Compositions comprising cells targeting cancer and methods of using the same

Abstract

The disclosure relates to cells expressing CAR-T receptor comprising a sequence specific for binding to cancer cells expressing NY-ESO-1. The disclosure also relates to methods of treating cancer comprising administering the same cells to subjects in need thereof.

Description

COMPOSITIONS COMPRISING CELLS TARGETING CANCER AND METHODSOF USING THE SAMECROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisional application No. 63 / 594,397, which was filed October 30, 2024, is titled “Compositions Comprising Cells Targeting Cancer and Methods of Using the Same,” and is incorporated herein by reference as if fully set forth.SEQUENCE LISTING

[0002] The Sequence Listing filed herewith has the filename STFD-004-PCT_SL.xml, was created on October 25, 2024, has a file size of 79,565 bytes, and is incorporated herein by reference as if fully set forth.FIELD

[0003] The disclosure relates to methods of treating hyperproliferative disease, cells comprising a CAR molecule, and pharmaceutical compositions.BACKGROUND

[0004] Many patients with malignancies are incurable with standard therapy. In addition, traditional treatment options often have serious side effects. Attempts have been made in cancer immunotherapy, however, several obstacles render this a very difficult goal to achieve clinical effectiveness. Although hundreds of so-called tumor antigens have been identified, these are generally derived from self and thus are poorly immunogenic. Furthermore, tumors use several mechanisms to render themselves hostile to the initiation and propagation of immune attack.

[0005] Recent developments using chimeric antigen receptor (CAR) modified autologous T cell (CART) therapy, which relies on redirecting T cells to a suitable cell-surface molecule on cancer cells such as B cell malignancies, show promising results in harnessing the power of the immune system to treat B cell malignancies and other cancers (see, e.g., Sadelain et al., Cancer Discovery 3:388-398 (2013)). The clinical results of the murine derived CART19 (i.e., “CTL019”) have shown promise in establishing complete remissions in patients suffering with CLL as well as in childhood ALL (see, e.g., Kalos et al., Sci Transl Med 3:95ra73 (2011), Porter et al., NEJM 365:725-733 (2011), Grupp et al., NEJM 368: 1509-1518 (2013)). Besides theability for the chimeric antigen receptor on the genetically modified T cells to recognize and destroy the targeted cells, a successful therapeutic T cell therapy needs to have the ability to proliferate and persist over time, in order to survey for leukemic relapse. The variable quality of T cells, resulting from anergy, suppression, or exhaustion, will have effects on CAR- transformed T cells’ performance, over which skilled practitioners have limited control at this time. To be effective, CAR-transformed patient T cells need to persist and maintain the ability to proliferate in response to the cognate antigen.SUMMARY OF EMBODIMENTS

[0006] In some embodiments, the disclosure relates to new chimeric antigen receptors (CARs), new CAR-T cells, new methods for manufacturing new CARs and new CAR-T cells, as well as new methods for treating diseases, including cancer, especially New York esophageal squamous cell carcinoma 1. In some embodiments, the cancer is esophageal cancer or testicular cancer.

[0007] In some embodiments, the disclosure relates to a method of treating a hyperproliferative disease, comprising administering to a patient in need thereof a pharmaceutical composition comprising a therapeutically effective amount of cells comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule that binds to cells expressing NYESO- 1. In some embodiments, the CAR molecule binds to cell expressing NYSEO-1 and an HLA molecule comprising HLA-A*02:01 or HLA-A*02:06, or a functional variant thereof. In some embodiments, the nucleic acid molecule encodes a peptide comprising EQWVANY (SEQ ID NO: 1), or a functional variant thereof comprising about 85% sequence identity to SEQ ID NO: 1. In some embodiments, the nucleic acid molecule encodes a peptide comprising GTHDKCENPKEQWVANYQNLNNVVFTNKELEDIYDESN (SEQ ID NO:2), or a functional variant thereof comprising from about 75% sequence identity to SEQ ID NO:2. In some embodiments, the nucleic acid molecule encodes a peptide comprising KEETKEVLKKFKEKVNQFYEHAFDIINKYGDKEIFNMMFMLLWRVFRSFRIDANNVE LIKFNIRVLDWIMAEADNDLSYFISQ (SEQ ID NO:3), or a functional variant thereof comprising from about 75% sequence identity to SEQ ID NO:3. In some embodiments, the nucleic acid molecule encodes a chimeric peptide comprising a first domain and a second domain, the first domain comprising one or a combination of: SEQ ID NO: 1, SEQ ID NO:2and SEQ ID N0:3, or a functional variant thereof. In some embodiments, the second domain comprising a single chain antibody or antibody fragment that binds to a cell expressing CD3.

[0008] The disclosure relates to methods of treating a disease comprising administering to a patient in need thereof a pharmaceutical composition comprising a population of cells comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule that binds to NYESO-1 at a dosage based on cells / kg body weight and / or the age of the patient. In some embodiments, the dosage is about 3x10^ cells / kg body weight for patients ::S50 kg body weight or about 1.5xl06cells for patients >50 kg body weight based on the total CAR+ cells in the pharmaceutical composition. In some embodiments, the dosage is about lOx 10^ cells / kg body weight for patients ::S50 kg body weight or about 5 xlO^ cells for patients >50 kg body weight based on the total CAR+ cells in the pharmaceutical composition. In some embodiments, the dosage is about 30x10^ cells / kg body weight for patients ::S50 kg body weight or about 15x10^ cells for patients >50 kg body weight based on the total CAR+ cells in the pharmaceutical composition.

[0009] In some embodiments, the disclosure provides a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule. In some embodiments, the said CAR molecule comprises: a first CAR comprising a first antigen binding domain which binds to NYESO-1; a first transmembrane domain; a first co-stimulatory signaling domain; and / or a first primary signaling domain.

[0010] In some embodiments of a nucleic acid encoding a CAR molecule disclosed herein, the nucleotide sequence encoding the first primary signaling domain is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% different from the nucleotide sequence encoding the second primary signaling domain. In some embodiments, the nucleotide sequence encoding the first primary signaling domain differs by at least 1 nucleotide, 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides or all nucleotides from the nucleotide sequence encoding the second primary signaling domain.

[0011] In some embodiments, the cell is an immune effector cell, e.g., T cell (e.g., CD3+, CD4+ or CD8+ T cell), or an NK cell. In some embodiments, the cell is a human cell.

[0012] In some embodiments, the disclosure relates to a method of providing anti-tumor immunity, comprising administering to a subject in need thereof, an effective amount of a cell, e.g., a population of immune effector cells, comprising, e.g., expressing, a CAR molecule disclosed herein, e.g., a dual CAR molecule disclosed herein.

[0013] In some embodiments, the disclosure relates to a method of treating a subject having a disease associated with an antigen. In some embodiments, the method comprises administering to the subject in need thereof, an effective amount of a cell, e.g., a population of immune effector cells, comprising, e.g., expressing, a CAR molecule disclosed herein, e.g., a dual CAR molecule.

[0014] In some embodiments, the disclosure provides a bispecific antigen binding domain. In some embodiments, the bispecific antigen binding domain comprises a first antigen binding domain which binds to NYESO-1 and a second antigen binding domain which binds to a costimulatory molecule. In some embodiments, the disclosure provides a monospecific antigen binding domain. In some embodiments, the monospecific antigen binding domain comprises a antigen binding domain which binds to NYESO-1 and is free of an antigen binding domain which binds to a co-stimulatory molecule. The disclosure also relates to a CAR molecule depicted in FIG. 10A or a nucleic acid molecule encoding the same.

[0015] In some embodiments, the disclosure relates to a cell comprising a CAR molecule comprises a first domain and a second domain. In some embodiments, the first domain comprises SEQ ID NO: 1, or functional variants thereof. In some embodiments, the first domain comprises SEQ ID NO:2, or functional variants thereof. In some embodiments, the first domain comprises SEQ ID NO:3, or functional variants thereof. In some embodiments, the first domain comprises a combination of two or more of : SEQ ID NO: 1, or functional variants thereof; SEQ ID NO:2 or functional variant thereof; and SEQ ID NO:3, or functional variant thereof.

[0016] The disclosure also relates to a chimeric antigen receptor (CAR) comprising a bi specific antigen binding domain described herein or a monospecific antigen binding domain described herein. In some embodiments, if the embodiment is monospecific, the CAR is monospecific with respect to the antigen to which it binds but may have one, two three, four, five, six or more subdomains, each binding domain binding to the antigen, antigenic determinant thereof or an epitope of the thereof.

[0017] In some embodiments, the disclosure relates to a nucleic acid molecule encoding a chimeric antigen receptor (CAR), which comprises a monospecific or bispecific antigen binding domain described herein.

[0018] In some embodiments of the bispecific antigen binding domain described herein, e.g., a CAR comprising the bispecific antigen binding domain, or nucleic acid encoding a CAR comprising the bispecific antigen binding domain, the first antigen binding domain can be upstream (e.g., in an NH2 -terminal orientation) of the second antigen binding domain, or the first antigen binding domain can be downstream (e.g., in a COOH-terminal orientation) of the second antigen binding domain.

[0019] In some embodiments, each of the first antigen binding domain and second antigen binding domains comprise a scFv, e.g., a light chain variable (VL) domain and a heavy chain variable (VH) domain. In some embodiments, the first antigen binding domain comprises an scFv comprising a first VH (VH1) and a first VL (VL1 ). In some embodiments, the second antigen binding domain comprises an scFv comprising a second VH (VH2) and a second VL (VL2).

[0020] In some embodiments, a bispecific antigen binding domain has any one of the following N terminal to C terminal configurations: VL1-VH1-VH2-VL2; VH1-VL1-VH2-VL2; VL1- VH1- VL2-VH2; VH1-VL1-VL2-VH2, VH2-VL2-VL1-VH1; VL2-VH2-VL1-VH1; VH2- VL2-VH1-VL1; or VL2-VH2-VH1-VL1.

[0021] In some embodiments, a CAR comprising a bispecific antigen binding domain comprises the amino acid sequence of SEQ ID NO: 2.

[0022] In some embodiments, the disclosure relates to a vector comprising a nucleic acid sequence encoding a CAR molecule disclosed herein, a nucleic acid encoding a bispecific antigen binding domain disclosed herein, or a nucleic acid encoding a CAR comprising a bispecific antigen binding domain disclosed herein.

[0023] The disclosure also relates to a pharmaceutical composition comprising a nucleic acid encoding a CAR molecule disclosed herein; or a pharmaceutical composition comprising: (i) a CAR molecule disclosed herein; and (ii) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises an pharmaceutically acceptable carrier, such as excipient, a carrier, a diluent and / or a stabilizer. The disclosure relates to a pharmaceutical composition comprising: (i) a therapeutically effective amount of a cell comprising a CAR molecule disclosed herein; and (ii) a pharmaceutically acceptable carrier.

[0024] In some embodiments, the disclosure relates to a pharmaceutical composition comprising a monospecific or bispecific antigen binding domain disclosed herein, a CAR comprising a monospecific or bispecific antigen binding domain disclosed herein, or a CAR nucleic acid sequence encoding a monospecific or bispecific antigen binding domain disclosed herein. In some embodiments, the pharmaceutical composition comprises an excipient, a carrier, a diluent and / or a stabilizer.

[0025] The disclosure also relates to a method of providing or inducing anti-tumor immunity, comprising administering to a subject in need thereof, a therapeutically effective amount of a cell, e.g., a population of immune effector cells, comprising, e.g., expressing, a CAR disclosed herein, e.g., a tandem, monospecific or bispecific CAR disclosed herein.

[0026] In some embodiments, the disclosure relates to a method of treating a subject having a disease associated with an antigen, comprising administering to the subject in need thereof, an effective amount of a cell, e.g., a population of immune effector cells, comprising, e.g., expressing, a CAR disclosed herein, e.g., a tandem CAR disclosed herein.

[0027] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. Such equivalents are intended to be encompassed by the following enumerated embodiments.

[0028] In some embodiments, the disclosure relates to a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule. In some embodiments, the CAR molecule comprises: a first CAR comprising a first antigen binding domain which binds to NYESO-1 or an antigenic determinant thereof; a first transmembrane domain; a first co-stimulatory signaling domain; and / or a first primary signaling domain. In some embodiments, the CAR molecule further comprises: a second CAR comprising a second antigen binding domain which binds to a second antigen; a second transmembrane domain; a second co-stimulatory domain; and / or a second primary signaling domain.

[0029] In some embodiments, the disclosure relates to nucleic acid molecule of any of the preceding embodiments. In some embodiments, the nucleotide sequence encoding the first transmembrane domain is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% different from the nucleotide sequence encoding the second transmembrane domain.

[0030] In some embodiments, the disclosure relates to a nucleic acid molecule of any of the preceding embodiments. In some embodiments, the nucleotide sequence encoding the firsttransmembrane domain differs by at least 1 nucleotide, 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides or all nucleotides from the nucleotide sequence encoding the second transmembrane domain.

[0031] In some embodiments, the disclosure relates to a nucleic acid molecule of any of the preceding embodiments. In some embodiments, the nucleotide sequence encoding the first costimulatory signaling domain differs by at least 1 nucleotide, 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 120 nucleotides, or all nucleotides from the nucleotide sequence encoding the second co- stimulatory signaling domain.

[0032] The disclosure also relates to a nucleic acid molecule of any of the preceding embodiments. In some embodiments, the nucleotide sequence encoding the first primary signaling domain is at least about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% different from the nucleotide sequence encoding the second primary signaling domain.

[0033] In some embodiments, the disclosure relates to a nucleic acid molecule of any of the preceding embodiments. In some embodiments, the nucleotide sequence encoding the first primary signaling domain differs by at least 1 nucleotide, 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides or all nucleotides from the nucleotide sequence encoding the second primary signaling domain.

[0034] In some embodiments, the disclosure relates to a nucleic acid molecule of any of the preceding embodiments. In some embodiments, the first CAR and / or the second CAR comprises a signal peptide, e.g., a peptide comprising a stretch of hydrophobic amino acids, e.g., 5-16 residues.

[0035] In some embodiments, the disclosure relates to a nucleic acid molecule of any of the preceding embodiments. In some embodiments, the signal peptide is chosen from a CD8alpha signal peptide, an interleukin 2 signal peptide, a human albumin signal peptide, a human chymotrypsinogen signal peptide, a human trypsinogen-2 signal peptide or other similar signal peptides disclosed in Stern B. et al. “Improving mammalian cell factories : The selection ofsignal peptide has a major impact on recombinant protein synthesis and secretion in mammalian cells.” (2007), which is incorporated herein by reference as if fully set forth.

[0036] In some embodiments, the disclosure relates to a nucleic acid molecule of any of the preceding embodiments. In some embodiments, the protease cleavage site or internal ribosomal entry site is situated between the first CAR and the second CAR.

[0037] In some embodiments, the disclosure relates to a nucleic acid molecule of any of the preceding embodiments. In some embodiments, the protease cleavage site is situated such that a cell can express a fusion protein comprising a first CAR and a second CAR. In some embodiments, the fusion protein is processed into two peptides by proteolytic cleavage.

[0038] In some embodiments, the disclosure relates to a nucleic acid molecule of any of the preceding embodiments. In some embodiments, the nucleotide sequence encoding the first CAR and the nucleotide sequence encoding the second CAR are disposed on a single nucleic acid construct.

[0039] In some embodiments, the disclosure relates to a nucleic acid molecule of any of the preceding embodiments. In some embodiments, the nucleotide encoding the first CAR and the nucleic acid encoding the second CAR are disposed on the same vector.

[0040] In some embodiments, the disclosure relates to a nucleic acid molecule of any of the preceding embodiments. In some embodiments, the nucleotide sequence encoding the first CAR and the nucleotide sequence encoding the second CAR are disposed on different nucleic acid constructs, e.g., the nucleotide sequence encoding the first CAR is disposed on a first nucleic acid construct, and the nucleotide sequence encoding the second CAR is disposed on a second nucleic acid construct.

[0041] In some embodiments, the disclosure relates to a nucleic acid molecule of any of the preceding embodiments. In some embodiments, the nucleotide sequence encoding the first CAR is disposed on a first vector.

[0042] In some embodiments, the disclosure relates to a nucleic acid molecule of any of the preceding embodiments. In some embodiments, the nucleotide sequence encoding the second CAR is disposed on a second vector.

[0043] In some embodiments, the nucleic acid molecule comprises a viral element, e.g., a viral packaging element.

[0044] In some embodiments, the disclosure relates to a vector comprising the nucleic acid molecule of any of embodiments described herein.

[0045] In some embodiments, the disclosure relates to a vector of any of the preceding embodiments. In some embodiments, the vector is chosen from a DNA, a RNA, a plasmid, a lentivirus vector, adenoviral vector, or a retrovirus vector.

[0046] In some embodiments, the disclosure relates to a cell (e.g., an immune effector cell) comprising a vector herein, or the nucleic acid molecule comprising a nucleic acid sequence encoding any of the preceding amino acid sequence disclosed herein.

[0047] In some embodiments, the disclosure relates a cell of any of the preceding embodiments. In some embodiments, the cell comprises a nucleic acid encoding the CAR molecule. In some embodiments, the disclosure relates to a cell of any of the preceding embodiments. In some embodiments, the cell comprises the nucleic acid molecule of any of the preceding embodiments.

[0048] In some embodiments, the disclosure relates a cell of any of the preceding embodiments. In some embodiments, the cell is a human cell.

[0049] In some embodiments, the disclosure relates to a method of making a cell (e.g., an immune effector cell). In some embodiments, the method comprises transducing an immune effector cell, e.g., a T cell or NK cell with a vector herein.

[0050] In some embodiments, the disclosure relates to a method of making a cell (e.g., an immune effector cell) comprising introducing a nucleic acid molecule of any one of the preceding embodiments, into an immune effector cell, e.g., a T cell or NK cell.

[0051] In some embodiments, the disclosure relates to a method of generating a population of RNA-engineered cells comprising introducing an in vitro transcribed RNA or synthetic RNA into a cell. In some embodiments, the RNA comprises a nucleic acid molecule of any of the preceding embodiments. In some embodiments of the bispecific antigen, the first antigen binding domain can be upstream (e.g., in an N-terminal orientation) of the second antigen binding domain, or the first antigen binding domain can be downstream (e.g., in a C-terminal orientation) of the second antigen binding domain.

[0052] In some embodiments, the disclosure relates to a bispecific antigen binding domain of any of the preceding embodiments. In some embodiments, each of the first antigen binding domain and second antigen binding domains comprise a scFv, e.g., a light chain variable (VL) domain and a heavy chain variable (VH) domain. In some embodiments, the VH can be upstream or downstream of the VL. In some embodiments, the first antigen binding domain comprises an scFv comprising a first VH (VH1) and a first VL (VL1).

[0053] In some embodiments, the disclosure relates to a bispecific antigen binding domain of any preceding embodiments. In some embodiments, the first antigen binding domain comprises an scFv comprising a first VH (VH1) and a first VL (VL1). In some embodiments, the second antigen binding domain comprises an scFv comprising a second VH (VH2) and a second VL(VL2).

[0054] In some embodiments, the disclosure relates to a bispecific antigen binding domain of any preceding embodiments. In some embodiments, the first antigen binding domain is arranged with VH1 upstream of VL1.

[0055] T In some embodiments, the disclosure relates to a bispecific antigen binding domain of any preceding embodiments. In some embodiments, the first antigen binding domain is arranged with VL1 upstream of VH1.

[0056] In some embodiments, the disclosure relates to a bispecific antigen binding domain of any preceding embodiments. In some embodiments, the second antigen binding domain is arranged with VH2 upstream of VL2.

[0057] In some embodiments, the disclosure relates to a bispecific antigen binding domain of any preceding embodiments. In some embodiments, the second antigen binding domain is arranged with VL2 upstream of VH2.

[0058] In some embodiments, the disclosure relates to a bispecific antigen binding domain of any preceding embodiments. In some embodiments, the antigen binding domain has the following N terminal to C terminal configuration: VL1-VH1-VH2-VL2.

[0059] In some embodiments, the disclosure relates to a bispecific antigen binding domain of any preceding embodiments. In some embodiments, the antigen binding domain has the following N terminal to C terminal configuration: VH1-VL1-VH2-VL2.

[0060] In some embodiments, the disclosure relates to a bispecific antigen binding domain of any preceding embodiments. In some embodiments, the antigen binding domain has the following N terminal to C terminal configuration: VL1-VH1-VL2-VH2.

[0061] In some embodiments, the disclosure relates to a monospecific or bispecific antigen binding domain of any preceding embodiments. In some embodiments, the antigen binding domain has the following N terminal to C terminal configuration: VH1-VL1-VL2-VH2.

[0062] In some embodiments, the disclosure relates to a monospecific or bispecific antigen binding domain of any preceding embodiments. In some embodiments, a linker is disposed between the first antigen binding domain and the second antigen binding domain.

[0063] In some embodiments, the disclosure relates to a monospecific or bispecific antigen binding domain of any preceding embodiments. In some embodiments, the linker is disposed between the scFv of the first antigen binding domain and the scFv of the second antigen binding domain.

[0064] In some embodiments, the disclosure relates to a monospecific or bispecific antigen binding domain of any preceding embodiments. In some embodiments, the linker is disposed between:VHl and VH2 if the construct has the configuration of VL1-VH1-VH2-VL2; VL1 and VH2 if the construct has the configu ration of VH1-VL1-VH2-VL2; VH1 and VL2 if the construct has the configuration ofVLl-VHl-VL2-VH2; or VL1 and VL2 if the construct has the configuration of VH1-VL1-VL2-VH2.

[0065] In some embodiments, the a monospecific or bispecific antigen binding domain of any one of any preceding embodiments comprises a linker. In some embodiments, the linker is long enough to avoid mispairing between the domains of the two scFvs. In some embodiments, the linker is a (Gly4-Ser)„ linker, wherein n is 1, 2, 3, 4, 5, or 6 (SEQ ID NO: 54). In some embodiments, the a monospecific or bispecific antigen binding domain comprises a linker, wherein the linker comprises of the amino acid sequence: LAEAAAK (SEQ ID NO: 40).

[0066] The monospecific or bispecific antigen binding domain of any preceding embodiments, wherein a linker is disposed between the VL and VH of the scFv of the first antigen binding domain, e.g., a linker described herein.

[0067] The monospecific or bispecific antigen binding domain of any preceding embodiments, wherein a linker is disposed between the VL and VH of the scFv of the second antigen binding domain, e.g., a linker described herein.

[0068] In some embodiments, the disclosure relates a monospecific or bispecific chimeric antigen receptor (CAR). In some embodiments, the bispecific CAR comprises the bispecific antigen binding domain of any one of the previously disclosed embodiments.

[0069] In some embodiments, the disclosure relates to a nucleic acid construct encoding a a monospecific or bispecific chimeric antigen receptor (CAR). In some embodiments, the nucleic acid construct encodes the bispecific antigen binding domain of any one chimeric antigen receptor (CAR), comprising a bispecific antigen binding domain which comprises: a first antigen binding domain which binds to NYESO-1 and a second antigen binding domain which binds to CD 137 and / or CD3, wherein the CAR comprises a transmembrane domain, a costimulatory domain and / or a primary signaling domain.

[0070] The nucleic acid molecule of any of the preceding embodiments, wherein the protease cleavage site is situated such that a cell can express a fusion protein comprising a first CAR and a second CAR, optionally wherein the fusion protein is processed into two peptides by proteolytic cleavage. A CAR comprising the bispecific antigen binding domain of any one of the preceding embodiments.

[0071] The CAR of the previous embodiment, comprising: a monospecific or bispecific antigen binding domain; a transmembrane domain; and a co-stimulatory signaling domain; a bispecific antigen binding domain; a transmembrane domain; and a primary signaling domain; or a bispecific antigen binding domain; a transmembrane domain; a co-stimulatory signaling domain; and a first primary signaling domain.

[0072] The CAR of any one of the previously disclosed embodiments, wherein the CAR comprises a transmembrane domain, wherein the transmembrane domain is chosen from the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137 or CD154.

