the modified T lymphocytes express or comprise an inducible caspase, e.g., an inducible caspase 9 (icaspase9), 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).

4,4, Methods of Using Modified T Lymphocytes [0120] The modified immune cells, e.g., the modified T lymphocytes, provided herein that comprise a CTLA4 or PD-1 transmembrane domain-containing polypeptide, e.g., CAR, can be used to treat an individual having one or more types of cells desired to be targeted by T lymphocytes, 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 certain embodiments, the cancer cells are cells of a solid tumor. In certain embodiments, the cells are cells of a lymphoma, a lung cancer, 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 neuroectodermal tumor, a solid germ cell tumor, a hepatoblastoma, a neuroblastoma, a non-rhabdomyosarcoma soft tissue sarcoma, an osteosarcoma, a retinoblastoma, 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 lymphocytic leukemia (small lymphocytic lymphoma), B-cell prolymphocytic leukemia, 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 effusion 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 lymphoma, blastic NK cell lymphoma, mycosis fungoides, Sezary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid

2017204617 06 Jul 2017 papulosis, angioimmunoblastic T lymphocyte lymphoma, peripheral T lymphocyte lymphoma (unspecified), anaplastic large cell lymphoma, Hodgkin lymphoma, or a non-Hodgkin lymphoma.

[0121] Efficacy of the modified T lymphocytes described herein, after administration to an individual having a disease or disorder remediable by T lymphocytes, e.g., an individual having cancer, can be assessed by one or more criteria, specific to the particular disease or disorder, known to those of ordinary skill in the art, to be indicative of progress of the disease or disorder. Generally, administration of the modified T lymphocytes described herein to such an individual is effective when one or more of said criteria detectably, e.g., significantly, moves from a disease state value or range to, or towards, a normal value or range. [0122] The modified T lymphocytes described herein may be formulated in any pharmaceutically-acceptable solution, preferably a solution suitable for the delivery 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 modified T lymphocytes, and may be mixed with auxiliary agents such as lubricants, preservatives, stabilizers, emulsifiers, salts for influencing osmotic pressure, buffers, and coloring. Pharmaceutical carriers suitable for use in formulating the modified T lymphocytes are known in the art and are described, for example, in WO 96/05309.

[0123] In certain embodiments, the modified T lymphocytes described herein are formulated into individual doses, wherein said individual doses comprise at least, at most, or about lxlO⁴, 5xl0⁴, lxlO⁵, 5xl0⁵, lxlO⁶, 5xl0⁶, lxlO⁷, 5xl0⁷, lxlO⁸, 5xl0⁸, lxlO⁹, 5xl0⁹, lxlO¹⁰, 5xlO¹⁰, or lxlO¹¹ modified T lymphocytes. In certain embodiments, the modified T lymphocytes are formulated for intravenous, intraarterial, parenteral, intramuscular, subcutaneous, intrathecal, or intraocular administration, or administration within a particular organ or tissue.

5. EXAMPLES

5.1. Example 1: Treatment of B Cell Lymphoma [0124] 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 17p deletion. T lymphocytes are obtained from the individual, transfected with a lentiviral vector comprising a nucleotide sequence that encodes a chimeric antigen receptor (CAR), and expanded using

CD3+CD28-coated beads to sufficient numbers for administration. The chimeric receptor

2017204617 06 Jul 2017 comprises an extracellular antigen-binding region that binds to CD 19; a transmembrane domain from CTLA4; an intracellular co-stimulatory domain from CD28; and an intracellular CD3ζ domain. The individual is administered between 10⁹ and 10¹⁰ of the T lymphocytes in a 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 CD 19+ B cells in the individual’s blood.

5.2. Example 2: Treatment of a B Cell Lymphoma [0125] 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 17p deletion. About

10⁶ T lymphocytes are obtained from the individual, transfected with a lentiviral vector comprising a nucleotide sequence that encodes a CAR. The CAR comprises an extracellular antigen-binding region that binds to CD 19; a transmembrane domain from PD-1; an intracellular co-stimulatory domain from CD28; and an intracellular CD3ζ domain. CARexpressing T cells are administered to the individual without prior expansion of the T cells.

The individual is administered between 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.

5.3. Example 3: Treatment of B Cell Lymphoma [0126] 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 that encodes a CAR, and expanded using CD3+CD28-coated beads to sufficient numbers for administration. The CAR comprises an extracellular antigen-binding region that binds to CD 19; a transmembrane domain from CTLA4; intracellular co-stimulatory domains from each of CD28, 4-1BB, and 0X40; and an intracellular ΟΏ3ζ 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 afterwards to establish a reduction of at least 90% of CD 19+ B cells in the individual’s blood.

