WO2022133030A1 - Combination therapy of a cell therapy and a bcl2 inhibitor - Google Patents

Combination therapy of a cell therapy and a bcl2 inhibitor Download PDF

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Publication number
WO2022133030A1
WO2022133030A1 PCT/US2021/063708 US2021063708W WO2022133030A1 WO 2022133030 A1 WO2022133030 A1 WO 2022133030A1 US 2021063708 W US2021063708 W US 2021063708W WO 2022133030 A1 WO2022133030 A1 WO 2022133030A1
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day
dose
bcl2 inhibitor
subject
inhibitor
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PCT/US2021/063708
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French (fr)
Inventor
Ronald DUBOWY
Heidi GILLENWATER
John-Michael WILLIFORD
Archana BRAHMANDAM
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Juno Therapeutics, Inc.
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Publication of WO2022133030A1 publication Critical patent/WO2022133030A1/en

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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
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
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    • 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
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    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • 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
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the present disclosure relates in some aspects to methods and uses of combination therapies involving a therapy such as an immunotherapy or a cell therapy, e.g., a T cell therapy, and the use of an inhibitor of BCL2 protein, for treating subjects with cancers such as leukemias and lymphomas, and related methods, uses, and articles of manufacture.
  • a therapy such as an immunotherapy or a cell therapy, e.g., a T cell therapy
  • an inhibitor of BCL2 protein for treating subjects with cancers such as leukemias and lymphomas
  • the T cell therapy includes cells that express recombinant receptors such as chimeric antigen receptors (CARs).
  • CARs chimeric antigen receptors
  • adoptive cell therapies including those involving the administration of cells expressing chimeric receptors specific for a disease or disorder of interest, such as chimeric antigen receptors (CARs) and/or other recombinant antigen receptors, as well as other adoptive immune cell and adoptive T cell therapies.
  • CARs chimeric antigen receptors
  • Subsets of cancers are resistant to or develop resistance to such therapies. Improved methods are therefore needed, for example, to overcome this resistance and increase the efficacy of such methods. Provided are methods and uses that meet such needs.
  • a method of treating cancer including (1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1; and (2) administering to the subject a BCL2 inhibitor in a dosing regimen including: (i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy; and (ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered
  • CLL chronic lymphocytic le
  • a method of treating cancer including administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the BCL2 inhibitor is administered in a dosing regimen including: (i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of a cell therapy, wherein the cell therapy includes a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) and was administered to the subject on Day 1; and (ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose
  • CLL chronic lymphocytic le
  • a method of treating cancer including administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen including: (i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy; and (ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at
  • the daily administration of the first dose of the BCL2 inhibitor begins about 1 day after or within about 1 day after initiation of administration of the cell therapy. In some embodiments, the daily administration of the first dose of the BCL2 inhibitor begins about 1 day after initiation of administration of the cell therapy. In some embodiments, the daily administration of the first dose of the BCL2 inhibitor begins within about 1 day after initiation of administration of the cell therapy. In some embodiments, the daily administration of the first dose of the BCL2 inhibitor begins at or after a time when activation-induced cell death (AICD) of the cells of the cell therapy has peaked following initiation of administration of the cell therapy.
  • AICD activation-induced cell death
  • the daily administration of the first dose of the BCL2 inhibitor begins at a time when activation-induced cell death (AICD) of the cells of the cell therapy has peaked following initiation of administration of the cell therapy. In some embodiments, the daily administration of the first dose of the BCL2 inhibitor begins after a time when activation- induced cell death (AICD) of the cells of the cell therapy has peaked following initiation of administration of the cell therapy.
  • AICD activation-induced cell death
  • the at least one subsequent dose includes a second dose
  • the dosing regimen includes: (i) daily administration of a first dose of the BCL2 inhibitor for a first predetermined period; and (ii) daily administration of a second dose of the BCL2 inhibitor for a second predetermined period beginning after the first predetermined period.
  • the at least one subsequent dose includes a second dose and a third dose
  • the dosing regimen includes: (i) daily administration of a first dose of the BCL2 inhibitor for a first predetermined period; (ii) daily administration of a second dose of the BCL2 inhibitor for a second predetermined period beginning after the first predetermined period; and (iii) daily administration of a third dose of the BCL2 inhibitor for a third predetermined period beginning after the second predetermined period.
  • the first predetermined period is from about Day 2 to about Day 30. In some embodiments, the first predetermined period is from about Day 2 to about Day 7.
  • the second predetermined period is from about Day 31 to about Day 90. In some embodiments, the second predetermined period is from about Day 8 to about Day 30. In some embodiments, the second predetermined period is from about Day 31 to about Day 37.
  • the third predetermined period is from about Day 31 to about Day 90. In some embodiments, the third predetermined period is from about Day 38 to about Day 90.
  • the first dose is at least about 20 mg of the BCL2 inhibitor. In some embodiments, no dose is greater than about 400 mg of the BCL2 inhibitor. In some embodiments, no dose of the dosing regimen is greater than about 400 mg of the BCL2 inhibitor.
  • the first dose is between about 20 mg and about 100 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 20 mg, about 50 mg, or about 100 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 20 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 100 mg of the BCL2 inhibitor.
  • the second dose is between about 50 mg and about 200 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 50 mg, about 100 mg, or about 200 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 50 mg, about 100 mg, about 150 mg, or about 200 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 150 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 200 mg of the BCL2 inhibitor.
  • the third dose is between about 100 mg and 400 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 100 mg, about 200 mg, or about 400 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 100 mg, about 200 mg, about 300 mg, or about 400 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 200 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 300 mg of the BCL2 inhibitor.In some embodiments, the third dose is about 400 mg of the BCL2 inhibitor.
  • the BCL2 inhibitor is venetoclax or navitoclax. In some embodiments, the BCL2 inhibitor is navitoclax.In some embodiments, the BCL2 inhibitor is venetoclax.
  • Also provided herein are methods of treating a cancer the method including: (1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and (2) administering to the subject a BCL2 inhibitor in a dosing regimen including: (i) daily administration of about 20 mg of the BCL2 inhibitor from about Day 2 to about Day 7 ; (ii) daily administration of about 50 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and (iii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • CD 19 cluster of differentiation 19
  • a method of treating a cancer including administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the subject has been administered a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, and wherein the BCL2 inhibitor is administered in a dosing regimen including: (i) daily administration of about 20 mg of the BCL2 inhibitor from about Day 2 to about Day 7 ; (ii) daily administration of about 50 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and (iii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • CD 19 cluster of differentiation 19
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • BCL2 inhibitor is administered in a dosing regimen including: (i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and (ii)
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • CD 19 cluster of differentiation 19
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • CD 19 cluster of differentiation 19
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • BCL2 inhibitor is administered in a dosing regimen including: (i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and (ii)
  • Also provided herein is a method of treating cancer, the method including administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen including (i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and (ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • CAR chimeric antigen receptor
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • CAR chimeric antigen receptor
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • BCL2 inhibitor is administering in a dosing regimen including:
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • the BCL2 inhibitor is administered for no more than about 24 months. In some embodiments, the BCL2 inhibitor is administered for no more than about 18 months. In some embodiments, the BCL2 inhibitor is administered for no more than about 12 months. In some embodiments, if the subject exhibits minimum residual disease (MRD) in peripheral blood (PB) at Day 90, the BCL2 inhibitor is administered for up to 12 months or for up to 24 months. In some embodiments, if the subject exhibits minimum residual disease (MRD) in peripheral blood (PB) at Day 90, the BCL2 inhibitor is administered for up to 12 months.
  • MRD minimum residual disease
  • the BCL2 inhibitor is administered for up to 18 months. In some embodiments, if the subject exhibits minimum residual disease (MRD) in peripheral blood (PB) at Day 90, the BCL2 inhibitor is administered for up to 24 months. In some embodiments, if the subject does not exhibit a complete response (CR) at Day 90, the BCL2 inhibitor is administered for up to 12 months or for up to 24 months. In some embodiments, if the subject does not exhibit a complete response (CR) at Day 90, the BCL2 inhibitor is administered for up to 12 months.
  • the BCL2 inhibitor is administered for up to 18 months. In some embodiments, if the subject does not exhibit a complete response (CR) at Day 90, the BCL2 inhibitor is administered for up to 24 months.
  • daily administration of the BCL2 inhibitor is discontinued after or about after Day 90. In some embodiments, the BCL2 inhibitor is administered daily until at or about Day 90.
  • the BCL2 inhibitor is venetoclax or navitoclax. In some embodiments, the BCL2 inhibitor is navitoclax.In some embodiments, the BCL2 inhibitor is venetoclax.
  • the subject has a chronic lymphocytic leukemia (CLL).
  • CLL chronic lymphocytic leukemia
  • r/r relapsed or refractory
  • SLL small lymphocytic lymphoma
  • the SLL is a relapsed or refractory (r/r) SLL.
  • the method further includes, prior to initiation of administration of the cell therapy, administering a lymphodepleting therapy to the subject.
  • the subject has been preconditioned with a lymphodepleting therapy prior to initiation of administration of the cell therapy.
  • the lymphodepleting therapy includes the administration of fludarabine and/or cyclophosphamide.
  • the lymphodepleting therapy includes the administration of fludarabine.
  • the lymphodepleting therapy includes the administration of cyclophosphamide.
  • the lymphodepleting therapy includes the administration of fludarabine and cyclophosphamide.
  • the lymphodepleting therapy includes administration of cyclophosphamide at about 200-400 mg/m 2 , optionally at or about 300 mg/m 2 , inclusive, and/or fludarabine at about 20-40 mg/m 2 , optionally 30 mg/m 2 , daily for 2-4 days.
  • the lymphodepleting therapy includes administration of cyclophosphamide at about 200-400 mg/m 2 , optionally at or about 300 mg/m 2 , inclusive, and/or fludarabine at about 20-40 mg/m 2 , optionally 30 mg/m 2 , daily for 3 days.