[0073] The CAR of any one of the previously disclosed embodiments, wherein the bispecific antigen binding domain is connected to the transmembrane domain by a hinge region, e.g., a hinge region described herein.

[0074] The CAR of any one of the previously disclosed embodiments, wherein the CAR comprises a co- stimulatory domain, wherein the co-stimulatory domain comprises a signaling domain of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la / CD18), ICOS (CD278) or 4-1BB (CD137).

[0075] The CAR of any one of the previously disclosed embodiments, wherein the co- stimulatory domain comprises a 4-1BB signaling domain. The CAR of any one of the previously disclosed embodiments, wherein the CAR comprises a primary signaling domain comprising a signaling domain of CD3 zeta.

[0076] The CAR of any one of the previously disclosed embodiments, wherein the CAR comprises an amino acid sequence SEQ ID NOs: 1, 2, and / or 3, or an amino acid sequence comprising at least about 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0077] A method of making a cell (e.g., an immune effector cell) comprising: transducing an immune effector cell, e.g., a T cell or NK cell, with a vector of embodiment 143; or introducing a CAR nucleic acid molecule of any one of embodiments 129 to 142, into an immune effector cell, e.g., a T cell or NK cell.

[0078] In some embodiments, the disclosure relates to a pharmaceutical composition comprising the nucleic acid encoding the CAR molecule of any one of the previously disclosed embodiments or the bispecific antigen binding domain of any one of embodiments, or the CAR of any one of above identified embodiments.

[0079] In some embodiments, the disclosure relates to a method of providing anti-tumor immunity. In some embodiments, the method comprises administering to a subject in need thereof a therapeutically effective amount of a cell, e.g., a population of immune effector cells, comprising, e.g., expressing, the nucleic acid encoding a CAR molecule of any one of embodiments herein, the bispecific antigen binding domain of any one of embodiments, the CAR of any one of embodiment or the CAR nucleic acid of any one of embodiments.

[0080] In some embodiments, the disclosure relates to a cell, e.g., a population of immune effector cells, comprising, e.g., expressing, the nucleic acid encoding a CAR molecule of any one of the preceding embodiments, the bispecific antigen binding domain of any one of the preceding embodiments of the CAR of any one of the preceding embodiments, or the CAR nucleic acid of for use in a method of providing anti -tumor immunity to a subject.

[0081] The method or the use of the previously disclosed two embodiments, wherein the cell is a T cell or an NK cell. The method or the use of the previously disclosed three embodiments, wherein the cell is a human cell. The method of any one of the preceding four embodiments, wherein the cell is an autologous cell or an allogeneic cell.

[0082] In some embodiments, the disclosure relates to a method of treating a subject having a disease associated with an antigen. In some embodiments, the method comprises administering to the subject in need thereof, an effective amount of a cell, e.g., a population of immune effector cells, comprising, e.g., expressing, a nucleic acid encoding a CAR molecule of any one of the disclosed embodiments, a bispecific antigen binding domain of any one of the disclosed embodiments, the CAR of any one of the disclosed embodiments or the CAR nucleic acid of any one of the disclosed embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0083] The following detailed description of embodiments of the present invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings certain embodiments. It isunderstood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. In the drawings:

[0084] FIGS. 1A through ID depicts a determination of the specificity of [TRACeR] _(MHC-I,A02)A(NY-ESO-1) with different peptides. FIG. 1 A. Alanine scan of the NY-ESO-1 antigen showing the requirement of SI, M4 and W5. Residues SI A, M4A, W5A, QB A are solvent exposed and residues LSI, L3A, 16A, and T7A have side chains buried in MHC-I groove. FIG. 1A includes SEQ ID NO: 57. FIG. IB. X-scan on the three key residues for TRACeR (left) and 1G4 TCR (right), MFI (mean fluorescence intensity) signal was normalized as a percentage of wild type binding signal. S1V peptide synthesis failed due to the hydrophobicity. FIG. 1C. Correlation plot of the binding levels between TRACeR construct with a panel of 97 individual HLA-I allotypes upon incubation with the wild-type NY-ESO-1 or the non-binder NY-ESO-1W5A peptides. The dashed line represents a conceptual 1 : 1 correlation (no difference between the peptides). FIG. ID. Alignment of MHC-I and MR1 structures. NY-ESO-1 peptide is shown in light grey (first position shown as stick) and the 5- OP-RU antigen is shown in grey. A schematic of the tandem constructs of the present disclosure. Each tandem CAR comprises a bispecific antigen binding domain comprising a NYESO-1 antigen binding domain and a CD3 antigen binding domain.

[0085] FIGS. 2A-2F depict TRACeR] _(MHC-I,A02)A(NY-ESO-l)-antiCD3 scFv BiTE mediate target-specific cancer killing and CAR-T killing. FIG. 2A shows a Schematic representation of the BiTE construct. [(TRACeR)]_(MHC-I,A02)A(NY-ESO-l)recognizes NY- ESO-1 antigen presented by surface MHC-I, and the anti-CD3 scFv was used for the T cell binding module. FIG. 2B shows in vitro tumor cell killing assays with TRACeR] (MHC- I,A02)A(NY-ESO-l)-antiCD3 BiTEs or control BiTEs. Activated human total CD3+ cells were mixed 2: 1 with tumor cell lines for 18 hours then analyzed for tumor specific killing by flow cytometry. Frequency of live tumor cells were normalized to PBS treated control cells. One of two independent repeats is shown. FIGS. 2B and 2C show anti-CD3 / anti-CD28 activated total T cells that were incubated with indicated tumor cell lines and treated with TRACeR BiTEs, control bispecific antibodies or PBS for 18 hours. Control bispecific antibodies were used at a concentration of 10 ng / mL. Cells were then isolated and analyzed by flow cytometry. Cells analyzed are live, CD3+CD4+ and CD3+CD8+ cells. FIG. 5C: CD69 and 4-1BB expression in T cells incubated with HBL-1 cells FIG. 2 D: CD69 and 4-1BB expression in T cellsincubated with HLY-1 cells. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test. FIG. 5 is a schematic representation of the [TRACeR] _(MHC-I,A02)A(NY-ESO-1)-CAR T cells recognizes NY-ESO-1 antigen presented by surface MHC-I. 2F. In vitro tumor cell killing assays with [TRACeR] (MHC- I,A02)A(NY-ESO-1)-CAR T cells or control 1G4 TCR. Engineered T cells were mixed 1 : 1 with tumor cell lines for 18 hours then analyzed for tumor specific killing by flow cytometry. Frequency of live tumor cells co-cultured with engineered T cells were normalized to those cocultured with untransduced T cells.

[0086] FIG. 3 depicts circular permutation scheme for redesigning TRACeR scaffold in schematic and crystal forms, along with cell binding data with NY-ESO-1 peptide bound to activating cells.

[0087] FIGS. 4A through 4C depict cross-allele specificity profiling of TRACeR] (MHC- I,A02)A(NY-ESO-1). FIG 4A. Scatterplot of the BLOSUM62 sequence similarity to A*02:01 for HLA residues interfacing with the TRACeR versus the predicted percent rank binding affinity. The black dashed line represents a 5% rank binding affinity denoting a weak binder per NetMHCPan. FIG. 4B. Polymorphisms (dark spheres) present in 10 HLAs (HLA-A*02:06, HLA-A*02:07, HLA-A*02:09, HLA-A*02: ll, HLA-A*02:131, HLA-A*02: 17, HLA- A*02: 18, HLA-A*02:240, HLA-A*02:448, HLA-A*02:642) relative to HLA-A*02:01 mapped onto the TRACeRNY-ESO-l,A02 crystal structure. FIG. 4C. Binding levels of TRACeR construct with a panel of 97 individual HLA-I allotypes upon incubation with the wild-type NY-ESO-1 or the non-binder NY-ESO-1W5A peptides.

[0088] FIGS. 5A and 5B depict Structural alignment of predicted model and crystal structure of TRACeRNY-F.so-i,A02.

[0089] FIG 5A. Alignment of TRACeR helical bundle (yellow and green) with the helical bundle from MANI N terminal domain (grey). FIG 5B. Alignment of TRACeR monomeric binding mode with computational model (model is shown in shadow.

[0090] FIG. 6 depicts SSM analysis of TRACeR] _(MHC-I,A02)A(NY-ESO-1), noncircular permuted. Site saturate mutagenesis library of [TRACeR] _(MHC-I,A02)A(NY- ESO-1) was stained with 10 nM pMHC monomer and top 0.5% binding population was collected as child pool. Enrichment ratio of each mutation was calculated as (sequence count percentage in child pool) / (sequence count percentage in mother pool), normalized based onthe enrichment ratio of wild type. Deep sequencing was performed with Illumina 2*300 Kit at Stanford PAN facility.

[0091] FIG. 7 depicts a comparison of TRACeR^^^0^ to TCR:pHLA-I structures in the Protein Data Bank. TCR:nonamer / HLA-I structural dataset was generated using a modified version of HLA3DB1as described previously. A selection resulted in 67 crystal structures. Complexes were analyzed using PDBePISA2as implemented in CCP4 (v. 8.0)3to obtain peptide / receptor and HLA / receptor interface area values. Scatter plot depicting the interface area between the immune receptor and the HLA (x-axis) or the peptide (y-axis) (black: TCRs, grey: scFvs). The corresponding interface areas of the TRACeR with HLA-A*02:01 and NY- ESO-1 peptide is shown as an triangle.

[0092] FIG. 8 depicts a purification of TRACeR^-^0^- antiCD3 construct. The dimer construct shows cancer killing.

[0093] FIG. 9A through 9E depicts designing dimerized TRACeR into monomer binder. FIG. 9A. Inspired by crystal structure, we redesigned the domain-swapped dimer into a monomer by connecting two monomers and removing one ARE site. Surface residues were redesigned with ProteinMPNN. FIG. 9B. Monomeric TRACeR purification SDS-PAGE gel and SEC curve (superdex 75). Monomeric TRACeR can be easily purified from E.coli and is highly soluble. FIG. 9C. Monomeric TRACeR is still peptide specific based on yeast surface display (staining concentration: 50 nM tetramer) FIG. 9D. Titration of monomeric TRACeR] _(MHC-I,A02)A(NY-ESO-1) on T2 cells pulsing NY-ESO-1 peptide or EBV peptide as negative control. FIG. 9E. The connection scheme to create the rewired monomer. The equivalence to the crystal structure is denoted by the inverted labels on the helices (Hl, H2 and H3).

[0094] FIG. 10A through 10C depict receptor design, expression and tetramer binding of engineered T cell receptors. FIG. 10A. Engineered CAR and TCR receptor construct design. FIG. 10B. Engineered receptor expression 13 days post sorting. Quantification of median fluorescent intensity of anti-myc signal. FIG. 10C. Binding of engineered T cells to cognate HLA-A*02:01 human NY-ESO-1 157-165 C165V SLLMWITQV (SEQ ID NO: 57) or control HLA-A*02:01 EBV LMP2 426-434 CLGGLLTMV (SEQ ID NO: 56) tetramers. Quantification of median fluorescent intensity of each tetramer signal.DETAILED DESCRIPTIONDefinitions

[0095] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains.

[0096] The terms “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0097] The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%„ ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. For recitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6,9, and 7.0 are explicitly contemplated.

[0098] As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in theart. For example, Berge et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences (1977) 66: 1-19.

[0099] The term “Chimeric Antigen Receptor,” a “CAR,” or a “CAR molecule” refers to a set of polypeptides, typically two in the simplest embodiments, which when in an immune effector cell, provides the cell with specificity for a target cell, typically a cancer cell, and with intracellular signal generation. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as “an intracellular signaling domain”) comprising a functional signaling domain derived from a stimulatory molecule and / or costimulatory molecule as defined below. In some embodiments, the set of polypeptides are contiguous with each other, e.g., are in the same polypeptide chain, e.g., comprise a chimeric fusion protein. In some embodiments, theset of polypeptides are not contiguous with each other, e.g., are in different polypeptide chains. In some embodiments, the set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen binding domain to an intracellular signaling domain.

[0100] In some embodiments, the stimulatory molecule is the zeta chain associated with the T cell receptor complex.

[0101] In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below.

[0102] In some embodiments, the costimulatory molecule is chosen from the costimulatory molecules described herein, e.g., 4-IBB (i.e., CD137), CD27 and / or CD28. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule.

[0103] In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule. In some embodiments, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecule(s) and a functional signaling domain derived from a stimulatory molecule.

[0104] In some embodiments, the CAR comprises an optional leader sequence at the aminoterminus (N-ter) of the CAR fusion protein. In some embodiments, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane.

[0105] The term “signaling domain” refers to the functional portion of a protein which acts by transmitting information within the cell to regulate cellular activity via defined signaling pathways by generating second messengers or functioning as effectors by responding to such messengers.

[0106] As used herein, the term “binding domain” refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term “binding domain” (also referred to herein as “antibody molecule”) encompasses antibodies and antibody fragments. In some embodiments an antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In some embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. Abispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0107] The term “antibody fragment” refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hinderance, stabilizing / destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, Fv fragments, scFv antibody fragments, disulfide-linkedFvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide brudge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23 : 1126-1136, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S. Patent No.: 6,703,199, which describes fibronectin polypeptide minibodies). The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising avariable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker- VL.

[0108] The term “complementarity determining region” or “CDR,” as used herein, refers to the sequences of amino acids within antibody variable regions which confer antigen specificity and binding affinity. The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat etal. (1991), “Sequences of Proteins of Immunological Interest,” 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme) and ImMunoGenTics (IMGT) numbering (Lefranc, M.-P, The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) (“IMGT” numbering scheme). For example, for classic formats, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1 ), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia, the CDR amino acids in the VH are numbered 26-32 (HCDR1 ), 52- 56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1 ), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1 ), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1 ), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Under IMGT, the CDR amino acid residues in the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3) (numbering according to “IMGT”). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0109] The portion of the CAR of embodiments herein comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv), a humanized antibody, or bispecific antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In some embodiments, the antigen binding domain of a CAR composition of embodiments herein comprises an antibody fragment. In a further embodiment, the CAR comprises an antibody fragment that comprises a scFv.

[0110] The term “antibody heavy chain” refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.

[0111] The “antibody light chain” refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (x) and lambda (1-) light chains refer to the two major antibody light chain isotypes.

[0112] The term “recombinant antibody” refers to an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology which is available and well known in the art.

[0113] The term “antigen” or “Ag” refers to a molecule that provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. A skilled artisan will understand that any DNA, which comprises a nucleic acid sequence or a partial nucleic acid sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleic acid sequence of a gene. It isreadily apparent that the present disclosure includes, but is not limited to, the use of partial nucleic acid sequences of more than one gene and that these nucleic acid sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated, synthesized, or can be derived from a biological sample, or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a fluid with other biological components.

[0114] The term “anti-cancer effect” refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, decrease in cancer cell proliferation, decrease in cancer cell survival, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-cancer effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of embodiments herein in prevention of the occurrence of cancer in the first place. The term “antitumor effect” refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival. The term “autologous” refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.

[0115] The term “allogeneic” refers to any material derived from a different animal of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some embodiments, allogeneic material from individuals of the same species may be sufficiently unlike genetically to interact antigenically.

[0116] The term “xenogeneic” refers to a graft derived from an animal of a different species.

[0117] The term “combination” refers to either a fixed combination in one dosage unit form, or a combined administration where a compound of the present disclosure and a combination partner (e.g. another drug as explained below, also referred to as “therapeutic agent” or “coagent”) may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e.g. synergistic effect. The single components may be packaged in a kit or separately. One orboth of the components (e.g., powders or liquids) may be reconstituted or diluted to a desired dose prior to administration. The terms “co-administrati on” or“combined administration” orthe like as utilized herein are meant to encompass administration ofthe selected combination partner to a single subject in need thereof (e.g. a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one therapeutic agent and includes both fixed and non-fixed combinations of the therapeutic agents. The term “fixed combination” means that the therapeutic agents, e.g. a compound of the present disclosure and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non- fixed combination” means that the therapeutic agents, e.g. a compound of the present disclosure and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g. the administration of three or more therapeutic agent.

[0118] The term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer and the like. The terms “tumor” and “cancer” are used interchangeably herein, e.g., both terms encompass solid and liquid tumors. As used herein, the term “cancer” or “tumor” includes premalignant, as well as malignant cancers and tumors.

[0119] As used herein, unless otherwise specified, the terms “prevent,” “preventing” and “prevention” refer to an action that occurs before the subject begins to suffer from the condition, or relapse of the condition. Prevention need not result in a complete prevention of the condition; partial prevention or reduction of the condition or a symptom of the condition, or reduction of the risk of developing the condition, is encompassed by this term.

[0120] Administered “in combination,” as used herein, means that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, e.g., the two or more treatments are delivered after the subject has been diagnosedwith the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In some embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered. In some embodiments, the CAR-expressing cell is administered at a dose and / or dosing schedule described herein, and the B-cell inhibitor, or agent that enhances the activity of the CDI 9 CAR-expressing cell is administered at a dose and / or dosing schedule described herein.

[0121] “Derived from” as that term is used herein, indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and a second molecule and does not connote or include a process or source limitation on a first molecule that is derived from a second molecule. For example, in the case of an intracellular signaling domain that is derived from a CD3zeta molecule, the intracellular signaling domain retains sufficient CD3zeta structure such that is has the required function, namely, the ability to generate a signal under the appropriate conditions. It does not connote or include a limitation to a particular process of producing the intracellular signaling domain, e.g., it does not mean that, to provide the intracellular signaling domain, one must start with a CD3zeta sequence and delete unwanted sequence, or impose mutations, to arrive at the intracellular signaling domain.

[0122] The term “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibodyor antibody fragment of embodiments herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CAR of embodiments herein can be replaced with other amino acid residues from the same side chain family and the altered CAR can be tested using the functional assays described herein.

[0123] The term “stimulation,” refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR / CD3 complex or CAR) with its cognate ligand (or tumor antigen in the case of a CAR) thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR / CD3 complex or signal transduction via the appropriate NK receptor or signaling domains of the CAR. Stimulation can mediate altered expression of certain molecules.

[0124] The term “stimulatory molecule,” refers to a molecule expressed by an immune cell, e.g., T cell, NK cell, or B cell, that provides the cytoplasmic signaling sequence(s) that regulates activation of the immune cell in a stimulatory way for at least some embodiment of the immune cell signaling pathway. In some embodiments, the signal is a primary signal that is initiated by, for instance, binding of a TCR / CD3 complex with an MHC molecule loaded with peptide, and which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A primary cytoplasmic signaling sequence (also referred to as a “primary signaling domain”) that acts in a stimulatory manner may contain a signaling motif which is known as immunoreceptor tyrosine-based activation motif or ITAM. Examples of an ITAM containing cytoplasmic signaling sequence that is of particular use in embodiments herein includes, but is not limited to, those derived from CD3 zeta, common FcR gamma (FCERI G), Fc gamma Rlla, FcR beta (Fc Epsilon RI b), CD3 gamma, CD3 delta , CD3 epsilon, CD79a, CD79b, DAPIO, and DAP12. In a specific CAR of embodiments herein,the intracellular signaling domain in any one or more CARS of embodiments herein comprises an intracellular signaling sequence, e.g., a primary signaling sequence of CD3-zeta. In a specific CAR of embodiments herein, the primary signaling sequence of CD3-zeta is the sequence, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.

[0125] The term “antigen presenting cell” or “APC” refers to an immune system cell such as an accessory cell (e.g., a B-cell, a dendritic cell, and the like) that displays a foreign antigen complexed with major histocompatibility complexes (MHC’s) on its surface. T-cells may recognize these complexes using their T-cell receptors (TCRs). APCs process antigens and present them to T-cells.

[0126] “Immune effector cell,” as that term is used herein, refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response. Examples of immune effector cells include T cells, e.g., alpha / beta T cells and gamma / delta T cells, B cells, natural killer (NK) cells, natural killer T (NK-T) cells, mast cells, and myeloid-derived phagocytes.

[0127] “Immune effector function or immune effector response,” as that term is used herein, refers to function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell. E.g., an immune effector function or response refers a property of a T or NK cell that promotes killing or the inhibition of growth or proliferation, of a target cell. In the case of a T cell, primary stimulation and co-stimulation are examples of immune effector function or response.

[0128] An “intracellular signaling domain,” as the term is used herein, refers to an intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes an immune effector function of the CAR containing cell, e.g., a CART cell or CAR-expressing NK cell. Examples of immune effector function, e.g., in a CART cell or CAR-expressing NK cell, include cytolytic activity and helper activity, including the secretion of cytokines.

[0129] In some embodiments, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from the molecules responsible for primary stimulation, or antigen dependent simulation. In some embodiments, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals, or antigen independent stimulation. For example, in the case of a CART, a primary intracellular signalingdomain can comprise a cytoplasmic sequence of a T cell receptor, and a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule.

[0130] A primary intracellular signaling domain can comprise a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAM- containing primary cytoplasmic signaling sequences include, but are not limited to, those derived from CD3 zeta, common FcR gamma (FCER1 G), Fc gamma Rlla, FcR beta (Fc Epsilon R1 b), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DAP1O and DAP12.

[0131] The term “zeta” or alternatively “zeta chain,” “CD3-zeta,” or “TCR-zeta” is defined as the protein provided as GenBan Acc. No. BAG36664. 1, or the equivalent residues from a nonhuman species, e.g., mouse, rodent, monkey, ape and the like, and a “zeta stimulatory domain” or alternatively a “CD3-zeta stimulatory domain” or a “TCR-zeta stimulatory domain” is defined as the amino acid residues from the cytoplasmic domain of the zeta chain or functional derivative thereof, that are sufficient to functionally transmit an initial signal necessary for T cell activation. In some embodiments, the cytoplasmic domain of zeta comprises residues 52 through 164 of GenBank Acc. No. BAG36664. 1 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like, that are functional orthologs thereof. In some embodiments, the “zeta stimulatory domain” or a “CD3-zeta stimulatory domain” is the sequence provided as SEQ ID NO: 96.

[0132] The term “costimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules include, but are not limited to an MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, 64ignalling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1(CDI la / CD18), 4-IBB (CD137), B7-H3, CDS, ICAM-1, ICOS 5 (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRFI), NKp44, NKp30, NKp46, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLAI, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDI Id, ITGAE, CD 103, ITGAL, CDI la, lb, ITGAX, CDI le, ITGBI, CD29, ITGB2,CD 18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAMI (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAMI, CRTAM, Ly9 (CD229), CD 160 (BY55), PSGLI, CDI00 (SEMA4D), CD69, SLAMF6 (NTB-A, LylO8), SLAM (SLAMFI, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD 19a, and a ligand that specifically binds with CD83.

[0133] A costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. A costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-IBB (CD 137), 0X40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-I (LFA-1), CD2, 25CSD, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and a ligand that specifically binds with CD83, and the like.

[0134] The intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment or derivative thereof.

[0135] The term “4-IBB” refers to a member of the TNFR superfamily with an amino acid sequence provided as GenBank Acc. No. AAA62478.2, or the equivalent residues from a nonhuman species, e.g., mouse, rodent, monkey, ape and the like; and a “4-IBB costimulatory domain” is defined as amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like. In some embodiments, the “4-IBB costimulatory domain” is the sequence provided as SEQ ID NO:4 or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape and the like.

[0136] The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleic acid sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0137] Unless otherwise specified, a “nucleic acid sequence encoding an amino acid sequence” includes all nucleic acid sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleic acid sequence that encodes a protein or a RNA may also include introns to the extent that the nucleic acid sequence encoding the protein may in some version contain an intron(s).

[0138] The term “effective amount” or “therapeutically effective amount” are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result.

[0139] The term “endogenous” refers to any material from or produced inside an organism, cell, tissue or system.

[0140] The term “exogenous” refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0141] The term “expression” refers to the transcription and / or translation of a particular nucleic acid sequence driven by a promoter.

[0142] The term “transfer vector” refers to a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.