5.4. Example 4: 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 p53 deletion. About 10⁶ T

2017204617 06 Jul 2017 lymphocytes are obtained from the individual, transfected with a lentiviral vector comprising a nucleotide sequence that encodes a CAR. The CAR comprises an extracellular antigenbinding region that binds to CD 19; a transmembrane domain from PD-1; intracellular costimulatory domains from each of CD28, 4-1BB, and 0X40; and an intracellular 0Ο3ζ domain. CAR-expressing T cells are administered to the individual without prior expansion of the T cells. The individual is administered between 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 CD 19+ B cells in the individual’s blood.

5.5. Example 5: Treatment of Prostate Cancer [0127] An individual presents with stage T2 prostate cancer, with no spread to regional or other lymph nodes (NO, 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 modified T lymphocytes that comprise a CAR, in 200 ml saline solution 15 by intravenous infusion over 30 minutes. The CAR comprises an extracellular antigenbinding region that binds to PSCA, a transmembrane domain from CTLA4, an intracellular costimulatory domain from CD28, and an intracellular Οϋ3ζ domain. The individual is reassessed 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.

5.6. Example 6: Treatment of Prostate Cancer [0128] An individual presents with stage T2 prostate cancer, with no spread to regional or other lymph nodes (NO, 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 modified T lymphocytes that comprise a CAR, in 200 ml saline solution 25 by intravenous infusion over 30 minutes. The CAR comprises an extracellular antigenbinding region that binds to PSCA, a transmembrane domain from PD-1, an intracellular costimulatory domain from CD28, and an intracellular 0Ο3ζ domain. The individual is reassessed 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.

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5.7. Example 7: Treatment of Prostate Cancer [0129] An individual presents with stage T2 prostate cancer, with no spread to regional or other lymph nodes (NO, MO). 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 modified T lymphocytes that comprise a CAR, in 200 ml saline solution by intravenous infusion over 30 minutes. The CAR comprises an extracellular antigen-binding region that binds to PSCA, a transmembrane domain from CTLA-4, intracellular co-stimulatory domains from each of CD28, 4-1BB, and 0X40, and an intracellular 6Ό3ζ domain. The individual is re-assessed for prostate cancer stage and spread 0 to lymph nodes, and histology of biopsied prostate tissue is performed, at 30, 60 and 90 days post-administration.

5.8. Example 8: Treatment of Prostate Cancer [0130] An individual presents with stage T2 prostate cancer, with no spread to regional or other lymph nodes (NO, 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 modified T lymphocytes that comprise a CAR, in 200 ml saline solution by intravenous infusion over 30 minutes. The CAR comprises an extracellular antigen-binding region that binds to PSCA, a transmembrane domain from PD-1, intracellular co-stimulatory domains from each of CD28, 4-1BB, and 0X40, and an intracellular Οϋ3ζ Ό 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 postadministration.

EQUIVALENTS [0131] The present disclosure is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the subject matter provided herein, in addition to those described, will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0132] Various publications, patents and patent applications are cited herein, the disclosures of which are incorporated by reference in their entireties.

Claims

1. A polynucleotide encoding a polypeptide comprising (i) a transmembrane domain from CTLA4 or PD-1, (ii) an intracellular domain of an endogenous protein expressed on the surface of lymphocytes that triggers the activation and/or proliferation of said lymphocytes, (iii) one or more co-stimulatory domains, and (iv) an extracellular domain that binds to an antigen, wherein if the transmembrane domain is from CTLA4, the intracellular domain and extracellular domain of said polypeptide are not from CTLA4; and if the transmembrane domain is from PD-1, the intracellular domain and extracellular domain of said polypeptide are not from PD-1.

2. The polynucleotide of claim 1, wherein said polypeptide is a chimeric antigen receptor (CAR).

3. The polynucleotide of claim 1 or claim 2, wherein a T lymphocyte expressing said polypeptide is activated or stimulated to proliferate when said polypeptide binds to said antigen.

4. The polynucleotide of any of claims 1-3, wherein said polypeptide, when expressed on the surface of a T lymphocyte, directs the T lymphocyte to kill a cell expressing said antigen.

5. The polynucleotide of any of claims 1-4, wherein the transmembrane domain is from CTLA4, and wherein the CTLA4 transmembrane domain is the polypeptide sequence encoded by exon 3 of a human CTLA4 gene.

