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WO2025096673A1 - Compositions comprenant des cellules ciblant le cancer et leurs procédés d'utilisation - Google Patents

Compositions comprenant des cellules ciblant le cancer et leurs procédés d'utilisation Download PDF

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Publication number
WO2025096673A1
WO2025096673A1 PCT/US2024/053742 US2024053742W WO2025096673A1 WO 2025096673 A1 WO2025096673 A1 WO 2025096673A1 US 2024053742 W US2024053742 W US 2024053742W WO 2025096673 A1 WO2025096673 A1 WO 2025096673A1
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Prior art keywords
cells
cell
seq
body weight
nucleic acid
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Inventor
Possu HUANG
Haotian DU
Rogelio A. HERNANDEZ-LOPEZ
Daniel Hoces BURGA
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Leland Stanford Junior University
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Leland Stanford Junior University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001184—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K39/001188—NY-ESO
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/32—T-cell receptors [TCR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4267—Cancer testis antigens, e.g. SSX, BAGE, GAGE or SAGE
    • A61K40/4269—NY-ESO
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2833—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against MHC-molecules, e.g. HLA-molecules
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by targeting or presenting multiple antigens
    • A61K2239/29—Multispecific CARs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/32—Immunoglobulins specific features characterized by aspects of specificity or valency specific for a neo-epitope on a complex, e.g. antibody-antigen or ligand-receptor
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the disclosure relates to methods of treating hyperproliferative disease, cells comprising a CAR molecule, and pharmaceutical compositions.
  • CAR chimeric antigen receptor
  • CART modified autologous T cell
  • CTL019 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)).
  • a successful therapeutic T cell therapy needs to have the ability to proliferate and persist over time, in order to survey for leukemic relapse.
  • 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.
  • CAR-transformed patient T cells need to persist and maintain the ability to proliferate in response to the cognate antigen.
  • 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.
  • the cancer is esophageal cancer or testicular cancer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 N0:3, or a functional variant thereof.
  • the second domain comprising a single chain antibody or antibody fragment that binds to a cell expressing CD3.
  • 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.
  • the dosage is about 3x10 ⁇ cells/kg body weight for patients ::S50 kg body weight or about 1.5xl0 6 cells for patients >50 kg body weight based on the total CAR+ cells in the pharmaceutical composition.
  • 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.
  • the disclosure provides a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule.
  • 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.
  • 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.
  • 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.
  • the cell is an immune effector cell, e.g., T cell (e.g., CD3+, CD4+ or CD8+ T cell), or an NK cell.
  • the cell is a human cell.
  • 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.
  • the disclosure relates to a method of treating a subject having a disease associated with an antigen.
  • 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.
  • the disclosure provides a bispecific antigen binding domain.
  • 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.
  • the disclosure provides a monospecific antigen binding domain.
  • 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.
  • the disclosure relates to a cell comprising a CAR molecule comprises a first domain and a second domain.
  • the first domain comprises SEQ ID NO: 1, or functional variants thereof.
  • the first domain comprises SEQ ID NO:2, or functional variants thereof.
  • the first domain comprises SEQ ID NO:3, or functional variants thereof.
  • 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.
  • 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.
  • CAR chimeric antigen receptor
  • 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.
  • 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.
  • 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.
  • 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.
  • the first antigen binding domain comprises an scFv comprising a first VH (VH1) and a first VL (VL1 ).
  • the second antigen binding domain comprises an scFv comprising a second VH (VH2) and a second VL (VL2).
  • 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.
  • a CAR comprising a bispecific antigen binding domain comprises the amino acid sequence of SEQ ID NO: 2.
  • 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.
  • the disclosure also relates to a pharmaceutical composition
  • 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.
  • 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.
  • the disclosure relates to a pharmaceutical composition
  • 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.
  • the pharmaceutical composition comprises an excipient, a carrier, a diluent and/or a stabilizer.
  • 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.
  • a cell e.g., a population of immune effector cells
  • a CAR disclosed herein e.g., a tandem, monospecific or bispecific CAR disclosed herein.
  • 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.
  • 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.
  • the disclosure relates to a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule.
  • 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.
  • 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.
  • the disclosure relates to nucleic acid molecule of any of the preceding 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.
  • the disclosure relates to a nucleic acid molecule of any of the preceding embodiments.
  • the nucleotide sequence encoding the first transmembrane 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.
  • the disclosure relates to a nucleic acid molecule of any of the preceding 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.
  • the disclosure also relates to a nucleic acid molecule of any of the preceding 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.
  • the disclosure relates to a nucleic acid molecule of any of the preceding 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.
  • the disclosure relates to a nucleic acid molecule of any of the preceding 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.
  • the disclosure relates to a nucleic acid molecule of any of the preceding 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 of signal 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.
  • the disclosure relates to a nucleic acid molecule of any of the preceding embodiments.
  • the protease cleavage site or internal ribosomal entry site is situated between the first CAR and the second CAR.
  • the disclosure relates to a nucleic acid molecule of any of the preceding embodiments.
  • the protease cleavage site is situated such that a cell can express a fusion protein comprising a first CAR and a second CAR.
  • the fusion protein is processed into two peptides by proteolytic cleavage.
  • the disclosure relates to a nucleic acid molecule of any of the preceding embodiments.
  • the nucleotide sequence encoding the first CAR and the nucleotide sequence encoding the second CAR are disposed on a single nucleic acid construct.
  • the disclosure relates to a nucleic acid molecule of any of the preceding embodiments.
  • the nucleotide encoding the first CAR and the nucleic acid encoding the second CAR are disposed on the same vector.
  • the disclosure relates to a nucleic acid molecule of any of the preceding 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.
  • the disclosure relates to a nucleic acid molecule of any of the preceding embodiments.
  • the nucleotide sequence encoding the first CAR is disposed on a first vector.
  • the disclosure relates to a nucleic acid molecule of any of the preceding embodiments.
  • the nucleotide sequence encoding the second CAR is disposed on a second vector.
  • the nucleic acid molecule comprises a viral element, e.g., a viral packaging element.
  • 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.
  • 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.
  • a cell e.g., an immune effector cell
  • the nucleic acid molecule comprising a nucleic acid sequence encoding any of the preceding amino acid sequence disclosed herein.
  • the disclosure relates a cell of any of the preceding embodiments.
  • the cell comprises a nucleic acid encoding the CAR molecule.
  • the disclosure relates to a cell of any of the preceding embodiments.
  • the cell comprises the nucleic acid molecule of any of the preceding embodiments.
  • the disclosure relates a cell of any of the preceding embodiments.
  • the cell is a human cell.
  • the disclosure relates to a method of making a cell (e.g., an immune effector cell).
  • the method comprises transducing an immune effector cell, e.g., a T cell or NK cell with a vector herein.
  • 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.
  • a cell e.g., an immune effector cell
  • 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.
  • the RNA comprises a nucleic acid molecule of any of the preceding embodiments.
  • 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.
  • the disclosure relates to a bispecific antigen binding domain of any of the preceding 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.
  • the VH can be upstream or downstream of the VL.
  • the first antigen binding domain comprises an scFv comprising a first VH (VH1) and a first VL (VL1).
  • VH1 first VH
  • VL1 first VL
  • the disclosure relates to a bispecific antigen binding domain of any preceding embodiments.
  • the first antigen binding domain comprises an scFv comprising a first VH (VH1) and a first VL (VL1).
  • the second antigen binding domain comprises an scFv comprising a second VH (VH2) and a second VL(VL2).
  • the disclosure relates to a bispecific antigen binding domain of any preceding embodiments.
  • the first antigen binding domain is arranged with VH1 upstream of VL1.
  • the disclosure relates to a bispecific antigen binding domain of any preceding embodiments.
  • the first antigen binding domain is arranged with VL1 upstream of VH1.
  • the disclosure relates to a bispecific antigen binding domain of any preceding embodiments.
  • the second antigen binding domain is arranged with VH2 upstream of VL2.
  • the disclosure relates to a bispecific antigen binding domain of any preceding embodiments.
  • the second antigen binding domain is arranged with VL2 upstream of VH2.
  • the disclosure relates to a bispecific antigen binding domain of any preceding embodiments.
  • the antigen binding domain has the following N terminal to C terminal configuration: VL1-VH1-VH2-VL2.
  • the disclosure relates to a bispecific antigen binding domain of any preceding embodiments.
  • the antigen binding domain has the following N terminal to C terminal configuration: VH1-VL1-VH2-VL2.
  • the disclosure relates to a bispecific antigen binding domain of any preceding embodiments.
  • the antigen binding domain has the following N terminal to C terminal configuration: VL1-VH1-VL2-VH2.
  • the disclosure relates to a monospecific or bispecific antigen binding domain of any preceding embodiments.
  • the antigen binding domain has the following N terminal to C terminal configuration: VH1-VL1-VL2-VH2.
  • the disclosure relates to a monospecific or bispecific antigen binding domain of any preceding embodiments.
  • a linker is disposed between the first antigen binding domain and the second antigen binding domain.
  • the disclosure relates to a monospecific or bispecific antigen binding domain of any preceding embodiments.
  • the linker is disposed between the scFv of the first antigen binding domain and the scFv of the second antigen binding domain.
  • the disclosure relates to a monospecific or bispecific antigen binding domain of any preceding 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.
  • the a monospecific or bispecific antigen binding domain of any one of any preceding embodiments comprises a linker.
  • the linker is long enough to avoid mispairing between the domains of the two scFvs.
  • the linker is a (Gly4-Ser) strong linker, wherein n is 1, 2, 3, 4, 5, or 6 (SEQ ID NO: 54).
  • 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).
  • the disclosure relates a monospecific or bispecific chimeric antigen receptor (CAR).
  • the bispecific CAR comprises the bispecific antigen binding domain of any one of the previously disclosed embodiments.
  • the disclosure relates to a nucleic acid construct encoding a a monospecific or bispecific chimeric antigen receptor (CAR).
  • 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.
  • CAR chimeric antigen receptor
  • 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.
  • 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.
  • 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.
  • the CAR comprises a co- stimulatory domain
  • 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).
  • 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 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.
  • a method of making a 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.
  • 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.
  • the disclosure relates to a method of providing anti-tumor immunity.
  • 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.
  • 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.
  • 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.
  • the cell is a T cell or an NK cell.
  • the disclosure relates to a method of treating a subject having a disease associated with an antigen.
  • 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.
  • a cell e.g., a population of immune effector cells
  • 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 is an amino acid sequence of the NY-ESO-1 antigen 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. 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.
  • Each tandem CAR comprises a bispecific antigen binding domain comprising a NYESO-1 antigen binding domain and a CD3 antigen binding domain.
  • 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.
  • 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.
  • 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 cells incubated 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.
  • 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.
  • 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 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 depict cross-allele specificity profiling of TRACeR] (MHC
  • 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.
  • FIGS. 5A and 5B depict Structural alignment of predicted model and crystal structure of TRACeRNY-F.so-i,A02.
  • 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.
  • 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 on the enrichment ratio of wild type. Deep sequencing was performed with Illumina 2*300 Kit at Stanford PAN facility.
  • 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 HLA3DB 1 as described previously. A selection resulted in 67 crystal structures.
  • Complexes were analyzed using PDBePISA 2 as implemented in CCP4 (v. 8.0) 3 to 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.
  • FIG. 8 depicts a purification of TRACeR ⁇ - ⁇ 0 ⁇ - antiCD3 construct.
  • the dimer construct shows cancer killing.
  • 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 Monomeric TRACeR is still peptide specific based on yeast surface display (staining concentration: 50 nM tetramer)
  • 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).
  • 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 DESCRIPTION
  • an element means one element or more than one element.
  • 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.
  • a CAR Chimeric Antigen Receptor
  • 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.
  • 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, the set 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.
  • the stimulatory molecule is the zeta chain associated with the T cell receptor complex.
  • the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below.
  • the costimulatory molecule is chosen from the costimulatory molecules described herein, e.g., 4-IBB (i.e., CD137), CD27 and/or CD28.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the antigen binding domain e.g., a scFv
  • 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.
  • binding domain refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence.
  • binding domain also referred to herein as “antibody molecule” encompasses antibodies and antibody fragments.
  • 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.
  • 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.
  • 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.
  • 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).
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a synthetic linker e.g., a short flexible polypeptide linker
  • 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.
  • CDR complementarity determining region
  • 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).
  • 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).
  • 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.
  • HCDR1 amino acid residues 26-35
  • HCDR2 amino acid residues 24-34
  • LCDR1 amino acid residues 24-34
  • LCDR1 amino acid residues 24-34
  • LCDR1 amino acid residues 24-34
  • LCDR1 amino acid residues 24-34
  • LCDR1 amino acid residues 24-34
  • LCDR1 amino acid residues 24-34
  • LCDR1 amino acid residues 24-34
  • LCDR1 amino acid residues 24-34
  • LCDR1 amino acid residues 24-34
  • LCDR1 amino acid residues 24-34
  • LCDR1 amino acid residues 24-34
  • LCDR1 amino acid residues 24-34
  • LCDR1 amino acid residues 24-
  • the CDR regions of an antibody can be determined using the program IMGT/DomainGap Align.