[0143] Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “transfer vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to further include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, a polylysine compound, liposome, and the like. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.

[0144] The term “expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleic acid sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids,plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno- associated viruses) that incorporate the recombinant polynucleotide.

[0145] The term “lentivirus” refers to a genus of the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a significant amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.

[0146] The term “lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Mil one et al., Mol. Then 17(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic, include but are not limited to, e.g., the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™ vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.

[0147] The “percent identity” or “percent homology” of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. “Identical” or “identity” as used herein in the context of two or more nucleic acids or amino acid sequences, may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may he performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0. Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity. Software for performing BLAST analyses is publicly available through the NationalCenter for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length Win the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negativescoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873- 5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to another if the smallest sum probability in comparison of the test nucleic acid to the other nucleic acid is less than about 1, less than about 0.1, less than about 0.01, and less than about 0.001. Two singlestranded polynucleotides are “the complement” of each other if their sequences can be aligned in an anti-parallel orientation such that every nucleotide in one polynucleotide is opposite its complementary nucleotide in the other polynucleotide, without the introduction of gaps, and without unpaired nucleotides at the 5’ or the 3’ end of either sequence. A polynucleotide is “complementary” to another polynucleotide if the two polynucleotides can hybridize to one another under moderately stringent conditions. Thus, a polynucleotide can be complementary to another polynucleotide without being its complement.

[0148] term “homologous” or “identity” refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA moleculesor two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomeric subunit; e.g., if a position in each of two DNA molecules is occupied by adenine, then they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions; e.g., if half (e.g., five positions in a polymer ten subunits in length) of the positions in two sequences are homologous, the two sequences are 50% homologous; if 90% of the positions (e.g., 9 of I 0), are matched or homologous, the two sequences are 90% homologous.

[0149] “Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2 or other antigen-binding subsequences of antibodies), which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies and antibody fragments thereof are human immunoglobulins (recipient antibody or antibody fragment) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antib ody / antibody fragment can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance. In general, the humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or a significant portion of the FR regions are those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region(Fe), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321 : 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992.

[0150] “Fully human” refers to an immunoglobulin, such as an antibody or antibody fragment, where the whole molecule is of human origin or consists of an amino acid sequence identical to a human form of the antibody or immunoglobulin.

[0151] “Murine” refers to mice or rats. For example, a murine antibody or fragment thereof contains the sequence of an antibody or fragment thereof that is isolated from a murine animal, e.g., mouse or rat.

[0152] The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0153] In the context of the present disclosure, the following abbreviations for the commonly occurring nucleic acid bases are used. “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0154] The term “operably linked” or “transcriptional control” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join two protein coding regions, are in the same reading frame.

[0155] The term “parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, intratumoral, or infusion techniques.

[0156] The term “nucleic acid” or “polynucleotide” refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementarity sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / ordeoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0157] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. A polypeptide includes a natural peptide, a recombinant peptide, or a combination thereof.

[0158] The term “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence.

[0159] The term “promoter / regulatory sequence” refers to a nucleic acid sequence that is required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that expresses the gene product in a tissue specific manner.

[0160] The term “constitutive” promoter refers to a nucleic acid sequence that, when operably linked with a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0161] The term “inducible” promoter refers to a nucleic acid sequence that, when operably linked with a polynucleotide that encodes or specifies a gene product, causes the gene product to 10 be produced in a cell substantially only when an inducer that corresponds to the promoter is present in the cell.

[0162] The term “tissue-specific” promoter refers to a nucleic acid sequence that, when operably linked with a polynucleotide that encodes or specifies a gene product, causes the geneproduct to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0163] The term “flexible polypeptide linker” or “linker” as used in the context of a scFv refers to a peptide linker that consists of amino acids such as glycine and / or serine residues used alone or in combination, to link variable heavy and variable light chain regions together. In some embodiments, the flexible polypeptide linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser) (SEQ ID NO: 52), repeated n times where n is a positive integer equal to or greater than 1. For example, n=l, n=2, n=3, n=4, n=5, n=6, n=7, n=8, n=9 or n=10. In some embodiments, the flexible polypeptide linker is (Gly4 Ser)3 (SEQ ID NO: 42). In some embodiments, the linkers include multiple repeats of (Gly2Ser), and (GlySer). In some embodiments, the polypeptide does not include a linker, e.g., (n=0). Also included within the scope of embodiments herein are linkers described in WO2012 / 138475, incorporated herein by reference).

[0164] As used herein, a 5’ cap (also termed an RNA cap, an RNA 7-methylguanosine cap or an RNA m7G cap) is a modified guanine nucleotide that has been added to the “front” or 5’ end of a eukaryotic messenger RNA shortly after the start of transcription. The 5’ cap consists of a terminal group which is linked to the first transcribed nucleotide. Its presence is critical for recognition by the ribosome and protection from RNAses. Cap addition is coupled to transcription, and occurs co-transcriptionally, such that each influences the other. Shortly after the start of transcription, the 5’ end of the mRNA being synthesized is bound by a capsynthesizing complex associated with RNA polymerase. This enzymatic complex catalyzes the chemical reactions that are required for mRNA capping. Synthesis proceeds as a multi-step biochemical reaction. The capping moiety can be modified to modulate functionality of mRNA such as its stability or efficiency of translation.

[0165] As used herein, “in vitro transcribed RNA” refers to RNA, preferably mRNA, that has been synthesized in vitro. Generally, the in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector comprises a template that is used to generate the in vitro transcribed RNA

[0166] As used herein, a “poly(A)” is a series of adenosines attached by polyadenylation to the mRNA. In some embodiments of a construct for transient expression, the polyA is between 50 and 5000 (SEQ ID NO: 58), preferably greater than 64, more preferably greater than 100, most preferably greater than 300 or 400. Poly(A) sequences can be modified chemically orenzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.

[0167] As used herein, “polyadenylation” refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule. In eukaryotic organisms, most messenger RNA (mRNA) molecules are polyadenylated at the 3’ end. The 3’ poly(A) tail is a long sequence of adenine nucleotides (often several hundred) added to the pre- mRNA through the action of an enzyme, polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal. The poly(A) tail and the protein bound to it aid in protecting mRNA from degradation by exonucleases. Polyadenylation is also important for transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation occurs in the nucleus immediately after transcription of DNA into RNA, but additionally can also occur later in the cytoplasm. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3’ end at the cleavage site.

[0168] As used herein, “transient” refers to expression of a non-integrated transgene for a period of hours, days or weeks, wherein the period of time of expression is less than the period of time for expression of the gene if integrated into the genome or contained within a stable plasmid replicon in the host cell.

[0169] As used herein, the terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the progression, severity and / or duration of a proliferative disorder, or the amelioration of one or more symptoms (preferably, one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more therapies (e.g., one or more therapeutic agents such as a CAR of embodiments herein). In specific embodiments, the terms “treat”, “treatment” and “treating” refer to the amelioration of at least one measurable physical parameter of a proliferative disorder, such as growth of a tumor, not necessarily discernible by the patient. In some embodiments, the terms “treat”, “treatment” and “treating” -refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In some embodiments, the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size or cancerous cell count.

[0170] The term “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the membrane of a cell.

[0171] The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human).

[0172] The term, a “substantially purified” cell refers to a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0173] The term “therapeutic” as used herein means a treatment. A therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.

[0174] The term “prophylaxis” as used herein means the prevention of or protective treatment for a disease or disease state.

[0175] In the context of the present disclosure, “tumor antigen” or “hyperproliferative disorder “antigen” or “antigen associated with a hyperproliferative disorder” refers to antigens that are common to specific hyperproliferative disorders. In certain embodiments, the hyperproliferative disorder antigens of the present disclosure are derived from, cancers including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non- Hodgkin’s lymphoma, non-Hodgkins lymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, New York esophageal squamous cell carcinoma 1. and the like.

[0176] The terms “transfected” or “transformed” or “transduced” refer to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0177] The term “specifically binds,” refers to an antibody, or a ligand, which recognizes and binds with a binding partner (e.g., a stimulatory tumor antigen) protein present in a sample, but which antibody or ligand does not substantially recognize or bind other molecules in the sample.

[0178] “Refractory” as used herein refers to a disease, e.g., cancer, that does not respond to a treatment. In some embodiments, a refractory cancer can be resistant to a treatment before or at the beginning of the treatment. In some embodiments, the refractory cancer can become resistant during a treatment. A refractory cancer is also called a resistant cancer.

[0179] A subject “responds” to treatment if a parameter of a cancer (e.g., a hematological cancer, e.g., cancer cell growth, proliferation and / or survival) in the subject is retarded or reduced by a detectable amount, e.g., about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as determined by any appropriate measure, e.g., by mass, cell count or volume. In one example, a subject responds to treatment if the subject experiences a life expectancy extended by about 5%, 10%, 20%, 30%, 40%, 50% or more beyond the life expectancy predicted if no treatment is administered. In another example, a subject responds to treatment, if the subject has an increased disease-free survival, overall survival or increased time to progression. Several methods can be used to determine if a patient responds to a treatment including, for example, criteria provided by NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®). For example, in the context of BALL, a complete response or complete responder, may involve one or more of < 5% BM blast, >1000 neutrophil / ANC ( / pL). >100,000 platelets ( / pL) with no circulating blasts or extramedullary disease (no lymphadenopathy, splenomegaly, skin / gum infiltration / testicular mass / CNS involvement), Trilineage hematopoiesis, and no recurrence for 4 weeks. A partial responder may involve one or more of >50% reduction in BM blast, >1000 neutrophil / ANC ( / pL). >100,000 platelets ( / pL). A non-responder can show disease progression, e.g.,> 25% in BM blasts. In some embodiments, a complete responder is defined as having 7% or greater CD27+ CD45RO- cells in the CD8+ population. In some embodiments, the percent of CAR+ cells at pre- harvest levels distinguish responders (e.g., complete responders and partial responders) from non-responders (NR).

[0180] The term “relapse” as used herein refers to reappearance of a cancer after an initial period of responsiveness (e.g., complete response or partial response). The initial period of responsiveness may involve the level of cancer cells falling below a certain threshold, e.g.,below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. The reappearance may involve the level of cancer cells rising above a certain threshold, e.g., above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, e.g., in the context of B-ALL, the reappearance may involve, e.g., a reappearance of blasts in the blood, bone marrow (> 5%), or any extramedullary site, after a complete response. A complete response, in this context, may involve < 5% BM blast. More generally, in an embodiment, a response (e.g., complete response or partial response) can involve the absence of detectable MRD (minimal residual disease). In some embodiments, the initial period of responsiveness lasts at least 1, 2, 3, 4, 5, or 6 days; at least 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10, or 12 months; or at least 1, 2, 3, 4, or 5 years.

[0181] “Regulatable chimeric antigen receptor (RCAR),”as that term is used herein, refers to a set of polypeptides, typically two in the simplest embodiments, which when in a RCARX cell, provides the RCARX cell with specificity for a target cell, typically a cancer cell, and with regulatable intracellular signal generation or proliferation, which can optimize an immune effector property of the RCARX cell. An RCARX cell relies at least in part, on an antigen binding domain to provide specificity to a target cell that comprises the antigen bound by the antigen binding domain. In some embodiments, an RCAR includes a dimerization switch that, upon the presence of a dimerization molecule, can couple an intracellular signaling domain to the antigen binding domain.

[0182] “Membrane anchor” or “membrane tethering domain,” as that term is used herein, refers to a polypeptide or moiety, e.g., a myristoyl group, sufficient to anchor an extracellular or intracellular domain to the plasma membrane.

[0183] “Switch domain,” as that term is used herein, e.g., when referring to an RCAR, refers to an entity, typically a polypeptide-based entity, that, in the presence of a dimerization molecule, associates with another switch domain. The association results in a functional coupling of a first entity linked to, e.g., fused to, a first switch domain, and a second entity linked to, e.g., fused to, a second switch domain. A first and second switch domain are collectively referred to as a dimerization switch. In some embodiments, the first and second switch domains are the same as one another, e.g., they are polypeptides having the same primary amino acid sequence, and are referred to collectively as a homodimerization switch. In some embodiments, the first and second switch domains are different from one another, e.g., they are polypeptides having different primary amino acid sequences, and are referred to collectively as a heterodimerization switch. In some embodiments, the switch is intracellular.In some embodiments, the switch is extracellular. In some embodiments, the switch domain is a polypeptide-based entity, e.g., FKBP or FRB-based, and the dimerization molecule is small molecule, e.g., a rapalogue. In some embodiments, the switch domain is a polypeptide-based entity, e.g., an scFv that binds a myc peptide, and the dimerization molecule is a polypeptide, a fragment thereof, or a multimer of a polypeptide, e.g., a myc ligand or multimers of a myc ligand that bind to one or more myc scFvs. In some embodiments, the switch domain is a polypeptide-based entity, e.g., myc receptor, and the dimerization molecule is an antibody or fragments thereof, e.g., myc antibody.

[0184] “Dimerization molecule,” as that term is used herein, e.g., when referring to an RCAR, refers to a molecule that promotes the association of a first switch domain with a second switch domain. In some embodiments, the dimerization molecule does not naturally occur in the subject, or does not occur in concentrations that would result in significant dimerization. In some embodiments, the dimerization molecule is a small molecule, e.g., rapamycin or a rapalogue, e.g, RAD001.

[0185] The term “bioequivalent” refers to an amount of an agent other than the reference compound (e.g., RAD00I), required to produce an effect equivalent to the effect produced by the reference dose or reference amount of the reference compound (e.g., RAD001). In some embodiments the effect is the level of mTOR inhibition, e.g., as measured by P70 S6 kinase inhibition, e.g., as evaluated in an in vivo or in vitro assay, e.g., as measured by an assay described herein, e.g., the Boulay assay, or measurement of phosphorylated S6 levels by western blot. In some embodiments, the effect is alteration of the ratio of PD- 1 positive / PD- 1 negative T cells, as measured by cell sorting. In some embodiments a bioequivalent amount or dose of an mTOR inhibitor is the amount or dose that achieves the same level of P70 S6 kinase inhibition as does the reference dose or reference amount of a reference compound. In some embodiments, a bioequivalent amount or dose of an mTOR inhibitor is the amount or dose that achieves the same level of alteration in the ratio of PD- 1 positive / PD- 1 negative T cells as does the reference dose or reference amount of a reference compound.

[0186] The term “low, immune enhancing, dose” when used in conjunction with an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD00I or rapamycin, or a catalytic mTOR inhibitor, refers to a dose of mTOR inhibitor that partially, but not fully, inhibits mTOR activity, e.g., as measured by the inhibition of P70 S6 kinase activity. Methods for evaluating Mtor activity, e.g., by inhibition of P70 S6 kinase, are discussed herein. The dose is insufficient toresult in complete immune suppression but is sufficient to enhance the immune response. In some embodiments, the low, immune enhancing, dose of mTOR inhibitor results in a decrease in the number of PD-1 positive T cells and / or an increase in the number of PD-1 negative T cells, or an increase in the ratio of PD-1 negative T cells / PD- 1 positive T cells. In some embodiments, the low, immune enhancing, dose of mTOR inhibitor results in an increase in the number of naive T cells. In some embodiments, the low, immune enhancing, dose of mTOR inhibitor results in one or more of the following: an increase in the expression of one or more of the following markers: CD62Lhigh, 5 CD127high, CD27+, and BCL2, e.g., on memory T cells, e.g., memory T cell precursors; a decrease in the expression of KLRG1, e.g., on memory T cells, e.g., memory T cell precursors; and an increase in the number of memory T cell precursors, e.g., cells with any one or combination of the following characteristics: increased CD62Lhigh, increased CD127high, increased CD27+, decreased KLRG1, and increased BCL2; wherein any of the changes described above occurs, e.g., at least transiently, e.g., as compared to a non-treated subject.

[0187] “Variant” used herein with respect to a nucleic acid means a nucleic acid sequence comprising (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid sequence that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid sequence that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto. “Variant” with respect to a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, truncation, conservative substitution of amino acids, or addition of at least one amino acid as compared to a reference sequence, but the peptide or polypeptide retains at least one biological activity of the reference sequence upon which it is based. Variant may also mean a protein with an amino acid sequence that is substantially identical to a referenced protein with an amino acid sequence that retains at least one biological activity. A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol. 157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. It is known in the art that amino acids of similar hydropathic indexes can be substitutedand still retain protein function. In one aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a peptide permits calculation of the greatest local average hydrophilicity of that peptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No. 4,554,101, incorporated fully herein by reference. Substitution of amino acids having similar hydrophilicity values can result in peptides retaining biological activity, for example immunogenicity, as is understood in the art. Substitutions may be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties. Nucleic acid molecules or nucleic acid sequences of the disclosure include those that encode amino acid sequences comprising one or more of: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and variants or functional fragments thereof that possess no less than about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the coding sequences of the foregoing. The term “variant” includes polypeptides conjugated to a non-natural chemical moieties or variants. In some embodiments, the polypeptide comprises a polymer, such as polyethylene glycol, and may be comprised of one or more additional derivatizations of cysteine, lysine, or other residues. In addition, variants of the instant disclosure may comprise a linker or polymer, wherein the amino acid to which the linker or polymer is conjugated may be a non-natural amino acid or may be conjugated to a naturally encoded amino acid utilizing techniques known in the art such as coupling to lysine or cysteine. Polymer modification of polypeptides has been reported. U.S. Pat. No. 4,904,584 discloses PEGylated lysine depleted polypeptides, wherein at least one lysine residue has been deleted or replaced with any other amino acid residue. WO 99 / 67291 discloses a process for conjugating a protein with PEG, wherein at least one amino acid residue on the protein is deleted and the protein is contacted with PEG under conditions sufficient to achieve conjugation to the protein.

[0188] The term variant also includes glycosylated variants, such as but not limited to, variants glycosylated at any amino acid position, N-linked or O-linked glycosylated forms of the polypeptide. In addition, splice variants are also included. The term variant also includes heterodimers, homodimers, heteromultimers, or homomultimers of any one or more polypeptide, protein, carbohydrate, polymer, small molecule, linker, ligand, or other biologically active molecule of any type, linked by chemical means or expressed as a fusion protein, as well as polypeptide variants containing, for example, specific deletions or other modifications yet maintain biological activity. In some embodiments, the first CAR comprises about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity (or homology) to the sequence identifiers used herein.

[0189] Ranges: throughout this disclosure, various embodiments can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of embodiments herein. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity, includes something with 95%, 96%, 97%, 98% or 99% sequence identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% sequence identity. This applies regardless of the breadth of the range.

[0190] The disclosure provides, at least in part, novel nucleic acid molecules encoding Chimeric Antigen Receptor (CAR) molecules comprising a first CAR comprising NYESO-1 CAR and a second CAR comprising a ligand to a costimulatory molecule or immunologically active protein, e.g., dual CARs as described herein. In some embodiments, the first CAR comprises an antigen binding domain, and a first transmembrane domain; a first co-stimulatory signaling domain; and / or a first primary signaling domain. In some embodiments, the second CAR comprises an antigen binding domain, and a second transmembrane domain; a second co-stimulatory signaling domain; and / or a second primary signaling domain. In some embodiments of a CAR molecule disclosed herein, the CAR molecule comprises two identical polypeptide sequences, e.g., of a first and second transmembrane domain; a first and secondco-stimulatory domain; and / or a first and second primary signaling domain, the polypeptide sequences of which are encoded by different nucleotide sequences. Also disclosed herein are methods of using said CARmolecules.

[0191] Without wishing to be bound by theory, it is believed that in some embodiments, a nucleic acid molecule encoding a CAR molecule, e.g., a dual CAR molecule, is optimized, e.g., codon optimized, to prevent recombination, e.g., homologous recombination. In some embodiments, a CAR molecule, e.g., a dual CAR molecule, comprises two domains, e.g., a first transmembrane domain and a second transmembrane domain, each of which comprises a similar amino acid sequence but is encoded by a different nucleotide sequence.

[0192] In some embodiments, a CAR molecule disclosed herein comprises a first CAR comprising a first antigen binding domain which binds a first transmembrane domain; a first co- stimulatory signaling domain; and / or a first primary signaling domain.

[0193] In some embodiments, the first antigen binding domain comprises one or more (e.g., all three) light chain complementarity determining region I (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of a binding domain described herein; and / or one or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR1 ), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of a binding domain described herein. In some embodiments, the antigen binding domain comprises a LC CDRI, LC CDR2 and LC CDR3 of a binding domain described herein; and / or a HC CDRI, HC CDR2 and HC CDR3 of a binding domain described herein.

[0194] In some embodiments, a CAR molecule disclosed herein comprises a first CAR comprising a first transmembrane domain and a second CAR comprising a second transmembrane domain. In some embodiments, the first transmembrane domain and the second transmembrane domain comprise the same amino acid sequence, e.g., as disclosed herein. In some embodiments, the first transmembrane domain and the second transmembrane domain are encoded by a first nucleotide sequence and a second nucleotide sequence, respectively. In some embodiments, the first nucleotide sequence and the second nucleotide sequence differ by at least one nucleotide.

[0195] In some embodiments, the first transmembrane domain and the second transmembrane domain are the same transmembrane domain, e.g., chosen from the alpha, beta or zeta chain ofthe T-cell receptor, CD28, CD3 epsilon, CD4S, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137 or CDIS4.

[0196] In some embodiments, the first transmembrane domain and the second transmembrane domain are different transmembrane domains, e.g., chosen from the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD4S, CD4, CDS, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137 or CDIS4.

[0197] In some embodiments, a nucleic acid molecule encoding a CAR molecule described herein comprises a first CAR comprising a first transmembrane domain and a second CAR comprising a second transmembrane domain. In some embodiments, the first transmembrane domain and the second transmembrane domain comprise the CD8 alpha transmembrane domain. In some embodiments, the first transmembrane domain and the second transmembrane domain comprise the amino acid sequence of SEQ ID NO: 5 or an amino acid sequence with at least about 90% identity thereto.

[0198] In some embodiments, a nucleotide sequence that encodes the first transmembrane domain and is comprised in the nucleic acid molecule is different from a nucleotide sequence that encodes the second transmembrane domain and is comprised in the nucleic acid molecule.

[0199] In some embodiments, a CAR molecule disclosed herein comprises a first CAR comprising a first co-stimulatory domain and a second CAR comprising a second costimulatory domain. In some embodiments, the first co-stimulatory domain and the second costimulatory domain comprise the same amino acid sequence, e.g., as disclosed herein. In some embodiments, the first co-stimulatory domain and the second co-stimulatory domain are encoded by a first nucleotide sequence and a second nucleotide sequence, respectively. In some embodiments, the first nucleotide sequence and the second nucleotide sequence differ by at least one nucleotide.

[0200] In some embodiments, the first co-stimulatory domain and the second co-stimulatory domain are the same co-stimulatory domain, e.g., chosen from a signaling domain of 0X40, CD2, CD27, CD28, CD5, ICAM-1, LFA-1 (CD1 la / CD18), ICOS (CD278) or 4-1BB (CD137).

[0201] In some embodiments, the first co-stimulatory domain and the second co-stimulatory domain are different co-stimulatory domains, e.g., chosen from a signaling domain of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD1 la / CD18), COS (CD278) or 4-1BB (CD137).

[0202] In some embodiments, a nucleic acid molecule encoding a CAR molecule described herein comprises a first CAR comprising a first co-stimulatory domain and a second CAR comprising a second co-stimulatory domain. In some embodiments, the first co-stimulatory domain and the second co-stimulatory domain comprise a 4-1BB co-stimulatory domain.

[0203] In some embodiments, a nucleotide sequence that encodes the first co-stimulatory domain and is comprised in the nucleic acid molecule is different from a nucleotide sequence that encodes the second co-stimulatory domain and is comprised in the nucleic acid molecule.