  • the lymphodepleting therapy includes administration of cyclophosphamide at about 200-400 mg/m 2 daily for 2-4 days. In some embodiments, the lymphodepleting therapy includes administration of cyclophosphamide at about 200-400 mg/m 2 daily for 3 days. In some embodiments, the lymphodepleting therapy includes administration of cyclophosphamide at or about 300 mg/m 2 daily for 2-4 days. In some embodiments, the lymphodepleting therapy includes administration of cyclophosphamide at or about 300 mg/m 2 daily for 3 days. In some embodiments, the lymphodepleting therapy includes administration of fludarabine at about 20-40 mg/m 2 daily for 2-4 days.
  • the lymphodepleting therapy includes administration of fludarabine at about 20-40 mg/m 2 daily for 3 days. In some embodiments, the lymphodepleting therapy includes administration of fludarabine at or about 30 mg/m 2 daily for 2-4 days. In some embodiments, the lymphodepleting therapy includes administration of fludarabine at or about 30 mg/m 2 daily for 3 days. In some embodiments, the lymphodepleting therapy includes administration of cyclophosphamide at or about 300 mg/m 2 and fludarabine at about 30 mg/m 2 daily for 3 days. In some embodiments, the cell therapy is administered at least at or about 2-7 days after the lymphodepleting therapy. In some embodiments, the cell therapy is administered at least at or about 2-7 days after the initiation of the lymphodepleting therapy.
  • the method further includes administering to the subject a bridging therapy including a BCL2 inhibitor.
  • the subject is administered a bridging therapy comprising a BCL2 inhibitor.
  • the bridging therapy is administered prior to initiation of administration of a lymphodepleting therapy.
  • the bridging therapy is administered at a time between collecting of autologous cells from the subject and prior to administering a lymphodepleting therapy to the subject.
  • the cells of the cell therapy are the autologous cells collected from the subject. In some embodiments, the collecting is by apheresis or leukapheresis.
  • the bridging therapy includes daily administration of a first dose of the BCL2 inhibitor for a first week, daily administration of a second dose of the BCL2 inhibitor for a second week, and daily administration of a third dose of the BCL2 inhibitor for a third week.
  • the bridging therapy includes daily administration of between about 20 mg and about 400 mg of the BCL2 inhibitor.
  • the second dose of the bridging therapy is an increased amount of BCL2 inhibitor compared to the first dose of the bridging therapy
  • the third dose of the bridging therapy is an increased amount of BCL2 inhibitor compared to the second dose of the bridging therapy.
  • the first dose of the bridging therapy is 20 mg of the BCL2 inhibitor
  • the second dose of the bridging therapy is 50 mg of the BCL2 inhibitor
  • the third dose of the bridging therapy is 100 mg of the BCL2 inhibitor.
  • the first dose of the bridging therapy is 50 mg of the BCL2 inhibitor
  • the second dose of the bridging therapy is 100 mg of the BCL2 inhibitor
  • the third dose of the bridging therapy is 200 mg of the BCL2 inhibitor.
  • the first dose of the bridging therapy is 100 mg of the BCL2 inhibitor
  • the second dose of the bridging therapy is 200 mg of the BCL2 inhibitor
  • the third dose of the bridging therapy is 400 mg of the BCL2 inhibitor.
  • the bridging therapy is ceased about or at least about 1 day prior to initiation of administration of a lymphodepleting therapy.
  • the BCL2 inhibitor is venetoclax or navitoclax. In some embodiments, the BCL2 inhibitor is navitoclax.In some embodiments, the BCL2 inhibitor is venetoclax.
  • the BCL2 inhibitor administered in the bridging therapy and the BCL2 inhibitor administered in the dosing regimen are the same BCL2 inhibitor.
  • the BCL2 inhibitor administered in the bridging therapy and the BCL2 inhibitor administered in the dosing regimen is venetoclax.
  • the BCL2 inhibitor administered in the bridging therapy and the BCL2 inhibitor administered in the dosing regimen are different BCL2 inhibitors.
  • the bridging therapy further includes administration of an anti-CD20 antibody to the subject. In some embodiments, if the subject has been previously treated with a BCL2 inhibitor, the bridging therapy further includes administration of an anti-CD20 antibody to the subject. In some embodiments, the bridging therapy further includes administration of a BTK inhibitor to the subject. In some embodiments, the BTK inhibitor is or comprises ibrutinib.
  • the subject has failed at least one prior line of therapy, and at least one of the at least one prior lines of therapy is a Bruton’s tyrosine kinase (BTK) inhibitor, or the subject is ineligible for treatment with a BTK inhibitor.
  • the subject has failed at least one prior line of therapy, and at least one of the at least one prior lines of therapy is a Bruton’ s tyrosine kinase (BTK) inhibitor.
  • the subject has failed a BTK inhibitor.
  • the subject is ineligible for treatment with a BTK inhibitor.
  • the subject has been previously treated with a Bruton’s tyrosine kinase (BTK) inhibitor.
  • BTK tyrosine kinase
  • the BTK inhibitor is or comprises ibrutinib.
  • the subject prior to the administration of the cell therapy, has been treated with one or more prior therapies for the CLL or the SLL, other than another dose of cells expressing a CAR or a lymphodepleting therapy.
  • the subject prior to the administration of the cell therapy, has relapsed following remission after treatment with, or become refractory to, failed and/or was intolerant to treatment with one or more prior therapies.
  • prior to the administration of the cell therapy the subject has relapsed following remission after treatment with one or more prior therapies.
  • prior to the administration of the cell therapy prior to the administration of the cell therapy, the subject has become refractory to treatment with one or more prior therapies.
  • prior to the administration of the cell therapy prior to the administration of the cell therapy, the subject has failed treatment with one or more prior therapies.
  • prior to the administration of the cell therapy the subject is intolerant to treatment with one or more prior therapies.
  • the subject prior to the administration of the cell therapy, has been treated with at least two prior therapies for the CLL or the SLL, other than another dose of cells expressing a CAR or a lymphodepleting therapy.
  • the subject prior to the administration of the cell therapy, has relapsed following remission after treatment with, or become refractory to, failed and/or was intolerant to treatment with two or more prior therapies.
  • prior to the administration of the cell therapy the subject has relapsed following remission after treatment with two or more prior therapies.
  • prior to the administration of the cell therapy prior to the administration of the cell therapy, the subject has become refractory to treatment with two or more prior therapies.
  • prior to the administration of the cell therapy prior to the administration of the cell therapy, the subject has failed treatment with two or more prior therapies.
  • prior to the administration of the cell therapy the subject is intolerant to treatment with two or more prior therapies.
  • the one or more prior therapies are selected from a kinase inhibitor; venetoclax; a combination therapy comprising fludarabine and rituximab; radiation therapy; and hematopoietic stem cell transplantation (HSCT).
  • the kinase inhibitor is an inhibitor of Bruton’s tyrosine kinase (BTK).
  • the kinase inhibitor is ibrutinib.
  • the one or more prior therapies are selected from a Bruton’s tyrosine kinase inhibitor; venetoclax; a combination therapy comprising fludarabine and rituximab; radiation therapy; and hematopoietic stem cell transplantation (HSCT).
  • the one or more prior therapies are selected from ibrutinib; venetoclax; a combination therapy comprising fludarabine and rituximab; radiation therapy; and hematopoietic stem cell transplantation (HSCT).
  • the one or more prior therapies includes an inhibitor of Bruton’s tyrosine kinase (BTK).
  • BTK tyrosine kinase
  • the BTK inhibitor is ibrutinib.
  • the one or more prior therapies includes ibrutinib.
  • the subject has failed a BTK inhibitor or is ineligible to receive a BTK inhibitor. In some embodiments, the subject has failed a BTK inhibitor. In some embodiments, failure of a BTK inhibitor is defined as having stable disease or progressive disease (PD) as best response, or progression after previous response, or discontinuation due to intolerance. In some embodiments, intolerance is defined as failure to tolerate treatment due to unmanageable toxicity. In some embodiments, a subject is ineligible for treatment with a BTK inhibitor. In some embodiments, a subject is ineligible for treatment with a BTK inhibitor due to a requirement for full-dose anticoagulation or history of arrhythmia.
  • PD progressive disease
  • intolerance is defined as failure to tolerate treatment due to unmanageable toxicity.
  • a subject is ineligible for treatment with a BTK inhibitor. In some embodiments, a subject is ineligible for treatment with a BTK inhibitor due to a requirement for full-dose anticoagulation or history of arrhythmia
  • the one or more prior therapies includes ibrutinib and a BCL2 inhibitor.
  • the BCL2 inhibitor is venetoclax.
  • the one or more prior therapies comprises ibrutinib and venetoclax.
  • the subject has not been previously treated with venetoclax.
  • the subject has been previously treated with venetoclax.
  • the subject is not intolerant to venetoclax at the time of initiation of administration of the cell therapy and/or at the time of initiation of administration of the BCL2 inhibitor.
  • the subject is not intolerant to venetoclax at the time of initiation of administration of the cell therapy.
  • the subject is not intolerant to venetoclax at the time of initiation of administration of the BCL2 inhibitor.
  • the subject is not intolerant to venetoclax at the time of initiation of administration of the cell therapy nor at the time of initiation of administration of the BCL2 inhibitor.
  • the subject is not intolerant to venetoclax at the time of initiation of administration of the cell therapy or at the time of initiation of administration of the BCL2 inhibitor.
  • more than six months have passed since the last dose of the previous treatment with the venetoclax if the subject’s best overall response (BOR) during or within 6 months of discontinuing the previous treatment with venetoclax was stable disease (SD) or progressive disease (PD). In some embodiments, more than six months have passed since the last dose of the previous treatment with the venetoclax if the subject’s best overall response (BOR) during or within 6 months of discontinuing the previous treatment with venetoclax was stable disease (SD). In some embodiments, more than six months have passed since the last dose of the previous treatment with the venetoclax if the subject’s best overall response (BOR) during or within 6 months of discontinuing the previous treatment with venetoclax was progressive disease (PD).