  • 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.
  • the antigen binding domain of a CAR composition of embodiments herein comprises an antibody fragment.
  • the CAR comprises an antibody fragment that comprises a scFv.
  • 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.
  • 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.
  • 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.
  • antigen 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.
  • 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.
  • an antigen need not be encoded solely by a full length nucleic acid sequence of a gene. It is readily 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.
  • 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.
  • an tumor effect refers to a biological effect which can be manifested by various means, including but not limited to, e.g., a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.
  • autologous refers to any material derived from the same individual to whom it is later to be re-introduced into the individual.
  • 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.
  • xenogeneic refers to a graft derived from an animal of a different species.
  • 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.
  • a combination partner e.g. another drug as explained below, also referred to as “therapeutic agent” or “coagent”
  • the single components may be packaged in a kit or separately.
  • One or both of the components e.g., powders or liquids
  • 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.
  • 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.
  • 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.
  • 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.
  • cocktail therapy e.g. the administration of three or more therapeutic agent.
  • 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.
  • 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.
  • prevention refers 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.
  • 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 diagnosed with the disorder and before the disorder has been cured or eliminated or treatment has ceased for other reasons.
  • 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.”
  • 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.
  • 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.
  • 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.
  • 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.
  • “Derived from” 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.
  • the term “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody or antibody fragment containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody or antibody fragment 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).
  • 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.
  • a stimulatory molecule e.g., a TCR/CD3 complex or CAR
  • its cognate ligand or tumor antigen in the case of a CAR
  • Stimulation can mediate altered expression of certain molecules.
  • the term “stimulatory molecule,” refers to a molecule expressed by an immune cell, e.g., T cell, NK cell, 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.
  • 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.
  • ITAM immunoreceptor tyrosine-based activation motif
  • 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.
  • 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.
  • 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.
  • MHC major histocompatibility complexes
  • T-cells may recognize these complexes using their T-cell receptors (TCRs).
  • TCRs T-cell receptors
  • APCs process antigens and present them to T-cells.
  • Immuno effector cell refers to a cell that is involved in an immune response, e.g., in the promotion of an immune effector response.
  • 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.
  • Immuno effector function or immune effector response refers to function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell.
  • 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.
  • primary stimulation and co-stimulation are examples of immune effector function or response.
  • intracellular signaling domain 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.
  • 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.
  • 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.
  • 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.
  • a primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor
  • a costimulatory intracellular signaling domain can comprise cytoplasmic sequence from co-receptor or costimulatory molecule.
  • a primary intracellular signaling domain can comprise a signaling motif which is known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • 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.
  • 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.
  • 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.
  • the “zeta stimulatory domain” or a “CD3-zeta stimulatory domain” is the sequence provided as SEQ ID NO: 96.
  • 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, ITGA
  • 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.
  • the intracellular signaling domain can comprise the entire intracellular portion, or the entire native intracellular signaling domain, of the molecule from which it is derived, or a functional fragment or derivative thereof.
  • the term “4-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.
  • 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.
  • 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.
  • 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.
  • 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).
  • ⁇ ективное 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.
  • endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • expression refers to the transcription and/or translation of a particular nucleic acid sequence driven by a promoter.
  • 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.
  • 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.
  • viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, and the like.
  • 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.
  • 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.
  • 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 LENTIMAXTM vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
  • the “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.
  • the residues of single sequence are included in the denominator but not the numerator of the calculation.
  • BLAST high scoring sequence pair
  • 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.
  • 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.
  • a polynucleotide can be complementary to another polynucleotide without being its complement.
  • homologous refers to the subunit sequence identity between two polymeric molecules, e.g., between two nucleic acid molecules, such as, two DNA molecules or two RNA molecules, or between two polypeptide molecules.
  • two nucleic acid molecules such as, two DNA molecules or two RNA molecules
  • polypeptide molecules between two polypeptide molecules.
  • 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.
  • “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.
  • 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.
  • CDR complementarity-determining region
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • 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.
  • 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.
  • an immunoglobulin constant region(Fe) typically that of a human immunoglobulin.
  • 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.
  • “Murine” refers to mice or rats.
  • 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.
  • isolated means altered or removed from the natural state.
  • 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.
  • A refers to adenosine
  • C refers to cytosine
  • G refers to guanosine
  • T refers to thymidine
  • U refers to uridine.
  • operably linked refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter.
  • 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.
  • 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.
  • 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.
  • nucleic acid 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.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).
  • peptide refers 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.
  • 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.
  • the flexible polypeptide linker is (Gly4 Ser)3 (SEQ ID NO: 42).
  • the linkers include multiple repeats of (Gly2Ser), and (GlySer).
  • 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.
  • RNA polymerase 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.
  • in vitro transcribed RNA refers to RNA, preferably mRNA, that has been synthesized in vitro.
  • 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
  • a “poly(A)” is a series of adenosines attached by polyadenylation to the mRNA.
  • 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 or enzymatically to modulate mRNA functionality such as localization, stability or efficiency of translation.
  • polyadenylation refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to a messenger RNA molecule.
  • mRNA messenger RNA
  • 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.
  • the poly(A) tail is added onto transcripts that contain a specific sequence, the polyadenylation signal.
  • 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.
  • 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.
  • adenosine residues are added to the free 3’ end at the cleavage site.
  • 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.
  • 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).
  • 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.
  • 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.
  • the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size or cancerous cell count.
  • 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.
  • subject is intended to include living organisms in which an immune response can be elicited (e.g., mammals, human).
  • 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.
  • 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.
  • the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.
  • terapéutica as used herein means a treatment.
  • a therapeutic effect is obtained by reduction, suppression, remission, or eradication of a disease state.
  • prophylaxis means the prevention of or protective treatment for a disease or disease state.
  • tumor antigen or “hyperproliferative disorder” or “antigen associated with a hyperproliferative disorder” refers to antigens that are common to specific hyperproliferative disorders.
  • 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.
  • 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
  • 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.
  • 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.
  • a binding partner e.g., a stimulatory tumor antigen
  • Refractory refers to a disease, e.g., cancer, that does not respond to a treatment.
  • a refractory cancer can be resistant to a treatment before or at the beginning of the treatment.
  • the refractory cancer can become resistant during a treatment.
  • a refractory cancer is also called a resistant cancer.
  • 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.
  • 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.
  • 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®).
  • 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).
  • 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.
  • a complete responder is defined as having 7% or greater CD27+ CD45RO- cells in the CD8+ population.
  • the percent of CAR+ cells at pre- harvest levels distinguish responders (e.g., complete responders and partial responders) from non-responders (NR).
  • 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%.
  • 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.
  • a response e.g., complete response or partial response
  • 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.
  • Regular chimeric antigen receptor 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.
  • 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.
  • 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.
  • Switch domain 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.
  • 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.
  • the switch domain is a polypeptide-based entity, e.g., an scFv that binds a myc peptide
  • 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.
  • the switch domain is a polypeptide-based entity, e.g., myc receptor
  • the dimerization molecule is an antibody or fragments thereof, e.g., myc antibody.
  • dimerization molecule refers to a molecule that promotes the association of a first switch domain with a second switch domain.
  • the dimerization molecule does not naturally occur in the subject, or does not occur in concentrations that would result in significant dimerization.
  • the dimerization molecule is a small molecule, e.g., rapamycin or a rapalogue, e.g, RAD001.
  • 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).
  • 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.
  • the effect is alteration of the ratio of PD- 1 positive/PD- 1 negative T cells, as measured by cell sorting.
  • 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.
  • 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.
  • the term “low, immune enhancing, dose” when used in conjunction with an 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 to result in complete immune suppression but is sufficient to enhance the immune response.
  • 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.
  • 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.
  • any of the changes described above occurs, e.g., at least transiently, e.g., as compared to a non-treated subject.
  • “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.
  • 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 substituted and 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.
  • 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.
  • 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.
  • variants includes polypeptides conjugated to a non-natural chemical moieties or variants.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the CAR molecule comprises two identical polypeptide sequences, e.g., of a first and second transmembrane domain; a first and second co-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.
  • a nucleic acid molecule encoding a CAR molecule is optimized, e.g., codon optimized, to prevent recombination, e.g., homologous recombination.
  • 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.
  • 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.
  • 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.
  • LC CDR1 light chain complementarity determining region 1
  • HC CDR2 heavy chain complementarity determining region 2
  • HC CDR3 heavy chain complementarity determining region 3
  • 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.
  • a CAR molecule disclosed herein comprises a first CAR comprising a first transmembrane domain and a second CAR comprising a second transmembrane domain.
  • the first transmembrane domain and the second transmembrane domain comprise the same amino acid sequence, e.g., as disclosed herein.
  • the first transmembrane domain and the second transmembrane domain are encoded by a first nucleotide sequence and a second nucleotide sequence, respectively.
  • the first nucleotide sequence and the second nucleotide sequence differ by at least one nucleotide.
  • the first transmembrane domain and the second transmembrane domain are the same transmembrane domain, e.g., chosen from the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD4S, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137 or CDIS4.
  • 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.
  • 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.
  • the first transmembrane domain and the second transmembrane domain comprise the CD8 alpha transmembrane domain.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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).
  • the nucleic acid is a plasmid.
  • the nucleic acid comprises a viral packaging element.
  • 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.
  • 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
  • Table 2 provides nucleotide and amino acid sequences for additional CAR components, e.g., signal peptide, linkers and P2A sites.
  • the disclosure relates to CARs comprising a bispecific antigen binding domain, e.g., tandem CARs.
  • a bispecific antigen binding domain comprises two antigen binding domains, e.g., a first antigen binding domain and a second antigen binding domain.
  • the first antigen binding domain is an antibody molecule, e.g., an antibody binding domain (e.g., a scFv).
  • the second antigen binding domain is an antibody molecule, e.g., an antibody binding domain (e.g., a scFv).
  • the VH can be upstream or downstream of the VL.4
  • the disclosure relates to a composition
  • 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.
  • 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.
  • 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:34 and/or SEQ ID NO:36.
  • 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.
  • the CAR comprises the sequence SLLMWITQV (SEQ ID NO: 57) or a functional variant comprising 88% sequence identity to SLLMWITQV (SEQ ID NO: 57).
  • the CAR comprises
  • 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.
  • 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.
  • 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 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.
  • 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.
  • 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.
  • 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-VL2-VH2;
  • 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.
  • the linker is a (Gly4-Ser)n linker, wherein n is 1, 2, 3, 4, 5, or 6 (SEQ ID NO: 54).
  • the linker comprises, e.g., consists of, the amino acid sequence: LAEAAAK (e.g., SEQ ID NO: 40).
  • a linker is disposed between the VL and VH of the first scFv.
  • a linker is disposed between the VL and VH of the second scFv.
  • any two or more of the linkers can be the same or different.
  • a bispecific CAR comprises VLs, VHs, and optionally one or more linkers in an arrangement as described herein.
  • each antibody molecule e.g., each antigen binding domain (e.g., each scFv) comprises a linker between the VH and the VL regions.
  • 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).
  • the VH and VL regions are connected without a linker.
  • 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.
  • 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).
  • the costimulatory domain is activated, and the cell proliferates.
  • the intracellular signaling domain is activated and cell-killing activity begins.
  • the CAR-expressing cell is only fully activated in the presence of both antigens.
  • the CAR e.g., dual CAR or tandem CAR
  • a messenger RNA mRNA
  • 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.
  • 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.
  • 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.
  • 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.
  • the DNA to be used for PCR is a human nucleic acid sequence.
  • 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.
  • 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.
  • 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.
  • 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 are substantially 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.
  • the RNA preferably has 5’ and 3’ UTRs.
  • 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.
  • the 5’ and 3’ UTRs can be the naturally occurring, endogenous 5’ and 3’ UTRs for the nucleic acid of interest.
  • 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.
  • the 5’ UTR can contain the Kozak sequence of the endogenous nucleic acid.
  • 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.
  • the 5’ UTR can be 5’UTR of an RNA virus whose RNA genome is stable in cells.
  • various nucleotide analogues can be used in the 3’ or 5’ UTR to impede exonuclease degradation of the mRNA.
  • a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed.
  • the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 of the transcribed.
  • the poly(A) tail is between 100 and 5000 adenosines (SEQ ID NO: 65).
  • 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).
  • E-PAP E. coli poly A polymerase
  • 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
  • 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.
  • ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase.
  • 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)).
  • RNAs produced by the methods disclosed herein can also contain an internal ribosome entry site (IRES) sequence.
  • 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.
  • 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).
  • non-viral methods can be used to deliver a nucleic acid encoding a CAR described herein into a cell or tissue or a subject.
  • the non-viral method includes the use of a transposon (also called a transposable element).
  • 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.
  • a transposon comprises a DNA sequence made up of inverted repeats flanking genes for transposition.
  • Exemplary methods of nucleic acid delivery using a transposon include a SleepingBeauty transposon system (SBTS) and a piggyBac (PB) transposon system.