[0204] In some embodiments, the present disclosure provides a nucleic acid molecule encoding a CAR molecule, e.g., comprising (i) a first CAR comprising a NYESO-1 antigen binding domain and (ii) a second CAR comprising a second antigen binding domain. In some embodiments, the nucleic acid comprises RNAor DNAIn some embodiments, the nucleic acid sequences encoding (i) and (ii) are situated in the same orientation, e.g., transcription of the nucleic acid sequences encoding (i) and (ii) proceeds in the same direction. In some embodiments, the nucleic acid sequences encoding (i) and (ii) are situated in different orientations. In some embodiments, a single promoter controls expression of the nucleic acid sequences encoding (i) and (ii). In some embodiments, a nucleic acid encoding a protease cleavage site (such as a T2A, P2A, E2A, or F2A cleavage site) is situated between the nucleic acid sequences encoding (i) and (ii). In some embodiments, the protease cleavage site is placed such that a cell can express a fusion protein comprising (i) and (ii), which protein is subsequently processed into two peptides by proteolytic cleavage. In some embodiments, the nucleic acid sequences encoding (i) is upstream of the nucleic acid sequences encoding (ii), or the nucleic acid sequences encoding (ii) is upstream of the nucleic acid sequences encoding (i). In some embodiments, a first promoter controls expression of the nucleic acid sequence encoding (i) and a second promoter controls expression of the nucleic acid sequence encoding (ii). In some embodiments, the nucleic acid is a plasmid. In some embodiments, the nucleic acid comprises a viral packaging element. In some embodiments, the present disclosure provides a cell, e.g., an immune effector cell, comprising the nucleic acid described herein, e.g., a nucleic acid comprising (i) and as described above. The cell may comprise a protease (e.g., endogenous or exogenous) that cleaves a T2A, P2A, E2A, or F2A cleavage site.

[0205] Exemplary nucleotide and amino acid sequences of a CAR molecule, e.g., dual CAR molecule disclosed herein is provided in Table 1 A.Table 1A: Single and Dual CAR sequences

[0206] Table 2 provides nucleotide and amino acid sequences for additional CAR components, e.g., signal peptide, linkers and P2A sites.Table 2: Additional CAR componentsTandem CARS

[0207] In some embodiments, the disclosure relates to CARs comprising a bispecific antigen binding domain, e.g., tandem CARs. In some embodiments, a bispecific antigen binding domain comprises two antigen binding domains, e.g., a first antigen binding domain and a second antigen binding domain. In some embodiments of the bispecific antigen binding domain, the first antigen binding domain is an antibody molecule, e.g., an antibody binding domain (e.g., a scFv). In some embodiments of the bispecific antigen binding domain, the second antigen binding domain is an antibody molecule, e.g., an antibody binding domain (e.g., a scFv). Within each antibody molecule, e.g., scFv, of the bispecific antigen binding domain, the VH can be upstream or downstream of the VL.4

[0208] In some embodiments, the disclosure relates to a composition comprising a CAR, or a nucleic acid sequence encoding a CAR, the CAR comprising an antigen binding domain that comprises an amino acid sequence comprising SEQ ID NO: 1, SEQ ID NO:2 and / or SEQ ID NO:3; or a functional variant of SEQ ID NO: 1, SEQ ID NO:2, and / or SEQ ID NO:3 that comprises about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1, SEQ ID NO:2 and / or SEQ ID NO:3, respectively. In some embodiments, the CAR comprises an amino acid sequence of Table 2 or is encoded by a nucleic acid of Table 2. In some embodiments, the CAR comprises a functional variant of the amino acid sequences of Table 2 that comprise that comprises about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acids of Table 2. In some embodiments, the CAR comprises SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:36, or a functional variant that comprises about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:32, SEQ ID NO:34and / or SEQ ID NO:36. In some embodiments, the CAR comprises SEQ ID NO:32, SEQ ID NO:34 or SEQ ID NO:36, wherein position 14 of each of the aforementioned seqeunces is a K in place of Q, or a functional variant that comprises about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:32, SEQ ID NO:34 and / or SEQ ID NO:36, wherein position 14 of each of the aforementioned seqeunces is a K in place of Q. In some embodiments, the CAR comprises the sequence SLLMWITQV (SEQ ID NO: 57) or a functional variant comprising 88% sequence identity to SLLMWITQV (SEQ ID NO: 57).

[0209] In some embodiments, the CAR comprisesDKEIAKEIFNMMFMLLWRVFRSQRIDANNVELIKFNIRVLDWIMAEADNDLCYFIGT HDKCENPKEQWVANYQNLNNVVFTNKELEDIYDLSNKEETKEVLKKFKEKVNQFY RHAFDIINKYG (SEQ ID NO: 60); orSVEEIKKEYEERLKRFDEFVERILKETGNKEIANMARMLLWRVERSYRIDKDNVELIK FNIRVIDWIMAEAENDLCYFIGTHDI<CENPI<EQWVANYQNLNNVVFTNI<ELEDIYDE SNKEETKEVLKKFKEKVNQFYEHAFDIINKYGDKEIFNMMFMLLWRVFRSFRIDANN VELIKFNIRVLDWIMAEADNDLSYFISQ (SEQ ID NO: 61); or a functional variant that comprises about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequences identified above.

[0210] In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VHI) upstream of its VL (VLI) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VL2) upstream of its VH (VH2), such that the overall bispecific antibody molecule has the arrangement VH1-VL1-VL2-VH2, from an N- to C- terminal orientation.

[0211] In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VLI) upstream of its VH (VHI) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH2) upstream of its VL (VL2), such that the overall bispecific antibody molecule has the arrangement VL1-VH1-VH2-VL2, from an N- to C- terminal orientation.

[0212] In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VL (VLI) upstream of its VH (VHI) and the downstream antibody or antibodyfragment (e.g., scFv) is arranged with its VL (VL2) upstream of its VH (VH2), such that the overall bispecific antibody molecule has the arrangement VL1-VH1-VL2-VH2, from an N- to C- terminal orientation. In yet other embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VHI) upstream of its VL (VLI) and the downstream antibody or antibody fragment (e.g., scFv) is arranged with its VH (VH2) upstream of its VL (VL2), such that the overall bispecific antibody molecule has the arrangement VH1-VL1-VH2- VL2, from an N- to C- terminal orientation.

[0213] In any of the aforesaid configurations, optionally, a linker is disposed between the two antibodies or antibody fragments (e.g., scFvs), e.g., between VLI and VL2 if the construct is arranged as VH1-VL1-VL2-VH2; between VHI and VH2 if the construct is arranged as VLI- VH1-VH2-VL2; between VHI and VL2 if the construct is arranged as VL1-VH1-VL2-VH2; or between VLI and VH2 if the construct is arranged as VHI -VLI -VH2-VL2. In general, the linker between the two scFvs should be long enough to avoid mispairing between the domains of the two scFvs. The linker may be a linker as described herein. In some embodiments, the linker is a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6 (SEQ ID NO: 54). In some embodiments, the linker is (Gly4-Ser)n (SEQ ID NO: 62), wherein n = 1, e.g., the linker has the amino acid sequence Gly4-Ser. In some embodiments, the linker is (Gly4-Ser)n, wherein n= 4 (SEQ ID NO: 51). In some embodiments, the linker comprises, e.g., consists of, the amino acid sequence: LAEAAAK (e.g., SEQ ID NO: 40).

[0214] In any of the aforesaid configurations, optionally, a linker is disposed between the VL and VH of the first scFv. Optionally, a linker is disposed between the VL and VH of the second scFv. In constructs that have multiple linkers, any two or more of the linkers can be the same or different. Accordingly, in some embodiments, a bispecific CAR comprises VLs, VHs, and optionally one or more linkers in an arrangement as described herein.

[0215] In some embodiments, each antibody molecule, e.g., each antigen binding domain (e.g., each scFv) comprises a linker between the VH and the VL regions. In some embodiments, the linker between the VH and the VL regions is a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6 (SEQ ID NO: 54). In some embodiments, the linker is (Gly4-Ser)n (SEQ ID NO: 62), wherein n = 1, e.g., the linker has the amino acid sequence Gly4-Ser (SEQ ID NO: 62). In some embodiments, the linker is (Gly4-Ser)n, wherein n= 4 (SEQ ID NO: 51). In some embodiments, the VH and VL regions are connected without a linker.Split CAR

[0216] In some embodiments, the CAR-expressing cell uses a split CAR. The split CAR approach is described in more detail in PCT publications WO2014 / 055442 and WO2014 / 055657, both of which are incorporated herein by reference. Briefly, a split CAR system comprises a cell expressing a first CAR having a first antigen binding domain and a costimulatory domain (e.g., 4-1BB), and the cell also expresses a second CAR having a second antigen binding domain and an intracellular signaling domain (e.g., CD3 zeta). When the cell encounters the first antigen, the costimulatory domain is activated, and the cell proliferates. When the cell encounters the second antigen, the intracellular signaling domain is activated and cell-killing activity begins. Thus, the CAR-expressing cell is only fully activated in the presence of both antigens.RNA Transfection

[0217] Disclosed herein are methods for producing an in vitro transcribed RNA CAR. The present disclosure also includes a CAR encoding RNA construct that can be directly transfected into a cell. A method for generating mRNA for use in transfection can involve in vitro transcription (IVT) of a template with specially designed primers, followed by polyA addition, to produce a construct containing 3’ and 5’ untranslated sequence (“UTR”), a 5’ cap and / or Internal Ribosome Entry Site (IRES), the nucleic acid to be expressed, and a polyA tail, typically 50- 2000 bases in length (SEQ ID NO: 63). RNA so produced can efficiently transfect different kinds of cells. In some embodiments, the template includes sequences for the CAR.

[0218] In one embodiment the CAR, e.g., dual CAR or tandem CAR, is encoded by a messenger RNA (mRNA). In one embodiment the mRNA encoding the CAR, e.g., dual CAR or tandem CAR, is introduced into an immune effector cell, e.g., a T cell or a NK cell, for production of a CAR- expressing cell, e.g., a CART cell or a CAR NK cell.

[0219] In some embodiments, the in vitro transcribed RNA of a CAR can be introduced to a cell as a form of transient transfection. The RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA.

[0220] The desired temple for in vitro transcription is a CAR of the present disclosure. For example, the template for the RNA CAR comprises an extracellular region comprising a single chain variable domain of an anti-tumor antibody; a hinge region, a transmembrane domain (e.g., a transmembrane domain of CD8a); and a cytoplasmic region that includes an intracellular signaling domain, e.g., comprising the signaling domain of CD3-zeta and the signaling domain of 4-1BB.

[0221] In some embodiments, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In some embodiments, the nucleic acid can include some or all of the 5’ and / or 3’ untranslated regions (UTRs). The nucleic acid can include exons and introns. In some embodiments, the DNA to be used for PCR is a human nucleic acid sequence. In some embodiments, the DNA to be used for PCR is a human nucleic acid sequence including the 5’ and 3’ UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.

[0222] PCR is used to generate a template for in vitro transcription of mRNA which is used for transfection. Methods for performing PCR are well known in the art. Primers for use in PCR are designed to have regions that are substantially complementarity to regions of the DNA to be used as a template for the PCR. “Substantially complementarity,” as used herein, refers to sequences of nucleotides where a majority or all of the bases in the primer sequence are complementarity, or one or more bases are non-complementarity, or mismatched. Substantially complementarity sequences are able to anneal or hybridize with the intended DNA target under annealing conditions used for PCR. The primers can be designed to be substantially complementarity to any portion of the DNA template. For example, the primers can be designed to amplify the portion of a nucleic acid that is normally transcribed in cells (the open reading frame), including 5’ and 3’ UTRs. The primers can also be designed to amplify a portion of a nucleic acid that encodes a particular domain of interest. In some embodiments, the primers are designed to amplify the coding region of a human cDNA, including all or portions of the 5’ and 3’ UTRs. Primers useful for PCR can be generated by synthetic methods that are well known in the art. “Forward primers” are primers that contain a region of nucleotides that aresubstantially complementarity to nucleotides on the DNA template that are upstream of the DNA sequence that is to be amplified. “Upstream” is used herein to refer to a location 5, to the DNA sequence to be amplified relative to the coding strand. “Reverse primers” are primers that contain a region of nucleotides that are substantially complementarity to a double-stranded DNA template that are downstream of the DNA sequence that is to be amplified. “Downstream” is used herein to refer to a location 3’ to the DNA sequence to be amplified relative to the coding strand.

[0223] Any DNA polymerase useful for PCR can be used in the methods disclosed herein. The reagents and polymerase are commercially available from a number of sources.

[0224] Chemical structures with the ability to promote stability and / or translation efficiency may also be used. The RNA preferably has 5’ and 3’ UTRs. In some embodiments, the 5’ UTR is between one and 3000 nucleotides in length. The length of 5’ and 3’ UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5 ’ and 3 ’ UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.

[0225] The 5’ and 3’ UTRs can be the naturally occurring, endogenous 5’ and 3’ UTRs for the nucleic acid of interest. Alternatively, UTR sequences that are not endogenous to the nucleic acid of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the nucleic acid of interest can be useful for modifying the stability and / or translation efficiency of the RNA For example, it is known that AU-rich elements in 3’ UTR sequences can decrease the stability of mRNA Therefore, 3’ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties ofUTRs that are well known in the art.

[0226] In some embodiments, the 5’ UTR can contain the Kozak sequence of the endogenous nucleic acid. Alternatively, when a 5’ UTR that is not endogenous to the nucleic acid of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5’ UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In some embodiments, the 5’ UTR can be 5’UTR of an RNA virus whose RNA genome is stablein cells. In some embodiments, various nucleotide analogues can be used in the 3’ or 5’ UTR to impede exonuclease degradation of the mRNA.

[0227] To enable synthesis of RNAfrom a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5’ end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In one preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters.

[0228] Consensus nucleic acid sequences for T7, T3 and SP6 promoters are known in the art.

[0229] In some embodiments, the mRNA has both a cap on the 5’ end and a 3’ poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3’ UTR results in normal sized mRNA which is not effective in eukaryotic transfection even if it is polyadenylated after transcription.

[0230] On a linear DNA template, phage T7 RNA polymerase can extend the 3’ end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

[0231] The conventional method of integration of polyA / T stretches into a DNA template is molecular cloning. However, polyA / T sequence integrated into plasmid DNA can cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other aberrations. This makes cloning procedures not only laborious and time consuming but often not reliable. That is why a method which allows construction of DNA templates with polyA / T 3’ stretch without cloning highly desirable.

[0232] The polyA / T segment of the transcriptional DNA template can be produced during PCR by using a reverse primer containing a polyT tail, such as 100T tail (SEQ ID NO: 64) (size can be 50-5000 T), or after PCR by any other method, including, but not limited to, DNA ligation or in vitro recombination. Poly(A) tails also provide stability to RNAs and reduce their degradation. Generally, the length of a poly(A) tail positively correlates with the stability ofthe transcribed. RNA In some embodiments, the poly(A) tail is between 100 and 5000 adenosines (SEQ ID NO: 65).

[0233] Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli poly A polymerase (E-PAP). In some embodiments, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides (SEQ ID NO: 66) results in about a two-fold increase in the translation efficiency of the RNA Additionally, the attachment of different chemical groups to the 3’ end can increase mRNA stability. Such attachment can contain modified / artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA 5’ caps on also provide stability to RNA molecules. In a preferred embodiment, RNAs produced by the methods disclosed herein include a 5’ cap. The 5’ cap is provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7: 1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0234] The RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. Any solutes suitable for cell electroporation, which can contain factors facilitating cellular permeability and viability such as sugars, peptides, lipids, proteins, antioxidants, and surfactants can be included.

[0235] RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-11 (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendorf, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Then, 12(8):861- 70 (2001).

[0236] Non-viral delivery methods

[0237] In some embodiments, non-viral methods can be used to deliver a nucleic acid encoding a CAR described herein into a cell or tissue or a subject. In some embodiments, the non-viral method includes the use of a transposon (also called a transposable element). In some embodiments, a transposon is a piece of DNA that can insert itself at a location in a genome, for example, a piece of DNA that is capable of self-replicatingand inserting its copy into a genome, or a piece of DNA that can be spliced out of a longer nucleic acid and inserted into another place in a genome. For example, a transposon comprises a DNA sequence made up of inverted repeats flanking genes for transposition.

[0238] Exemplary methods of nucleic acid delivery using a transposon include a SleepingBeauty transposon system (SBTS) and a piggyBac (PB) transposon system. See, e.g., Aronovich et al. Hum. Mol. Genet. 2O.R1(2O1 l):R14-20; Singh et al. Cancer Res. 15(2008):2961-2971; Huang et al. Mol. Then 16(2008): 580-589; Grabundzija et al. Mol. Then 18(2010): 1200-1209; Kebriaei et al. Blood. 122.21(2013): 166; Williams. Molecular Therapy 16.9(2008): 1515-16; Bell et al. Nat. Protoc. 2.12(2007):3153-65; and Ding et al. Cell. 122.3(2005):473-83, all of which are incorporated herein by reference.

[0239] The SBTS includes two components: 1) a transposon containing a transgene and 2) a source of transposase enzyme. The transposase can transpose the transposon from a carrier plasmid (or other donor DNA) to a target DNA, such as a host cell chromosome / genome. For example, the transposase binds to the carrier plasmid / donor DNA, cuts the transposon (including transgene(s)) out of the plasmid, and inserts it into the genome of the host cell. See, e.g., Aronovich et al.

[0240] Exemplary transposons include a pT2-based transposon. See, e.g., Grabundzija et al. Nucleic Acids Res. 41.3(2013): 1829-47; and Singh et al. Cancer Res. 68.8(2008): 2961-2971, all of which are incorporated herein by reference. Exemplary transposases include a Tcl / mariner-type transposase, e.g., the SB10 transposase or the SB11 transposase (a hyperactive transposase which can be expressed, e.g., from a cytomegalovirus promoter). See, e.g., Aronovich et al.; Kebriaei et al.; and Grabundzija et al., all of which are incorporated herein by reference.

[0241] Use of the SBTS permits efficient integration and expression of a transgene, e.g., a nucleic acid encoding a CAR described herein. Provided herein are methods of generating a cell, e.g., T cell or NK cell, that stably expresses a CAR described herein, e.g., using a transposon system such as SBTS. In accordance with methods described herein, in someembodiments, one or more nucleic acids, e.g., plasmids, containing the SBTS components are delivered to a cell (e.g., Tor NK cell). For example, the nucleic acid(s) are delivered by standard methods of nucleic acid (e.g., plasmid DNA) delivery, e.g., methods described herein, e.g., electroporation, transfection, or lipofection. In some embodiments, the nucleic acid contains a transposon comprising a transgene, e.g., a nucleic acid encoding a CAR described herein. In some embodiments, the nucleic acid contains a transposon comprising a transgene (e.g., a nucleic acid encoding a CAR described herein) as well as a nucleic acid sequence encoding a transposase enzyme. In some embodiments, a system with two nucleic acids is provided, e.g., a dual-plasmid system, e.g., where a first plasmid contains a transposon comprising a transgene, and a second plasmid contains a nucleic acid sequence encoding a transposase enzyme. For example, the first and the second nucleic acids are co-delivered into a host cell.

[0242] In some embodiments, cells, e.g., Tor NK cells, are generated that express a CAR described herein by using a combination of gene insertion using the SBTS and genetic editing using a nuclease (e.g., Zinc finger nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR / Cas system, or engineered meganuclease reengineered homing endonucleases ). In some embodiments, use of a non-viral method of delivery permits reprogramming of cells, e.g., Tor NK cells, and direct infusion of the cells into a subject. Advantages of non-viral vectors include but are not limited to the ease and relatively low cost of producing sufficient amounts required to meet a patient population, stability during storage, and lack of immunogenicity.Nucleic Acid Constructs Encoding a CAR

[0243] The present disclosure also provides nucleic acid molecules encoding one or more CAR constructs described herein. In some embodiments, the nucleic acid molecule is provided as a messenger RNA transcript. In some embodiments, the nucleic acid molecule is provided as a DNA construct.

[0244] The nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

[0245] The present disclosure also provides vectors in which a DNA of the present disclosure is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce nonproliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[0246] In some embodiments, the vector comprising the nucleic acid encoding the desired CAR of embodiments herein is an adenoviral vector (A5 / 35). In some embodiments, the expression of nucleic acids encoding CARs can be accomplished using of transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See below June et al. 2009Nature Reviews Immunology 9. 1 0: 704-716, is incorporated herein by reference.

[0247] In brief summary, the expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter, and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[0248] The expression constructs of the present disclosure may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. In some embodiments, the disclosure provides a gene therapy vector.

[0249] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0250] Further, the expression vector may be provided to a cell in the form of a viral vector Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1 -4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0251] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subj ect either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In some embodiments, lentivirus vectors are used.

[0252] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription. Exemplary promoters include the CMV IE gene, EF-la, ubiquitin C, or phosphoglycerokinase (PGK) promoters.

[0253] An example of a promoter that is capable of expressing a CAR transgene in a mammalian T cell is the EF-1 alpha (EFla) promoter. The native EFla promoter drives expression of the alpha subunit of the elongation factor- 1 complex, which is responsible for the enzymatic delivery of aminoacyl tRNAs to the ribosome. The EFla promoter has been extensively used in mammalian expression plasmids and has been shown to be effective in driving CAR expression from transgenes cloned into a lentiviral vector. See, e.g., Milone et al., Mol. Ther. 17(8): 1453- 1464 (2009). In some embodiments, the EFla promoter comprises the sequence as known in the art.

[0254] Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the elongation factor-lex promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, embodiments herein should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of embodiments herein. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0255] In order to assess the expression of a CAR polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co- transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[0256] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, betagalactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82). Suitable expression systems are well known and may be prepared using known techniques or obtained commercially. In general, the construct with the minimal 5’ flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions maybe linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.

[0257] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[0258] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1 -4, Cold Spring Harbor Press, NY). A preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[0259] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNAand RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus 1, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[0260] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of state-of-the-art targeted delivery of nucleic acids are available, such as delivery of polynucleotides with targeted nanoparticles or other suitable sub-micron sized delivery system.

[0261] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In some embodiments, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and theoligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / DNA or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0262] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, MO; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL.). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10).

[0263] However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine- nucleic acid complexes.

[0264] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present disclosure, in order to confirm thepresence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of embodiments herein.

[0265] The present disclosure further provides a vector comprising a CAR encoding nucleic acid molecule. In some embodiments, a CAR vector can be directly transduced into a cell, e.g., a T cell or NK cell. In some embodiments, the vector is a cloning or expression vector, e.g., a vector including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs. In some embodiments, the vector is capable of expressing the CAR construct in mammalian T cells or NK cells. In some embodiments, the mammalian T cell is a human T cell.Methods of Manufacture / Production

[0266] The present disclosure also provides methods of making a cell disclosed herein, e.g., methods of engineering a T cell or NK cell to express a nucleic acid molecule encoding one or more CAR constructs described herein. In some embodiments, the manufacturing methods disclosed herein are used to manufacture a cell comprising a nucleic acid molecule encoding two

[0267] CARs disclosed herein (e.g., a tandem and / or dual CAR disclosed herein). In some embodiments, the manufacturing methods disclosed herein are used to manufacture a cell comprising a nucleic acid molecule encoding a diabody CAR disclosed herein, e.g., an anti- NYESO-l / anti-CD3 diabody CAR disclosed herein. In some embodiments, the manufacturing methods disclosed herein are used to manufacture a cell comprising two nucleic acid molecules, each of which encodes a CAR disclosed herein (e.g., one nucleic acid molecule encoding an anti-NYESO-1 CAR and one nucleic acid molecule encoding an anti-CD3 CAR). In some embodiments, provided herein is a population of cells (for example, immune effector cells, for example, T cells or NK cells) made by any of the manufacturing processes described herein.