  • SD stable disease
  • PD progressive disease
  • the subject prior to administration of the cell therapy and/or prior to administration of the BCL2 inhibitor, has measurable disease in the lymph nodes and/or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, prior to administration of the cell therapy, the subject has measurable disease in the lymph nodes. In some embodiments, prior to administration of the cell therapy the subject has evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen.
  • PB peripheral blood
  • BM bone marrow
  • the subject prior to administration of the cell therapy, has measurable disease in the lymph nodes and evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, prior to administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes. In some embodiments, prior to administration of the BCL2 inhibitor, the subject has evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, prior to administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes and evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen.
  • PB peripheral blood
  • BM bone marrow
  • the subject prior to administration of the cell therapy and prior to administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes. In some embodiments, prior to administration of the cell therapy and prior to administration of the BCL2 inhibitor, the subject has evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, prior to administration of the cell therapy and prior to administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes and evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen.
  • PB peripheral blood
  • BM bone marrow
  • liver or spleen.
  • the subject has measurable disease in the lymph nodes and/or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, at the time of administration of the cell therapy, the subject has measurable disease in the lymph nodes. In some embodiments, at the time of administration of the cell therapy the subject has evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen.
  • PB peripheral blood
  • BM bone marrow
  • liver or spleen.
  • the subject has measurable disease in the lymph nodes and evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, at the time of administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes. In some embodiments, at the time of administration of the BCL2 inhibitor, the subject has evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, at the time of administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes and evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen.
  • the subject has measurable disease in the lymph nodes. In some embodiments, at the time of administration of the cell therapy and at the time of administration of the BCL2 inhibitor, the subject has evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, at the time of administration of the cell therapy and at the time of administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes and evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen.
  • the subject prior to administration of the cell therapy and/or prior to administration of the BCL2 inhibitor, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 10 4 . In some embodiments, prior to administration of the cell therapy, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 10 4 . In some embodiments, prior to administration of the BCL2 inhibitor, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 10 4 . In some embodiments, prior to administration of the cell therapy and prior to administration of the BCL2 inhibitor, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 10 4 .
  • the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 10 4 . In some embodiments, at the time of administration of the cell therapy, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 10 4 . In some embodiments, at the time of administration of the BCL2 inhibitor, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 10 4 . In some embodiments, at the time of administration of the cell therapy and prior to administration of the BCL2 inhibitor, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 10 4 .
  • the subject has Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.
  • ECG Eastern Cooperative Oncology Group
  • the dose of engineered cells has a defined ratio of CD4 + cells expressing the CAR to CD8 + cells expressing the CAR. In some embodiments, the ratio is between approximately 1:3 and approximately 3:1. In some embodiments, the dose of engineered cells has a defined ratio of CD4 + cells expressing the CAR to CD8 + cells expressing the CAR. In some embodiments, the ratio is between approximately 1:2 and approximately 2:1. In some embodiments, the dose of engineered cells has a defined ratio of CD4 + cells expressing the CAR to CD8 + cells expressing the CAR that is or is approximately 1:1.
  • the dose of engineered cells includes between about 2.5 x 10 7 total CAR-expressing cells and about 1.0 x 10 8 total CAR-expressing cells. In some embodiments, the dose of engineered cells includes at or about 2.5 x 10 7 total CAR-expressing cells. In some embodiments, the dose of engineered cells includes at or about 5 x 10 7 total cells or total CAR-expressing cells. In some embodiments, the dose of engineered cells includes at or about 7.5 x 10 7 total cells or total CAR- expressing cells. In some embodiments, the dose of engineered cells includes at or about 1 x 10 8 total cells or total CAR-expressing cells.
  • administration of the cell therapy includes administering a plurality of separate compositions, wherein the plurality of separate compositions includes a first composition of one of the CD4 + T cells and the CD8 + T cells and a second composition of the other of the CD4 + T cells and the CD8 + T cells.
  • the first composition includes the CD8 + T cells and the second composition includes the CD4 + T cells.
  • the first composition includes the CD4 + T cells and the second composition includes the CD8 + T cells.
  • the first composition and the second composition are administered 0 to 12 hours apart, 0 to 6 hours apart, or 0 to 2 hours apart. In some embodiments, the first composition and the second composition are administered 0 to 12 hours apart. In some embodiments, the first composition and the second composition are administered 0 to 6 hours apart. In some embodiments, the first composition and the second composition are administered 0 to 2 hours apart. In some embodiments, the administration of the first composition and the administration of the second composition are carried out on the same day, between about 0 and about 12 hours apart, between about 0 and about 6 hours apart, or between about 0 and 2 hours apart. In some embodiments, the administration of the first composition and the administration of the second composition are carried out on the same day.
  • the administration of the first composition and the administration of the second composition are carried out on between about 0 and about 12 hours apart. In some embodiments, the the administration of the first composition and the administration of the second composition are carried out between about 0 and about 6 hours apart. In some embodiments, the the administration of the first composition and the administration of the second composition are carried out between about 0 and 2 hours apart. In some embodiments, the initiation of administration of the first composition and the initiation of administration of the second composition are carried out between about 1 minute and about 1 hour apart, or between about 5 minutes and about 30 minutes apart. In some embodiments, the initiation of administration of the first composition and the initiation of administration of the second composition are carried out between about 1 minute and about 1 hour apart.
  • the initiation of administration of the first composition and the initiation of administration of the second composition are carried out between about 5 minutes and about 30 minutes apart.
  • the first composition and the second composition are administered no more than 2 hours, no more than 1 hour, no more than 30 minutes, no more than 15 minutes, no more than 10 minutes, or no more than 5 minutes apart.
  • the first composition and the second composition are administered no more than 2 hours apart.
  • the first composition and the second composition are administered no more than 1 hour apart.
  • the first composition and the second composition are administered no more than 30 minutes apart.
  • the first composition and the second composition are administered no more than 15 minutes apart.
  • the first composition and the second composition are administered no more than 10 minutes apart.
  • the first composition and the second composition are administered no more than 5 minutes apart.
  • the first composition is administered prior to the second composition.
  • the first composition is enriched for CD8+ cells.
  • the first composition is enriched for CD4+ cells.
  • the first composition is essentially free of CD4+ cells.
  • the first composition is essentially free of CD8+ cells.
  • the second composition is essentially free of CD4+ cells.
  • the second composition is essentially free of CD8+ cells.
  • the CAR comprised by the CD4 + T cells and the CAR comprised by the CD8 + T cells comprises a CAR that is the same and/or wherein the CD4 + T cells and the CD8 + T cells are genetically engineered to express a CAR that is the same.
  • the CAR comprised by the CD4 + T cells and the CAR comprised by the CD8 + T cells is the same.
  • the CD4 + T cells and the CD8 + T cells are genetically engineered to express a CAR that is the same.
  • the CAR contains an extracellular antigen-binding domain specific for CD19, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule.
  • the CAR contains an extracellular antigen-binding domain specific for CD19, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4- IBB, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is a CD3zeta.
  • the cytoplasmic signaling domain is derived from 4-1BB. In some embodiments, the cytoplasmic signaling domain is derived from CD3zeta. In some embodiments, the CAR has an extracellular antigen-binding domain specific for CD19, a transmembrane domain, a cytoplasmic signaling domain derived from a 4-1BB, and a cytoplasmic signaling domain derived from a CD3zeta.
  • the CAR contains, in order, an extracellular antigen-binding domain specific for CD19, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, and a cytoplasmic signaling domain derived from a primary signaling ITAM- containing molecule.
  • the antigen-binding domain is an scFv.
  • the scFv contains a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37).
  • the scFv contains a CDRH1 sequence of DYGVS (SEQ ID NO: 38), a CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and a CDRH3 sequence of YAMDYWG (SEQ ID NO: 40).
  • the scFv contains a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37), a CDRH1 sequence of DYGVS (SEQ ID NO: 38), a CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and a CDRH3 sequence of YAMDYWG (SEQ ID NO: 40).
  • the scFv contains a variable heavy chain region of FMC63 and a variable light chain region of FMC63.
  • the scFv contains a CDRL1 sequence of FMC63, a CDRL2 sequence of FMC63, a CDRL3 sequence of FMC63, a CDRH1 sequence of FMC63, a CDRH2 sequence of FMC63, and a CDRH3 sequence of FMC63.
  • the scFv binds to the same epitope as or competes for binding with any of the foregoing.
  • the scFv comprises a VH set forth in SEQ ID NO: 41 and a VL set forth in SEQ ID NO: 42.
  • the VH and VL are separated by a flexible linker.
  • the flexible linker is or contains the sequence set forth in SEQ ID NO: 59.
  • the scFv is or contains the sequence set forth in SEQ ID NO:43.
  • the costimulatory signaling domain is a signaling domain of CD28 or 4-1BB. In some embodiments, the costimulatory signaling domain is a signaling domain of 4-1BB. In some embodiments, the costimulatory signaling domain is a signaling domain of CD28. In some embodiments, the costimulatory signaling domain contains the sequence set forth in SEQ ID NO: 12 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
  • the costimulatory signaling domain contains a variant of the sequence set forth in SEQ ID NO: 12 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the costimulatory signaling domain contains the sequence set forth in SEQ ID NO:
  • the primary signaling domain is a CD3zeta signaling domain.
  • the primary signaling domain contains the sequence set forth in SEQ ID NO: 13 or 14 or 15, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
  • the primary signaling domain is a variant of the sequence set forth in SEQ ID NO: 13 or 14 or 15 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
  • the primary signaling domain is a CD3zeta signaling domain.
  • the primary signaling domain contains the sequence set forth in SEQ ID NO: 13 or 14 or 15.