  • SBTS SleepingBeauty transposon system
  • PB piggyBac
  • 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.
  • a target DNA such as a host cell chromosome/genome.
  • 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.
  • 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.
  • a transgene e.g., a nucleic acid encoding a CAR described herein.
  • a transgene e.g., a nucleic acid encoding a CAR described herein.
  • one or more nucleic acids, e.g., plasmids, containing the SBTS components are delivered to a cell (e.g., Tor NK cell).
  • 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.
  • the nucleic acid contains a transposon comprising a transgene, e.g., a nucleic acid encoding a CAR described herein.
  • 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.
  • 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.
  • a first plasmid contains a transposon comprising a transgene
  • a second plasmid contains a nucleic acid sequence encoding a transposase enzyme.
  • the first and the second nucleic acids are co-delivered into a host cell.
  • cells e.g., Tor NK cells
  • 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 ).
  • ZFNs Zinc finger nucleases
  • TALENs Transcription Activator-Like Effector Nucleases
  • 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.
  • the present disclosure also provides nucleic acid molecules encoding one or more CAR constructs described herein.
  • the nucleic acid molecule is provided as a messenger RNA transcript.
  • the nucleic acid molecule is provided as a DNA construct.
  • 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.
  • 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.
  • the vector comprising the nucleic acid encoding the desired CAR of embodiments herein is an adenoviral vector (A5/35).
  • 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.
  • 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.
  • 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.
  • the disclosure provides a gene therapy vector.
  • the nucleic acid can be cloned into a number of types of vectors.
  • 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.
  • 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.
  • 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).
  • Additional promoter elements e.g., enhancers, regulate the frequency of transcriptional initiation.
  • 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.
  • tk thymidine kinase
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline.
  • 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.
  • EFla EF-1 alpha
  • 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).
  • the EFla promoter comprises the sequence as known in the art.
  • CMV immediate early cytomegalovirus
  • 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.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • 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.
  • 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.
  • Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences.
  • 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 may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter- driven transcription.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • 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.
  • a preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
  • 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.
  • 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.
  • an exemplary delivery vehicle is a liposome.
  • lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo).
  • 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 the oligonucleotide, 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.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • 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.
  • Lipids suitable for use can be obtained from commercial sources.
  • DMPC dimyristyl phosphatidylcholine
  • DCP dicetyl phosphate
  • Choi cholesterol
  • DMPG dimyristyl phosphatidylglycerol
  • Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C.
  • 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).
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine- nucleic acid complexes are also contemplated.
  • 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.
  • the present disclosure further provides a vector comprising a CAR encoding nucleic acid molecule.
  • a CAR vector can be directly transduced into a cell, e.g., a T cell or NK cell.
  • 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.
  • the vector is capable of expressing the CAR construct in mammalian T cells or NK cells.
  • the mammalian T cell is a human T cell.
  • 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.
  • the manufacturing methods disclosed herein are used to manufacture a cell comprising a nucleic acid molecule encoding two
  • CARs disclosed herein e.g., a tandem and/or dual CAR disclosed herein.
  • 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.
  • 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).
  • 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.
  • the methods disclosed herein may manufacture immune effector cells engineered to express one or more CARs in less than 24 hours.
  • 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.
  • 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 on the 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
  • CAR chimeric anti
  • 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.
  • 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).
  • the methods further comprise adding an adjuvant or a transduction enhancement reagent in the cell culture medium to enhance transduction efficiency.
  • the adjuvant or transduction enhancement reagent comprises a cationic polymer.
  • the adjuvant or transduction enhancement reagent is selected from: LentiBOOS TTM (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 LentiTransTM.
  • the adjuvant is LentiBOOSTTM (Sirion Biotech).
  • the adjuvant is F108 (Poloxamer 338 or Pluronic® F-38).
  • the adjuvant is Pluronic F127 (Poloxamer 407 or Synperonic PE/F-127).
  • the population of cells is collected from an apheresis sample (for example, a leukapheresis sample) from a subject.
  • 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 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
  • the selected T cells are then seeded for CART manufacturing using the activation process described herein.
  • the selected T cells undergo one or more rounds of freeze-thaw before being seeded for CART manufacturing.
  • 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
  • the selected T cells are then shipped as a frozen sample (for example, a cryopreserved sample) to a cell manufacturing facility.
  • the selected T cells are later thawed and seeded for CART manufacturing using the activation process described herein.
  • cells for example, T cells
  • a vector for example, a lentiviral vector
  • a CAR e.g. one or more CARs
  • brief CD3 and CD28 stimulation may promote efficient transduction of self-renewing T cells.
  • 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.
  • cells for example, T cells
  • a vector for example, a lentiviral vector
  • a CAR e.g. one or more CARs
  • brief CD3 and CD28 stimulation may promote efficient transduction of self-renewing T cells.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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).
  • 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).
  • a cell sorting machine for example, a CliniMACS® Prodigy® device.
  • 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).
  • a cell sorting machine for example, a CliniMACS® Prodigy® device.
  • the agent that stimulates a CD3/TCR complex is an agent that stimulates CD3.
  • 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.
  • the agent that stimulates a costimulatory molecule is an agent that stimulates CD28.
  • 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).
  • the agent that stimulates a CD3/TCR complex is an antibody.
  • the agent that stimulates a CD3/TCR complex is an anti-CD3 antibody.
  • 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).
  • the agent that stimulates a costimulatory molecule is an antibody.
  • the agent that stimulates a costimulatory molecule is an anti- CD28 antibody.
  • the agent that stimulates a CD3/TCR complex or the agent that stimulates a costimulatory molecule does not comprise a bead.
  • the agent that stimulates a CD3/TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymeric nanomatrix.
  • the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymeric nanomatrix.
  • the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule comprising T Cell TransActTM.
  • the matrix comprises or consists of a polymeric, for example, biodegradable or biocompatible inert material, for example, which is non-toxic to cells.
  • the matrix is composed of hydrophilic polymer chains, which obtain maximal mobility in aqueous solution due to hydration of the chains.
  • 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.
  • the mobile matrix is a polymer of dextran.
  • 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).
  • a nucleic acid molecule e.g. one or more nucleic acid molecules
  • 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).
  • 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.
  • a population of cells in the case of a co-transduction of lentiviral vectors, 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.
  • 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 of cells 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the cells described above.
  • 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.
  • 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.
  • 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.
  • the population of cells is not expanded ex vivo.
  • 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.
  • the population 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.
  • 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.
  • 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.
  • 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.
  • the activation process is conducted in serum free cell media.
  • 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).
  • 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).
  • hetIL-15 comprises the amino acid sequence of NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVT
  • 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.
  • the activation process is conducted in cell media comprising a LSD 1 inhibitor.
  • the activation process is conducted in cell media comprising a MALTI inhibitor.
  • the serum free cell media comprises a serum replacement.
  • the serum replacement is CTSTM Immune Cell Serum Replacement (ICSR).
  • the level of ICSR can be, for example, up to 5%, for example, about 1%, 2%, 3%, 4%, or 5%.
  • using cell media, for example, Rapid Media, comprising ICSR, for example, 2% ICSR may improve cell viability during a manufacture process described herein.
  • 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 lO 7 cells/mL; (d) contacting T cells with an agent that stimulates T 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(
  • a population of cells for example, immune effector cells, for example, T cells or NK cells
  • a population of cells made by any of the manufacturing processes described herein (e.g., the Activation Process described herein).
  • 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,
  • 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).
  • the population of cells at the end of the manufacturing process 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).
  • naive T cells for example, CD45RA+ CD45RO- CCR7+ T cells
  • 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%.
  • 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,
  • 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).
  • the population of cells at the end of the manufacturing process 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).
  • 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)
  • 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 is no more than 40, 45, 50, 55, 60, 65, 70, 75, or 80%.
  • 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).
  • a higher level for example, at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% higher
  • 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).
  • 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).
  • 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
  • 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, an adenoviral 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.
  • 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).
  • the cells are engineered to comprise a nucleic acid molecule encoding a tandem or dual CAR disclosed herein
  • the cells are engineered to comprise a nucleic acid molecule encoding a diabody CAR disclosed herein
  • the cells are engineered to comprise two nucleic acid molecules, each of which encodes a CAR disclosed herein.
  • the population of cells (for example, T cells) is collected from an apheresis sample (for example, a leukapheresis sample) from a subject.
  • 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.
  • T cells for example, CD4+ T cells and/or CD8+ T cells
  • 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
  • the CART cells are cryopreserved and later thawed and administered to the subject.
  • the selected T cells undergo one or more rounds of freeze-thaw before being seeded for CART manufacturing.
  • 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
  • the selected T cells are then seeded for CART manufacturing using the cytokine process described herein.
  • the selected T cells undergo one or more rounds of freeze-thaw before being seeded for CART manufacturing.
  • 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 are then shipped as a frozen sample (for example, a cryopreserved sample) to a cell manufacturing facility.
  • the selected T cells are later thawed and seeded for CART manufacturing using the cytokine process described herein.
  • 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)
  • a vector for example, a lentiviral vector
  • CAR e.g., one or more CARs
  • 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.
  • 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).
  • 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).
  • 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)).
  • 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).
  • the population of cells is contacted with IL-15 (for example, hetIL-15 (ILI 5/sIL-l 5Ra)) and IL-21.
  • 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).
  • the population of cells is contacted with IL-21 and IL- 6 (for example, IL-6/sIL-6Ra).
  • the population of cells is contacted with IL-7, IL- 15 (for example, hetIL-15 (IL15/sIL-15Ra)), and IL-21.
  • the population of cells is further contacted with a LSD1 inhibitor.
  • the population of cells is further contacted with a MALT1 inhibitor.
  • 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,
  • 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.
  • 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).
  • a nucleic acid molecule e.g. one or more nucleic acid molecules
  • 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).
  • 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.
  • 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 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 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.
  • 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.
  • the population of cells is harvested for storage or administration.
  • 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.
  • 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.
  • 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.
  • the population of cells is not expanded ex vivo.
  • 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.
  • the population 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 one or more cytokines described above.
  • 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.
  • 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.
  • 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.
  • 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.
  • an agent that stimulates a CD3/TCR complex for example, an anti-CD3 antibody
  • an agent that stimulates a costimulatory molecule on the surface of the cells for example, an anti-CD28 antibody
  • 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 that stimulates 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.
  • an agent that stimulates a CD3/TCR complex for example, an anti-CD3 antibody
  • an agent that stimulates a costimulatory molecule on the surface of the cells for 20, 21, 22, 23, 24, 25, 26, 27, or 28 hours.
  • 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).
  • an agent that binds a CD3/TCR complex for example, an anti-CD3 antibody
  • an agent that binds a costimulatory molecule on the surface of the cells for example, an anti-CD28 antibody
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an optimized viscosity of the starting sample for example, cell sample, for example, thawed cell sample
  • certain isotonic solutions for example, PBS
  • 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 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.
  • Ficoll is not a preferred reagent for isolating cells for therapeutic use, as Ficoll is not qualified for clinical use.
  • Ficoll contains glycol, which has toxic potential to the cells.
  • Ficoll density gradient centrifugation of thawed apheresis products after cryopreservation yields a suboptimal T cell product, for example, as described in the Examples herein.
  • 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.
  • immune effector cells for example, T cells
  • dehydrate during cryopreservation to become denser than fresh cells.
  • immune effector cells for example, T cells
  • 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.
  • the density gradient centrifugation method described herein includes the use of a density gradient medium comprising iodixanol.
  • the density gradient medium comprises about 60% iodixanol in water.
  • 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
  • the selection comprises a positive selection, for example, selection for the desired immune effector cells.
  • the selection comprises a negative selection, for example, selection for unwanted cells, for example, removal of unwanted cells.
  • 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.
  • a flow-through device also referred to as a cell processing system
  • 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.
  • 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.
  • 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.
  • a source of cells e.g., T cells or natural killer (NK) cells
  • T cells can be obtained from a subject.
  • 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.
  • immune effector cells e.g., T cells
  • 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 FicollTM separation.
  • 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.
  • 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.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • 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.
  • 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.
  • 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.
  • buffers such as, for example, Ca-free, Mg -free PBS, PlasmaLyte A, or other saline solution with or without buffer.
  • the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.
  • T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM 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.
  • the population of T regulatory depleted cells contains less than 30%, 25%, 20%, 15%, 1 0%, 5%, 4%, 3%, 2%, 1% of CD25+ cells.
  • T regulatory cells e.g., CD25+ T cells
  • T regulatory cells are removed from the population using an anti-C25 antibody, or fragment thereof, or a CD25-binding ligand, IL-2.
  • 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.
  • the anti-CD25 antibody, or fragment thereof is conjugated to a substrate as described herein.
  • the T regulatory cells e.g., CD25+ T cells
  • 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.