[0268] In some embodiments, the methods disclosed herein may manufacture immune effector cells engineered to express one or more CARs in less than 24 hours. Without wishing to be bound by theory, the methods provided herein preserve the undifferentiated phenotype of T cells, such as naive T cells, during the manufacturing process. These CAR-expressing cells with an undifferentiated phenotype may persist longer and / or expand better in vivo after infusion. In some embodiments, CART cells produced by the manufacturing methods provided herein comprise a higher percentage of stem cell memory T cells, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using single-cell or bulk RNA-seq or flow cytometry using markers known in the art. In some embodiments, CART cells produced by the manufacturing methods provided herein comprise a lower percentage of effector T cells, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using single-cell RNA-seq. In some embodiments, CART cells produced by the manufacturing methods provided herein better preserve the sternness of T cells, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using scRNA-seq. In some embodiments, CART cells produced by the manufacturing methods provided herein show a lower level of hypoxia, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using scRNA-seq. In some embodiments, CART cells produced by the manufacturing methods provided herein show a lower level of autophagy, compared to CART cells produced by the traditional manufacturing process, e.g., as measured using scRNA-seq. In some embodiments, the methods disclosed herein do not involve using a bead, such as Dynabeads® (for example, CD3 / CD28 Dynabeads®), and do not involve a de-beading step. In some embodiments, the CART cells manufactured by the methods disclosed herein may be administered to a subject with minimal ex vivo expansion, for example, less than 2 days, less than 1 day, less than 12 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, or no ex vivo expansion. Accordingly, the methods described herein provide a fast manufacturing process of making improved CAR-expressing cell products for use in treating a disease in a subject.

[0269] In some embodiments, the present disclosure provides methods of making a population of cells (for example, T cells) that express a chimeric antigen receptor (CAR) (e.g., one or more CARs, e.g., two CARs) comprising: (i) contacting a population of cells (for example, T cells, for example, T cells isolated from a frozen or fresh leukapheresis product) with an agent that stimulates a CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule onthe surface of the cells; (ii) contacting the population of cells (for example, T cells) with a nucleic acid molecule(s) (for example, a DNA or RNA molecule) encoding the CAR(s), thereby providing a population of cells (for example, T cells) comprising the nucleic acid molecule, and harvesting the population of cells (for example, T cells) for storage (for example, reformulating the population of cells in cryopreservation media) or administration, wherein: (a) step (ii) is performed together with step (i) or no later than 20 hours after the beginning of step (i), for example, no later than 12, 13, 14, 15, 16, 17, or 18 hours after the beginning of step (i), for example, no later than 18 hours after the beginning of step (i), and step (iii) is performed no later than 26 hours after the beginning of step (i), for example, no later than 22, 23, or 24 hours after the beginning of step (i), for example, no later than 24 hours after the beginning of step (i); (b) step (ii) is performed together with step (i) or no later than 20 hours after the beginning of step (i), for example, no later than 12, 13, 14, 15, 16, 17, or 18 hours after the beginning of step (i), for example, no later than 18 hours after the beginning of step (i), and step (iii) is performed no later than 30 hours after the beginning of step (ii), for example, no later than 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the beginning of step (ii); and / or (c) the population of cells from step (iii) are not expanded, or expanded by no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%, for example, no more than 10%, as assessed by the number of living cells compared to the population of cells at the beginning of step (i); or d) the population of cells from step (iii) are fewer, or less by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, for example, as assessed by the number of living cells compared to the population of cells at the beginning of step (i). In some embodiments, the nucleic acid molecule in step (ii) is a DNA molecule. In some embodiments, the nucleic acid molecule in step (ii) is an RNA molecule. In some embodiments, the nucleic acid molecule in step (ii) is on a viral vector, for example, a viral vector chosen from a lentivirus vector, an adenoviral vector, or a retrovirus vector. In some embodiments, the nucleic acid molecule in step (ii) is on a non-viral vector. In some embodiments, the nucleic acid molecule in step (ii) is on a plasmid. In some embodiments, the nucleic acid molecule in step (ii) is not on any vector. In some embodiments, step (ii) comprises transducing the population of cells (for example, T cells) a viral vector(s) comprising a nucleic acid molecule encoding the CAR(s). In some embodiments of the aforementioned methods, the methods further comprise adding an adjuvant or a transduction enhancement reagent in the cell culture medium to enhance transduction efficiency. In some embodiments, the adjuvant or transduction enhancement reagent comprisesa cationic polymer. In some embodiments, the adjuvant or transduction enhancement reagent is selected from: LentiBOOS T™ (Sirion Biotech), vectofusin-1, F108 (Poloxamer 338 or Pluronic® F-38), hexadimethrine bromide (Polybrene), PEA, Pluronic F68, Pluronic F127(Poloxamer 407 or Synperonic PE / F-127), Protamine Sulfate, Synperonic or LentiTrans™. In some embodiments, the adjuvant is LentiBOOST™ (Sirion Biotech). In other embodiments, the adjuvant is F108 (Poloxamer 338 or Pluronic® F-38). In other embodiments, the adjuvant is Pluronic F127 (Poloxamer 407 or Synperonic PE / F-127).

[0270] In some embodiments, the population of cells (for example, T cells) is collected from an apheresis sample (for example, a leukapheresis sample) from a subject. In some embodiments, the apheresis sample (for example, a leukapheresis sample) is collected from the subject and shipped as a frozen sample (for example, a cryopreserved sample) to a cell manufacturing facility. Then the frozen apheresis sample is thawed, and T cells (for example, CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorting machine (for example, a CliniMACS® Prodigy® device). The selected T cells (for example, CD4+ T cells and / or CD8+ T cells) are then seeded for CART manufacturing using the activation process described herein. In some embodiments, the selected T cells (for example, CD4+ T cells and / or CD8+ T cells) undergo one or more rounds of freeze- thaw before being seeded for CART manufacturing.

[0271] In some embodiments, the apheresis sample (for example, a leukapheresis sample) is collected from the subject and shipped as a fresh product (for example, a product that is not frozen) to a cell manufacturing facility. T cells (for example, CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorting machine (for example, a CliniMACS® Prodigy® device). The selected T cells (for example, CD4+ T cells and / or CD8+ T cells) are then seeded for CART manufacturing using the activation process described herein. In some embodiments, the selected T cells (for example, CD4+ T cells and / or CD8+ T cells) undergo one or more rounds of freeze-thaw before being seeded for CART manufacturing.

[0272] In some embodiments, the apheresis sample (for example, a leukapheresis sample) is collected from the subject. T cells (for example, CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorting machine (for example, a CliniMACS® Prodigy® device). The selected T cells (for example, CD4+ T cells and / or CD8+ T cells) are then shipped as a frozen sample (for example, a cryopreserved sample) to a cellmanufacturing facility. The selected T cells (for example, CD4+ T cells and / or CD8+ T cells) are later thawed and seeded for CART manufacturing using the activation process described herein.

[0273] In some embodiments, cells (for example, T cells) are contacted with anti-CD3 and anti- CD28 antibodies and, for example, immediately followed by transduction with a vector (for example, a lentiviral vector) (e.g. one or more vectors) encoding a CAR (e.g. one or more CARs). 24 hours after culture initiation, the cells are washed and formulated for storage or administration.

[0274] Without wishing to be bound by theory, brief CD3 and CD28 stimulation may promote efficient transduction of self-renewing T cells. Compared to traditional CART manufacturing approaches, the activation process provided herein does not involve prolonged ex vivo expansion. Similar to the cytokine process, the activation process provided herein also preserves undifferentiated T cells during CART manufacturing.

[0275] In some embodiments, cells (for example, T cells) are contacted with anti-CD3 and anti- CD28 antibodies for, for example, 12 hours, followed by transduction with a vector (for example, a lentiviral vector) (e.g. one or more vectors) encoding a CAR (e.g. one or more CARs). 24 hours after culture initiation, the cells are washed and formulated for storage or administration.

[0276] Without wishing to be bound by theory, brief CD3 and CD28 stimulation may promote efficient transduction of self-renewing T cells. Compared to traditional CART manufacturing approaches, the activation process provided herein does not involve prolonged ex vivo expansion. Similar to the cytokine process, the activation process provided herein also preserves undifferentiated T cells during CART manufacturing.

[0277] In some embodiments, the population of cells is contacted with an agent that stimulates a CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells. In some embodiments, T cells are selected using anti-CD4 and anti-CDS beads by positive selection, using, for example, a cell sorting machine (for example, a CliniMACS® Prodigy® device).

[0278] In some embodiments, T cells are selected using anti-CD45RA and anti-CCR7 beads by positive selection, using, for example, a cell sorting machine (for example, a CliniMACS® Prodigy® device).

[0279] In some embodiments, T cells are selected using anti-CD45RA and anti-CD27 beads by positive selection, using, for example, a cell sorting machine (for example, a CliniMACS® Prodigy® device).

[0280] In some embodiments, T cells are selected using anti-CD3 and anti-CD28 beads by positive selection, using, for example, a cell sorting machine (for example, a CliniMACS® Prodigy® device).

[0281] In some embodiments, T cells are selected using anti-lineage beads (except for T cell) by negative selection, using, for example, a cell sorting machine (for example, a CliniMACS® Prodigy® device).

[0282] In some embodiments, the agent that stimulates a CD3 / TCR complex is an agent that stimulates CD3. In some embodiments, the agent that stimulates a costimulatory molecule is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIMI, CD2, CD226, or any combination thereof. In some embodiments, the agent that stimulates a costimulatory molecule is an agent that stimulates CD28. In some embodiments, the agent that stimulates a CD3 / TCR complex is chosen from an antibody (for example, a single- domain antibody (for example, a heavy chain variable domain antibody), a peptibody, a Fab fragment, or a scFv), a small molecule, or a ligand (for example, a naturally- existing, recombinant, or chimeric ligand). In some embodiments, the agent that stimulates a CD3 / TCR complex is an antibody. In some embodiments, the agent that stimulates a CD3 / TCR complex is an anti-CD3 antibody. In some embodiments, the agent that stimulates a costimulatory molecule is chosen from an antibody (for example, a single-domain antibody (for example, a heavy chain variable domain antibody), a peptibody, a Fab fragment, or a scFv), a small molecule, or a ligand (for example, a naturally-existing, recombinant, or chimeric ligand). In some embodiments, the agent that stimulates a costimulatory molecule is an antibody. In some embodiments, the agent that stimulates a costimulatory molecule is an anti- CD28 antibody. In some embodiments, the agent that stimulates a CD3 / TCR complex or the agent that stimulates a costimulatory molecule does not comprise a bead. In some embodiments, the agent that stimulates a CD3 / TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymeric nanomatrix. In some embodiments, the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymeric nanomatrix. In some embodiments, the agent that stimulates a CD3 / TCR complex and the agent that stimulates a costimulatory molecule comprising T Cell TransAct™.

[0283] In some embodiments, the matrix comprises or consists of a polymeric, for example, biodegradable or biocompatible inert material, for example, which is non-toxic to cells. In some embodiments, the matrix is composed of hydrophilic polymer chains, which obtain maximal mobility in aqueous solution due to hydration of the chains. In some embodiments, the mobile matrix may be of collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions. A polysaccharide may include for example, cellulose ethers, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectins, xanthan, guar gum or alginate. Other polymers may include polyesters, polyethers, polyacrylates, polyacrylamides, polyamines, polyethylene imines, polyquaternium polymers, polyphosphazenes, polyvinylalcohols, polyvinylacetates, polyvinylpyrrolidones, block copolymers, or polyurethanes. In some embodiments, the mobile matrix is a polymer of dextran.

[0284] In some embodiments, the population of cells is contacted with a nucleic acid molecule (e.g. one or more nucleic acid molecules) encoding a CAR (e.g. one or more CARs). In some embodiments, the population of cells is transduced with a DNA molecule (e.g. one or more DNA) encoding a CAR (e.g. one or more CARs).

[0285] In some embodiments, in the case of a co-transduction of two nucleic acid molecules (e.g., lentiviral vectors), each of which encodes a CAR disclosed herein (e.g., one nucleic acid molecule encoding an anti-CD22 CAR and one nucleic acid molecule encoding an anti -CD 19 CAR, as disclosed herein), each of the vectors containing nucleic acid molecules encoding the

[0286] CAR can be added to the reaction mixture (e.g., containing a cell population), e.g., at 1 : 1 ratio.

[0287] Without wishing to be bound by theory, it is believed that, in some embodiments, using different MOIs for the vectors containing nucleic acid molecules that encode distinct CAR molecules may affect the final composition of the cellular population.

[0288] In some embodiments, in the case of a co-transduction of lentiviral vectors, a population of cells is contacted with the first viral vector at an MOI that is higher than, equal to, or less than an MOI at which the population of cells is contacted with the second viral vector. In some embodiments, the population of cells is contacted with the first viral vector at an MOI that is higher than an MOI at which the population of cells is contacted with the second viral vector.

[0289] In some embodiments, the population of cells is contacted with the first viral vector at first MOI and with the second viral vector at a second MOI, such that a resultant population ofcells comprises a first population of cells that comprise the first CAR but not the second CAR, a second population of cells that comprise the second car, but not the first CAR, and a third population of cells that comprise both the first CAR and the second CAR. Co-transduction methods are as described in the art, e.g., in WO 2021 / 108661 which is incorporated by reference in its entirety.

[0290] In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs simultaneously with contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 20 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 19 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 18 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 17 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 16 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates acostimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 15 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 14 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 14 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 13 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 12 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 11 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 10 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 9 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s)encoding the CAR(s) occurs no later than 8 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 7 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 6 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 5 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 4 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 3 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR(s) occurs no later than 2 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contactingthe population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 1 hour after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with the nucleic acid molecule(s) encoding the CAR(s) occurs no later than 30 minutes after the beginning of contacting the population of cells with the agent that stimulates aCD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above.

[0291] In some embodiments, the population of cells is harvested for storage or administration. In some embodiments, the population of cells is harvested for storage or administration no later than 72, 60, 48, 36, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 26 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 25 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or New York esophageal squamous cell carcinoma 1 administration no later than 24 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 23 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is harvested for storage or administration no later than 22 hours after the beginning of contacting the population of cells with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above.

[0292] In some embodiments, the population of cells is not expanded ex vivo.

[0293] In some embodiments, the population of cells is expanded by no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, thepopulation of cells is expanded by no more than 5%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 10%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 15%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 20%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 25%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 30%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 35%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 40%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the agent that stimulates a CD3 / TCR complex and / or the agent that stimulates a costimulatory molecule on the surface of the cells described above.

[0294] In some embodiments, the population of cells is expanded by no more than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 36, or 48 hours, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the one or more cytokines described above.

[0295] In some embodiments, the activation process is conducted in serum free cell media. In some embodiments, the activation process is conducted in cell media comprising one or more cytokines chosen from: IL-2, IL- 15 (for example, hetIL-15 (IL-15 / sIL-l 5Ra)), or IL-6 (for example, IL-6 / sIL-6Ra). In some embodiments, hetIL-15 comprises the amino acid sequence of NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIH DTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSITCPPP MS VEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDP ALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKS P STGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQG(SEQ ID NO: 50).

[0296] In some embodiments, hetIL-15 comprises an amino acid sequence having at least about 70, 75, 80, 85,90, 95, or 99% identity to SEQ ID NO: 109. In some embodiments, the activation process is conducted in cell media comprising a LSD 1 inhibitor. In some embodiments, the activation process is conducted in cell media comprising a MALTI inhibitor. In some embodiments, the serum free cell media comprises a serum replacement. In some embodiments, the serum replacement is CTS™ Immune Cell Serum Replacement (ICSR). In some embodiments, the level of ICSR can be, for example, up to 5%, for example, about 1%, 2%, 3%, 4%, or 5%. Without wishing to be bound by theory, using cell media, for example, Rapid Media, comprising ICSR, for example, 2% ICSR may improve cell viability during a manufacture process described herein.

[0297] In some embodiments, the present disclosure provides methods of making a population of cells (for example, T cells) that express a chimeric antigen receptor (CAR) comprising: (a) providing an apheresis sample (for example, a fresh or cryopreserved leukapheresis sample) collected from a subject; (b) selecting T cells from the apheresis sample (for example, using negative selection, positive selection, or selection without beads); (c) seeding isolated T cells at, for example, I x lC to l x lO7cells / mL; (d) contacting T cells with an agent that stimulatesT cells, for example, an agent that stimulates a CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells (for example, contacting T cells with anti-CD3 and / or anti-CD28 antibody, for example, contacting T cells with TransAct); (e) contacting T cells with a nucleic acid molecule(s) (for example, a DNA or RNA molecule) encoding the CAR(s) (for example, contacting T cells with a virus comprising a nucleic acid molecule(s) encoding the CAR(s)) for, for example, 6-48 hours, for example, 20-28 hours; and (f) washing and harvesting T cells for storage (for example, reformulating T cells in cry opreservation media) or administration. In some embodiments, step (f) is performed no later than 30 hours after the beginning of step (d) or (e), for example, no later than 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours after the beginning of step (d) or (e).

[0298] In some embodiments, provided herein is a population of cells (for example, immune effector cells, for example, T cells or NK cells) made by any of the manufacturing processes described herein (e.g., the Activation Process described herein).

[0299] In some embodiments, the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, in the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) (1) is the same as, (2) differs, for example, by no more than 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15%, from, or (3) is increased, for example, by at least 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, as compared to, the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ cells, in the population of cells at the beginning of the manufacturing process (for example, at the beginning of the cytokine process or the activation process described herein). In some embodiments, the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) shows a higher percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells (for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50% higher), compared with cells made by an otherwise similar method which lasts, for example, more than 26 hours (for example, which lasts more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or which involves expanding the population of cells in vitro for, for example, more than 3 days (for example, expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

[0300] In some embodiments, the percentage of naive cells, for example, naive T cells, for example, CD45RA+ CD45RO- CCR7+ T cells, in the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) is not less than 20, 25, 30, 35, 40, 45, 50, 55, or 60%.

[0301] In some embodiments, the percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memory T cells, CD45RO+ central memory T cells, and / or CCR7+ central memory T cells, in the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) (1) is the same as, (2) differs, for example, by no more than 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, or 15% from, or (3) is decreased, for example, by at least 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, as compared to, the percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memory T cells, in the population of cells at the beginning of the manufacturing process (for example, at the beginning of the cytokine process or the activation process described herein). In some embodiments, the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) shows a lower percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memory T cells (for example, at least 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,25,30,35,40, 45, or 50% lower), compared with cells made by an otherwise similar method which lasts, for example, more than 26 hours (for example, which lasts more than 5,6,7,8,9,10,11, or 12 days) or which involves expanding the population of cells in vitro for, for example, more than 3 days (for example, expanding the population of cells in vitro for 3,4,5,6,7,8,9,10,11,12,13,14, or 15 days).

[0302] In some embodiments, the percentage of central memory cells, for example, central memory T cells, for example, CD95+ central memory T cells, in the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) is no more than 40, 45, 50, 55, 60, 65, 70, 75, or 80%.

[0303] In some embodiments, the population of cells at the end of the manufacturing process (for example, at the end of the cytokine process or the activation process described herein) after being administered in vivo, persists longer or expands at a higher level (for example, at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher), compared with cells made by an otherwise similar method which lasts, for example, more than 26 hours (for example,which lasts more than 5, 6, 7, 8, 9, 10, 11, or 12 days) or which involves expanding the population of cells in vitro for, for example, more than 3 days (for example, expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

[0304] In some embodiments, the population of cells has been enriched for IL6R-expressing cells (for example, cells that are positive for IL6Ra and / or IL6R ) prior to the beginning of the manufacturing process (for example, prior to the beginning of the cytokine process or the activation process described herein). In some embodiments, thepopulation of cells comprises, for example, no less than 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% of!L6R-expressing cells (for example, cells that are positive for IL6Ra and / or IL6R ) at the beginning of the manufacturing process (for example, at the beginning of the cytokine process or the activation process described herein).Cytokine Process

[0305] In some embodiments, the present disclosure provides methods of making a population of cells (for example, T cells) that express a chimeric antigen receptor (CAR) (e.g., one or more CARs, e.g., two CARs) comprising: (1) contacting a population of cells with a cytokine chosen from IL-2, IL-7, IL- 15, IL-21, IL-6, or a combination thereof, (2) contacting the population of cells (for example, T cells) with a nucleic acid molecule(s) (for example, a DNA or RNA molecule) encoding the CAR(s), thereby providing a population of cells (for example, T cells) comprising the nucleic acid molecule, and (3) harvesting the population of cells (for example, T cells) for storage (for example, reformulating the population of cells in cryopreservation media) or administration, wherein: (a) step (2) is performed together with step (1) or no later than 5 hours after the beginning of step (1), for example, no later than 1, 2, 3, 4, or 5 hours after the beginning of step (1), and step (3) is performed no later than 26 hours after the beginning of step (1), for example, no later than 22, 23, or 24 hours after the beginning of step (1), for example, no later than 24 hours after the beginning of step (1), or (b) the population of cells from step (3) are not expanded, or expanded by no more than 5, 10, 15, 20, 25, 30, 35, or 40%, for example, no more than 10%, for example, as assessed by the number of living cells, compared to the population of cells at the beginning of step (1). In some embodiments, the nucleic acid molecule in step (2) is a DNA molecule. In some embodiments, the nucleic acid molecule in step (2) is an RNA molecule. In some embodiments, the nucleic acid molecule in step (2) is on a viral vector, for example, a viral vector chosen from a lentivirus vector, anadenoviral vector, or a retrovirus vector. In some embodiments, the nucleic acid molecule in step (2) is on a non-viral vector. In some embodiments, the nucleic acid molecule in step (2) is on a plasmid. In someembodiments, the nucleic acid molecule in step (2) is not on any vector. In some embodiments, step (2) comprises transducing the population of cells (for example, T cells) with a viral vector comprising a nucleic acid molecule(s) encoding the CAR(s). In some embodiments, the cells are engineered to comprise a nucleic acid molecule encoding a tandem or dual CAR disclosed herein In some embodiments, the cells are engineered to comprise a nucleic acid molecule encoding a diabody CAR disclosed herein

[0306] In some embodiments, the cells are engineered to comprise two nucleic acid molecules, each of which encodes a CAR disclosed herein. In some embodiments, the population of cells (for example, T cells) is collected from an apheresis sample (for example, a leukapheresis sample) from a subject. In some embodiments, the apheresis sample (for example, a leukapheresis sample) is collected from the subject and shipped as a frozen sample (for example, a cryopreserved sample) to a cell manufacturing facility. The frozen apheresis sample is then thawed, and T cells (for example, CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorting machine (for example, a CliniMACS® Prodigy® device). The selected T cells (for example, CD4+ T cells and / or CD8+ T cells) are then seeded for CART manufacturing using the cytokine process described herein. In some embodiments, at the end of the cytokine process, the CART cells are cryopreserved and later thawed and administered to the subject. In some embodiments, the selected T cells (for example, CD4+ T cells and / or CD8+ T cells) undergo one or more rounds of freeze-thaw before being seeded for CART manufacturing.

[0307] In some embodiments, the apheresis sample (for example, a leukapheresis sample) is collected from the subject and shipped as a fresh product (for example, a product that is not frozen) to a cell manufacturing facility. T cells (for example, CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorting machine (for example, a CliniMACS® Prodigy® device). The selected T cells (for example, CD4+ T cells and / or CD8+ T cells) are then seeded for CART manufacturing using the cytokine process described herein. In some embodiments, the selected T cells (for example, CD4+ T cells and / or CD8+ T cells) undergo one or more rounds of freeze-thaw before being seeded for CART manufacturing.

[0308] In some embodiments, the apheresis sample (for example, a leukapheresis sample) is collected from the subject. T cells (for example, CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorting machine (for example, a CliniMACS® Prodigy® device). The selected T cells (for example, CD4+ T cells and / or CD8+ T cells) are then shipped as a frozen sample (for example, a cryopreserved sample) to a cell manufacturing facility. The selected T cells (for example, CD4+ T cells and / or CD8+ T cells) are later thawed and seeded for CART manufacturing using the cytokine process described herein.

[0309] In some embodiments, after cells (for example, T cells) are seeded, one or more cytokines (for example, one or more cytokines chosen from IL-2, IL-7, IL- 15 (for example, hetIL-15 (IL15 / sIL-15Ra)), IL-21, or IL-6 (for example, IL-6 / sIL-6R)) as well as a vector (for example, a lentiviral vector) (e.g. one or more vectors) encoding a CAR (e.g., one or more CARs) are added to the cells. After incubation for 20-24 hours, the cells are washed and formulated for storage or administration.