  • the primary signaling domain contains the sequence set forth in SEQ ID NO:
  • the primary signaling domain contains the sequence set forth in SEQ ID NO: 13.
  • the CAR further contains a spacer between the transmembrane domain and the scFv.
  • the spacer is a polypeptide spacer that contains or consists of all or a portion of an immunoglobulin hinge or a modified version thereof.
  • the spacer is a polypeptide spacer that contains or consists of all or a portion of an IgG4 hinge, or a modified version thereof.
  • the spacer is about 15 amino acids or less.
  • the spacer does not contain a CD28 extracellular region. In some embodiments, the spacer does not contain a CD8 extracellular region.
  • the spacer does not contain a CD28 extracellular region or a CD8 extracellular region. In some embodiments, the spacer is about 15 amino acids or less and does not contain a CD28 extracellular region or a CD8 extracellular region. In some embodiments, the spacer is at or about 12 amino acids in length.
  • the spacer contains or consists of the sequence of SEQ ID NO: 1, a sequence encoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
  • the spacer contains or consists of the sequence of SEQ ID NO: 1.
  • the spacer contains the sequence of SEQ ID NO: 1.
  • the spacer consists of the sequence of SEQ ID NO: 1.
  • the spacer contains or consists of the formula X1PPX2P, where Xi is glycine, cysteine or arginine and X2 is cysteine or threonine.
  • the scFv comprises, in order, a VH, a linker, and a VL-
  • the linker contains the sequence set forth in SEQ ID NO: 59.
  • the scFv comprises, in order, a VH, a linker containing the sequence set forth in SEQ ID NO:59, and a VL-
  • the antigen binding domain contains an scFv that contains a variable heavy chain region of FMC63 and a variable light chain region of FMC63;
  • the spacer is a polypeptide spacer that contains the sequence set forth in SEQ ID NO: 1;
  • the antigen binding domain contains an scFv that contains a variable heavy chain region of FMC63 and a variable light chain region of FMC63;
  • the spacer is a polypeptide spacer that contains the sequence set forth in SEQ ID NO: 1;
  • the costimulatory domain contains the sequence set forth in SEQ ID NO: 12;
  • the primary signaling domain contains the sequence set forth in SEQ ID NO: 13.
  • the engineered cells are primary T cells obtained from a subject. In some embodiments, the engineered cells are autologous to the subject.
  • the subject is a human subject.
  • an article of manufacture containing a composition for a cell therapy, or one of a plurality of compositions for a cell therapy, comprising T cells expressing a CAR that binds CD19 and an inhibitor of BCL2 protein, and instructions for administering the cell therapy and the BCL2 inhibitor to the subject, wherein the instructions specify administering the cell therapy and the BCL2 inhibitor according to any of the methods provided herein.
  • the BCL2 inhibitor is venetoclax or navitoclax. In some embodiments, the BCL2 inhibitor is venetoclax.
  • Also provided herein is use of a cell therapy and a BCL2 inhibitor for the treatment of a subject having a CLL or a SLL. Also provided herein is use of a cell therapy and a BCL2 inhibitor for the treatment of a subject having a CLL. Also provided herein is use of a cell therapy and a BCL2 inhibitor for the treatment of a subject having a CLL. Also provided herein is a cell therapy and a BCL2 inhibitor for the treatment of a subject having a CLL or a SLL. Also provided herein is a cell therapy and a BCL2 inhibitor for the treatment of a subject having a CLL.
  • a cell therapy and a BCL2 inhibitor for the treatment of a subject having a SLL are also provided herein. Also provided herein is use of a cell therapy in the manufacture of a medicament for the treatment of a subject having a CLL or a SLL, wherein the medicament is for use with a BCL2 inhibitor. Also provided herein is use of a cell therapy in the manufacture of a medicament for the treatment of a subject having a CLL, wherein the medicament is for use with a BCL2 inhibitor. Also provided herein is use of a cell therapy in the manufacture of a medicament for the treatment of a subject having a SLL, wherein the medicament is for use with a BCL2 inhibitor.
  • a BCL2 inhibitor in the manufacture of a medicament for the treatment of a subject having a CLL or a SLL, wherein the medicament is for use with a cell therapy. Also provided herein is use of a BCL2 inhibitor in the manufacture of a medicament for the treatment of a subject having a CLL, wherein the medicament is for use with a cell therapy. Also provided herein is use of a BCL2 inhibitor in the manufacture of a medicament for the treatment of a subject having a SLL, wherein the medicament is for use with a cell therapy.
  • a cell therapy including a dose of engineered cells containing T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) and a BCL2 inhibitor for use in a method of treating a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL), where the method comprises: (1) administering to the subject the cell therapy on Day 1; and (2) administering to the subject the BCL2 inhibitor in a dosing regimen including: (i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy; and (ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared
  • a BCL2 inhibitor for use in a method of treating a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) that has been treated with a cell therapy including a dose of engineered T cells containing T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the method comprises administering the BCL2 inhibitor to the subject in dosing regimen including: (i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy, wherein the cell therapy was administered to the subject on Day 1; and (ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor
  • a cell therapy including a dose of engineered cells containing T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) for use in a method of treating a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL), wherein the method comprises administering the cell therapy to the subject on Day 1, and the subject is to be administered a BCL2 inhibitor in a dosing regimen including: (i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy; and (ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the
  • a BCL2 inhibitor in the manufacture of a medicament for treatment of a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) that has been treated with a cell therapy including a dose of engineered T cells containing T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19), wherein the medicament is to be administered to the subject in dosing regimen including: (i) daily administration of a first dose of the medicament for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy, wherein the cell therapy was administered to the subject on Day 1; and (ii) daily administration of at least one subsequent dose of the medicament, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the medicament, wherein: each of the at least one subsequent dose is an increased amount of the medicament compared to the preceding dose; and the medicament is
  • a cell therapy including a dose of engineered cells containing T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) in the manufacture of a medicament for the treatment of a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) that is to be administered a BCL2 inhibitor in a dosing regimen including: (i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the medicament, wherein the medicament is administered to the subject on Day 1; and (ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the B
  • CAR chimeric antigen receptor
  • FIG. 1A shows the cell viability of human CD19-expressing lymphoma and leukemia target cell lines co-cultured with anti-CD19 CAR T cells at increasing ratios of effector cells to target cells (E:T).
  • FIG. IB shows the size of spheroids generated from the non-Hodgkin lymphoma (NHL) RL cell line over time, co-cultured with CD19-targeting CAR T cells at E:T ratios of 0.25:1, 0.5:1 and 1: 1.
  • FIG. 2 shows the fold change in cell count of human CD19-expressing leukemia and lymphoma target cell lines co-cultured for 120 hours with CD19-targeting CAR T cells at an E:T ratio of 2.5:1.
  • FIG. 3 shows the cell viability of anti-CD19 CAR-expressing T cells treated for 96 hours with an exemplary BCL2 inhibitor.
  • FIG. 4A shows the cell count of human CD19-expressing lymphoma cell lines cultured alone, with CD19-targeting CAR T cells at an E:T ratio of 2.5:1, with an exemplary BCL2 inhibitor, or with both.
  • FIG. 4B shows the cell count of CD19-expressing Granta-519 lymphoma target cells cultured alone, with anti-CD19 CAR T cells at a suboptimal E:T ratio of 1:1, with an exemplary BCL2 inhibitor, or both.
  • FIG. 5A shows the cell count of RL cells cultured alone, with CD19-targeting CAR T cells at an E:T ratio of 1:1, with an exemplary BCL2 inhibitor, or with both.
  • FIG. SB shows the tumor volume of RL spheroids cultured alone, with CD19-targeting CAR T cells at an E:T ratio of 1:1, with an exemplary BCL2 inhibitor, or with both.
  • FIG. SC shows the tumor volume of RL spheroids cultured alone or with CD19-targeting CAR T cells for 9 days, in the presence of increasing concentrations of an exemplary BCL2 inhibitor.
  • FIG. A shows the number of RL cells cultured for 8 days alone or with anti-CD19 CAR T cells (E:T ratio of 1:1) that had been previously subjected to chronic stimulation by incubation with anti- idiotypic antibody-coated beads, in the presence or absence of an exemplary BCL2 inhibitor.
  • FIG. 6B shows the tumor volume of RL spheroids cultured for 8 days alone or with anti- CD19 CAR T cells (E:T ratio of 1:1) that had been previously subjected to chronic stimulation by incubation with anti-idiotypic antibody-coated beads, in the presence or absence of an exemplary BCL2 inhibitor.
  • FIG. 7A shows the expression, indicated by mean fluorescence intensity (MFI), of BCL2 by anti-CD19 CAR-expressing T cell compositions generated from three healthy human donors, compared to a fluorescence minus one (FMO) control.
  • MFI mean fluorescence intensity
  • FIG. 7B shows the percent of CAR+caspase 3+ cells among CD 19 CAR T cell compositions generated from three healthy human donors (each dot represents an individual donor), following chronic stimulation by incubation with anti-idiotypic antibody-coated beads and exposure to increasing concentrations of an exemplary BCL2 inhibitor.
  • FIGS. 7C and 7D show cell viability and expansion kinetics of CD19-targeting CAR T cells, respectively, following chronic stimulation by incubation with anti-idiotypic antibody-coated beads and exposure to an exemplary BCL2 inhibitor.
  • FIG. 8 shows JeKo-1 mantle cell lymphoma (MCL) target cells cultured alone, with CD19- targeting CAR T cells, with an exemplary BCL2 inhibitor, or with both.
  • MCL mantle cell lymphoma
  • FIG. 9 shows the IC50 of an exemplary BCL2 inhibitor against anti-CD19 CAR T cells in culture with 5%, 10%, or 20% serum.
  • FIGS. 10A and 10B show the tumor burden and body weight, respectively, of NOD scid gamma (NSG) mice injected with JeKo-1 MCL cells at Day -7 and treated daily with an exemplary BCL2 inhibitor from Day 0 to Day 21.