  • the population of immune effector cells to be depleted includes about 6 x 10 9 CD25+ T cells. In other embodiments, the population of immune effector cells to be depleted include about 1 x 10 9 to lx 10 10 CD25+ T cell, and any integer value in between. In some embodiments, the resulting population T regulatory depleted cells has 2 x 10 9 T regulatory cells, e.g., CD25+ cells, or less (e.g., 1 x 10 9 , 5 x 10 8 , 1 x 10 8 , 5 x 10 7 , 1 x 10 7 , or less CD25+ cells).
  • the T regulatory cells e.g., CD25+ cells
  • a depletion tubing set such as, e.g., tubing 162- OL
  • the CliniMAC system is run on a depletion setting such as, e.g., DEPLETION2. 1.
  • 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.
  • a monoclonal antibody cocktail can include antibodies to CD14, CD20, CDllb, CD16, HLA-DR, and CD8.
  • 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
  • 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.
  • check point inhibitor expressing cells are removed simultaneously with the T regulatory, e.g, CD25+ cells.
  • 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.
  • 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.
  • 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.
  • the time period is about 30 minutes.
  • the time period ranges from 30 minutes to 36 hours or longer and all integer values there between.
  • the time period is at least 1, 2, 3, 4, 5, or 6 hours.
  • the time period is 10 to 24 hours.
  • the incubation time period is 24 hours.
  • TIL tumor infiltrating lymphocytes
  • use of longer incubation times can increase the efficiency of capture of CD8+ T cells.
  • T cells 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.
  • subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points.
  • 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.
  • the concentration of cells and surface e.g., particles such as beads
  • 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.
  • a concentration of 1 billion cells/ml is used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used.
  • 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, 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.
  • the cells 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.
  • the concentration of cells used is 5 X 10 ⁇ /ml. In other embodiments, the concentration used can be from about 1 X 10 5 /ml to 1 X 10 6 /ml, and any integer value in between.
  • 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.
  • T cells for stimulation can also be frozen after a washing step.
  • the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population.
  • the cells may be suspended in a freezing solution.
  • 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.
  • 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.
  • a blood sample or an apheresis product is taken from a generally healthy subject.
  • 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.
  • the T cells may be expanded, frozen, and used at a later time.
  • samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments.
  • the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, 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.
  • 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
  • T cells are obtained from a patient directly following treatment that leaves the subject with functional T cells.
  • the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo.
  • these cells may be in a preferred state for enhanced engraftment and in vivo expansion.
  • mobilization for example, mobilization with GM-CSF
  • 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.
  • the immune effector cells expressing a CAR molecule are obtained from a subject that has received a low, immune enhancing dose of an mTOR inhibitor.
  • 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.
  • 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 effector cells, 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.
  • an mTOR inhibitor that increases the number of PDI negative immune effector cells, 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.
  • 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.
  • RNA-interfering agents e.g., siRNA, shRNA, miRNA
  • DGK-deficient cells can be generated by treatment with DGK inhibitors described herein.
  • 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.
  • RNA- interfering agents e.g., siRNA, shRNA, miRNA
  • Ikaros-deficient cells can be generated by treatment with Ikaros inhibitors, e.g., lenalidomide.
  • 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.
  • DGK and Ikaros-deficient cells can be generated by any of the methods described herein.
  • the NK cells are obtained from the subject.
  • the NK cells are an NK cell line, e.g., NK-92 cell line (Conkwest).
  • the immune effector cell can be an allogenic immune effector cell, e.g., T cell or NK cell.
  • 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.
  • TCR functional T cell receptor
  • HLA human leukocyte antigen
  • 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.
  • 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.
  • 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 gene editing systems as described herein.
  • 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.
  • TCR TCR alpha constant chain gene
  • TRBC TCR beta constant chain gene
  • a T cell described herein can be, e.g., engineered such that it does not express a functional HLA on its surface.
  • 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.
  • HLA cell surface expression
  • Such cells can be created through the use of one or more gene editing systems as described herein.
  • the gene editing system targets a sequence encoding a component of one or more HLA molecules.
  • the gene editing system targets a sequence encoding a factor which affects the expression of one or more HLA molecules.
  • the gene editing system targets a regulator of MHC class 1 expression, for example a sequence encoding beta-2 microglobulin (B2M).
  • the gene editing system targets a sequence encoding a regulator of MHC class II molecule expression, for example, CIITA.
  • gene editing systems targeting both a regulator of MHC class 1 expression (for example, B2M) and a regulator ofMHC class II molecule expression are introduced into the cells, such that at least MHC class 1 molecule and at least one MHC class II molecule expression is downregulated.
  • 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.
  • 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).
  • siRNA siRNA
  • shRNA clustered regularly interspaced short palindromic repeats
  • TALEN clustered regularly interspaced short palindromic repeats
  • ZFN zinc finger endonuclease
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • siRNA and shRNAs in T cells can be achieved using any conventional expression system, e.g., such as a lentiviral expression system.
  • shRNAs that downregulate expression of components of the TCR are described, e.g., in US Publication No.: 2012/0321667.
  • 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
  • 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 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.
  • 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 et al. (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.
  • the CRISPR sequence sometimes called a CRISPR locus, comprises alternating repeats and spacers.
  • 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.
  • 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.
  • the Cse (Cas subtype, E.coli) proteins 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.
  • 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.
  • the RNA can guide the Cas protein, e.g., a Cas protein lacking nuclease activity (e.g., dCas9), to a TCR and/or HLA promoter, sterically blocking RNA polymerases.
  • TALEN to inhibit, e.g., TCR and/or HLA
  • 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.
  • TALENs are produced artificially by fusing a TAL effector DNA binding domain to a DNA cleavage domain.
  • Transcription activator-like effects can be engineered to bind any desired DNA sequence, including a portion of the HLA or TCR gene.
  • TALEs Transcription activator-like effects
  • a restriction enzyme can be produced which is specific to any desired DNA sequence, including a HLA or TCR sequence.
  • 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 12 th and 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.
  • TALEN To produce a TALEN, a TALE protein is fused to a nuclease (N), which is a wild-type or mutated Fokl endonuclease.
  • N nuclease
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • ZFN 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.
  • a ZFN comprises a Fok: 1 nuclease domain (or derivative thereof) fused to a DNA-binding domain.
  • the DNA-binding domain comprises one or more zinc fingers.
  • 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.
  • 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.
  • a ZFN 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.
  • 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.
  • Immune Effector Cells e.g., T Cells
  • 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.
  • 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.
  • 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.
  • a protein kinase C activator e.g., bryostatin
  • a ligand that binds the accessory molecule is used for co-stimulation of an accessory molecule on the surface of the T cells.
  • 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.
  • an anti-CD3 antibody and an anti-CD28 antibody are examples of an anti-CD28 antibody.
  • 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.
  • the primary stimulatory signal and the costimulatory signal for the T cell may be provided by different protocols.
  • the agents providing each signal may be in solution or 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).
  • one agent may be coupled to a surface and the other agent in solution.
  • 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.
  • aAPCs artificial antigen presenting cells
  • the two agents are immobilized on beads, either on the same bead, i.e., “cis,” or to separate beads, i.e., “trans.”
  • 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.
  • a 1 : 1 ratio of each antibody bound to the beads for CD4+ T cell expansion and T cell growth is used.
  • 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.
  • 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 preferred embodiment, 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.
  • 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.
  • 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.
  • 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.
  • the ratio of particles to cells can be varied depending on the day of stimulation.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • ratios will vary depending on particle size and on cell size and type.
  • the most typical ratios for use are in the neighborhood of 1 : 1, 2: 1 and 3 : 1 on the first day.
  • the cells such as T cells
  • the beads and the cells are subsequently separated, and then the cells are cultured.
  • the agent-coated beads and cells prior to culture, are not separated but are cultured together.
  • 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.
  • 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.
  • the cells for example, 10 ⁇ to 10 ⁇ T cells
  • beads for example, DYNABEADS® M-450 CD3/CD28 T paramagnetic beads at a ratio of 1 : 1
  • a buffer for example PBS (without divalent cations such as, calcium and magnesium).
  • 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.
  • any cell number is within the context of the present disclosure.
  • 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.
  • 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.
  • greater than 100 million cells/ml is used.
  • a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml is used.
  • a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used.
  • 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.
  • 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.
  • using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.
  • cells transduced with a nucleic acid encoding a CAR are expanded, e.g., by a method described herein.
  • 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).
  • the cells are expanded for a period of 4 to 9 days.
  • the cells are expanded for a period of 8 days or less, e.g., 7, 6 or 5 days.
  • 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.
  • 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.
  • proinflammatory cytokine production e.g., IFN-y and/or GM-CSF levels
  • 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.
  • the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between.
  • the mixture may be cultured for 21 days.
  • the beads and the T cells are cultured together for about eight days.
  • 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.
  • 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
  • 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).
  • 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.
  • the cells are expanded in the presence IL-15 and/or IL-7 (e.g., IL-15 and IL-7).
  • T cells that have been exposed to varied stimulation times may exhibit different characteristics.
  • 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+).
  • TH, CD4+ helper T cell population
  • TC cytotoxic or suppressor T cell population
  • 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.
  • infusing a subject with a T cell population comprising predominately of TH cells may be advantageous.
  • an antigen-specific subset of TC cells has been isolated it may be beneficial to expand this subset to a greater degree.
  • a CAR e.g., a dual CAR or a tandem CAR
  • 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.
  • 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 endogenous TCR-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.
  • CAR+ T cells following antigen stimulation can be measured by flow cytometry.
  • 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.
  • 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.
  • 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.
  • Animal models can also be used to measure a CART activity.
  • 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).
  • 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.
  • Dose dependent CAR treatment response can be evaluated. See, e.g., Milone et al., Molecular Therapy 17(8): 1453-1464 (2009).
  • 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.
  • 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 CountBrightTM 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.
  • 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 using the 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.
  • 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).
  • 51Cr as NaCrO4, New England Nuclear, Boston, MA
  • 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.
  • 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.
  • the cancer or disease includes New York esophageal squamous cell carcinoma.
  • 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.
  • 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.
  • CAR-modified cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.
  • 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.
  • 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.
  • CAR chimeric antigen receptor
  • the infused cell is able to kill cancer cells in the recipient.
  • the CAR-expressing cells e.g., Tor NK cells
  • the CAR-expressing cells 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.
  • the CAR-modified cells may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal.
  • the mammal is a human.
  • 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.
  • the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.
  • 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.
  • other factors such as FLT3-L, IL-1, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.
  • compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient 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.
  • 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.
  • a proliferative disease such as a cancer or malignancy or a precancerous condition such as a myelodysplasia, a myelodysplastic syndrome or a preleukemia.
  • NHL Non-Hodgkin lymphoma
  • 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.
  • 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.
  • Diffuse large B-cell lymphoma 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.
  • DLBCL Diffuse large B-cell lymphoma
  • 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.
  • chemotherapy e.g., cyclophosphamide, doxorubicin, vincristine, prednisone, etoposide
  • antibodies e.g., Rituxan
  • radiation or stem cell transplants.
  • 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.
  • a follicular lymphoma tumor is made up of follicles containing a mixture of centrocytes (also called cleaved 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.
  • 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.
  • alkyating agents e.g., alky
  • 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.
  • 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.
  • 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.
  • 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.
  • stage B where three or more lymph nodes are involved
  • stage C wherein anemia or thrombocytopenia, or both are present.
  • 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.
  • the CAR-expressing cells of the present disclosure are used to treat cancers or leukemias, e.g., with leukemia stem cells.
  • the leukemia stem cells can be CD34+/CD38-leukemia cells.
  • 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
  • BALL B-cell acute lymphoblastic leukemia
  • TALL T-cell acute lympho
  • 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.
  • the disclosure provides a method of bone marrow ablation comprising administering a CAR-expressing cell, e.g., a dual CAR or tandem CAR-expressing cell, of embodiments herein to a subject in need of bone marrow ablation.
  • a CAR-expressing cell e.g., a dual CAR or tandem CAR-expressing cell
  • 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.
  • 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.
  • the method of the disclosure provides a cellular conditioning regimen prior to bone marrow or stem cell transplantation.
  • bone marrow transplantation comprises transplantation of a stem cell.
  • the bone marrow transplantation may comprise transplantation of autologous or allogeneic cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 thro
  • 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.
  • 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.
  • 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.
  • a bone marrow conditioning therapy comprises administering a bone marrow or stem cell transplant to the subject following the eradication of native bone marrow.
  • 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.
  • 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.
  • 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 tumor cells in the recipient.
  • CAR-modified cells e.g., T cells or NK cells
  • the cells e.g., T cells or NK cells
  • the cells persist in the patient for at least four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, thirteen months, fourteen month, fifteen months, sixteen months, seventeen months, eighteen months, nineteen months, twenty months, twenty-one months, twenty-two months, twenty-three months, two years, three years, four years, or five years after administration of the T cell to the patient.
  • the disclosure also includes 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) cell is infused to a recipient in need thereof.
  • CAR chimeric antigen receptor
  • the infused cell is able to kill cancer cells in the recipient.
  • the CAR-expressing cells e.g., T cells or NK cells
  • the CAR-expressing cells are 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., T cells or NK cell, to the patient.