[0310] Different from traditional CART manufacturing approaches, the cytokine process provided herein does not involve CD3 and / or CD28 stimulation, or ex vivo T cell expansion. T cells that are contacted with anti-CD3 and anti-CD28 antibodies and expanded extensively ex vivo tend to show differentiation towards a central memory phenotype. Without wishing to be bound by theory, the cytokine process provided herein preserves or increases the undifferentiated phenotype of T cells during CART manufacturing, generating a CART product that may persist longer after being infused into a subject.

[0311] In some embodiments, the population of cells is contacted with one or more cytokines (for example, one or more cytokines chosen from IL-2, IL-7, IL- 15 (for example, hetIL-15 (IL15 / sIL-15Ra)), IL-21, or IL-6 (for example, IL-6 / sIL-6Ra).

[0312] In some embodiments, the population of cells is contacted with IL-2. In some embodiments, the population of cells is contacted with IL-7. In some embodiments, the population of cells is contacted with IL- 15 (for example, hetIL-(ILI 5 / sIL-l 5Ra)). In some embodiments, the population of cells is contacted with IL-21. In some embodiments, the population of cells is contacted with IL-6 (for example, IL-6 / sIL-6Ra). In some embodiments, the population of cells is contacted with IL-2 and IL-7. In some embodiments, the population of cells is contacted with IL-2 and IL-15 (for example, hetIL-15 (IL15 / sIL-15Ra)). In some embodiments, the population of cells is contacted with IL-2 and IL-21. In some embodiments,the population of cells is contacted with IL-2 and IL-6 (for example, IL-6 / sIL-6Ra). In some embodiments, the population of cells is contacted with IL-7 and IL- 15 (for example, hetIL-15 (ILI 5 / sIL-l 5Ra)). In some embodiments, the population of cells is contacted with IL-7 and IL-6. In some embodiments, the population of cells is contacted with IL-7 and IL-6 (for example, IL-6 / sIL-6Ra). In some embodiments, the population of cells is contacted with IL-15 (for example, hetIL-15 (ILI 5 / sIL-l 5Ra)) and IL-21. In some embodiments, the population of cells is contacted with IL-15 (for example, hetIL-15 (IL15 / sIL-15Ra)) and IL-6 (for example, IL-6 / sIL- 6Ra). In some embodiments, the population of cells is contacted with IL-21 and IL- 6 (for example, IL-6 / sIL-6Ra). In some embodiments, the population of cells is contacted with IL-7, IL- 15 (for example, hetIL-15 (IL15 / sIL-15Ra)), and IL-21. In some embodiments, the population of cells is further contacted with a LSD1 inhibitor. In some embodiments, the population of cells is further contacted with a MALT1 inhibitor.

[0313] In some embodiments, the population of cells is contacted with 20, 30, 40, 50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240, 250,260,270, 280, 290, or 300 U / ml of IL-2. In some embodiments, the population of cells is contacted with 1,2, 3, 4, 5, 6, 7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20ng / ml of IL-7. In some embodiments, the population of cells is contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20 ng / ml of IL-15.

[0314] In some embodiments, the population of cells is contacted with a nucleic acid molecule (e.g. one or more nucleic acid molecules) encoding a CAR (e.g., one or more CARs). In some embodiments, the population of cells is transduced with a DNA molecule (e.g. one or more DNA molecules) encoding a CAR (e.g. one or more CARs).

[0315] In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR(s) occurs simultaneously with contacting the population of cells with the one or more cytokines described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR(s) occurs no later than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 hours after the beginning of contacting the population of cells with the one or more cytokines described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR(s) occurs no later than 5 hours after the beginning of contacting the population of cells with the one or more cytokines described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR(s) occurs no later than 4 hours after the beginningof contacting the population of cells with the one or more cytokines described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR(s) occurs no later than 3 hours after the beginning of contacting the population of cells with the one or more cytokines described above. In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR(s) occurs no later than 2 hours after the beginning of contacting the population of cells with the one or more cytokines described above.

[0316] In some embodiments, contacting the population of cells with the nucleic acid molecule encoding the CAR(s) occurs no later than 1 hour after the beginning of contacting the population of cells with the one or more cytokines described above. In some embodiments, the population of cells is harvested for storage or administration. In some embodiments, the population of cells is harvested for storage or administration no later than 72, 60, 48, 36, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 hours after the beginning of contacting the population of cells with the one or more cytokines described above. In some embodiments, the population of cells is harvested for storage or administration no later than 26 hours after the beginning of contacting the population of cells with the one or more cytokines described above. In some embodiments, the population of cells is harvested for storage or administration no later than 25 hours after the beginning of contacting the population of cells with the one or more cytokines described above. In some embodiments, the population of cells is harvested for storage or administration no later than 24 hours after the beginning of contacting the population of cells with the one or more cytokines described above. In some embodiments, the population of cells is harvested for storage or administration no later than 23 hours after the beginning of contacting the population of cells with the one or more cytokines described above. In some embodiments, the population of cells is harvested for storage or administration no later than 22 hours after the beginning of contacting the population of cells with the one or more cytokines described above.

[0317] In some embodiments, the population of cells is not expanded ex vivo. In some embodiments, the population of cells is expanded by no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the one or more cytokines described above. In some embodiments, the population of cells is expanded by no more than 5%, for example, as assessed by the number of living cells, compared to thepopulation of cells before it is contacted with the one or more cytokines described above. In some embodiments, the population of cells is expanded by no more than 10%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the one or more cytokines described above. In some embodiments, the population of cells is expanded by no more than 15%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the one or more cytokines described above. In some embodiments, the population of cells is expanded by no more than 20%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the one or more cytokines described above. In some embodiments, the population of cells is expanded by no more than 25%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the one or more cytokines described above. In some embodiments, the population of cells is expanded by no more than 30%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the one or more cytokines described above. In some embodiments, the population of cells is expanded by no more than 35%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the one or more cytokines described above. In some embodiments, the population of cells is expanded by no more than 40%, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the one or more cytokines described above.

[0318] In some embodiments, the population of cells is expanded by no more than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 36, or 48 hours, for example, as assessed by the number of living cells, compared to the population of cells before it is contacted with the one or more cytokines described above.

[0319] In some embodiments, the population of cells is not contacted in vitro with an agent that stimulates a CD3 / TCR complex (for example, an anti-CD3 antibody) and / or an agent that stimulates a costimulatory molecule on the surface of the cells (for example, an anti-CD28 antibody), or if contacted, the contacting step is less than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 hours.

[0320] In some embodiments, the population of cells is contacted in vitro with an agent that stimulates a CD3 / TCR complex (for example, an anti-CD3 antibody) and / or an agent thatstimulates a costimulatory molecule on the surface of the cells (for example, an anti-CD28 antibody) for 20, 21, 22, 23, 24, 25, 26, 27, or 28 hours.

[0321] In some embodiments, the population of cells manufactured using the cytokine process provided herein shows a higher percentage of naive cells among CAR-expressing cells (for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60% higher), compared with cells made by an otherwise similar method which further comprises contacting the population of cells with, for example, an agent that binds a CD3 / TCR complex (for example, an anti-CD3 antibody) and / or an agent that binds a costimulatory molecule on the surface of the cells (for example, an anti-CD28 antibody).

[0322] In some embodiments, the cytokine process provided herein is conducted in cell media comprising no more than 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8% serum. In some embodiments, the cytokine process provided herein is conducted in cell media comprising a LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof.Elutriation

[0323] In some embodiments, the methods described herein feature an elutriation method that removes unwanted cells, for example, monocytes and blasts, thereby resulting in an improved enrichment of desired immune effector cells suitable for CAR expression. In some embodiments, the elutriation method described herein is optimized for the enrichment of desired immune effector cells suitable for CAR expression from a previously frozen sample, for example, a thawed sample. In some embodiments, the elutriation method described herein provides a preparation of cells with improved purity as compared to a preparation of cells collected from the elutriation protocols known in the art. In some embodiments, the elutriation method described herein includes using an optimized viscosity of the starting sample, for example, cell sample, for example, thawed cell sample, by dilution with certain isotonic solutions (for example, PBS), and using an optimized combination of flow rates and collection volume for each fraction collected by an elutriation device. Exemplary elutriation methods that could be applied in the present disclosure are described on pages 48-51 of WO 2017 / 117112, herein incorporated by reference in its entirety.Density Gradient Centrifugation

[0324] Manufacturing of adoptive cell therapeutic product requires processing the desired cells, for example, immune effector cells, away from a complex mixture of blood cells and blood elements present in peripheral blood apheresis starting materials. Peripheral blood- derived lymphocyte samples have been successfully isolated using density gradient centrifugation through Ficoll solution. However, Ficoll is not a preferred reagent for isolating cells for therapeutic use, as Ficoll is not qualified for clinical use. In addition, Ficoll contains glycol, which has toxic potential to the cells. Furthermore, Ficoll density gradient centrifugation of thawed apheresis products after cryopreservation yields a suboptimal T cell product, for example, as described in the Examples herein. For example, a loss of T cells in the final product, with a relative gain of non-T cells, especially undesirable B cells, blast cells and monocytes was observed in cell preparations isolated by density gradient centrifugation through Ficoll solution.

[0325] Without wishing to be bound by theory, it is believed that immune effector cells, for example, T cells, dehydrate during cryopreservation to become denser than fresh cells. Without wishing to be bound by theory, it is also believed that immune effector cells, for example, T cells, remain denser longer than the other blood cells, and thus are more readily lost during Ficoll density gradient separation as compared to other cells. Accordingly, without wishing to be bound by theory, a medium with a density greater than Ficoll is believed to provide improved isolation of desired immune effector cells in comparison to Ficoll or other mediums with the same density as Ficoll, for example, 1.077 g / mL.

[0326] In some embodiments, the density gradient centrifugation method described herein includes the use of a density gradient medium comprising iodixanol. In some embodiments, the density gradient medium comprises about 60% iodixanol in water.

[0327] In some embodiments, the density gradient centrifugation method described herein includes the use of a density gradient medium having a density greater than Ficoll. In some embodiments, the density gradient centrifugation method described herein includes the use of a density gradient medium having a density greater than 1.077 g / mL, for example, greater than 1.077 g / mL, greater than 1.1 g / mL, greater than 1.15 g / mL, greater than 1.2 g / mL, greater than 1.25 g / mL, greater than 1.3 g / mL, greater than 1.31 g / mL. In some embodiments, the density gradient medium has a density of about 1.32 g / mL. Additional embodiments of density gradient centrifugation are described on pages 51-53 of WO 2017 / 117112, herein incorporated by reference in its entirety.Enrichment by Selection

[0328] Provided herein are methods for selection of specific cells to improve the enrichment of the desired immune effector cells suitable for CAR expression. In some embodiments, the selection comprises a positive selection, for example, selection for the desired immune effector cells. In some embodiments, the selection comprises a negative selection, for example, selection for unwanted cells, for example, removal of unwanted cells. In some embodiments, the positive or negative selection methods described herein are performed under flow conditions, for example, by using a flow-through device, for example, a flow-through device described herein. Exemplary positive and negative selections are described on pages 53-57 of WO 2017 / 117112, herein incorporated by reference in its entirety. Selection methods can be performed under flow conditions, for example, by using a flow-through device, also referred to as a cell processing system, to further enrich a preparation of cells for desired immune effector cells, for example, T cells, suitable for CAR expression. Exemplary flow-through devices are described on pages 57- 70 of WO 2017 / 117112, herein incorporated by reference in its entirety. Exemplary cell separation and debeading methods are described on pages 70-78 of WO 201 7 / 117112, herein incorporated by reference in its entirety.

[0329] Selection procedures are not limited to ones described on pages 57-70 of WO 2017 / 117112. Negative T cell selection via removal of unwanted cells with CD19, CD14 and CD26 Miltenyi beads in combination with column technology (CliniMACS® Plus or CliniMACS® Prodigy®) or positive T cell selection with a combination of CD4 and CD8 Miltenyi beads and column technology (CliniMACS® Plus or CliniMACS® Prodigy®) can be used. Alternatively, column-free technology with releasable CD3 beads (GE Healthcare) can be used. In addition, bead-free technologies such as ThermoGenesis X-series devices can be utilized as well.

[0330] Methods of making a cell disclosed herein include those known in the art, e.g., as described in CN 108103105, CN 108085342, CN 108018312, CN 107287164, WO 18052947, WO 17123956, WO 17114497, WO 17103596, WO 17068421, WO 17023803, WO 17015427, WO 16196388, WO 16168595, WO 14186469, WO 17165245, WO 18106732, WO 17015490, WO 18075813, WO 18102761, WO 17127755, WO 17214333, WO 18059549, WO 17190100, WO 16180778, WO 18057823, and / or CN 106957822, each one of which is hereby incorporated by reference in its entirety.Sources of Cells

[0331] Prior to expansion and genetic modification or other modification, a source of cells, e.g., T cells or natural killer (NK) cells, can be obtained from a subject. The term “subject” is intended to include living organisms in which an immune response can be elicited (e.g., mammals). Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present disclosure disclosure, immune effector cells, e.g., T cells, can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In one preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, the cells collected by apheresis may be washed to remove the plasma fraction and, optionally, to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium can lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow- through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer’s instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg -free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[0332] In some embodiments, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. The methods described herein can include, e.g., selection of a specific subpopulation of immune effector cells, e.g., T cells,that are a T regulatory cell-depleted population, CD25+ depleted cells, using, e.g., a negative selection technique, e.g., described herein. Preferably, the population of T regulatory depleted cells contains less than 30%, 25%, 20%, 15%, 1 0%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.

[0333] In some embodiments, T regulatory cells, e.g., CD25+ T cells, are removed from the population using an anti-C25 antibody, or fragment thereof, or a CD25-binding ligand, IL-2. In some embodiments, the anti-CD25 antibody, or fragment thereof, or CD25-binding ligand is conjugated to a substrate, e.g., a bead, or is otherwise coated on a substrate, e.g., a bead. In some embodiments, the anti-CD25 antibody, or fragment thereof, is conjugated to a substrate as described herein.

[0334] In some embodiments, the T regulatory cells, e.g., CD25+ T cells, are removed from the population using CD25 depletion reagent from Militenyi™. In some embodiments, the ratio of cells to CD25 depletion reagent is le7 cells to 20 uL, or le7 cells to 15 uL, or le7 cells to 10 uL, or le7 cells to 5 uL, or le7 cells to 2.5 uL, or le7 cells to 1.25 uL.

[0335] In some embodiments, the population of immune effector cells to be depleted includes about 6 x 109CD25+ T cells. In other embodiments, the population of immune effector cells to be depleted include about 1 x 109to lx 1010CD25+ T cell, and any integer value in between. In some embodiments, the resulting population T regulatory depleted cells has 2 x 109T regulatory cells, e.g., CD25+ cells, or less (e.g., 1 x 109, 5 x 108, 1 x 108, 5 x 107, 1 x 107, or less CD25+ cells).

[0336] In some embodiments, the T regulatory cells, e.g., CD25+ cells, are removed from the population using the CliniMAC system with a depletion tubing set, such as, e.g., tubing 162- OL In some embodiments, the CliniMAC system is run on a depletion setting such as, e.g., DEPLETION2. 1.

[0337] The methods described herein can include more than one selection step, e.g., more than one depletion step. Enrichment of a T cell population by negative selection can be accomplished, e.g., with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail can include antibodies to CD14, CD20, CDllb, CD16, HLA-DR, and CD8.

[0338] Also provided are methods that include removing cells from the population which express a check point inhibitor, e.g, a check point inhibitor described herein, e.g, one or more of PD1 + cells, LAG3+ cells, and TIM3+ cells, to thereby provide a population of T regulatory depleted, e.g., CD25+ depleted cells, and check point inhibitor depleted cells, e.g., PD1 +, LAG3+ and / or TIM3+ depleted cells. Exemplary check point inhibitors include B7-H1, B&-1, CD160, PIH, 2B4, PDI, TIM3, CEACAM (e.g, CEACAM-1, CEACAM-3 and / or CEACAM- 5), LAG3, TIGIT, CTLA-4, BTLA and LAIRI. In some embodiments, check point inhibitor expressing cells are removed simultaneously with the T regulatory, e.g, CD25+ cells. For example, an anti-C25 antibody, or fragment thereof, and an anti-check point inhibitor antibody, or fragment thereof, can be attached to the same bead which can be used to remove the cells, or an anti-CD25 antibody, or fragment thereof, and the anti-check point inhibitor antibody, or fragment there, can be attached to separate beads, a mixture of which can be used to remove the cells. In some embodiments, the removal of T regulatory cells, e.g, CD25+ cells, and the removal of the check point inhibitor expressing cells is sequential, and can occur, e.g, in either order.

[0339] Methods described herein can include a positive selection step. For example, T cells can be isolated by incubation with anti-CD3 / anti-CD28 (e.g, 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a time period sufficient for positive selection of the desired T cells. In some embodiments, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In some embodiments, the incubation time period is 24 hours. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies onthe beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points.

[0340] In some embodiments, a T cell population can be selected that expresses one or more of IFN-r, TNFa, IL-1 7A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other appropriate molecules, e.g., other cytokines. Methods for screening for cell expression can be determined, e.g., by the methods described in PCT Publication No.: WO 2013 / 126712.

[0341] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (e.g., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, In some embodiments, a concentration of about 10 billion cells / ml, 9 billion / ml, 8 billion / ml, 7 billion / ml, 6 billion / ml, or 5 billion / ml is used. In some embodiments, a concentration of 1 billion cells / ml is used. In some embodiments, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used.

[0342] Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

[0343] In a related embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In some embodiments, the concentration of cells used is 5 X 10^ / ml. In other embodiments, the concentration used can be from about 1 X 105 / ml to 1 X 106 / ml, and any integer value in between.

[0344] In other embodiments, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2- 1 0°C or at room temperature.

[0345] T cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media containing 10% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% Dextrose 5%, 0.45% NaCl, 1 0% Dextran 40 and 5% Dextrose, 20% Human Serum Albumin, and 7.5% DMSO or other suitable cell freezing media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80°C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at - 20° or in liquid nitrogen.

[0346] In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present disclosure.

[0347] Also contemplated in the context of embodiments herein is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, isolated and frozen for later use in immune effector cell therapy for any number of diseases or conditions that would benefit from immune effector cell therapy, such as those described herein. In one embodiment a blood sample or an apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the T cells may be expanded, frozen, and used at a later time. In certain embodiments, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further embodiment, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited totreatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, 5-azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.

[0348] In a further embodiment of the present disclosure, T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present disclosure to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, in certain embodiments, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and / or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0349] In some embodiments, the immune effector cells expressing a CAR molecule, e.g., a CAR molecule described herein, are obtained from a subject that has received a low, immune enhancing dose of an mTOR inhibitor. In some embodiments, the population of immune effector cells, e.g., T cells or NK cells, to be engineered to express a CAR, are harvested after a sufficient time, or after sufficient dosing of the low, immune enhancing, dose of an mTOR inhibitor, such that the level of PDI negative immune effector cells, e.g., T cells or NK cells, or the ratio of PDI negative immune effector cells, e.g., T cells / NK cells / PDI positive immune effector cells, e.g.,T cells or NK cells, in the subject or harvested from the subject has been, at least transiently, increased.

[0350] In some embodiments, population of immune effector cells, e.g., T cells or NK cells, which have, or will be engineered to express a CAR can be treated ex vivo by contact with an amount of an mTOR inhibitor that increases the number of PDI negative immune effectorcells, e.g., T cells or increases the ratio of PDI negative immune effector cells, e.g., T cells / NK cells / PDI positive immune effector cells, e.g., T cells or NK cells.

[0351] In some embodiments, a T cell population is diaglycerol kinase (DGK)-deficient. DGK- deficient cells include cells that do not express DGK RNA or protein, or have reduced or inhibited DGK activity. DGK-deficient cells can be generated by genetic approaches, e.g., administering RNA-interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or prevent DGK expression. Alternatively, DGK-deficient cells can be generated by treatment with DGK inhibitors described herein.

[0352] In some embodiments, a T cell population is Ikaros-deficient. Ikaros-defi cient cells include cells that do not express Ikaros RNA or protein, or have reduced or inhibited Ikaros activity, Ikaros-deficient cells can be generated by genetic approaches, e.g., administering RNA- interfering agents, e.g., siRNA, shRNA, miRNA, to reduce or prevent Ikaros expression. Alternatively, Ikaros-deficient cells can be generated by treatment with Ikaros inhibitors, e.g., lenalidomide.

[0353] In some embodiments, a T cell population is DGK-deficient and Ikaros-deficient, e.g., does not express DGK and Ikaros, or has reduced or inhibited DGK and Ikaros activity. Such DGK and Ikaros-deficient cells can be generated by any of the methods described herein.

[0354] In some embodiments, the NK cells are obtained from the subject. In some embodiments, the NK cells are an NK cell line, e.g., NK-92 cell line (Conkwest).Allogenic CART

[0355] In some embodiments described herein, the immune effector cell can be an allogenic immune effector cell, e.g., T cell or NK cell. For example, the cell can be an allogenic T cell, e.g., an allogenic T cell lacking expression of a functional T cell receptor (TCR) and / or human leukocyte antigen (HLA), e.g., HLA class 1 and / or HLA class II.

[0356] AT cell lacking a functional TCR can be, e.g., engineered such that it does not express any functional TCR on its surface, engineered such that it does not express one or more subunits that comprise a functional TCR or engineered such that it produces very little functional TCR on its surface. Alternatively, the T cell can express a substantially impaired TCR, e.g., by expression of mutated or truncated forms of one or more of the subunits of the TCR. The term “substantially impaired TCR” means that this TCR will not elicit an adverse immune reaction in a host. Such cells can be created throughout the use of one or more geneediting systems as described herein. In some embodiments, the gene editing system targets a sequence encoding a component of the TCR, for example a sequence in the TCR alpha constant chain gene (TRAC) or its regulatory elements. In some embodiments, the gene editing system targets a sequence encoding a component of the TCR, for example a sequence in the TCR beta constant chain gene (TRBC) or its regulatory elements.

[0357] A T cell described herein can be, e.g., engineered such that it does not express a functional HLA on its surface. For example, a T cell described herein, can be engineered such that cell surface expression HLA, e.g., HLA class 1 and / or HLA class II, is downregulated. Such cells can be created through the use of one or more gene editing systems as described herein. In some embodiments, the gene editing system targets a sequence encoding a component of one or more HLA molecules. In some embodiments, the gene editing system targets a sequence encoding a factor which affects the expression of one or more HLA molecules. In some embodiments, the gene editing system targets a regulator of MHC class 1 expression, for example a sequence encoding beta-2 microglobulin (B2M). In some embodiments, the gene editing system targets a sequence encoding a regulator of MHC class II molecule expression, for example, CIITA. In some embodiments, gene editing systems targeting both a regulator of MHC class 1 expression (for example, B2M) and a regulator ofMHC class II molecule expression (e.g., CIITA) are introduced into the cells, such that at least MHC class 1 molecule and at least one MHC class II molecule expression is downregulated.

[0358] In some embodiments, the T cell can lack a functional TCR and a functional HLA, e.g., HLA class 1 and / or HLA class II. Modified T cells that lack expression of a functional TCR and / or HLA can be obtained by any suitable means, including a knock out or knock down of one or more subunit of TCR or HLA. For example, the T cell can include a knock down of TCR and / or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR) transcription- activator like effector nuclease (TALEN), or zinc finger endonuclease (ZFN).