  • NSG NOD scid gamma
  • FIGS. 11A to 11C show tumor burden (FIG. 11 A) and survival in NSG mice injected wth JeKo-1 MCL cells at Day -7 and treated with an exemplary BCL2 inhibitor daily from Day 0 to Day 21 (FIG. 1 IB), anti-CD19 CAR T cells on Day -1 and Day 0 (FIG. 11C), or both (FIG. 11C).
  • FIG. 12 shows the number of CD4+CAR+ and CD8+CAR+ cells in the blood of NSG mice injected with JeKo-1 MCL cells and treated with CD19-targeting CAR T cells, in the presence or absence of an exemplary BCL2 inhibitor, on Days 7, 13, and 19.
  • FIGS. 13A and 13B show the number of CD3+CAR+ T cells and RL target cells, respectively, following a 6-day co-culture.
  • Various concentrations of an exemplary BCL2 inhibitor were provided in the co-culture at its initiation, or 24, 48, or 72 hours after the initiation of co-culture.
  • FIGS. 14A and 14B show the survival of NSG mice injected with Granta-519 cells at Day - 14 and treated with anti-CD19 CAR T cells on Day 0 and an exemplary BCL2 inhibitor at 25 mg/kg (FIG. 14A) or 100 mg/kg (FIG. 14B) daily, beginning at either Day -1 or Day 5.
  • FIG. 15 shows gene expression data of tumor biopsies from 36 DLBCL patients enrolled in a clinical trial for a CD19-targeting CAR T cell therapy.
  • a hypothetical threshold was set assuming, at 3 months after admistration of the cell therapy, 30% of subjects (11/36) would not respond and 70% of subjects (25/36) would respond. Actual responses are shown, designated as complete reponse (CR), partial response (PR), progressive disease (PD), or status unavailable (Not Available).
  • FIG. 16 shows exemplary dosing regimens for beating a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) with a combination therapy of CAR-expressing T cells and the exemplary BCL2 inhibitor venetoclax (D: Day; DC1: Dosing cohort 1; DC2: Dosing cohort 2; DC3: Dosing cohort 3; LD: lymphodepleting therapy).
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • combination therapies for treating a subject having a cancer involving administration of an immunotherapy or a cell therapy (e.g. a T cell therapy, such as CAR-T cell) for treating a cancer and an inhibitor of BCL2 protein (a BCL2 inhibitor).
  • an immunotherapy or cell therapy e.g. a T cell therapy, such as CAR-T cell
  • the immunotherapy or cell therapy includes any such therapy that specifically binds to an antigen associated with, expressed by, or present on cells of the cancer.
  • the immunotherapy or cell therapy is or involves a therapy that results in cytolytic effector-mediated killing of cancer cells, such as by target cell apoptosis (hereinafter “cytotoxic therapy”).
  • combination therapies involving administration of an immunotherapy involving T cell function or activity, such as a T-cell engaging therapy or a T cell therapy (e.g., CAR-expressing T cells), and administration of a BCL2 inhibitor.
  • the cytotoxic therapy is a T cell therapy, such as CAR-T cells.
  • the provided combination therapies and methods improve responses to the immunotherapy or cell therapy by activity of the BCL2 inhibitor to increase susceptibility of the cancer cells, such as tumor cells, to apoptosis.
  • the cancer cells are thereby rendered more sensitive to cytolytic effector-mediated killing.
  • kits that contain a composition comprising the therapy and/or a composition comprising a BCL2 inhibitor, e.g., venetoclax, and uses of such compositions and combinations to treat or prevent cancers, such as a B cell malignancy (e.g. CLL or SLL).
  • a BCL2 inhibitor e.g., venetoclax
  • Cell therapies such as T cell-based therapies, for example, adoptive T cell therapies (including those involving the administration of cells expressing chimeric receptors specific for a cancer of interest, such as chimeric antigen receptors (CARs) and/or other recombinant antigen receptors, as well as other adoptive immune cell and adoptive T cell therapies) can be effective in the treatment of diseases and disorders such as a B cell malignancies.
  • CARs chimeric antigen receptors
  • the engineered expression of recombinant receptors, such as chimeric antigen receptors (CARs) on the surface of T cells enables the redirection of T cell specificity.
  • CAR-T cells for example anti-CD19 CAR-T cells
  • have produced durable, complete responses in both leukemia and lymphoma patients (Porter et al. (2015) Sci Transl Med., 7:303ral39; Kochenderfer (2015) J. Clin. Oncol., 33: 540-9; Lee et al. (2015) Lancet, 385:517-28; Maude et al. (2014) N Engl J Med, 371:1507-17). 10112]
  • available approaches to adoptive cell therapy may not always be entirely satisfactory.
  • optimal efficacy can depend on the ability of the administered cells to recognize and bind to a target, e.g., target antigen, and to exert various effector functions, including cytotoxic killing of cancer cells and secretion of various factors such as cytokines.
  • target e.g., target antigen
  • certain cancer cells exhibit resistance to certain therapies, such as immunotherapies and cell therapies.
  • results herein demonstrate that certain cancers are resistant to CAR T cell- mediated killing while others are more sensitive.
  • the provided methods, combinations and uses provide for or achieve improved or more durable responses or efficacy as compared to alternative methods, such as alternative methods involving only the administration of the immunotherapy or cell therapy but not in combination with a BCL2 inhibitor (e.g. venetoclax).
  • the methods are advantageous by virtue of administering a BCL2 inhibitor (e.g. venetoclax) shortly after (e.g. within 1 or 2 days of) administration of an immunotherapy or a cell therapy (e.g. T cell therapy, such as CAR-T cell), thereby sensitizing the tumor and/or making the tumor less resistant to, or more susceptible to, treatment with the immunotherapy or cell therapy.
  • a BCL2 inhibitor e.g. venetoclax
  • T cell therapy such as CAR-T cell
  • the immunotherapy or cell therapy is a cell therapy (e.g. T cell therapy, such as CAR-T cell), and it is further found that the advantageous effect of sensitizing the tumor and/or making the tumor less resistant to, or more suspectible to, treatment with the cell therapy can be achieved by initiating administration of the BCL2 inhibitor (e.g. venetoclax) in a window of time after initiation of administration of the cell therapy to minimize or avoid a detrimental effect of the inhibitor on cells of the cell therapy.
  • the BCL2 inhibitor e.g. venetoclax
  • a BCL2 inhibitor e.g. venetoclax
  • AICD activation induced cell death
  • delaying administration of the BCL2 inhibitor e.g. venetoclax
  • a time after AICD is at or has reached its peak, or has decreased after having peaked can avoid detrimental effects on the cells of the cell therapy while substantially improving, e.g. synergistically increasing, T cell-mediated killing of the tumor by cells of the cell therapy (e.g. CAR-T cells).
  • the immunotherapy or cell therapy is a cell therapy (e.g. T cell therapy, such as CAR-T cell), and it is further found that the advantageous effect of sensitizing the tumor and/or making the tumor less resistant to, or more suspectible to, treatment with the cell therapy can be achieved by initiating administration of the BCL2 inhibitor (e.g. venetoclax) in a window of time shortly after initiation of administration of the cell therapy, such as at a time before symptoms of cytokine release syndrome (CRS) have begun or peaked.
  • the BCL2 inhibitor e.g. venetoclax
  • administering the BCL2 inhibitor e.g. venetoclax
  • initiation of administration of the cell therapy e.g.
  • 1-2 days after, such as 1 day after may avoid exacerbating CRS symptoms and/or may improve clinical outcomes.
  • initiating administration of the BCL2 inhibitor (e.g. venetoclax) in a window of time shortly after initiation of administration of the cell therapy (e.g. 1-2 days after, such as 1 day after) minimizes the amount of time between a bridging therapy with a BCL2 inhibitor (e.g. venetoclax) and administration of the BCL2 inhibitor dosing regimen (venetoclax), and may thereby improve clinical outcomes.
  • the BCL2 inhibitor e.g. venetoclax
  • the provided methods are based on observations that certain cancers that are resistant to CAR T cell-mediated killing exhibit high expression of the prosurvival tumor suppressor p53 and other genes involved in prosurvival cell cycle pathways. It is found herein that the presence of a BCL2 inhibitor, e.g. venetoclax, improves T cell-mediated killing of cancer cells by a T cell therapy (e.g. CAR T cells), particularly among cancers that exhibit resistance following exposure to the T cell therapy (e.g. CAR T cells) alone. Such results were observed with low doses of the BCL2 inhibitor, which did not exhibit activity against the tumor when administered alone. These results evidence that administration of a BCL2 inhibitor, including at subtherapeutic doses, may improve responses to certain effector-mediated immunotherapies, such as T cell engagers or T cell therapies.
  • a BCL2 inhibitor e.g. venetoclax
  • a BCL2 inhibitor e.g. venetoclax
  • a T cell therapy e.g. CAR T cells
  • cancers that exhibit resistance to T cell therapy-mediated cell death e.g. CAR T cells
  • the cytotoxic effects of a BCL2 inhibitor, e.g. venetoclax, and a T cell therapy may be synergistic to result in cell death of cells otherwise resistant to treatment with the T cell therapy alone.
  • administration of the inhibitor may sensitize otherwise resistant cancer cells to CAR T cell-mediated killing when it is administered within or at about 1-2 days after administration of the cell therapy.
  • the inhibitor e.g. venetoclax
  • tumor cells were relatively resistant to cell death mediated by CAR T cells.
  • a therapeutic dose of venetoclax given concurrently or after 24 hours reduced CAR-T cell numbers and activity
  • a therapeutic dose of venetoclax administered 48 or 72 hours after initiation of co-culture of tumor cells and CAR T cells was able to sensitize the tumor cells to CAR T cell-mediated killing, without deleterious effects on the CAR T cells being observed.
  • Venetoclax (ABT-199) is a small molecule inhibitor (SMI) that blocks the activity of the B- cell lymphoma 2 (BCL2) protein.