  • the CAR cells e.g., T cells or NK cells
  • the CAR cells of the disclosure may be a type of vaccine for ex vivo immunization and/or in vivo therapy in a mammal.
  • the mammal is a human.
  • 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 cell can be administered to a mammalian recipient to provide a therapeutic benefit.
  • the mammalian recipient may be a human and the CAR cell can be autologous with respect to the recipient.
  • the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.
  • 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.
  • other factors such as flt3-L, IL-1, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.
  • compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient 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.
  • the CAR expressing cells of the disclosure are used to treat a cancer, wherein the cancer is a hematological cancer.
  • Hematological cancer conditions are the types of cancer such as leukemia and malignant lymphoproliferative conditions that affect blood, bone marrow and the lymphatic system.
  • Leukemia can be classified as acute leukemia and chronic leukemia.
  • Acute leukemia can be further classified as acute myelogenous leukemia (AML) and acute lymphoblastic leukemia (ALL).
  • Chronic leukemia includes chronic myelogenous leukemia (CML) and chronic lymphoid leukemia (CLL).
  • CML chronic myelogenous leukemia
  • CLL chronic lymphoid leukemia
  • Other related conditions include myelodysplastic syndromes (MDS, formerly known as “preleukemia”) which are a diverse collection of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells and risk of transformation to AML.
  • MDS myelodysplastic syndromes
  • Lymphoma is a group of blood cell tumors that develop from lymphocytes.
  • Exemplary lymphomas include non-Hodgkin lymphoma and Hodgkin lymphoma.
  • compositions and CART cells or CAR expressing NK cells of the present disclosure are particularly useful for treating B cell malignancies, such as nonHodgkin lymphomas, e.g., DLBCL, Follicular lymphoma, or CLL.
  • B cell malignancies such as nonHodgkin lymphomas, e.g., DLBCL, Follicular lymphoma, or CLL.
  • Non-Hodgkin lymphoma 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.
  • 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.
  • Diffuse large B-cell lymphoma 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.
  • DLBCL Diffuse large B-cell lymphoma
  • 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 ofDLBCL 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.
  • chemotherapy e.g., cyclophosphamide, doxorubicin, vincristine, prednisone, etoposide
  • antibodies e.g., Rituxan
  • radiation or stem cell transplants.
  • Follicular lymphoma 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.
  • a follicular lymphoma tumor is made up of follicles containing a mixture of centrocytes (also called cleaved 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, anthracy cline-containing regimens, e.g., a combination therapy called CHOP- cyclophosphamide, doxorubicin, vincristine, prednisone/prednisolone, antibodies (e.g., rituximab), radioimmunotherapy, and hematopoietic stem cell transplantation.
  • 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.
  • 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.
  • 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.
  • 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.
  • stage B where three or more lymph nodes are involved
  • stage C wherein anemia or thrombocytopenia, or both are present.
  • the CAR expressing cells of the present disclosure are used to treat cancers or leukemias, e.g., with leukemia stem cells.
  • the leukemia stem cells are CD34+/CD38-leukemia cells.
  • 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 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-
  • BALL B-cell acute lymphoblastic leukemia
  • TALL T-cell acute lympho
  • the CAR 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.
  • the CAR-expressing cell described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed.
  • the CAR therapy and/or other therapeutic agents, procedures or modalities can be administered during periods of active disorder, or during a period of remission or less active disease.
  • the CAR therapy can be administered before the other treatment, concurrently with the treatment, post-treatment, or during remission of the disorder.
  • the CAR therapy and the additional agent can be administered in an amount or dose that is higher, lower or the same than the amount or dosage of each agent used individually, e.g., as a monotherapy.
  • the administered amount or dosage of the CAR therapy, the additional agent (e.g., second or third agent), or all is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as a monotherapy.
  • the amount or dosage of the CAR therapy, the additional agent (e.g., second or third agent), or all, that results in a desired effect (e.g., treatment of cancer) lower e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower
  • a desired effect e.g., treatment of cancer
  • the amount or dosage of each agent used individually e.g., as a monotherapy, required to achieve the same therapeutic effect.
  • a CAR-expressing cell described herein may be used in a treatment regimen in combination with surgery, cytokines, radiation, or chemotherapy such as cytoxan, fludarabine, histone deacetylase inhibitors, demethylating agents, or peptide vaccine, such as that described in Izumoto et al. 2008 J Neurosurg 108:963-971.
  • a CAR-expressing cell described herein may be used in combination with a CD20xCD3 bispecific antibody.
  • compounds of the present disclosure are combined with other therapeutic agents, such as other anti-cancer agents, anti -allergic agents, anti-nausea agents (or [0453] anti-emetics), pain relievers, cytoprotective agents, and combinations thereof.
  • other therapeutic agents such as other anti-cancer agents, anti -allergic agents, anti-nausea agents (or [0453] anti-emetics), pain relievers, cytoprotective agents, and combinations thereof.
  • General Chemotherapeutic agents considered for use in combination therapies include anastrozole (Arimidex®), bicalutamide (Casodex®), bleomycin sulfate (Blenoxane®), busulfan (Myleran®), busulfan injection (Busulfex®), capecitabine (Xeloda®), N4- pentoxycarbonyl-5- deoxy-5-fluorocytidine, carboplatin (Paraplatin®), carmustine (BiCNU®), chlorambucil (Leukeran®), cisplatin (Platinol®), cladribine (Leustatin®), cyclophosphamide (Cytoxan® or Neosar®), cytarabine, cytosine arabinoside (Cytosar-U®), cytarabine liposome injection (DepoCyt®), dacarbazine (DTIC-Dome®), d
  • Anti-cancer agents of particular interest for combinations with the compounds of the present disclosure include: anti-tumor antibiotics; tyrosine kinase inhibitors; alkylating agents; anti-microtubule or anti-mitotic agents; or oncolytic viruses.
  • Exemplary tyrosine kinase inhibitors include but are not limited to Erlotinib hydrochloride (Tarceva®); Linifanib (N-[4-(3-amino-lH-indazol-4-yl)phenyl]-N’-(2-fluoro-5- methylphenyl)urea, also known as ABT 869, available from Genentech); Sunitinib malate (Sutent®); Bosutinib (4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4- methylpiperazin-l-l)propoxy]quinoline-3-carbonitrile, also known as SKI-606, and described in US Patent No.
  • Tarceva® Linifanib (N-[4-(3-amino-lH-indazol-4-yl)phenyl]-N’-(2-fluoro-5- methylphenyl)urea, also
  • alkylating agents include, without limitation, Oxaliplatin (Eloxatin®); Temozolomide (Temodar® and Temodal®); Dactinomycin (also known as actinomycin-D, Cosmegen®); Melphalan (also known as L-PAM, L-sarcolysin, and phenylalanine mustard, Alkeran®); Altretamine (also known as hexamethylmelamine (HMM), Hexalen®); Carmustine, (BiCNU®); Bendamustine (Treanda®); Busulfan (Busulfex® and Myleran®); Carboplatin (Paraplatin®); Lomustine (also known as CCNU, CeeNU®); Cisplatin (also known as CDDP, Platinol® and Platinol®-AQ); Chlorambucil (Leukeran®); Cyclophosphamide (Cytoxan® and Neosar®); dacarbazine
  • Exemplary anti-tumor antibiotics include, e.g., Doxorubicin (Adriamycin® and Rubex®); Bleomycin (lenoxane®); Daunorubicin (dauorubicin hydrochloride, daunomycin, and rubidomycin hydrochloride, Cerubidine®); Daunorubicin liposomal (daunorubicin citrate liposome, DaunoXome®); Mitoxantrone (DHAD, Novantrone®); Epirubicin (EllenceTM); Idarubicin (Idamycin®, Idamycin PFS®); Mitomycin C (Mutamycin®); Geldanamycin; Herbimycin; Ravidomycin; and Desacetylravidomycin.
  • Doxorubicin Adriamycin® and Rubex®
  • Bleomycin lenoxane®
  • Daunorubicin daunorubicin hydrochloride, daunomycin, and
  • anti-microtubule or anti-mitotic agents include, without limitation, Vinca Alkaloids (such as Vinorelbine tartrate (Navelbine®), Vincristine (Oncovin®), and Vindesine (Eldisine®)); Taxanes (such as paclitaxel and docetaxel); and Estramustine (Emcyl® or Estracyt®).
  • Vinca Alkaloids such as Vinorelbine tartrate (Navelbine®), Vincristine (Oncovin®), and Vindesine (Eldisine®)
  • Taxanes such as paclitaxel and docetaxel
  • Estramustine Emcyl® or Estracyt®
  • a CAR-expressing cell described herein is administered in combination with an oncolytic virus.
  • oncolytic viruses are capable of selectively replicating in and triggering the death of or slowing the growth of a cancer cell. In some cases, oncolytic viruses have no effect or a minimal effect on non-cancer cells.
  • An oncolytic virus includes but is not limited to an oncolytic adenovirus, oncolytic Herpes Simplex Viruses, oncolytic retrovirus, oncolytic parvovirus, oncolytic vaccinia virus, oncolytic Sinbis virus, oncolytic influenza virus, or oncolytic RNA virus (e.g., oncolytic reovirus, oncolytic Newcastle Disease Virus (NDV), oncolytic measles virus, or oncolytic vesicular stomatitis virus (VSV)).
  • oncolytic adenovirus e.g., oncolytic Herpes Simplex Viruses, oncolytic retrovirus, oncolytic parvovirus, oncolytic vaccinia virus, oncolytic Sinbis virus, oncolytic influenza virus, or oncolytic RNA virus (e.g., oncolytic reovirus, oncolytic Newcastle Disease Virus (NDV), oncolytic measles virus, or oncolytic vesicular stomatitis virus (V
  • the oncolytic virus is a virus, e.g., recombinant oncolytic virus, described in US2010/0178684 Al, which is incorporated herein by reference in its entirety.
  • a recombinant oncolytic virus comprises a nucleic acid sequence (e.g., heterologous nucleic acid sequence) encoding an inhibitor of an immune or inflammatory response, e.g., as described in US2010/01 78684 Al, incorporated herein by reference in its entirety.
  • the recombinant oncolytic virus e.g., oncolytic NDV
  • a pro-apoptotic protein e.g., apoptin
  • a cytokine e.g., GM-CSF, interferon-gamma, interleukin-2 (IL-2), tumor necrosis factor-alpha
  • the oncolytic virus is a chimeric oncolytic NDV described in US 8591881 B2, US 2012/0122185 Al, or US 2014/0271677 Al, each of which is incorporated herein by reference in their entireties.
  • the oncolytic virus comprises a conditionally replicative adenovirus (CRAd), which is designed to replicate exclusively in cancer cells. See, e.g., Alemany et al. Nature Biotechnol. 18(2000):723-27.
  • CRAd conditionally replicative adenovirus
  • an oncolytic adenovirus comprises one described in Table 1 on page 725 of Alemany et al., incorporated herein by reference in its entirety.
  • Exemplary oncolytic viruses include but are not limited to the following: Group B Oncolytic Adenovirus (ColoAdl) (PsiOxus Therapeutics Ltd.) (see, e.g., Clinical Trial Identifier: NCT02053220); ONCOS-102 (previously called CGTG-102), which is an adenovirus comprising granulocyte-macrophage colony stimulating factor (GM-CSF) (Oncos Therapeutics) (see, e.g., Clinical Trial Identifier: NCT0I 598129); VCN-01, which is a genetically modified oncolytic human adenovirus encoding human PH20 hyaluronidase (VCN Biosciences, S.L.) (see, e.g., Clinical Trial Identifiers: NCT02045602 and NCT02045589); Conditionally Replicative Adenovirus ICOVIR-5, which is a virus derived from wild- type human adenovirus ser
  • an oncolytic virus described herein is administering by injection, e.g., subcutaneous, intra-arterial, intravenous, intramuscular, intrathecal, or intraperitoneal injection. In some embodiments, an oncolytic virus described herein is administered intratumorally, transdermally, transmucosally, orally, intranasally, or via pulmonary administration.
  • a CAR expressing cell described herein are administered to a subject in combination with a molecule targeting GITR and/or modulating GITR functions, such as a GITR agonist and/or a GITR antibody that depletes regulatory T cells (Tregs).
  • a molecule targeting GITR and/or modulating GITR functions such as a GITR agonist and/or a GITR antibody that depletes regulatory T cells (Tregs).
  • the GITR binding molecules and/or molecules modulating GITR functions e.g., GITR agonist and/or Treg depleting GITR antibodies
  • the GITR agonist can be administered prior to apheresis of the cells.
  • the subject has CLL.
  • Exemplary GITR agonists include, e.g., GITR fusion proteins and anti-GITR antibodies (e.g., bivalent anti -GITR antibodies) such as, e.g., a GITR fusion protein described in U.S. Patent No.: 6,111,090, European Patent No.: 090505B1, U.S Patent No.: 8,586,023, PCT Publication Nos.: WO 2010/003118 and 2011/090754, or an anti-GITR antibody described, e.g., in U.S. Patent No.: 7,025,962, European Patent No.: 1947183B1, U.S. Patent No.: 7,812,135, U.S. Patent No.: 8,388,967, U.S.