6. The polynucleotide of any of claims 1-4, wherein the transmembrane domain is from CTLA4, and wherein the CTLA4 transmembrane domain is or comprises the amino acid sequence of SEQ ID NO: 1.

7. The polynucleotide of any of claims 1-4, wherein the transmembrane domain is from CTLA4, and wherein the CTLA4 transmembrane domain is or comprises the amino acid sequence of SEQ ID NO:2.

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8. The polynucleotide of any of claims 1-4, wherein the transmembrane domain is from CTLA4, and wherein the CTLA4 transmembrane domain is or comprises the amino acid sequence of SEQ ID NO:3.

9. The polynucleotide of any of claims 1-4, wherein the transmembrane domain is from CTLA4, and wherein the CTLA4 transmembrane domain is or comprises the amino acid sequence of SEQ ID NO:4.

10. The polynucleotide of any of claims 1-4, wherein the transmembrane domain is from CTLA4, and wherein the CTLA4 transmembrane domain is or comprises the amino acid sequence of SEQ ID NO:5.

11. The polynucleotide of any of claims 1-4, wherein the transmembrane domain is from PD-1, and wherein the PD-1 transmembrane domain is or comprises the polypeptide sequence of SEQ ID NO:6.

12. The polynucleotide of any of claims 1-4, wherein the transmembrane domain is from PD-1, and wherein the PD-1 transmembrane domain is or comprises the polypeptide sequence of SEQ ID NO:7.

13. The polynucleotide of any of claims 1-4, wherein the transmembrane domain is from PD-1, and wherein the PD-1 transmembrane domain is or comprises the polypeptide sequence of SEQ ID NO:8

14. The polynucleotide of any of claims 1-13, wherein said extracellular domain is or comprises a receptor, or a portion of a receptor, that binds to said antigen.

15. The polynucleotide of any of claims 1-13, wherein said extracellular domain is or comprises an antibody or an antigen-binding portion thereof.

16. The polynucleotide of any of claims 1-13, wherein said extracellular domain comprises a single-chain Fv domain.

17. The polynucleotide of claim 16, wherein said single-chain Fv domain comprises a V/, linked to Nh by a flexible linker, wherein said Nl and Nh are from an antibody that binds said antigen.

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18. The polynucleotide of any of claims 1-17, wherein said antigen is an antigen on a tumor cell.

19. The polynucleotide of claim 18, wherein said tumor cell is a cell in a solid tumor.

20. The polynucleotide of claim 18, wherein said tumor cell is a cell of a blood cancer.

21. The polynucleotide of any of claims 1-20, wherein said antigen is a tumorassociated antigen or a tumor-specific antigen.

22. The polynucleotide of claim 21, wherein said tumor-associated antigen or tumor-specific antigen is Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanomaassociated 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 (melanoma antigen recognized by T lymphocytes; MART-1), myo-Dl, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, the dimeric form of the pyruvate kinase isoenzyme type M2 (tumor M2-PK), CD 19, 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, STEAP1 (sixtransmembrane epithelial antigen of the prostate 1), an abnormal ras protein, an abnormal p53 protein, integrin ανβ3 (CD61), galactin, K-Ras (V-Ki-ras2 Kirsten rat sarcoma viral oncogene), or Ral-B.

23. The polynucleotide of any of claims 1-22, wherein said extracellular domain is joined to said transmembrane domain by a linker, spacer or hinge polypeptide sequence.

24. The polynucleotide of claim 23, wherein said linker, spacer or hinge polypeptide sequence is from CD28.

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25. The polynucleotide of claim 23, wherein said linker, spacer or hinge polypeptide sequence is from CTLA4.

26. The polynucleotide of claim 23, wherein said linker, spacer or hinge polypeptide is from PD-1.

27. The polynucleotide of any of claims 1-26, wherein said intracellular domain is an intracellular domain of a protein that is expressed on the surface of T cells and triggers activation and/or proliferation of said T cells.

28. The polynucleotide of claim 27, wherein said intracellular domain is a Οϋ3ζ intracellular signaling domain.

29. The polynucleotide of claim 27, wherein said intracellular domain is from a lymphocyte receptor chain, a TCR/CD3 complex protein, an Fc receptor subunit or an IL-2 receptor subunit.

30. The polynucleotide of any one of claims 1-29, wherein said one or more costimulatory domains comprises one or more of: a co-stimulatory CD27 polypeptide sequence, a co-stimulatory CD28 polypeptide sequence, a co-stimulatory 0X40 (CD 134) polypeptide sequence, a co-stimulatory 4-1BB (CD137) polypeptide sequence, and a co-stimulatory inducible T-cell costimulatory (ICOS) polypeptide sequence.