  • SMI small molecule inhibitor
  • Venetoclax is approved for use (1) in chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL); and (2) in combination with azacitidine, decitabine, or low-dose cytarabine for newly diagnosed acute myeloid leukemia (AML) in adult who are at least 75 years of age, or who have comorbidities that preclude use of intensive induction chemotherapy.
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • AML acute myeloid leukemia
  • Navitoclax (ABT-263), another prosurvival BCL2 family protein inhibitor, is a SMI that blocks the activity of the BCL2, B-cell lymphoma extra-large (BCLXL), and BCL2-like protein 2 (BCLW) proteins.
  • BCL2 inhibitors include, but are not limited to ABT-737, AT-101/GDC-0199 (Gossypol), apogossypol, TW-37, G3139 (Genasense), GX15-070 (obatoclax), sabutoclax, HA14-1, antimycin A, BH3I-1, YC137, maritoclax, clitocine, UMI-77, WEHI-539, and 544563.
  • BCL2 family proteins include both pro-apoptotic and anti-apoptotic proteins, and the balance of signaling between these two groups of proteins can determine whether a cell is more sensitized to or more resistant to apoptosis.
  • the overexpression of one or more prosurvival BCL2 family proteins can result in increased anti-apoptotic signaling and resistance to cell death.
  • overexpression of BCL2 may be caused by a (14;18)(q32;q21) translocation.
  • overexpression of BCL2 may be caused by amplification of the gene encoding the BCL2 protein.
  • Resistance to cell death may, in some cases, occur when the signaling of prosurvival (anti-apoptotic) BCL2 proteins (e.g. BCL2, BCLXL, BCLB, BCLW, BFL1, MCL1) outweighs the signaling of pro-apoptotic BCL2 proteins (e.g. BAX, BAK, BIG, BIM, NOXA, PUMA), such as when one or more prosurvival BCL2 proteins are overexpressed.
  • prosurvival BCL2 family proteins can support and increase cancer cell survival.
  • increased expression of prosurvival BCL2 family proteins blocks pro-apoptotic BCL2 family proteins and inhibits a cancer cell’s intrinsic (mitochondrial) apoptotic pathway.
  • the methods involve combination therapy of a therapy that targets or is directed to killing of cells of a cancer, e.g. an immunotherapy or cell therapy, such as a CAR T cell therapy, and an inhibitor of BCL2 protein.
  • the inhibitor inhibits activity of a BCL2 family protein that is a prosurvival (anti-apoptotic) BCL2 family protein such as BCL2, B-cell lymphoma extra-large (BCLXL), BCL2 related protein Al (BFL1), BCL2-like protein 2 (BCLW), BCL2-like protein 10 (BCLB), induced myeloid leukemia cell differentiation protein (MCL1), or combinations thereof.
  • the cancer is one in which the prosurvival BCL2 family protein is overexpressed.
  • the inhibitor does not inhibit or reduce the activity of a BCL2 family protein that is a proapoptopic BCL2 family protein such as BCL2 associated X (BAX), BCL2 antagonist/killer 1 (BAK), DIVA, BCLXS, BCL2 interacting killer (BIK), BCL2-like protein 11 (BIM), BCL2 associated agonist of cell death (BAD), or combinations thereof.
  • the cancer is one in which the proapoptopic BCL2 family protein is underexpressed or its activity is inhibited.
  • overexpression of a prosurvival BCL2 family protein is implicated in a number of cancers, including bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, head and neck cancers, lung cancer, ovarian cancer, pancreatic cancer, renal cancer, skin cancer, and hematologic malignancies, such as leukemias and lymphomas (see e.g., WO 2005/049593 and W02005/024636).
  • overexpression or aberrant expression of one or more prosurvival BCL2 family proteins is a mechanism underlying leukemias, lymphomas, and solid tumors, whereby overexpression dampens pro-apoptotic signaling to promote the survival of cancer cells (see e.g., Campbell, K.J. and Tait, S.W.G. (2016) Open Biol., 8:180002).
  • existing methods of employing BCL2 inhibitors e.g. venetoclax
  • venetoclax involve use of the inhibitors as therapeutics for treating a variety of cancers.
  • venetoclax is indicated for treatment of certain cancers, such as B cell lymphomas, with a dose of 400 to 800 milligrams per day after a ramp-up period and for a duration of time that can extend for months to years.
  • BCL2 prosurvival BCL2 family proteins
  • CAR-expressing T cells therapy may undergo activation-induced cell death (AICD).
  • AICD activation-induced cell death
  • CAR T cells may be prone to upregulation of expression of Fas, FasL, DR5, and TRAIL, such as upon excessive T cell stimulation, thereby resulting in programmed cell death.
  • BCL2 inhibitor e.g. venetoclax
  • an immunotherapy or a cell therapy, including a T cell therapy such as a CAR-T cell therapy is advantageous, even at therapeutic doses of the inhibitor capable of exerting deleterious effects on CAR T cells.
  • the inhibitor e.g. venetoclax
  • the methods are advantageous by virtue of administering a BCL2 inhibitor at a therapeutic dose subsequent to administration of a T cell therapy (e.g.
  • the provided methods and uses provide for or achieve improved or more durable responses or efficacy as compared to certain alternative methods.
  • the observations herein indicate that the combination of a therapy, e.g. an immunotherapy or a cell therapy, including a T cell therapy such as a CAR-T cell therapy is advantageous, even at subtherapeutic or lower doses of the inhibitor.
  • a therapy e.g. an immunotherapy or a cell therapy, including a T cell therapy such as a CAR-T cell therapy
  • the results herein also show that certain doses of the inhibitor do not impact viability of T cells.
  • the methods are advantageous by virtue of administering a BCL2 inhibitor at a subtherapeutic dose, such as a dose in which the inhibitor alone would not be expected to or would not reduce tumor burden in the subject.
  • the provided methods and uses provide for or achieve improved or more durable responses or efficacy as compared to certain alternative methods.
  • the provided methods enhance or modulate the cytotoxic activity, such as via perforin- and/or granzyme-mediated apoptosis, toward one or more cells of a cancer of T cells against cancer cells, such as associated with administration of a T cell engaging therapy or a T cell therapy (e.g. CAR-expressing T cells).
  • a BCL2 inhibitor e.g. venetoclax
  • combination therapy of the inhibitor in methods involving T cells such as involving administration of adoptive T cell therapy, achieves improved function of the T cell therapy.
  • combination of the cell therapy e.g., administration of engineered T cells such as CAR T cells
  • the BCL2 inhibitor improves or enhances one or more functions and/or effects of the T cell therapy, such as cytotoxicity and/or therapeutic outcomes, e.g., ability to kill or reduce the burden of tumor or other disease or target cell.
  • such effects are observed despite that the tumor or disease or target cell itself is insensitive, resistant and/or otherwise not sufficiently responsive to the therapy, e.g. immunotherapy or cell therapy, such as immunotherapy including T cell engaging therapy or T cell therapy (e.g. CAR T cells), or to the dose of the inhibitor when each is administered alone.
  • the cancer is insensitive to or has become resistant to treatment with a therapy for treating the cancer that is directed to or targets killing of the cancer, e.g. immunotherapy or cell therapy, including a T cell engaging therapy or a T cell therapy (e.g. CAR T cell therapy).
  • the cancer is insensitive to or has become resistant to such therapies by virtue of the cells of the cancer suppressing pro-apoptotic signaling.
  • the cancer is insensitive to or has become resistant to CAR T cells targeting the cancer-associated antigen, e.g. CD19.
  • the provided combination therapy achieves synergistic effects and activity compared to a therapy involving only administration of the therapy, e.g. immunotherapy or cell therapy, or of the BCL2 inhibitor given at the same dosing regimen, e.g. dose and frequency.
  • the provided methods, uses and combination therapies include administration of a BCL2 inhibitor, in combination with a therapy for treating the cancer that is directed to or targets killing of the cancer, e.g. immunotherapy or cell therapy, such as an immunotherapy, including a T cell engaging therapy or a T cell therapy (e.g. CAR T cell therapy) in a subject that has already been administered the BCL2 inhibitor or another prosurvival BCL2 family protein inhibitor.
  • a therapy for treating the cancer e.g. immunotherapy or cell therapy, such as an immunotherapy, including a T cell engaging therapy or a T cell therapy (e.g. CAR T cell therapy) in a subject that has already been administered the BCL2 inhibitor or another prosurvival BCL2 family protein inhibitor.
  • the combination therapy, methods and uses include continued administration of the BCL2 inhibitor, e.g., venetoclax, in combination with a T cell therapy (e.g.
  • the combination therapy, methods, and uses include or involve administration of a lower dose of the BCL2 inhibitor, e.g., venetoclax, than the previous treatment.
  • the methods and combinations result in improvements in T cell- mediated cytotoxicity against cancer cells. In some embodiments, the methods and combinations result in improvements in T cell-mediated cytotoxicity against cancer cells, optionally by increasing perforin- and/or granzyme-mediated apoptosis. In some embodiments, the methods and combinations result in improvements in T cell-mediated cytotoxicity against cancer cells by increasing perforin-mediated apoptosis. In some embodiments, the methods and combinations result in improvements in T cell- mediated cytotoxicity against cancer cells by increasing granzyme-mediated apoptosis.
  • the combination with the BCL2 inhibitor while improving the cytotoxicity of the T cells, does not reduce the ability of the cells to become activated, secrete one or more desired cytokines, expand and/or persist, e.g., as measured in an in vitro assay as compared to such cells cultured under conditions otherwise the same but in the absence of the inhibitor.
  • the provided embodiments involve initiating the administration of a BCL2 inhibitor, e.g., venetoclax, after administration of an immunotherapy or a cell therapy (e.g. CAR T cell therapy) in a dosing regimen.