  • anti-GITR antibodies e.g., bivalent anti -GITR antibodies
  • a CAR expressing cell described herein is administered to a subject in combination with an mTOR inhibitor, e.g., an mTOR inhibitor described herein, e.g., a rapalog such as everolimus.
  • an mTOR inhibitor e.g., an mTOR inhibitor described herein, e.g., a rapalog such as everolimus.
  • the mTOR inhibitor is administered prior to the CAR-expressing cell.
  • the mTOR inhibitor can be administered prior to apheresis of the cells.
  • a CAR expressing cell described herein is administered to a subject in combination with a GITR agonist, e.g., a GITR agonist described herein.
  • a GITR agonist e.g., a GITR agonist described herein.
  • the GITR agonist is administered prior to the CAR-expressing cell.
  • the GITR agonist can be administered prior to apheresis of the cells.
  • a CAR expressing cell described herein is administered to a subject in combination with a protein tyrosine phosphatase inhibitor, e.g., a protein tyrosine phosphatase inhibitor described herein.
  • a protein tyrosine phosphatase inhibitor e.g., a protein tyrosine phosphatase inhibitor described herein.
  • the protein tyrosine phosphatase inhibitor is an SHP-1 inhibitor, e.g., an SHP-1 inhibitor described herein, such as, e.g., sodium stibogluconate.
  • the protein tyrosine phosphatase inhibitor is an SHP-2 inhibitor.
  • cells e.g., T cells or NK cells, which have, or will be engineered to express a CAR
  • an mTOR inhibitor that increases the number of PDI negative immune effector cells, e.g., T cells/NK 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.
  • administering is initiated prior to administration of an CAR expressing cell described herein, e.g., T cells or NK cells.
  • an mTOR inhibitor e.g., an allosteric inhibitor, e.g., RAD00I, or a catalytic inhibitor
  • an CAR expressing cell described herein e.g., T cells or NK cells.
  • the CAR cells are administered after a sufficient time, or sufficient dosing, of an mTOR inhibitor, such that the level of PDI negative immune effector cells, e.g., T cells/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, has been, at least transiently, increased.
  • an mTOR inhibitor such that the level of PDI negative immune effector cells, e.g., T cells/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, has been, at least transiently, increased.
  • the cell e.g., T cell or NK cell, to be engineered to express a CAR
  • the cell is harvested after a sufficient time, or after sufficient dosing of the low, immune enhancing, dose of an mTOR inhibitor, such that the level of 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/PD 1 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.
  • the level of PDI negative immune effector cells e.g., T cells or NK cells
  • the ratio of PDI negative immune effector cells e.g., T cells/NK cells/PD 1 positive immune effector cells, e.g., T cells or NK cells
  • the mTOR inhibitor is administered for an amount of time sufficient to decrease the proportion of PD- 1 positive T cells, increase the proportion of PD- 1 negative T cells, or increase the ratio of PD- 1 negative T cells/PD- 1 positive T cells, in the peripheral blood of the subject, or in a preparation of T cells isolated from the subject.
  • the dose of an mTOR inhibitor is associated with mTOR inhibitor of at least 5 but no more than 90%, e.g., as measured by p70 S6K inhibition. In some embodiments, the dose of an mTOR inhibitor is associated with mTOR inhibition of at least 10% but no more than 40%, e.g., as measured by p70 S6K inhibition.
  • the kinase inhibitor is a CDK4 inhibitor, e.g., a CDK4 inhibitor described herein, e.g., a CD4/6 inhibitor, such as, e.g., 6-Acetyl-8-cyclopentyl-5-methyl-2-(5- piperazin-l-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one, hydrochloride (also referred to as palbociclib or PD0332991).
  • a CDK4 inhibitor described herein e.g., a CD4/6 inhibitor, such as, e.g., 6-Acetyl-8-cyclopentyl-5-methyl-2-(5- piperazin-l-yl-pyridin-2-ylamino)-8H-pyrido[2,3-d]pyrimidin-7-one, hydrochloride (also referred to as palbociclib or PD0332991).
  • the kinase inhibitor is aBTK inhibitor, e.g., a BTK inhibitor described herein, such as, e.g., ibrutinib.
  • the kinase inhibitor is an mTOR inhibitor, e.g., an mTOR inhibitor described herein, such as, e.g., rapamycin, a rapamycin analog, OSI-027.
  • the mTOR inhibitor can be, e.g., an mTORCl inhibitor and/or an mT0RC2 inhibitor, e.g., an mTORCl inhibitor and/or mT0RC2 inhibitor described herein.
  • the kinase inhibitor is a MNK inhibitor, e.g., a MNK inhibitor described herein, such as, e.g., 4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d] pyrimidine.
  • the MNK inhibitor can be, e.g., a MNKla, MNK1 b, MNK2a and/or MNK2b inhibitor.
  • the kinase inhibitor is a DGK inhibitor, e.g., a DGK inhibitor described herein, such as, e.g., DGKinhl (D5919) orDGKinh2 (D5794).
  • the kinase inhibitor is a CDK4 inhibitor selected from aloisine A; flavopiridol or HMR-1275, 2-(2-chlorophenyl)-5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-l-methyl-4-piperidinyl]-4- chromenone; crizotinib (PF-02341066; 2-(2-Chlorophenyl)-5,7-dihydroxy-8-[(2R,3S)-2- (hydroxymethyl)-l- methyl-3-pyrrolidinyl]- 4H-l-benzopyran-4-one, hydrochloride (P276-00); l-methyl-5-[[2-[5- (trifluoromethyl)-lH-imidazol-2-yl]-4-pyridinyl]oxy ]-N-[ 4-
  • the kinase inhibitor is a CDK4 inhibitor, e.g., palbociclib (PD0332991), and the palbociclib is administered at a dose of about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (e.g., 75 mg, 100 mg or 125 mg) daily for a period of time, e.g., daily for 14-21 days of a 28 day cycle,
  • a CDK4 inhibitor e.g., palbociclib (PD0332991)
  • the palbociclib is administered at a dose of about 50 mg, 60 mg, 70 mg, 75 mg, 80 mg, 90 mg, 100 mg, 105 mg, 110 mg, 115 mg, 120 mg, 125 mg, 130 mg, 135 mg (e.g., 75 mg, 100 mg or 125 mg) daily for a period of time, e.g., daily for 14-21 days of
  • the kinase inhibitor is a BTK inhibitor selected from ibrutinib (PCI-32765); GDC-0834; RN-486; CGI-560; CGI-1764; HM-71224; CC-292; ONO-4059; CNX-774; and LFM-A13.
  • the kinase inhibitor is a BTK inhibitor, e.g., ibrutinib (PCI- 32765), and the ibrutinib is administered at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600 mg (e.g., 250 mg, 420 mg or 560 mg) daily for a period of time, e.g., daily for 21 day cycle cycle, or daily for 28 day cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of ibrutinib are administered.
  • PCI- 32765 BTK inhibitor
  • the ibrutinib is administered at a dose of about 250 mg, 300 mg, 350 mg, 400 mg, 420 mg, 440 mg, 460 mg, 480 mg, 500 mg, 520 mg, 540 mg, 560 mg, 580 mg, 600
  • the kinase inhibitor is an mTOR inhibitor selected from temsirolimus; ridaforolimus (lR,2R,4S)-4-[(2R)-2 [(1R,9S,12S,1 5R,l 6E,1 8R,19R,21R, 23S,24E,26E,28Z,30S,32S,35R)-l,l 8-dihydroxy-19,30-dimethoxy-l 5,17,21,23, 29,35- hexamethyl-2,3,10,1 4,20-pentaoxo-l 1 ,36-dioxa-4-azatricyclo[30.3.1.
  • the kinase inhibitor is an mTOR inhibitor, e.g., rapamycin, and the rapamycin is administered at a dose of about 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg (e.g., 6 mg) daily for a period of time, e.g., daily for 21 day cycle, or daily for 28 day cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of rapamycin are administered.
  • the kinase inhibitor is an mTOR inhibitor, e.g., everolimus and the everolimus is administered at a dose of about 2 mg, 2.5 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg (e.g., 10 mg) daily for a period of time, e.g., daily for 28 day cycle. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more cycles of everolimus are administered.
  • the kinase inhibitor is an MNK inhibitor selected from CGP052088; 4-amino-3-(p-fluorophenylamino)-pyrazolo [3,4-d] pyrimidine (CGP57380); cercosporamide; ETC-1780445-2; and 4-amino-5-(4-fluoroanilino)-pyrazolo [3,4-d] pyrimidine.
  • Drugs that inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling can also be used.
  • the cell compositions of the present disclosure may be administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, and/or antibodies such as OKT3 or CAMPATH.
  • the cell compositions of the present disclosure are administered following B- cell ablative therapy such as agents that react with CD20, e.g., Rituxan.
  • subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • subjects receive an infusion of the expanded immune cells of the present disclosure.
  • expanded cells are administered before or following surgery.
  • Some patients may experience allergic reactions to the compounds of the present disclosure and/or other anti-cancer agent(s) during or after administration; therefore, antiallergic agents are often administered to minimize the risk of an allergic reaction.
  • Suitable antiallergic agents include corticosteroids, such as dexamethasone (e.g., Decadron®), beclomethasone (e.g., Beclovent®), hydrocortisone (also known as cortisone, hydrocortisone sodium succinate, hydrocortisone sodium phosphate, and sold under the tradenames Ala- Cort®, hydrocortisone phosphate, Solu-Cortef®, Hydrocort Acetate® and Lanacort®), prednisolone (sold under the tradenames Delta-Cortel®, Orapred®, Pediapred® and Prelone®), prednisone (sold under the tradenames Deltasone®, Liquid Red®, Meticorten® and Orasone®), methylprednisolone (also known as 6-methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, sold under the tradenames Duralone®
  • anti-emetics are used in preventing nausea (upper stomach) and vomiting.
  • Suitable anti-emetics include aprepitant (Emend®), ondansetron (Zofiran®), granisetron HC1 (Kytril®), lorazepam (Ativan®, dexamethasone (Decadron®), prochlorperazine (Compazine®), casopitant (Rezonic® and Zunrisa®), and combinations thereof.
  • Medication to alleviate the pain experienced during the treatment period is often prescribed to make the patient more comfortable.
  • Common over-the-counter analgesics such Tylenol®, are often used.
  • opioid analgesic drugs such as hydrocodone/paracetamol or hydrocodone/acetaminophen (e.g., Vicodin®), morphine (e.g., Astramorph® or Avinza®), oxycodone (e.g., OxyContin® or Percocet®), oxymorphone hydrochloride (Opana®), and fentanyl (e.g., Duragesic®) are also useful for moderate or severe pain.
  • hydrocodone/paracetamol or hydrocodone/acetaminophen e.g., Vicodin®
  • morphine e.g., Astramorph® or Avinza®
  • oxycodone e.g., OxyContin® or Percocet®
  • OxyContin® oxymorphone
  • cytoprotective agents such as neuroprotectants, free-radical scavengers, cardioprotectors, anthracycline extravasation neutralizers, nutrients and the like
  • Suitable cytoprotective agents include Amifostine (Ethyol®), glutamine, dimesna (Tavocept®), mesna (Mesnex®), dexrazoxane (Zinecard® or Totect®), xaliproden (Xaprila®), and leucovorin (also known as calcium leucovorin, citrovorum factor and folinicacid).
  • the present disclosure provides pharmaceutical compositions comprising at least one compound of the present disclosure (e.g., a compound of the present disclosure) or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier suitable for administration to a human or animal subject, either alone or together with other anti-cancer agents.
  • a pharmaceutically acceptable carrier suitable for administration to a human or animal subject, either alone or together with other anti-cancer agents.
  • the present disclosure provides methods of treating human or animal subjects suffering from a cellular proliferative disease, such as cancer.
  • the present disclosure provides methods of treating a human or animal subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure (e.g., a compound of the present disclosure) or a pharmaceutically acceptable salt thereof, either alone or in combination with other anti-cancer agents.
  • a compound of the present disclosure e.g., a compound of the present disclosure
  • a pharmaceutically acceptable salt thereof either alone or in combination with other anti-cancer agents.
  • compositions will either be formulated together as a combination therapeutic or administered separately.
  • the compound of the present disclosure and other anti-cancer agent(s) may be administered 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 compound of the present disclosure and the other anticancer agent(s) is generally administered sequentially in any order by infusion or orally.
  • the dosing regimen may vary depending upon the stage of the disease, physical fitness of the patient, safety profiles of the individual drugs, and tolerance of the individual drugs, as well as other criteria well-known to the attending physician and medical practitioner(s) administering the combination.
  • the compound of the present disclosure and other anti-cancer agent(s) may be administered within minutes of each other, hours, days, or even weeks apart depending upon the particular cycle being used for treatment.