31. The polynucleotide of any of claims 1-30, wherein said polypeptide comprises, in order, from N-terminus to C-terminus: (i) the extracellular domain that binds to an antigen; (ii) a CD28 or CTLA4 hinge polypeptide sequence; (iii) the transmembrane domain from CTLA4 or PD-1; (iv) the one or more costimulatory domains; and (v) the intracellular domain.

32. The polynucleotide of any one of claims 1-13, wherein said polypeptide comprises, in order, from N-terminus to C-terminus: (i) the extracellular domain comprising a single-chain Fv domain comprising a V/, linked to Nh by a flexible linker, wherein said Vt and Nh are from an antibody that binds said antigen; (ii) a CD28 hinge polypeptide sequence;

(iii) the transmembrane domain from CTLA4; (iv) the one or more costimulatory domains, wherein the one or more costimulatory domains is a CD28 costimulatory domain; and (v) the intracellular domain comprising a CD3ζ intracellular signaling domain.

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33. The polynucleotide of any one of claims 1-13, wherein said polypeptide comprises, in order, from N-terminus to C-terminus: (i) the extracellular domain comprising a single-chain Fv domain comprising a Nl linked to V//by a flexible linker, wherein said V/, and V// are from an antibody that binds said antigen; (ii) a CTLA4 hinge polypeptide sequence; (iii) the transmembrane domain from CTLA4; (iv) the one or more costimulatory domains, wherein the one or more costimulatory domains is a CD28 costimulatory domain; and (v) the intracellular domain comprising a 6Ό3ζ intracellular signaling domain.

34. The polynucleotide of any one of claims 1-13, wherein said polypeptide comprises, in order, from N-terminus to C-terminus: (i) the extracellular domain comprising a single-chain Fv domain comprising a V/, linked to Nh by a flexible linker, wherein said Vt and N_H are from an antibody that binds said antigen; (ii) a CD28 hinge polypeptide sequence; (iii) the transmembrane domain from PD-1; (iv) the one or more costimulatory domains, wherein the one or more costimulatory domains is a CD28 costimulatory domain; and (v) the intracellular domain comprising a 6Ό3ζ intracellular signaling domain.

35. The polynucleotide of any one of claims 1-13, wherein said polypeptide comprises, in order, from N-terminus to C-terminus: (i) the extracellular domain comprising a single-chain Fv domain comprising a Nl linked to Nh by a flexible linker, wherein said Nl and Nh are from an antibody that binds said antigen; (ii) a CTLA4 hinge polypeptide sequence; (iii) the transmembrane domain from PD-1; (iv) the one or more costimulatory domains, wherein the one or more costimulatory domains is a CD28 costimulatory domain; and (v) the intracellular domain comprising a CD3ζ intracellular signaling domain.

36. The polynucleotide of any one of claims 1-13, wherein said polypeptide comprises, in order, from N-terminus to C-terminus: (i) the extracellular domain comprising a single-chain Fv domain comprising a VL linked to VH by a flexible linker, wherein said VL and VH are from an antibody that binds said antigen; (ii) a PD-1 hinge polypeptide sequence; (iii) the transmembrane domain from CTLA4; (iv) the one or more costimulatory domains, wherein the one or more costimulatory domains is a CD28 costimulatory domain; and (v) the intracellular domain comprising a CD3ζ intracellular signaling domain.

37. The polynucleotide of any one of claims 1-13, wherein said polypeptide comprises, in order, from N-terminus to C-terminus: (i) the extracellular domain comprising a

2017204617 31 Jul 2019 single-chain Fv domain comprising a VL linked to VH by a flexible linker, wherein said VL and VH are from an antibody that binds said antigen; (ii) a PD-1 hinge polypeptide sequence; (iii) the transmembrane domain from PD-1; (iv) the one or more costimulatory domains, wherein the one or more costimulatory domains is a CD28 costimulatory domain; and (v) the intracellular domain comprising a €Ό3ζ intracellular signaling domain.

38. A method of producing a modified T lymphocyte, comprising obtaining a T lymphocyte, then transforming said T lymphocyte with a polynucleotide according to any one of claims 1 to 37.

39. A modified T lymphocyte produced by the method of claim 38.

40. A polynucleotide according to any one of claims 1 to 37 when used for producing a modified T lymphocyte.