  • a BCL2 inhibitor e.g., venetoclax
  • the initiation of the administration of a BCL2 inhibitor, e.g., venetoclax is after administration of the immunotherapy or cell therapy, such as between about 1 day after and about 2 days after (e.g. 1 day after) administration of the therapy for treating the cancer.
  • the initiation of the administration of a BCL2 inhibitor is not until activation-induced cell death (AICD) of the cells of the immunotherapy or cell therapy has peaked.
  • the initiation of the administration of a BCL2 inhibitor e.g., venetoclax
  • administration of a prosurvival BCL2 family protein inhibitor, such as a BCL2 inhibitor, e.g., venetoclax is not until about 1 day after administration of the immunotherapy or cell therapy.
  • administration of a prosurvival BCL2 family protein inhibitor is not until about 1 day after administration of the cytotoxic therapy.
  • administration of the immunotherapy or cell therapy e.g. CAR T cell therapy
  • administration of the BCL2 inhibitor begins on Day 2.
  • the method includes administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising daily administration of a first dose for a first predetermined period and daily administration of at least one subsequent dose for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor.
  • each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor (e.g. venetoclax) compared to the preceding dose.
  • the at least one subsequent dose comprises a second dose, a third dose, a fourth dose, a fifth dose, a sixth dose, a seventh dose, an eighth dose, a ninth dose, and/or a tenth dose.
  • the at least one subsequent dose comprises a second dose.
  • the at least one subsequent dose comprises a second dose and a third dose.
  • the at least one subsequent dose comprises a second, third, and fourth dose, n some embodiments, the at least one subsequent dose comprises a second, third, fourth, and fifth dose.
  • administration of the subsequent dose may begin immediately after the predetermined period of the preceding dose.
  • the subsequent dose e.g. the second dose
  • administration of the BCL2 inhibitor may be paused for one or more days between one or more predetermined periods of any given dose.
  • the method includes administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising (i) daily administration of a first dose for a first predetermined period and (ii) daily administration of a second dose for a second predetermined period beginning after the first predetermined period, wherein the second dose is an increased amount of the BCL2 inhibitor compared to the first dose.
  • a BCL2 inhibitor e.g. venetoclax
  • the method includes administering to the subject a BCL2 (e.g. venetoclax) inhibitor in a dosing regimen comprising (i) daily administration of a first dose for a first predetermined period, (ii) daily administration of a second dose for a second predetermined period beginning after the first predetermined period, and (iii) daily administration of a third dose for a third predetermined period beginning after the second predetermined period, wherein the second dose is an increased amount of the BCL2 inhibitor compared to the first dose and the third dose is an increased amount of the inhibitor compared to the third dose.
  • a BCL2 e.g. venetoclax
  • the BCL2 inhibitor e.g. venetoclax
  • the BCL2 inhibitor is provided at no more than or no more than about 100 mg per day through Day 30.
  • providing the BCL2 inhibitor (e.g. venetoclax) at no more than or no more than about 100 mg per day through Day 30 can minimize neutropenia and/or thrombocytopenia in the subject.
  • the method includes administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising (i) daily administration of a first dose for a first predetermined period and (ii) daily administration of a second dose for a second predetermined period beginning after the first predetermined period, wherein the second dose comprises an increased amount of the BCL2 inhibitor compared to the first dose and the BCL2 inhibitor is provided at no more than or no more than about 100 mg per day through Day 30.
  • a BCL2 inhibitor e.g. venetoclax
  • the method includes administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising (i) daily administration of a first dose for a first predetermined period, (ii) daily administration of a second dose for a second predetermined period beginning after the first predetermined period, and (iii) daily administration of a third dose for a third predetermined period beginning after the second predetermined period, wherein the second dose comprises an increased amount of the BCL2 inhibitor compared to the first dose, the third dose comprises an increased amount of the BCL2 inhibitor compared to the second dose, and the BCL2 inhibitor is provided at no more than or no more than about 100 mg per day through Day 30.
  • a BCL2 inhibitor e.g. venetoclax
  • the first predetermined period is from about Day 2 to about Day 7. In some embodiments, the first predetermined period is from about Day 2 to about Day 30. In some embodiments, the first dose is about 20 mg, about 50 mg, or about 100 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 20 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 100 mg of the BCL2 inhibitor.
  • the first predetermined period is from about Day 2 to about Day 7, and the first dose is about 20 mg of the BCL2 inhibitor. In some embodiments, the first predetermined period is from about Day 2 to about Day 7, and the first dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the first predetermined period is from about Day 2 to about Day 7, and the first dose is about 100 mg of the BCL2 inhibitor.
  • the first dose predetermined period is from about Day 2 to about Day 30, and the first dose is about 20 mg of the BCL2 inhibitor. In some embodiments, the first predetermined period is from about Day 2 to about Day 30, and the first dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the first predetermined period is from about Day 2 to about Day 30, and the first dose is about 100 mg of the BCL2 inhibitor.
  • the second predetermined period is from about Day 8 to about Day 30. In some embodiments, the second predetermined period is from about Day 31 to Day 37. In some embodiments, the second predetermined period is from about Day 31 to Day 90.
  • the second dose is about 50 mg, about 100 mg, or about 200 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 200 mg of the BCL2 inhibitor.
  • the second predetermined period is from about Day 8 to about Day 30, and the second dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 8 to about Day 30, and the second dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 8 to about Day 30, and the second dose is about 200 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 to Day 37, and the second dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 to Day 37, and the second dose is about 100 mg of the BCL2 inhibitor.
  • the second predetermined period is from about Day 31 to Day 37, and the second dose is about 200 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 to Day 90, and the second dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 to Day 90, and the second dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 and Day 90, and the second dose is about 200 mg of the BCL2 inhibitor.
  • administration of a first dose for a first predetermined period comprises daily administration of about 20 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 7 ; and administration of a second dose for a second predetermined period comprises daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 8 to about Day 30.
  • administration of a first dose for a first predetermined period comprises daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 7; and administration of a second dose for a second predetermined period comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 8 to about Day 30.
  • administration of a first dose for a first predetermined period comprises daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 30; and administration of a second dose for a second predetermined period comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 90.
  • administration of a first dose for a first predetermined period comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 30; and administration of a second dose for a second predetermined period comprises daily administration of about 200 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 90.
  • administration of a first dose for a first predetermined period comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 30; and administration of a second dose for a second predetermined period comprises daily administration of about 200 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 37.
  • administration of a third dose for a third predetermined period comprises daily administration of the BCL2 inhibitor to the subject from about Day 31 to Day 90. In some embodiments, administration of a third dose for a third predetermined period comprises daily administration of the BCL2 inhibitor to the subject from about Day 38 to Day 90. In some embodiments, the third dose is about 100 mg, about 200 mg, or about 400 mg of the BCL inhibitor. In some embodiments, the third dose is about 100 mg of the BCL inhibitor. In some embodiments, the third dose is about 200 mg of the BCL inhibitor. In some embodiments, the third dose is about 400 mg of the BCL inhibitor.
  • the third predetermined period is from about Day 31 to Day 90, and the third dose is about 100 mg of the BCL inhibitor. In some embodiments, the third predetermined period is from about Day 31 to Day 90, and the third dose is about 200 mg of the BCL inhibitor. In some embodiments, the third predetermined period is from about Day 38 to Day 90, and the third dose is about 400 mg of the BCL inhibitor.
  • administration of a first dose for a first predetermined period comprises daily administration of about 20 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 7 ; administration of a second dose for a second predetermined period comprises daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 8 to about Day 30; and administration of a third dose for a third predetermined period comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 90.
  • administration of a first dose for a first predetermined period comprises daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 2 toabout Day 7; administration of a second dose for a second predetermined period comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 8 to about Day 30; and administration of a third dose for a third predetermined period comprises daily administration of about 200 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 90.
  • administration of a first dose for a first predetermined period comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 30; administration of a second dose for a second predetermined period comprises daily administration of about 200 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 37; and administration of a third dose for a third predetermined period comprises daily administration of about 400 mg of the BCL2 inhibitor to the subject from about Day 38 to about Day 90.
  • the combination therapy involves administration of the inhibitor for up to or or about 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more (e.g. 24 months) after the subject has received administration of the therapy, e.g. CAR T cell therapy.
  • the combination therapy involves administration of the inhibitor up to or or about 3 months after the subject has received administration of the therapy, e.g. CAR T cell therapy.
  • the combination therapy involves administration of the inhibitor up to or or about 6 months after the subject has received administration of the therapy, e.g. CAR T cell therapy.
  • the combination therapy involves administration of the inhibitor up to or or about 9 months after the subject has received administration of the therapy, e.g. CAR T cell therapy. In some cases, the combination therapy involves administration of the inhibitor up to or or about 12 months after the subject has received administration of the therapy, e.g. CAR T cell therapy. In some cases, the combination therapy involves administration of the inhibitor up to or or about 15 months after the subject has received administration of the therapy, e.g. CAR T cell therapy. In some cases, the combination therapy involves administration of the inhibitor up to or or about 18 months after the subject has received administration of the therapy, e.g. CAR T cell therapy.
  • the combination therapy involves administration of the inhibitor up to or or about 21 months after the subject has received administration of the therapy, e.g. CAR T cell therapy. In some cases, the combination therapy involves administration of the inhibitor up to or or about 24 months after the subject has received administration of the therapy, e.g. CAR T cell therapy.
  • the combination therapy involves administration of the inhibitor up to or or about 12 months after the subject has received administration of the therapy, e.g. CAR T cell therapy.
  • the combination therapy involves administration of the inhibitor up to or or about 12 months after the subject has received administration of the therapy, e.g. CAR T cell therapy.
  • the combination therapy involves administration of the inhibitor up to or or about 18 months after the subject has received administration of the therapy, e.g. CAR T cell therapy.
  • the combination therapy involves administration of the inhibitor up to or or about 18 months after the subject has received administration of the therapy, e.g. CAR T cell therapy.