  • the cycle could include administration of one drug more often than the other during the treatment cycle and at different doses per administration of the drug.
  • kits that include one or more compound of the present disclosure and a combination partner as disclosed herein are provided.
  • Representative kits include (a) a compound of the present disclosure or a pharmaceutically acceptable salt thereof, (b) at least one combination partner, e.g., as indicated above, whereby such kit may comprise a package insert or other labeling including directions for administration.
  • a compound of the present disclosure may also be used to advantage in combination with known therapeutic processes, for example, the administration of hormones or especially radiation.
  • a compound of the present disclosure may in particular be used as a radiosensitizer, especially for the treatment of tumors which exhibit poor sensitivity to radiotherapy.
  • the subject can be administered an agent which reduces or ameliorates a side effect associated with the administration of a CAR-expressing cell.
  • Side effects associated with the administration of a CAR-expressing cell include, but are not limited to CRS, and hemophagocytic lymphohistiocytosis (HLH), also termed Macrophage Activation Syndrome (MAS).
  • Symptoms of CRS include high fevers, nausea, transient hypotension, hypoxia, and the like.
  • CRS may include clinical constitutional signs and symptoms such as fever, fatigue, anorexia, myalgias, arthalgias, nausea, vomiting, and headache.
  • CRS may include clinical skin signs and symptoms such as rash.
  • CRS may include clinical gastrointestinal signs and symptoms such as nausea, vomiting and diarrhea.
  • CRS may include clinical respiratory signs and symptoms such as tachypnea and hypoxemia.
  • CRS may include clinical cardiovascular signs and symptoms such as tachycardia, widened pulse pressure, hypotension, increased cardiac output (early) and potentially diminished cardiac output (late).
  • CRS may include clinical coagulation signs and symptoms such as elevated cl-dimer, hypofibrinogenemia with or without bleeding.
  • CRS may include clinical renal signs and symptoms such as azotemia.
  • CRS may include clinical hepatic signs and symptoms such as transaminitis and hyperbilirubinemia.
  • the methods described herein can comprise administering a CAR-expressing cell described herein to a subject and further administering one or more agents to manage elevated levels of a soluble factor resulting from treatment with a CAR-expressing cell.
  • the soluble factor elevated in the subject is one or more oflFN-y, TNFa, IL-2 and IL-6.
  • the factor elevated in the subject is one or more ofLL-1, GM- CSF, IL- 10, IL-8, IL-5 and fraktalkine. Therefore, an agent administered to treat this side effect can be an agent that neutralizes one or more of these soluble factors.
  • the agent that neutralizes one or more of these soluble forms is an antibody or antigen binding fragment thereof. Examples of such agents include, but are not limited to a steroid (e.g., corticosteroid), an inhibitor of TNFa, and an inhibitor ofLL-6.
  • TNFa inhibitor is an anti-TNFa antibody molecule such as, infliximab, adalimumab, certolizumab pegol, and golimumab.
  • TNFa inhibitor is a fusion protein such as entanercept.
  • Small molecule inhibitor of TNFa include, but are not limited to, xanthine derivatives (e.g. pentoxifylline) and bupropion.
  • an IL-6 inhibitor is an anti-IL-6 antibody molecule such as tocilizumab (toe), sarilumab, elsilimomab, CNTO 328, ALD518/BMS- 945429, CNTO 136, CPSL2364, CDP6038, VX:30, ARGX-109, FE301, and FMI 01.
  • the anti-IL-6 antibody molecule is tocilizumab.
  • An example of an IL-IR based inhibitor is anakinra.
  • the subject is administered a corticosteroid, such as, e.g. methylprednisolone, hydrocortisone, among others.
  • a corticosteroid such as, e.g. methylprednisolone, hydrocortisone, among others.
  • the subject is administered a vasopressor, such as, e.g., norepinephrine, dopamine, phenylephrine, epinephrine, vasopressin, or a combination thereof.
  • a vasopressor such as, e.g., norepinephrine, dopamine, phenylephrine, epinephrine, vasopressin, or a combination thereof.
  • the subject can be administered an antipyretic agent.
  • the subject can be administered an analgesic agent.
  • the subject can be administered an agent which enhances the activity of a CAR-expressing cell.
  • the agent can be an agent which inhibits an inhibitory molecule.
  • Inhibitory molecules e.g., Programmed Death 1 (PD1)
  • PD1 can, in some embodiments, decrease the ability of a CAR-expressing cell to mount an immune effector response.
  • 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.
  • an inhibitory nucleic acid e.g, an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, or a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription-activator-like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN)
  • an inhibitory nucleic acid e.g, an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, or a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription-activator-like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN)
  • CRISPR clustered regularly interspaced short palindromic repeats
  • TALEN transcription-activator-like effector nuclease
  • ZFN zinc finger endonu
  • the inhibitory molecule is inhibited within a CAR-expressing cell.
  • a dsRNA molecule that inhibits expression of the inhibitory molecule is linked to the nucleic acid that encodes a component, e.g., all of the components, of the CAR.
  • the inhibitor of an inhibitory signal can be, e.g., an antibody or antibody fragment that binds to an inhibitory molecule.
  • the agent can be an antibody or antibody fragment that binds to PDI, PD-LI, PD-L2 or CTLA4 (e.g., ipilimumab (also referred to as MDX-010 and MDX- 101, and marketed as Yervoy®; Bristol-Myers Squibb; Tremelimumab (IgG2 monoclonal antibody available from Pfizer, formerly known as ticilimumab, CP-675,206).).
  • the agent is an antibody or antibody fragment that binds to TIM3.
  • the agent is an antibody or antibody fragment that binds to LAG3.
  • the agent is an antibody or antibody fragment that binds to CEACAM (e.g.,
  • CEACAM- 1, CEACAM- 3 and/or CEACAM-5 are examples of CEACAM- 1, CEACAM- 3 and/or CEACAM-5.
  • PDI is an inhibitory member of the CD28 family of receptors that also includes CD28, CTLA-4, ICOS, and BTLA. PDI is expressed on activated B cells, T cells and myeloid cells (Agata et al. 1996 Int. Immunol 8:765-75). Two ligands for PDI, PD-LI and PD-L2 have been shown to downregulate T cell activation upon binding to PDI (Freeman et a. 2000 J Exp Med 192: 1027-34; Latchman et al. 2001 Nat Immunol 2:261-8; Carter et al. 2002 Eur JImmunol.
  • PD-LI is abundant in human cancers (Dong et al. 2003 J Mol Med 81 :281 - 7; Blank et al. 2005 Cancer Immunol. Immunother 54:307-314; Konishi et al. 2004 Clin Cancer Res 10:5094). Immune suppression can be reversed by inhibiting the local interaction of PDI with PD-LI.
  • Antibodies, antibody fragments, and other inhibitors of PDI, PD-LI and PD-L2 are available in the art and may be used combination with a CAR described herein.
  • nivolumab also referred to as BMS-936558 or MDXI 1 06; Bristol-Myers Squibb
  • BMS-936558 or MDXI 1 06 is a fully human IgG4 monoclonal antibody which specifically blocks PDI.
  • Nivolumab clone 5C4
  • Pidilizumab CT-011; Cure Tech
  • W02009/10161 1 is a humanized IgGlk monoclonal antibody that binds to PDIPidilizumab and other humanized anti-PD 1 monoclonal antibodies are disclosed in W02009/10161 1.
  • Pembrolizumab (formerly known as lambrolizumab and also referred to as Keytruda, MK.03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PDI. Pembrolizumab and other humanized anti-PD 1 antibodies are disclosed in US 8,354,509 and W02009/1 14335.
  • MEDI4736 (Medimmune) is a human monoclonal antibody that binds to PDLI, and inhibits interaction of the ligand with PDI.
  • MDPL3280A (Genentech / Roche) is a human Fe optimized IgGl monoclonal antibody that binds to PD-LI.
  • MDPL3280A and other human monoclonal antibodies to PD-LI are disclosed in U.S. Patent No.: 7,943,743 and U.S Publication No.: 20120039906.
  • Other anti- PD-LI binding agents include YW243.55.S70 (heavy and light chain variable regions are shown in SEQ ID NOs: 20 and 21 in W02010/077634) and MDX-1 105 (also referred to as BMS-936559, and, e.g., anti-PD-LI binding agents disclosed in W02007/005874).
  • the agent which enhances the activity of a CAR-expressing cell is a CEACAM inhibitor (e.g., CEACAM-1, CEACAM-3, and/or CEACAM-5 inhibitor).
  • the inhibitor of CEACAM is an anti-CEACAM antibody molecule.
  • Exemplary anti-CEACAM-1 antibodies are described in WO 2010/125571, WO 2013/082366 WO2014/059251 and WO 2014/022332, e.g., a monoclonal antibody 34B1, 26H7, and 5F4; or a recombinant form thereof, as described in, e.g., US 2004/0047858, US 7,132,255 and WO 99/052552.
  • the anti-CEACAM antibody binds to CEACAM-5 as described in, e.g., Zheng et al. PLoS One. 2010 Sep 2;5(9). pii: el25295 (DOI:IO: 1371/journal. pone.0021146), or cross reacts with CEACAM-1 and CEACAM-5 as described in, e.g., WO 2013/054331 and US 2014/0271618.
  • CEACAM carcinoembryonic antigen cell adhesion molecules
  • CEACAM-1 has been described as a heterophilic ligand for TIM-3 and as playing a role in TIM-3 -mediated T cell tolerance and exhaustion (see e.g., WO 2014/022332; Huang, et al. (2014) Nature doi: 10.1038/naturel3848).
  • co-blockade ofCEACAM-1 and TIM-3 has been shown to enhance an anti-tumor immune response in xenograft colorectal cancer models (see e.g., WO 2014/022332; Huang, et al. (2014), supra).
  • co- blockade of CEACAM-1 and PD-1 reduce T cell tolerance as described, e.g., in WO2014/059251.
  • CEACAM inhibitors can be used with the other immunomodulators described herein (e.g., anti -PD-1 and/or anti-TIM-3 inhibitors) to enhance an immune response against a cancer, e.g., a melanoma, a lung cancer (e.g., NSCLC), a bladder cancer, a colon cancer an ovarian cancer, and other cancers as described herein.
  • a cancer e.g., a melanoma
  • a lung cancer e.g., NSCLC
  • bladder cancer e.g., a colon cancer an ovarian cancer
  • other cancers as described herein.
  • LAG3 lymphocyte activation gene-3 or CD223
  • CD223 lymphocyte activation gene-3
  • Antibodies, antibody fragments, and other inhibitors of LAG3 and its ligands are available in the art and may be used combination with a CAR, e.g., a CAR described herein.
  • BMS-986016 Bristol-Myers Squib
  • IMP701 Immutep
  • IMP73 1 Immutep and GlaxoSmithKline
  • LAG3 inhibitors include IMP321 (Immutep), which is a recombinant fusion protein of a soluble portion of LAG3 and 1g that binds to MHC class II molecules and activates antigen presenting cells (APC).
  • IMP321 Immutep
  • APC antigen presenting cells
  • the agent which enhances the activity of a CAR-expressing cell can be, e.g., a fusion protein comprising a first domain and a second domain, wherein the first domain is an inhibitory molecule, or fragment thereof, and the second domain is a polypeptide that is associated with a positive signal, e.g., a polypeptide comprising an intracellular signaling domain as described herein.
  • the polypeptide that is associated with a positive signal can include a costimulatory domain of CD28, CD27, ICOS, e.g., an intracellular signaling domain of CD28, CD27 and/or ICOS, and/or a primary signaling domain, e.g., of CD3 zeta, e.g., described herein.
  • the fusion protein is expressed by the same cell that expressed the CAR.
  • the agent which enhances activity of a CAR-expressing cell described herein is miR- 17-92.
  • compositions of the present disclosure may comprise a CAR- expressing cell, e.g., a plurality of CAR-expressing cells, as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • Compositions of the present disclosure are in one embodiment formulated for intravenous administration. In some embodiments, compositions of the present disclosure can be formulated for intracranial administration and/or spinal injection.
  • compositions of the present disclosure may be administered in a manner appropriate to the disease to be treated (or prevented).
  • the quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.
  • the pharmaceutical composition is substantially free of, e.g., there are no detectable levels of a contaminant, e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-Gnucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus.
  • a contaminant e.g., selected from the group consisting of endotoxin, mycoplasma, replication competent lentivirus (RCL), p24, VSV-Gnucleic acid, HIV gag, residual anti-CD3/anti-CD28 coated beads, mouse antibodies, pooled human serum, bovine serum albumin, bovine serum, culture media components, vector packaging cell or plasmid components, a bacterium and a fungus.
  • the bacterium is at least one selected from the group consisting of Alcaligenes faecalis, Candida albicans, Escherichia coli, Haemophilus influenza, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumonia, and Streptococcus pyogenes group A [0511]
  • an immunologically effective amount “an anti -turn or effective amount,” “a tumor-inhibiting effective amount,” or “therapeutic amount”
  • the precise amount of the compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject).