[0359] In some embodiments, the allogeneic cell can be a cell which does not express or expresses at low levels an inhibitory molecule, e.g. by any method described herein. For example, the cell can be a cell that does not express or expresses at low levels an inhibitory molecule, e.g., that can decrease the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PDI, PD-LI, CTLA4, TIM3,CEACAM (e.g., CEACAM-1, CEACAM-3 and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIRI, CD 160, 2B4 and TGF beta. Inhibition of an inhibitory molecule, e.g., by inhibition at the DNA, RNA or protein level, can optimize a CAR-expressing cell performance. In some embodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription-activator like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), e.g., as described herein, can be used. siRNA and shRNA to inhibit, e.g., TCR or HLA

[0360] In some embodiments, TCR expression and / or HLA expression can be inhibited using siRNA or shRNA that targets a nucleic acid encoding a TCR and / or HLA in a T cell. Expression of siRNA and shRNAs in T cells can be achieved using any conventional expression system, e.g., such as a lentiviral expression system.

[0361] Exemplary shRNAs that downregulate expression of components of the TCR are described, e.g., in US Publication No.: 2012 / 0321667. Exemplary siRNA and shRNA that downregulate expression of HLA class 1 and / or HLA class II genes are described, e.g., in U.S. publication No.: US 2007 / 0036773.CRISPR to inhibit, e.g., TCR or HLA

[0362] “CRISPR” or “CRISPR to TCR and / or HLA” or “CRISPR to inhibit TCR and / or HLA” as used herein refers to a set of clustered regularly interspaced short palindromic repeats, or a system comprising such a set of repeats. “Cas”, as used herein, refers to a CRISPR-associated protein. A “CRISPR / Cas” system refers to a system derived from CRISPR and Cas which can be used to silence or mutate a TCR and / or HLA gene.

[0363] Naturally-occurring CRISPR / Cas systems are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008) Science 322: 1843- 1845.

[0364] The CRISPR / Cas system has been modified for use in gene editing (silencing, enhancing or changing specific genes) in eukaryotes such as mice or primates. Wiedenheft etal. (2012) Nature 482: 331-8. This is accomplished by introducing into the eukaryotic cell a plasmid containing a specifically designed CRISPR and one or more appropriate Cas.

[0365] The CRISPR sequence, sometimes called a CRISPR locus, comprises alternating repeats and spacers. In a naturally-occurring CRISPR, the spacers usually comprise sequences foreign to the bacterium such as a plasmid or phage sequence; in the TCR and / or HLA CRISPR / Cas system, the spacers are derived from the TCR or HLA gene sequence.

[0366] RNA from the CRISPR locus is constitutively expressed and processed by Cas proteins into small RNAs. These comprise a spacer flanked by a repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Horvath et al. (2010) Science 327: 167-170; Makarova et al. (2006) Biology Direct 1: 7. The spacers thus serve as templates for RNA molecules, analogously to siRNAs. Pennisi (2013) Science 341 : 833-836.

[0367] As these naturally occur in many different types of bacteria, the exact arrangements of the CRISPR and structure, function and number of Cas genes and their product differ somewhat from species to species. Haft et al. (2005) PLoS Comput. Biol. 1: e60; Kunin et al. (2007) Genome Biol. 8: R61; Mojica et al. (2005) J. Mai. Eval. 60: 174-182; Bolotin et al. (2005) Microbial. 151 : 2551-2561; Pourcel et al. (2005) Microbial. 151 : 653-663; and Stern et al. (2010) Trends. Genet. 28: 335-340. For example, the Cse (Cas subtype, E.coli) proteins (e.g., Cas A) form a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer- repeat units that Cascade retains. Brouns et al. (2008) Science 321 : 960-964. In other prokaryotes, Cas6 processes the CRISPR transcript. The CRISPR-based phage inactivation in E. coli requires Cascade and Cas3, but not Casl or Cas2. The Cmr (Cas RAMP module) proteins in Pyrococcus furiosus and other prokaryotes form a functional complex with small CRISPR RNAs that recognizes and cleaves complementarity target RNAs. A simpler CRISPR system relies on the protein Cas9, which is a nuclease with two active cutting sites, one for each strand of the double helix. Combining Cas9 and modified CRISPR locus RNA can be used in a system for gene editing. Pennisi (2013) Science 341 : 833-836.

[0368] The CRISPR / Cas system can thus be used to edit a TCR and / or HLA gene (adding / deleting one or more base pairs), or for introducing a premature stop which thus decreases expression of a target gene or chromosomal sequence such as a TCR and / or HLA. The CRISPR / Cas system can alternatively be used like RNA interference, turning off TCR and / or HLA gene in a reversible fashion. In a mammalian cell, for example, the RNA can guidethe Cas protein, e.g., a Cas protein lacking nuclease activity (e.g., dCas9), to a TCR and / or HLA promoter, sterically blocking RNA polymerases.

[0369] Artificial CRISPR / Cas systems can be generated which inhibit, for example, TCR and / or HLA, using technology known in the art, e.g., that described in U.S. Publication No.20140068797. CRISPR systems which may be useful in embodiments described herein include those described in, for example, PCT application publication WO2017 / 093969, the contents of which are incorporated herein by reference in their entirety.TALEN to inhibit, e.g., TCR and / or HLA

[0370] “TALEN” or “TALEN to HLA and / or TCR” or “TALEN to inhibit HLA and / or TCR” refers to a transcription activator-like effector nuclease, an artificial nuclease which can be used to edit the HLA and / or TCR gene.

[0371] TALENs are produced artificially by fusing a TAL effector DNA binding domain to a DNA cleavage domain. Transcription activator-like effects (TALEs) can be engineered to bind any desired DNA sequence, including a portion of the HLA or TCR gene. By combining an engineered TALE with a DNA cleavage domain, a restriction enzyme can be produced which is specific to any desired DNA sequence, including a HLA or TCR sequence. These can then be introduced into a cell, wherein they can be used for genome editing. Boch (2011) Nature Biotech. 29: 135-6; and Boch et al. (2009) Science 326: 1509-12; Moscou et al. (2009) Science 326: 3501.

[0372] TALEs are proteins secreted by Xanthomonas bacteria. The DNA binding domain contains a repeated, highly conserved 33-34 amino acid sequence, with the exception of the 12thand 13th amino acids. These two positions are highly variable, showing a strong correlation with specific nucleotide recognition. They can thus be engineered to bind to a desired DNA sequence.

[0373] To produce a TALEN, a TALE protein is fused to a nuclease (N), which is a wild-type or mutated Fokl endonuclease. Several mutations to Fokl have been made for its use in TALENs; these, for example, improve cleavage specificity or activity. Cermak et al. (2011) Nucl. Acids Res. 39: e82; Miller et al. (2011) Nature Biotech. 29: 143-8; Hockemeyer et al. (2011) Nature Biotech. 29: 731-734; Wood et al. (2011) Science 333: 307; Doyon et al. (2010) Nature Methods 8: 74-79; Szczepek et al. (2007) Nature Biotech. 25: 786-793; and Guo et al. (2010) J. Mai. Biol. 200: 96.

[0374] The Fokl domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the Fokl cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity. Miller et al. (2011) Nature Biotech. 29: 143-8.

[0375] A HLA or TCR TALEN can be used inside a cell to produce a double-stranded break (DSB). A mutation can be introduced at the break site if the repair mechanisms improperly repair the break via non-homologous end joining. For example, improper repair may introduce a frame shift mutation. Alternatively, foreign DNA can be introduced into the cell along with the TALEN; depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to correct a defect in the HLA or TCR gene or introduce such a defect into a wt HLA or TCR gene, thus decreasing expression of HLA or TCR.

[0376] TALENs specific to sequences in HLA or TCR can be constructed using any method known in the art, including various schemes using modular components. Zhang et al. (2011) Nature Biotech. 29: 149-53; Geibler et al. (2011) Zo5 ONE 6: el 9509.Zinc finger nuclease to inhibit, e.g., HLA and / or TCR

[0377] “ZFN” or “Zinc Finger Nuclease” or “ZFN to HLA and / or TCR” or “ZFN to inhibit HLA and / or TCR” refer to a zinc finger nuclease, an artificial nuclease which can be used to edit the HLA and / or TCR gene.

[0378] Like a TALEN, a ZFN comprises a Fok: 1 nuclease domain (or derivative thereof) fused to a DNA-binding domain. In the case of a ZFN, the DNA-binding domain comprises one or more zinc fingers. Carroll et al. (2011) Genetics Society of America 188: 773-782; and Kim et al. 20 (1996) Proc. Natl. Acad. Sci. USA 93: 1156-1160.

[0379] A zinc finger is a small protein structural motif stabilized by one or more zinc ions. A zinc finger can comprise, for example, Cys2His2, and can recognize an approximately 3-bp sequence. Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides which recognize about 6, 9, 12, 15 or 18-bp sequences. Various selection and modular assembly techniques are available to generate zinc fingers (and combinations thereof) recognizing specific sequences, including phage display, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells.

[0380] Like a TALEN, a ZFN must dimerize to cleave DNA Thus, a pair of ZFNs are required to target non-palindromic DNA sites. The two individual ZFNs must bind opposite strands of the DNA with their nucleases properly spaced apart. Bitinaite et al. (1998) Proc. Natl. Acad. Set. USA 95: 10570-5. Also like a TALEN, a ZFN can create a double-stranded break in the DNA, which can create a frame-shift mutation if improperly repaired, leading to a decrease in the expression and amount ofBILA and / or TCR in a cell. ZFNs can also be used with homologous recombination to mutate in the HLA or TCR gene.

[0381] ZFNs specific to sequences in HLA AND / OR TCR can be constructed using any method known in the art. Cathomen et al. (2008)MoL Ther. 16: 1200-7; and Guo et al. (2010) J. Mai. Biol. 400: 96.Activation and Expansion of Immune Effector Cells (e.g., T Cells)

[0382] Immune effector cells such as T cells may be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0383] Generally, a population of immune effector cells may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3 / TCR complex associated signal and a ligand that stimulates a costimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4+ T cells or CD8+ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besarn;;on, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol Meth. 227(1- 2):53-63, 1999).

[0384] In certain embodiments, the primary stimulatory signal and the costimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In some embodiments, the agent providing the costimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In some embodiments, the agents may be in soluble form, and then cross- linked to a surface, such as a cell expressing Fe receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present disclosure.

[0385] In some embodiments, the two agents are immobilized on beads, either on the same bead, i.e., “cis,” or to separate beads, i.e., “trans.” By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof and the agent providing the costimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts. In some embodiments, a 1 : 1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell growth is used. In certain embodiments of the present disclosure, a ratio of anti CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1 : 1. In one particular embodiment an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1 : 1. In some embodiments, the ratio of CD3:CD28 antibody bound to the beads ranges from 100: 1 to 1 : 100 and all integer values there between. In one embodiment of the present disclosure, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In some embodiments, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2: 1. In one particular embodiment, a 1 : 100 CD3 :CD28 ratio of antibody bound to beads is used. In some embodiments, a 1 :75 CD3:CD28 ratio of antibody bound to beads is used. In a further embodiment, a 1 :50 CD3:CD28 ratio of antibody bound to beads is used. In some embodiments, a 1 :30 CD3:CD28 ratio of antibody bound to beads is used. In one preferredembodiment, a 1 : 10 CD3:CD28 ratio of antibody bound to beads is used. In some embodiments, a 1 :3 CD3:CD28 ratio of antibody bound to the beads is used. In yet one embodiment, a 3: 1 CD3:CD28 ratio of antibody bound to the beads is used.

[0386] Ratios of particles to cells from 1 :500 to 500: 1 and any integer values in between may be used to stimulate T cells or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many. In certain embodiments the ratio of cells to particles ranges from 1 : 100 to 100: 1 and any integer values in-between and in further embodiments the ratio comprises 1:9 to 9: 1 and any integer values in between, can also be used to stimulate T cells. The ratio of anti-CD3- and anti-CD28-coupled particles to T cells that result in T cell stimulation can vary as noted above, however certain preferred values include 1 : 100, 1 :50, 1 :40, 1 :30, 1 :20, 1 : 10, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 : 1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, and 15: 1 with one preferred ratio being at least 1 : 1 particles per T cell. In some embodiments, a ratio of particles to cells of 1 : 1 or less is used. In one particular embodiment, a preferred particle: cell ratio is 1 : 5. In further embodiments, the ratio of particles to cells can be varied depending on the day of stimulation. For example, In some embodiments, the ratio of particles to cells is from 1 : 1 to 10: 1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1 : 1 to 1 : 10 (based on cell counts on the day of addition). In one particular embodiment, the ratio of particles to cells is 1 : 1 on the first day of stimulation and adjusted to 1 :5 on the third and fifth days of stimulation. In some embodiments, particles are added on a daily or every other day basis to a final ratio of 1 : 1 on the first day, and 1 :5 on the third and fifth days of stimulation. In some embodiments, the ratio of particles to cells is 2: 1 on the first day of stimulation and adjusted to 1 : 10 on the third and fifth days of stimulation. In some embodiments, particles are added on a daily or every other day basis to a final ratio of 1 : 1 on the first day, and 1 : 10 on the third and fifth days of stimulation. One of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present disclosure. In particular, ratios will vary depending on particle size and on cell size and type. In some embodiments, the most typical ratios for use are in the neighborhood of 1 : 1, 2: 1 and 3 : 1 on the first day.

[0387] In further embodiments of the present disclosure, the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and thenthe cells are cultured. In an alternative embodiment, prior to culture, the agent-coated beads and cells are not separated but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[0388] By way of example, cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3x28 beads) to contact the T cells. In one embodiment the cells (for example, 10^ to 10^ T cells) and beads (for example, DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads at a ratio of 1 : 1) are combined in a buffer, for example PBS (without divalent cations such as, calcium and magnesium). Again, those of ordinary skill in the art can readily appreciate any cell concentration may be used. For example, the target cell may be very rare in the sample and comprise only 0.01% of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest. Accordingly, any cell number is within the context of the present disclosure. In certain embodiments, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and particles. For example, In some embodiments, a concentration of about 10 billion cells / ml, 9 billion / ml, 8 billion / ml, 7 billion / ml, 6 billion / ml, or 5 billion / ml is used. In some embodiments, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet one embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such populations of cells may have therapeutic value and would be desirable to obtain in certain embodiments. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

[0389] In some embodiments, cells transduced with a nucleic acid encoding a CAR, e.g., a CAR described herein, are expanded, e.g., by a method described herein. In some embodiments, the cells are expanded in culture for a period of several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13 or 14 days). In some embodiments, the cells are expanded for a period of 4 to 9 days. In some embodiments, the cells are expanded for a period of 8 days or less, e.g., 7, 6 or 5 days. In some embodiments, the cells, e.g., a cell comprising, e.g., expressing, a dual CAR or a tandem CAR described herein, are expanded in culture for 5 days, and the resulting cells are more potent than the same cells expanded in culture for 9 days under the same culture conditions. Potency can be defined, e.g., by various T cell functions, e.g. proliferation, target cell killing, cytokine production, activation, migration, or combinations thereof.

[0390] In some embodiments, the cells, e.g., the cells comprising, e.g., expressing, a dual CAR or a tandem CAR described herein, are expanded in culture for 5 days, and the resulting cells exhibit higher proinflammatory cytokine production, e.g., IFN-y and / or GM-CSF levels, as compared to the same cells expanded in culture for 9 days under the same culture conditions. In some embodiments, the cells comprising, e.g., expressing, a dual CAR or a tandem CAR described herein, are expanded for 5 days show at least a one, two, three, four, five, ten fold or more increase in pg / ml of proinflammatory cytokine production, e.g., IFN-y and / or GM- CSF levels, as compared to the same cells expanded in culture for 9 days under the same culture conditions. In one embodiment of the present disclosure, the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In some embodiments, the mixture may be cultured for 21 days. In some embodiments, the beads and the T cells are cultured together for about eight days. In some embodiments, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired such that culture time of T cells can be 60 days or more. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, GM-CSF, IL-1 0, IL-12, IL-15, TGF , and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X- Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures ofcells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C) and atmosphere (e.g., air plus 5% CO2).

[0391] In some embodiments, the cells are expanded in an appropriate media (e.g., media described herein) that includes one or more interleukin that result in at least a 200-fold (e.g., 200-fold, 250- fold, 300-fold, 350-fold) increase in cells over a 14 day expansion period, e.g., as measured by a method described herein such as flow cytometry. In some embodiments, the cells are expanded in the presence IL-15 and / or IL-7 (e.g., IL-15 and IL-7).

[0392] T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4+) that is greater than the cytotoxic or suppressor T cell population (TC, CD8+). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of TC cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.

[0393] Further, in addition to Cn4 and ens markers, other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process.

[0394] Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes. Once a CAR, e.g., a dual CAR or a tandem CAR, is constructed, various assays can be used to evaluate the activity of the molecule, such as but not limited to, the ability to expand T cells following antigen stimulation, sustain T cell expansion in the absence of restimulation, and anti-cancer activities in appropriate in vitro and animal models. Assays to evaluate the effects of a CAR, e.g., a dual CAR or a tandem CAR, are described in further detail below.

[0395] Western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers anders. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Very briefly, T cells (1 : 1 mixture of cn4+ and ens+ T cells) expressing the CARs are expanded in vitro for more than 10 days followed by lysis and SnS-PAGE under reducing conditions. CARs containing the full length TCR-1;: cytoplasmic domain and the endogenousTCR-1;: chain are detected by western blotting using an antibody to the TCR-1;: chain. The same T cell subsets are used for SnS-PAGE analysis under non-reducing conditions to permit evaluation of covalent dimer formation.

[0396] In vitro expansion of CAR+ T cells following antigen stimulation can be measured by flow cytometry. For example, a mixture of cn4+ and ens+ T cells are stimulated with aCn3 / aCn28 aAPCs followed by transduction with lentiviral vectors expressing GFP under the control of the promoters to be analyzed. Exemplary promoters include the CMV IE gene, EFla, ubiquitin C, or phosphoglycerokinase (PGK) promoters. GFP fluorescence is evaluated on day 6 of culture in the cn4+ and / or ens+ T cell subsets by flow cytometry. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Alternatively, a mixture of cn4+ and ens+ T cells are stimulated with aCn3 / aCn28 coated magnetic beads on day 0, and transduced with CAR on day 1 using a bicistronic lentiviral vector expressing CAR along with eGFP using a 2A ribosomal skipping sequence. Cultures are re-stimulated with either cnl9+ K562 cells (K562-CD19), wild-type K562 cells (K562 wild type) or K562 cells expressing hCD32 and 4- IBBL in the presence of antiCD3 and anti-CD28 antibody (K562-BBL-3 / 28) following washing. Exogenous IL-2 is added to the cultures every other day at 1 00 lU / ml. GFP+ T cells are enumerated by flow cytometry using bead-based counting. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Similar assays can be performed using anti-CD20 T cells (see, e.g. Gill et al Blood 2014; 123:2343) or with anti-CD20 CART cells.

[0397] Sustained CAR+ T cell expansion in the absence of re-stimulation can also be measured. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, mean T cell volume (fl) is measured on day 8 of culture using a Coulter Multisizer III particle counter, a Nexcelom Cellometer Vision, or Millipore Scepter following stimulation with aCD3 / aCD28 coated magnetic beads on day 0, and transduction with the indicated CAR on day 1.

[0398] Animal models can also be used to measure a CART activity. For example, xenograft model using human CD19-specific CAR+ T cells to treat a primary human pre-BALL in immunodeficient mice can be used. See, e.g., Milone et al., Molecular Therapy 17(8): 1453- 1464 (2009). Very briefly, after establishment of ALL, mice are randomized as to treatment groups. Different numbers of aCD19-l;: and aCD19-BB-l;: engineered T cells are coinjected at a 1 : 1 ratio into NOD-SCID-y- / - mice bearing B-ALL. The number of copies of aCD19-l;: and aCD19-BB-l;: vector in spleen DNA from mice is evaluated at various times following T cell injection. Animals are assessed for leukemia at weekly intervals. Peripheral blood CD 19+B-ALL blast cell counts are measured in mice that are injected with aCD19-l;: CAR+ T cells or mock-transduced T cells. Survival curves for the groups are compared using the log-rank test. In addition, absolute peripheral blood CD4+ and CD8+ T cell counts 4 weeks following T cell injection in NOD-SCID-y-1- mice can also be analyzed. Mice are injected with leukemic cells and 3 weeks later are injected with T cells engineered to express CAR by a bicistronic lentiviral vector that encodes the CAR linked to eGFP T cells are normalized to 45-50% input GFP+ T cells by mixing with mock-transduced cells prior to injection, and confirmed by flow cytometry. Animals are assessed for leukemia at I-week intervals. Survival curves for the CAR+ T cell groups are compared using the log-rank test. Similar experiments can be done with dual CARTs or tandem CARTs.

[0399] Dose dependent CAR treatment response can be evaluated. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). For example, peripheral blood is obtained 35-70 days after establishing leukemia in mice injected on day 21 with CART cells, an equivalent number of mock-transduced T cells, or no T cells. Mice from each group are randomly bled for determination of peripheral blood CD 19+ ALL blast counts and then killed on days 35 and 49. The remaining animals are evaluated on days 57 and 70. Similar experiments can be done with dual CARTs or tandem CARTs.

[0400] Assessment of cell proliferation and cytokine production has been previously described, e.g., at Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, assessment of CAR-mediated proliferation is performed in microtiter plates by mixing washed T cells with K562 cells expressing CD19 (K19) or CD32 and CD137 (KT32-BBL) for a final T-cell :K562 ratio of 2: 1. K562 cells are irradiated with gamma-radiation prior to use. Anti-CD3 (clone OKT3) and anti- CD28 (clone 9.3) monoclonal antibodies are added to cultures with KT32-BBL cells to serve as a positive control for stimulating T-cell proliferation since these signals support long-term CD8+ T cell expansion ex vivo. T cells are enumerated in cultures using CountBright™ fluorescent beads (Invitrogen, Carlsbad, CA) and flow cytometry as described by the manufacturer. CAR+ T cells are identified by GFP expression using T cells that are engineered with eGFP-2 A linked CAR-expressing lentiviral vectors. For CAR+ T cells not expressing GFP, the CAR+ T cells are detected with biotinylated recombinant CD 19 protein and a secondary avidin-PE conjugate. CD4+ and CD8+ expression on T cells are also simultaneously detected with specific monoclonal antibodies (BD Biosciences). Cytokine measurements are performed on supernatants collected 24 hours following re-stimulation usingthe human TH1 / TH2 cytokine cytometric bead array kit (BD Biosciences, San Diego, CA) according the manufacturer’s instructions or using a Luminex 30-plex kit (Invitrogen). Fluorescence is assessed using a BD Fortessa flow cytometer, and data is analyzed according to the manufacturer’s instructions. Similar experiments can be done with dual CARTs or tandem CARTs.

[0401] Cytotoxicity can be assessed by a standard 51Cr-release assay. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009). Briefly, target cells (K562 lines and primary pro- B-ALL cells) are loaded with 51Cr (as NaCrO4, New England Nuclear, Boston, MA) at 37°C for 2 hours with frequent agitation, washed twice in complete RPMI and plated into microtiter plates. Effector T cells are mixed with target cells in the wells in complete RPMI at varying ratios of effector celktarget cell (E:T). Additional wells containing media only (spontaneous release, SR) or a 1% solution of triton-X 100 detergent (total release, TR) are also prepared.

[0402] After 4 hours of incubation at 37°C, supernatant from each well is harvested. Released 5 ICr is then measured using agamma particle counter (Packard Instrument Co., Waltham, MA). Each condition is performed in at least triplicate, and the percentage of lysis is calculated using the formula:% Lysis =(ER-SR) / (TR- SR), where ER represents the average 51Cr released for each experimental condition.

[0403] Imaging technologies can be used to evaluate specific trafficking and proliferation of CARs in tumor-bearing animal models. Such assays have been described, for example, in Barrett et al., Human Gene Therapy 22: 1575-1586 (2011). Briefly, NOD / SCID / yc-1- (NSG) mice are injected IV with Nalm-6 cells followed 7 days later with T cells 4 hour after electroporation with the CAR constructs. The T cells are stably transfected with a lentiviral construct to express firefly luciferase, and mice are imaged for bioluminescence. Alternatively, therapeutic efficacy and specificity of a single injection of CAR+ T cells in Nalm-6 xenograft model can be measured as the following: NSG mice are injected with Nalm-6 transduced to stably express firefly luciferase, followed by a single tail-vein injection of T cells electroporated with a CAR 7 days later. Animals are imaged at various time points post injection. For example, photon-density heat maps of firefly luciferase positive leukemia in representative mice at day 5 (2 days before treatment) and day 8 (24 hr post CAR+PBLs) can be generated.