  • Described herein is a method of treating a disease, e.g., ALL, comprising administering to a patient in need thereof a pharmaceutical composition comprising a population of cells, e.g., T cells or NK cells, comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule that binds to a first antigen and a second antigen at a dosage based on cell/kg body weight and the total CAR+ cells in the pharmaceutical composition.
  • the total CAR+ cells are measured as the population of CAR cells expressing both the first CAR and second CAR as described throughout.
  • CAR-expressing cells are generated using lentiviral viral vectors, such as lentivirus.
  • CAR-expressing cells e.g., CARTs
  • CAR-expressing cells e.g., CARTs
  • Transient expression of CARs can be effected by RNA CAR vector delivery.
  • the CAR RNA is transduced into the cell, e.g., NK cell or T cell, by electroporation.
  • CART infusion breaks should not last more than ten to fourteen days.
  • one or more CAR-expressing cells as disclosed herein can be administered or delivered to the subject via a biopolymer scaffold, e.g., a biopolymer implant.
  • Biopolymer scaffolds can support or enhance the delivery, expansion, and/or dispersion of the CAR-expressing cells described herein.
  • a biopolymer scaffold comprises a biocompatible (e.g., does not substantially induce an inflammatory or immune response) and/or a biodegradable polymer that can be naturally occurring or synthetic.
  • biopolymers include, but are not limited to, agar, agarose, alginate, alginate/calcium phosphate cement (CPC), beta-galactosidase ( -GAL), (1 , 2, 3,4,6- pentaacetyl a-D-galactose), cellulose, chitin, chitosan, collagen, elastin, gelatin, hyaluronic acid collagen, hydroxyapatite, poly(3-hydroxybutyrate-co-3 -hydroxy -hexanoate) (PHBHHx), poly(lactide), poly(caprolactone) (PCL), poly(lactide-co-glycolide) (PLG), polyethylene oxide (PEO), poly(lactic-co-glycolic acid) (PLGA), polypropylene oxide (PPO), polyvinyl alcohol) (PVA), silk, soy protein, and soy protein isolate, alone or in combination with any other polymer composition, in any combination with any other polymer
  • the biopolymer can be augmented or modified with adhesion- or migration-promoting molecules, e.g., collagen-mimetic peptides that bind to the collagen receptor of lymphocytes, and/or stimulatory molecules to enhance the delivery, expansion, or function, e.g., anti-cancer activity, of the cells to be delivered.
  • adhesion- or migration-promoting molecules e.g., collagen-mimetic peptides that bind to the collagen receptor of lymphocytes, and/or stimulatory molecules to enhance the delivery, expansion, or function, e.g., anti-cancer activity, of the cells to be delivered.
  • the biopolymer scaffold can be an injectable, e.g., a gel or a semi-solid, or a solid composition.
  • CAR-expressing cells described herein are seeded onto the biopolymer scaffold prior to delivery to the subject.
  • the biopolymer scaffold further comprises one or more additional therapeutic agents described herein (e.g., another CAR- expressing cell, an antibody, or a small molecule) or agents that enhance the activity of a CAR- expressing cell, e.g., incorporated or conjugated to the biopolymers of the scaffold.
  • the biopolymer scaffold is injected, e.g., intratumorally, or surgically implanted at the tumor or within a proximity of the tumor sufficient to mediate an anti-tumor effect. Additional examples of biopolymer compositions and methods for their delivery are described in Stephan et al., Nature Biotechnology, 2015, 33:97-101; and WO2014/1 10591.
  • NY-ESO-1 is a well-known cancer testis antigen, that is reexpressed in a variety of tumors and homogeneously expressed in the rare and aggressive soft- tissue cancers, myxoid round cell liposarcoma (94%) and synovial sarcoma (70%). Multiple TCRs and TCR-like antibodies have been developed for this target, allowing for a systematic comparison with our platform.
  • the beads are comprised of 97 HLA allotypes encoded with different colors 49, 50 and can be loaded with peptides of choice 45, 29 .
  • NY-ESO-1157-165 is expected to bind two types of the SABs carrying HLA-A*02:01 and HLA-A*02:06 in a similar conformation, based on NetMHCpan4.1 51 and HLA3DB predictions.
  • MHC-I TRACeR The sensitivity of MHC-I TRACeR binding to position 1 of the peptide may also explain the unique metabolite sensitivity achieved with MR1 TRACeR. Comparing the structures of MHC-I and MR1, we observed that the small molecule metabolite in MR1 is located close to the peptide position 1 in MHC-I (FIG. ID). TRACeR ’s apparent access to this region of the antigen pocket on MHC grants it a significant structural advantage, enabling the achievement of unparalleled antigen specificity.
  • Example 2 In vivo activity of dual and tandem CAR-Ts targeting NYESO-1 and CD3 [0532] TRACeR N M TM A0 directed cytotoxicity as Chimeric Antigen Receptors (CARs) [0533] TRACeR ⁇ H ⁇ c E -i°Ao outperformed other existing binders of the HLA-A*02:01/NY-ESO- 1 complex in binding specificity, implying that our binder would be relevant for therapeutic applications. To test this, we implemented our TRACeR ⁇ Cc E -i°Ao as a chimeric antigen receptor on T cells.
  • TRACeR ⁇ c E - °Ao a chimeric antigen receptor (FIG. 2E).
  • TRACeR ⁇ c E - °Ao a chimeric antigen receptor
  • the monomeric TRACeR ⁇ H ⁇ c E -i°A02 was fused to a CD8a hinge-transmembrane domain, a 4 IBB co-stimulatory domain and a CD3 ⁇ signaling domain.
  • DLBCL cell lines were either directly received from the ATCC or verified by ATCC STR profiling.
  • Cells were cultured at 37°C in a 5% CO2 incubator with Advanced RPMI (Gibco), 5% heat inactivated FBS (Gibco), Glutamax (Gibco), and penicillin/streptomycin (Gibco).
  • Cells were maintained between 200,000 - 1 million cells/mL and split every two days. Lines were routinely tested for mycoplasma contamination using the Universal Mycoplasma Detection Kit (ATCC).
  • ATCC Universal Mycoplasma Detection Kit
  • T cells were cultured at 1 million cells/mL and activated with a 1 : 1 ratio of Dynabeads Human T- Activator CD3/CD28 beads (Gibco) for 72 hours, after which they were debeaded and expanded in IL-2-containing media.
  • CD8+ T cells were isolated from fresh leukopacks of anonymous donors (AllCells, LP, FR, 5B) by negative selection using EasySepTM Human CD8+ T Cell Isolation Kit (STEMCELL Technologies #17953). Isolated CD8+ T cells were cryopreserved in CELLBANKER 1 (AMSBIO #11910).
  • T cells were cultured in human T cell medium (HTCM) consisting of X- VIVO 15 (Lonza #04-418Q), 5% Human AB serum, and 10 mM neutralized N-acetyl L-Cysteine (SigmaAldrich #A9165) supplemented with 30 units/mL IL-2 (NCI BRB Preclinical Repository) for all experiments.
  • HTCM human T cell medium
  • Sytox Blue (Invitrogen) viability stain was added to each well.
  • Cells were analyzed by the high throughput sampler of a LSR Fortessa (BD). Tumors were gated from immune cells, and the fraction live of each well was normalized to wells that received no antibody.
  • T cells incubated with off-target tumor cell lines SUDHL4 and SUDHL5 also displayed some activation in the presence of our TRACeR BiTE, albeit at a lower level. This is likely due to activity of dimerized anti-CD3 scFv, which can facilitate cross-linking of CD3 on T cells and facilitate T cell activation independent of target recognition/killing.
  • TRACeR] _(MHC-I,A02) A (NY-ESO-1) variants and the NY-ESO-1 -specific TCR 1G443 were synthesized as gene fragments (Twist Bioscience).
  • Anti -NY-ESO-1 CAR T cells were built by fusing a TRACeR] (MHC- I,A02) A (NY-ESO-1) binder to the hinge region of the human CD8a chain and transmembrane and cytoplasmic regions of the human 4- IBB, and CD3z signaling domains. CARs were expressed under the control of a spleen focus-forming virus (SFFV) promoter, followed by the human CD8a signal sequence.
  • SFFV spleen focus-forming virus
  • a myc tag was added in the N-terminus of the CAR for sorting CAR-expressing T cells by flow cytometry.
  • the 1G4 TCR was also expressed under the SFFV promoter. Signal sequences of both alpha and beta TCR chains were preserved. 1G4 beta chain was followed by a furin cleavage site, a SGSG (SEQ ID NO: 68) spacer and P2A sequence.
  • a myc tag was inserted between the signal sequence and the N-terminus of the alpha chain. All gene fragments were cloned in the BamHI restriction site of the pHR-SFFV plasmid (addgene #79121). Receptor design, expression and tetramer binding of engineered T cell receptors.
  • Pantropic VSV-G pseudotyped lentivirus was produced by transfecting Lenti-X 293T cells (Takara # 632180) with a pHR’SIN:CSW transgene expression vector and the viral packaging plasmids pCMVdR8.91 and pMD2.G (addgene #12259) using Fugene HD (Promega #E2312). After 48 hr, viral supernatant was harvested and added to activated CD8+ primary T cells. T cells were exposed to the virus for 24 hr, when viral supernatant was replaced with fresh HTCM media supplemented with IL-2.
  • T cells were stained in flow buffer (PBS 5%FBS) with Alexa Fluor 647 anti-myc antibody (Cell Signaling Technology, #2233) and sorted on myc+ events in a FACSymphony S6 Sorter (BD Biosciences). Untransduced T cells were used to set up the sorting gates but were not sorted. T cells were expanded for at least 9 days keeping a cell density of approximately 0.5 x 10 6 cells/mL, until they stopped growing and became quiescent (resting). Resting T cells were used in all cytotoxic assays.
  • the percentage of specific lysis of target cells was determined by comparing the number of target cells alive in the co-culture with engineered T cells (CAR or 1G4) compared to average number of target cells alive coculture with untransduced T cell. All flow cytometry data analysis was performed in FlowJo X software (TreeStar) and GraphPad Prism 9 (Dotmatic). The following equation was used to calculate the percent of specific lysis.
  • HLA-A*2:01/NY-ESO-l/MAM-TRACeR complex was prepared by mixing MAM- TRACeR and HLA-A*2:01/NY-ESO-1 at 1 : 1.3 molar ratio, mixture was incubated at 4 °C for 1 hour. Unbound proteins were separated by size-exclusion chromatography using Superdex200 increase 10/300 GL column, run at 0.5 mg/min flow rate with running buffer, 25 mM Tris pH 8.0 and 150 mM NaCl. Complex was concentrated to 10.4 mg/ml for crystallography, sample was mixed at 1 : 1 ratio with reservoir well solution and incubated at 20 °C.
  • Crystals were obtained in 200 mM Sodium sulfate and 20% w/v PEG 3350 using sitting drop vapor diffusion method. Crystals were harvested after transferring to cryogenic solution containing reservoir solution and 20% v/v Glycerol. Crystals were screened and data was collected on National Synchrotron Light Source (FMX, 17-ID-2) at Brookhaven National Laboratory (BNL) using Dectris Eiger 16M detector. Data was processed using XDS62 and structure of HLA-A*02:01/NY-ESO-l/MAM-TRACeR complex was solved by molecular replacement method using Molrep63 and Refmac564 software of ccp4 package suits65.
  • FMX National Synchrotron Light Source
  • BNL Brookhaven National Laboratory
  • HLA-A*02:01/NY-ESO-1 (PDB ID, 1S9W)32 and model generated from AlphaFold structure prediction tool for MAM-TRACeR were used as search model.
  • Complex structure was refined by Buster Global Phasing method66 and Phenix67 and COOT68 was used for model building.

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Abstract

La divulgation concerne des cellules exprimant un récepteur CAR-T comprenant une séquence spécifique pour la liaison à des cellules cancéreuses exprimant NY-ESO-1. L'invention concerne également des méthodes de traitement du cancer comprenant l'administration desdites cellules à des sujets en ayant besoin.
PCT/US2024/053742 2023-10-30 2024-10-30 Compositions comprenant des cellules ciblant le cancer et leurs procédés d'utilisation Pending WO2025096673A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170066827A1 (en) * 2014-03-05 2017-03-09 Ucl Business Plc Chimeric antigen receptor
US20180037630A1 (en) * 2015-02-12 2018-02-08 University Health Network Chimeric Antigen Receptors
WO2019089650A1 (fr) * 2017-10-31 2019-05-09 Allogene Therapeutics, Inc. Procédés et compositions pour le dosage de lymphocytes t allogéniques à récepteur antigénique chimérique

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170066827A1 (en) * 2014-03-05 2017-03-09 Ucl Business Plc Chimeric antigen receptor
US20180037630A1 (en) * 2015-02-12 2018-02-08 University Health Network Chimeric Antigen Receptors
WO2019089650A1 (fr) * 2017-10-31 2019-05-09 Allogene Therapeutics, Inc. Procédés et compositions pour le dosage de lymphocytes t allogéniques à récepteur antigénique chimérique

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