[0404] Other assays, including those described in the Example section herein as well as those that are known in the art can also be used to evaluate the dual CART or tandem CART constructs disclosed herein.Methods of Treatment

[0405] The present disclosure provides, among other things, compositions and methods for treating a cancer or a disease associated with expression of an amino acid sequence comprising SEQ ID NO: 1, SEQ ID NO:2, and / or SEQ ID NO:3, or functional variants thereof. The cancer or disease includes New York esophageal squamous cell carcinoma.

[0406] Some embodiments pertain to a method of treating cancer in a subject. The method comprises administering to the subject a CAR-expressing cell, e.g., a dual CAR or tandem CAR -expressing cell of the present disclosure such that the cancer is treated in the subject. An example of a cancer that is treatable by the CAR-expressing cell, e.g., dual CAR or tandem CAR -expressing cell of embodiments herein is New York esophageal squamous cell carcinoma 1. An example of a cancer that is treatable by the CAR-expressing cell, e.g., dual CAR or tandem CAR -expressing cell of embodiments herein includes but is not limited to a hematological cancer described herein. Embodiments herein include a type of cellular therapy where cells are genetically modified to express a chimeric antigen receptor (CAR) and the CAR-expressing cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Unlike antibody therapies, CAR-modified cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control. In various embodiments, the T cells administered to the patient, or their progeny, persist in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty- one months, twenty -two months, twenty -three months, two years, three years, four years, or five years after administration of the cell to the patient.

[0407] Embodiments herein also include a type of cellular therapy where immune effector cells, e.g., NK cells or T cells are modified, e.g., by in vitro transcribed RNA, to transiently express a chimeric antigen receptor (CAR) and the CAR-expressing (e.g., CART or CAR- expressing NK) cell is infused to a recipient in need thereof. The infused cell is able to kill cancer cells in the recipient. Thus, in various embodiments, the CAR-expressing cells, e.g., TorNK cells, administered to the patient, is present for less than one month, e.g., three weeks, two weeks, one week, after administration of the CAR-expressing cell, e.g., Tor NK cell, to the patient.

[0408] In some embodiments, the CAR-modified cells, e.g., fully human CAR- expressing cells, may be a type of vaccine for ex vivo immunization and / or in vivo therapy in a mammal. In some embodiments, the mammal is a human.

[0409] With respect to ex vivo immunization, at least one of the following occurs in vitro prior to administering the cell into a mammal: i) expansion of the cells, ii) introducing a nucleic acid encoding a CAR to the cells or iii) cry opreservation of the cells.

[0410] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR- modified cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the CAR-modified cell can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.

[0411] The procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described in U.S. Pat. No. 5,199,942, incorporated herein by reference, can be applied to the cells of the present disclosure. Other suitable methods are known in the art, therefore the present disclosure is not limited to any particular method of ex vivo expansion of the cells. Briefly, ex vivo culture and expansion of T cells comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammal from peripheral blood harvest or bone marrow explants; and (2) expanding such cells ex vivo. In addition to the cellular growth factors described in U.S. Pat. No. 5,199,942, other factors such as FLT3-L, IL-1, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.

[0412] In addition to using a cell-based vaccine in terms of ex vivo immunization, the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.

[0413] Embodiment herein may be used to treat a proliferative disease such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia.

[0414] Non-Hodgkin lymphoma (NHL) is a group of cancers of lymphocytes, formed from either B or T cells. NHLs occur at any age and are often characterized by lymph nodes that are larger than normal, weight loss, and fever. Different types of NHLs are categorized as aggressive (fast-growing) and indolent (slow-growing) types. B-cell non-Hodgkin lymphomas include Burkitt lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and mantle cell lymphoma. Examples of T- cell non-Hodgkin lymphomas include mycosis fungoides, anaplastic large cell lymphoma, and precursor T-lymphoblastic lymphoma. Lymphomas that occur after bone marrow or stem cell transplantation are typically B-cell non-Hodgkin lymphomas. See, e.g., Maloney. NEJM. 366.21(2012):2008-16. Diffuse large B-cell lymphoma (DLBCL) is a form of NHL that develops from B cells. DLBCL is an aggressive lymphoma that can arise in lymph nodes or outside of the lymphatic system, e.g., in the gastrointestinal tract, testes, thyroid, skin, breast, bone, or brain. Three variants of cellular morphology are commonly observed in DLBCL: centroblastic, immunoblastic, and anaplastic. Centroblastic morphology is most common and has the appearance of medium-to-large-sized lymphocytes with minimal cytoplasm. There are several subtypes of DLBCL. For example, primary central nervous system lymphoma is a type of DLBCL that only affects the brain is called and is treated differently than DLBCL that affects areas outside of the brain. Another type of DLBCL is primary mediastinal B-cell lymphoma, which often occurs in younger patients and grows rapidly in the chest. Symptoms of DLBCL include a painless rapid swelling in the neck, armpit, or groin, which is caused by enlarged lymph nodes. For some subjects, the swelling may be painful. Other symptoms of DLBCL include night sweats, unexplained fevers, and weight loss. Although most patients with DLBCL are adults, this disease sometimes occurs in children. Treatment for DLBCL includes chemotherapy (e.g., cyclophosphamide, doxorubicin, vincristine, prednisone, etoposide), antibodies (e.g., Rituxan), radiation, or stem cell transplants.

[0415] Follicular lymphoma is a type of non-Hodgkin lymphoma and is a lymphoma of follicle center B-cells (centrocytes and centroblasts), which has at least a partially follicular pattern. Follicular lymphoma cells express the B-cell markers CD10, CD19, CD20, and CD22. Follicular lymphoma cells are commonly negative for CDS. Morphologically, a follicular lymphoma tumor is made up of follicles containing a mixture of centrocytes (also calledcleaved follicle center cells or small cells) and centroblasts (also called large noncleaved follicle center cells or large cells). The follicles are surrounded by non-malignant cells, mostly T-cells. The follicles contain predominantly centrocytes with a minority of centroblasts. The World Health Organization (WHO) morphologically grades the disease as follows: grade 1 (<5 centroblasts per high-power field (hpf); grade 2 (6-15 centroblasts / hpf); grade 3 (>15 centroblasts / hpf). Grade 3 is further subdivided into the following grades: grade 3A (centrocytes still present); grade 3B (the follicles consist almost entirely of centroblasts). Treatment of follicular lymphoma includes chemotherapy, e.g., alkyating agents, nucleoside analogs, anthracycline-containing regimens, e.g., a combination therapy called CHOP- cyclophosphamide, doxorubicin, vincristine, prednisone / prednisolone, antibodies (e.g., rituximab), radioimmunotherapy, and hematopoietic stem cell transplantation.

[0416] CLL is a B-cell malignancy characterized by neoplastic cell proliferation and accumulation in bone morrow, blood, lymph nodes, and the spleen. The median age at time of diagnosis of CLL is about 65 years. Current treatments include chemotherapy, radiation therapy, biological therapy, or bone marrow transplantation. Sometimes symptoms are treated surgically (e.g., splenectomy removal of enlarged spleen) or by radiation therapy (e.g., debulking swollen lymph nodes). Chemotherapeutic agents to treat CLL include, e.g., fludarabine, 2- chlorodeoxyadenosine (cladribine), chlorambucil, vincristine, pentostatin, cyclophosphamide, alemtuzumab (Campath-IH), doxorubicin, and prednisone. Biological therapy for CLL includes antibodies, e.g., alemtuzumab, rituximab, and ofatumumab; as well as tyrosine kinase inhibitor therapies. A number of criteria can be used to classify stage of CLL, e.g., the Rai or Binet system. The Rai system describes CLL has having five stages: stage O where only lymphocytosis is present; stage 1 where lymphadenopathy is present; stage II where splenomegaly, lymphadenopathy, or both are present; stage III where anemia, organomegaly, or both are present (progression is defined by weight loss, fatigue, fever, massive organomegaly, and a rapidly increasing lymphocyte count); and stage IV where anemia, thrombocytopenia, organomegaly, or a combination thereof are present. Under the Binet staging system, there are three categories: stage A where lymphocytosis is present and less than three lymph nodes are enlarged (this stage is inclusive of all Rai stage O patients, one-half of Rai stage 1 patients, and one-third of Rai stage II patients); stage B where three or more lymph nodes are involved; and stage C wherein anemia or thrombocytopenia, or both are present. These classification systems can be combined with measurements of mutation of theimmunoglobulin genes to provide a more accurate characterization of the state of the disease. The presence of mutated immunoglobulin genes correlates to improved prognosis.

[0417] New York esophageal squamous cell carcinoma 1 (NY-ESO-1) is a known cancer testis gene with exceptional immunogenicity and prevalent expression in many cancer types. These characteristics have made it an appropriate vaccine candidate with the potential application against various malignancies.

[0418] In some embodiments, the CAR-expressing cells of the present disclosure are used to treat cancers or leukemias, e.g., with leukemia stem cells. For example, the leukemia stem cells can be CD34+ / CD38-leukemia cells.

[0419] The present disclosure provides, among other things, compositions and methods for treating cancer. In some embodiments, the cancer is a hematologic cancer including but is not limited to one or more acute leukemias including but not limited to B-cell acute lymphoblastic leukemia (BALL), e.g., pediatric BALL and / or adult BALL, T-cell acute lymphoblastic leukemia (TALL), small lymphocytic lymphoma (SLL), acute lymphoblastic leukemia (ALL); one or more chronic leukemias including but not limited to chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL); additional hematologic cancers or hematologic conditions including, but not limited to mantle cell lymphoma (MCL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and “preleukemia” which is a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells.

[0420] The CAR-modified cells of the present disclosure may be administered either alone, or as a pharmaceutical composition in combination with diluents and / or with other components such as IL-2 or other cytokines or cell populations.Bone Marrow Ablation

[0421] In some embodiments, the disclosure provides a method of bone marrow ablation comprising administering a CAR-expressing cell, e.g., a dual CAR or tandem CAR-expressingcell, of embodiments herein to a subject in need of bone marrow ablation. For example, the present method may be used to eradicate some or all of the existing bone marrow of a subject having a disease or disorder in which bone marrow transplantation or bone marrow reconditioning is a beneficial treatment strategy. In some embodiments, the bone marrow ablation method, comprising the administration of a CAR-expressing cell, e.g., a dual CAR or tandem CAR -expressing cell, described elsewhere herein, is performed in a subject prior to bone marrow transplantation. Thus, In some embodiments, the method of the disclosure provides a cellular conditioning regimen prior to bone marrow or stem cell transplantation. In some embodiments, bone marrow transplantation comprises transplantation of a stem cell. The bone marrow transplantation may comprise transplantation of autologous or allogeneic cells.

[0422] The present disclosure provides a method of treating a disease or disorder comprising administering a CAR-expressing cell, e.g., a dual CAR or tandem CAR-expressing cell herein to eradicate at least a portion of existing bone marrow. The method may be used as at least a portion of a treatment regimen for treating any disease or disorder where bone marrow transplantation is beneficial. That is, the present method may be used in any subject in need of a bone marrow transplant. In some embodiments, bone marrow ablation comprising administration of a CAR-expressing cell, e.g., a dual CAR or tandem CAR -expressing cell, is useful in the treatment of AML. In certain embodiments, bone marrow ablation by way of the present method is useful in treating a hematological cancer, a solid tumor, a hematologic disease, a metabolic disorder, HIV, HTLV, a lysosomal storage disorder, and an immunodeficiency.

[0423] Compositions and methods disclosed herein may be used to eradicate at least a portion of existing bone marrow to treat hematological cancers including, but not limited to cancers described herein, e.g., leukemia, lymphoma, myeloma, ALL, AML, CLL, CML, Hodgkin lymphoma, Non-Hodgkin lymphoma (e.g., DLBCL or follicular lymphoma), and multiple myeloma.

[0424] Compositions and methods disclosed herein may be used to treat hematologic diseases including, but not limited to myelodysplasia, anemia, paroxysmal nocturnal hemoglobinuria, aplastic anemia, acquired pure red cell anemia, Diam on -Blackfan anemia, Fanconi anemia, cytopenia, amegakaryotic thrombocytopenia, myeloproliferative disorders, polycythemia vera, essential thrombocytosis, myelofibrosis, hemoglobinopathies, sickle cell disease, thalassemia major, among others.

[0425] Different from traditional CART manufacturing approaches, the cytokine process provided herein does not involve CD3 and / or CD28 stimulation, or ex vivo T cell expansion. T cells that are contacted with anti-CD3 and anti-CD28 antibodies and expanded extensively ex vivo tend to show differentiation towards a central memory phenotype. Without wishing to be bound by theory, the cytokine process provided herein preserves or increases the undifferentiated phenotype of T cells during CART manufacturing, generating a CART product that may persist longer after being infused into a subject.

[0426] In some embodiments, the present disclosure provides a method of treating cancer comprising bone marrow conditioning, where at least a portion of bone marrow of the subject is eradicated by the CAR-expressing cell, e.g., dual CAR or tandem CAR-expressing cell of embodiments herein. For example, in certain instances, the bone marrow of the subject comprises a malignant precursor cell that can be targeted and eliminated by the activity of the CAR-expressing cell, e.g., the dual CAR or tandem CAR -expressing cell. In some embodiments, a bone marrow conditioning therapy comprises administering a bone marrow or stem cell transplant to the subject following the eradication of native bone marrow. In some embodiments, the bone marrow reconditioning therapy is combined with one or more other anti-cancer therapies, including, but not limited to anti-tumor CAR therapies, chemotherapy, radiation, and the like.

[0427] In some embodiments, eradication of the administered CAR expressing cell may be required prior to infusion of bone marrow or stem cell transplant. Eradication of the CAR - expressing cell may be accomplished using any suitable strategy or treatment, including, but not limited to, use of a suicide gene, limited CAR persistence using RNA encoded CARs, or anti-T cell modalities including antibodies or chemotherapy.

[0428] The disclosure includes a type of cellular therapy where cells (e.g., T cells or NK cells) are genetically modified to express a chimeric antigen receptor (CAR) and the CAR expressing cell (e.g., T cell or NK cells) is infused to a recipient in need thereof. The infused cell is able to kill tumo...

Claims

CLAIMS1. A method of treating a hyperproliferative disease, comprising administering to a patient in need thereof a pharmaceutical composition comprising a therapeutically effective amount of cells comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule that binds to cells expressing NYESO-1.

2. The method of claim 1, wherein the CAR molecule binds to cell expressing NYSEO-1 and an HLA molecule comprising HLA-A*02:01 or HLA-A*02:06, or a function variant thereof.

3. The method of claim 1 or 2, wherein the nucleic acid molecule encodes a peptide comprising EQWVANY (SEQ ID NO: 1), or a functional variant thereof comprising about 85% sequence identity to SEQ ID NO: 1.

4. The method of any of claims 1 through 3, wherein the nucleic acid molecule encodes a peptide comprising GTHDKCENPKEQWVANYQNLNNVVFTNKELEDIYDESN (SEQ ID NO:2), or a functional variant thereof comprising from about 75% sequence identity to SEQ ID N0:2.

5. The method of any of claims 1 through 4, wherein the nucleic acid molecule encodes a peptide comprising KEETKEVLKKFKEKVNQFYEHAFDIINKYGDKEIFNMMFMLLWRVFRSFRIDANNVE LIKFNIRVLDWIMAEADNDLSYFISQ (SEQ ID NO:3), or a functional variant thereof comprising from about 75% sequence identity to SEQ ID NO:3.

6. The method of any of claims 1 through 5, wherein the nucleic acid molecule encodes a chimeric peptide comprising a first domain and a second domain, the first domain comprising one or a combination of: SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO:3, or a functional variant thereof; and the second domain comprising a single chain antibody or antibody fragment that binds to a cell expressing CD3.

7. The method of any of claims 1 through 6, wherein the dosage is about 3xl04cells / kg body weight for patients with less than or equal to 50 kg body weight based on the total CAR- T cells in the pharmaceutical composition.

8. The method of any of claims 1 through 7, wherein the dosage is from about 1.5 x 106cells for patients with more than 50 kg body weight based on the total CAR-T cells in the pharmaceutical composition.

9. The method of any of claims 1 through 8, wherein the hyperproliferative disorder is testicular cancer, esophageal cancer, myxoid round cell liposarcoma or synovial sarcoma.

10. The method of any of claims 1 through 9, wherein the dosage is from about 3.0 x 105to 1.5 x 106cells.

11. The method of any of claims 1 through 9, wherein the dosage is about 10 x 104cells / kg body weight for patients with less than or equal to about 50 kg body weight based on the total CAR-T cells in the pharmaceutical composition.

12. The method of any of claims 1 through 9, wherein the dosage is from about 5 xlO6cells for patients with more than about 50 kg body weight based on the total CAR-T cells in the pharmaceutical composition.

13. The method of any of claims 1 through 9, wherein the dosage is about 3. Ox 104cells / kg body weight for patients with less than or equal to 50 kg body weight based on the total CAR- T cells in the pharmaceutical composition.

14. The method of claim 1, wherein the dosage is about 1.5 x 106cells for patients with more than 50 kg body weight based on the total CAR-T cells in the pharmaceutical composition.

15. The method of any one of claims 1 to 14, wherein the dosage is about IxlO4cells / kg body weight, 2x 104cells / kg body weight, 3 x 104cells / kg body weight, 4 x 104cells / kg bodyweight, 5 xlO4cells / kg body weight, 6 xlO4cells / kg body weight, 7 xlO4cells / kg body weight, 8 xlO4cells / kg body weight, 9 xlO4cells / kg body weight, 10 xlO4cells / kg body weight, 11 x 104cells / kg body weight, 12 x 104cells / kg body weight, 13 x 104cells / kg body weight, 14 x104cells / kg body weight, 15 x 104cells / kg body weight, 16 x 104cells / kg body weight, 17 x104cells / kg body weight, 18 x 104cells / kg body weight, 19 x 104cells / kg body weight, 20 x104cells / kg body weight, 21 x 104cells / kg body weight, 22 x 104cells / kg body weight, 23 x104cells / kg body weight, 24 x 104cells / kg body weight, 25 x 104cells / kg body weight, 26 x104cells / kg body weight, 27 x 104cells / kg body weight, 28 x 104cells / kg body weight, 29 x104cells / kg body weight, 30 x 104cells / kg body weight, 40 x 104cells / kg body weight, 50 x 104cells / kg body weight, 60 x 104cells / kg body weight, 70 x 104cells / kg body weight, 80 x 104cells / kg body weight, or about 90 x 104cells / kg body weight.

16. The method of any one of claims 1 to 13, wherein the dosage is about 0.5 x 106cells, 1 xlO6cells, 1. 5 x 106cells, 2 x 106cells, 2.5 xlO6cells, 3 xlO6cells, 3.5 x 106cells, 4 xlO6cells, 4.5 x 106cells, 5 x 106cells, 5.5 x 106cells, 6 x 106cells, 6.5 x 106cells, 7 x 106cells, 7.5 x 106cells, 8 x 106cells, 8.5 x 106cells, 9 x 106cells, 9.5 x 106cells, 10 x 106cells, 10.5 x 106cells, 11 x 106cells, 11.5 x 106cells, 12 x 106cells, 12.5 xlO6cells, 13 x 106cells, 13.5 xlO6cells, 14 xlO6cells, 14.5 xl 06cells, 15 x 106cells, 16 x 106cells, 17 x 106cells, 18 x 106cells, 19 x 106cells, 20 x 106cells, 21 x 106cells, 22 x 106cells, 23 x 106cells, 24 x 106cells, 25 x 106cells, 26 xl 06cells, 27 x 106cells, 28xl06cells, 29 x 106cells, 30 x 106cells, 31 x 106cells, 32 x 106cells, 33 x 106cells, 34xl06cells, 35 x 106cells, 36 x 106cells, 37 x 106cells, 38 x 106cells, 39 x 106cells, 40 x 106cells, 41 x 106cells, 42x 106cells, 43 x 106cells, 44 x 106cells, or 45 x 106cells.

17. The method of any of claims 1 through 16, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding an amino acid sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO: 1.

18. A cell comprising a CAR molecule that binds to a cell expressing NYESO-1 and an HLA molecule comprising HLA-A*02:01 or HLA-A*02:06, or functional variants thereof.

19. A cell comprising a nucleic acid molecule encoding a peptide comprising EQWVANY (SEQ ID NO:1), or a functional variant thereof comprising about 85% sequence identity to SEQ ID NO: 1.

20. The cell of claim 19, wherein the nucleic acid molecule encodes a peptide comprising GTHDKCENPKEQWVANYQNLNNVVFTNKELEDIYDESN (SEQ ID NO:2), or a functional variant thereof comprising from about 75% sequence identity to SEQ ID NO:2.

21. The cell of any of claims 19 or 20, wherein the nucleic acid molecule encodes a peptide comprising KEETKEVLKKFKEKVNQFYEHAFDIINKYGDKEIFNMMFMLLWRVFRSFRIDANNVE LIKFNIRVLDWIMAEADNDLSYFISQ (SEQ ID NO:3), or a functional variant thereof comprising from about 75% sequence identity to SEQ ID NO:3; and / or SEQ ID NO:5, or a functional variant thereof; comprising from about 75% sequence identity to SEQ ID NO:5.

22. The cell of any of claims 19 through 21, wherein the nucleic acid molecule encodes a chimeric peptide comprising a first domain and a second domain, the first domain comprising one or a combination of: SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, or a functional variant thereof; and the second domain comprises a CD3^ signaling domain.

23. The cell of any of claims 19 through 22, wherein the cells is a T cell or an NK cell.

24. The cell of any of claims 19 through 23, wherein the cell the nucleic acid molecule comprising a nucleic acid that encodes a CD8a hinge-transmembrane domain, a 4 IBB costimulatory domain and a CD3^ signaling domain.

25. The cell of claim 24 wherein the nucleic acid molecule encodes SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, or a functional variant thereof comprising from about 75% sequence identity to SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively.

26. A pharmaceutical composition comprising a therapeutically effective amount of a peptide comprising SEQ ID NO: 1, or a functional variant thereof comprising from about 85% sequence identity to SEQ ID NO: 1; and a pharmaceutically acceptable carrier.

27. The pharmaceutical composition of claim 26 further comprising a cell that comprises SEQ ID NO: 1, or a functional variant thereof comprising from about 85% sequence identity to SEQ ID NO: 1.

28. The pharmaceutical composition of claim 26 or 27, wherein the peptide comprises one or a combination of SEQ ID NO:2 and SEQ ID NO:3, or a functional variant thereof comprising an amino acid sequence comprising from about 75% sequence identity to about 99% sequence identity to SEQ ID NO:2 and SEQ ID NO:3, respectively.

29. The pharmaceutical composition of any of claims 26 through 28, wherein the peptide further comprises one or a combination of: a CD8a hinge-transmembrane domain, a 41BB costimulatory domain and a CD3^ signaling domain.

30. A pharmaceutical composition comprising a therapeutically effective amount of a cell comprising a peptide comprising SEQ ID NO: 1 or a functional variant thereof comprising at least about 85% sequence identity to SEQ ID NO: 1; and a pharmaceutically acceptable carrier.

31. The pharmaceutical composition of claim 30 comprising one or a combination of SEQ ID NO:2 and SEQ ID NO:3, or a functional variant thereof comprising an amino acid sequence comprising from about 75% sequence identity to about 99% sequence identity to SEQ ID NO:2 and SEQ ID NO:3, respectively.

32. The pharmaceutical composition of either of claims 30 or 31, wherein the peptide further comprises a CD8a hinge-transmembrane domain, a 4 IBB co-stimulatory domain and a CD3^ signaling domain.

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