  • the combination therapy involves administration of the inhibitor up to or or about 24 months after the subject has received administration of the therapy, e.g. CAR T cell therapy.
  • the combination therapy involves administration of the inhibitor up to or or about 24 months after the subject has received administration of the therapy, e.g. CAR T cell therapy.
  • a BCL2 inhibitor e.g., venetoclax
  • a BCL2 inhibitor is administered for no more than three months after administration of the therapy for treating the cancer that is directed to or targets killing of the cancer, e.g. immunotherapy or cell therapy, such as an immunotherapy, including a T cell engaging therapy or a T cell therapy (e.g. CAR T cell therapy).
  • inhibitor is administered in combination with a T cell therapy (e.g. CAR T cells) and administration of the inhibitor is continued in a dosing regimen that includes continued administration of the inhibitor until a time after which the cells of the T cell therapy have reached peak levels in the subject and/or are persisting in the subject.
  • advantages of the provided embodiments also include the ability to modulate the dosing or administration of the inhibitor, e.g., venetoclax, or removing or discontinuing the administration of the inhibitor, e.g., venetoclax, depending on the tolerability in the subject.
  • the provided embodiments e.g., involving combination treatment with a BCL2 inhibitor, e.g., venetoclax, can help reduce tumor burden and/or mitigate cancer cell resistance to certain therapies, such as cell therapies, e.g. CAR T cell therapy.
  • the provided methods can potentiate CAR-T cell therapy, which, in some aspects, can improve outcomes for treatment of subjects that have a cancer that is resistant or refractory to other therapies, is an aggressive or high-risk cancer, and/or that is or is likely to exhibit a relatively lower response rate to a CAR-T cell therapy when administered without the inhibitor.
  • administering a BCL2 inhibitor, e.g., venetoclax according to the provided methods could increase the activity of CAR-expressing cells for treating a cancer, e.g. B cell malignancy such as CLL or NHL, e.g.
  • DLBCL or SLL by increasing T cell cytotoxicity by reducing the resistance of cancer cells to the CAR T cell therapy, optionally by increasing perforin- and/or granzyme-mediated apoptosis of the cancer cells.
  • anti-tumor activity of administered CAR+ T cells against human lymphoma cell is improved.
  • anti-tumor activity of administered CAR+ T cells against CLL cells is improved.
  • anti-tumor activity of administered CAR+ T cells against SLL cells is improved.
  • a proliferative disease e.g. a cancer
  • a combination therapy of (1) an inhibitor of BCL2 protein e.g. venetoclax
  • an immunotherapy or a cell therapy e.g. a T cell therapy (e.g. CAR-expressing T cells).
  • the immunotherapy or cell therapy such as an adoptive immune cell therapy comprising T cells (e.g. CAR-expressing T cells) specifically recognizes and/or binds to an antigen associated with, expressed by or present on cells of the cancer.
  • kits that contain a composition comprising the T cell therapy and/or a composition comprising the BCL2 inhibitor, and uses of such compositions and combinations to treat or prevent conditions or diseases such as cancers, including hematologic malignancies (e.g. CLL or SLL).
  • methods can include administration of the BCL2 inhibitor (e.g. venetoclax) subsequently to the administration (e.g., 1 or 2 days after initiation of the administration) of the immunotherapy or cell therapy, wherein the therapy specifically binds to an antigen associated with, expressed by or present on cells of the cancer (e.g. CLL or SLL).
  • the BCL2 inhibitor e.g. venetoclax
  • the therapy specifically binds to an antigen associated with, expressed by or present on cells of the cancer (e.g. CLL or SLL).
  • the methods include administering the BCL2 inhibitor in a dosing regimen comprising (i) daily admistration of a first dose for a first predetermined period and (ii) daily administration of at least one subsequent dose for a predetermined period beginning after a preceding predetermined period, wherein each of the at least one dose is a greater amount of the BCL2 inhibitor compared to the preceding dose.
  • a dosing regimen comprising (i) daily admistration of a first dose for a first predetermined period and (ii) daily administration of at least one subsequent dose for a predetermined period beginning after a preceding predetermined period, wherein each of the at least one dose is a greater amount of the BCL2 inhibitor compared to the preceding dose.
  • no more than 100 mg per day of the BCL2 inhibitor is provided though Day 30, where the immunotherapy or cell therapy (e.g. CAR T cells) is administered on Day 1.
  • methods can include administration of the BCL2 inhibitor (e.g. venetoclax) about 1 day after initiation of the administration of the immunotherapy or cell (e.g. CAR T cells), wherein the therapy specifically binds to an antigen associated with, expressed by or present on cells of the cancer (e.g.
  • the BCL2 inhibitor e.g. venetoclax
  • the therapy specifically binds to an antigen associated with, expressed by or present on cells of the cancer
  • the inhibitor is administered in a dosing regimen comprising (i) daily admistration of a first dose for a first predetermined period and (ii) daily administration of at least one subsequent dose for a predetermined period beginning after a preceding predetermined period, wherein each of the at least one dose is a greater amount of the BCL2 inhibitor compared to the preceding dose and no more than 100 mg per day of the BCL2 inhibitor is provided through Day 30, where the immunotherapy or cell therapy (e.g. CAR T cells) is administered on Day 1.
  • a dosing regimen comprising (i) daily admistration of a first dose for a first predetermined period and (ii) daily administration of at least one subsequent dose for a predetermined period beginning after a preceding predetermined period, wherein each of the at least one dose is a greater amount of the BCL2 inhibitor compared to the preceding dose and no more than 100 mg per day of the BCL2 inhibitor is provided through Day 30, where the immunotherapy or cell therapy (e.g. CAR T cells) is administered on Day
  • the BCL2 inhibitor is administered to the subject as a single agent therapy (e.g. monotherapy) in combination with an immunotherapy or a cell therapy (e.g. CAR T celsl).
  • administration as a monotherapy consists of a single type of treatment alone, to treat a disease or condition, except where otherwise provided.
  • an inhibitor of a prosurvival BCL2 family protein is provided as a monotherapy with an immunotherapy or a cell therapy, such that no other treatment is provided to treat a disease or condition beyond provision of (1) the BCL2 inhibitor and (2) the immunotherapy or the cell therapy.
  • the subject is administered a BCL2 inhibitor (e.g. venetoclax) as a bridging therapy, prior to administration of the immunotherapy or cell therapy (e.g. CAR T cells).
  • the bridging therapy comprises administration a BCL2 inhibitor (e.g. venetoclax) at 20 mg per day for a first week, at 50 mg per day for a second week, and at 100 mg per day for a third week.
  • the bridging therapy further comprises administration of an anti-CD20 antibody.
  • the bridging therapy further comprises administration of an anti-CD20 antibody if the subject has been previously treated with a BCL2 inhibitor (e.g. venetoclax).
  • the bridging therapy does not further comprise administration of the BCL2 inhibitor.
  • the bridging therapy further comprises administration of a BTK inhibitor (e.g. ibrutinib.)
  • the bridging therapy further comprises administration of the BTK inhibitor (e.g. ibrutinib) if the subject was receiving treatment with ibrutinib prior to treatment with the combination therapy.
  • the subject is administered a BTK inhibitor (e.g. ibrutinib) until autologous cells are collected from the subject and again during the bridging therapy.
  • the immunotherapy or cell therapy is adoptive cell therapy.
  • the cell therapy is or comprises a transgenic TCR therapy or a recombinant-receptor expressing cell therapy (optionally T cell therapy), which optionally is a chimeric antigen receptor (CAR)-expressing cell therapy.
  • the therapy targets CD19 or is a B cell targeted therapy.
  • the cytotoxic therapy is a cell therapy comprising T cells expressing a chimeric antigen-receptor (CAR), wherein the antigen-binding domain of the CAR binds to CD 19.
  • the cells and dosage regimens for administering the cells can include any as described in the following Subsection A under “Administration of an Immunotherapy or Cell Therapy.”
  • the immunotherapy or cell therapy is capable of mediating and/or inducing a cell’s intrinsic, mitochondrial-mediated apoptotic pathway. In some embodiments, the immunotherapy or cell therapy is capable of mediating and/or inducing a cell’s perforin- and or granzyme-mediated apoptotic pathway. In some embodiments, the cancer cells are resistant to the instrinsic apoptotic pathways. In some embodiments, the BCL2 inhibitor sensitizes cells to apoptosis via the intrinsic apoptotic pathways. In some embodiments, the BCL2 inhibitor sensitizes cells to apoptosis, as mediated or induced by the immunotherapy or cell therapy. In some ways, the BCL2 inhibitor lowers the apoptotic resistance of cells to the immunotherapy or cell therapy.
  • the immunotherapy or cell therapy is an adoptive cell therapy (e.g. a T cell therapy).
  • the adoptive cell therapy comprises cells that are autologous to the subject.
  • the cells that are autologous to the subject are engineered to express a chimeric antigen receptor (CAR).
  • CAR-expressing autologous T cells are provided to the subject.
  • the immunotherapy or cell therapy such as a T cell therapy e.g. CAR-expressing T cells) or a T cell-engaging therapy, and the BCL2 inhibitor are provided as pharmaceutical compositions for administration to the subject.
  • the pharmaceutical compositions contain therapeutically effective amounts of one or both of the agents for combination therapy, e.g., T cells for adoptive cell therapy and a BCL2 inhibitor as described.
  • the pharmaceutical compositions contain therapeutically effective amounts of a BCL2 inhibitor.
  • the pharmaceutical compositions contain subtherapeutically effective amounts of one or both of the agents for combination therapy, e.g., T cells for adoptive cell therapy and a BCL2 inhibitor as described.
  • the pharmaceutical compositions contain subtherapeutically effective amounts of a BCL2 inhibitor.
  • the agents are formulated for administration in separate pharmaceutical compositions.
  • any of the pharmaceutical compositions provided herein can be formulated in dosage forms appropriate for each route of administration.
  • the combination therapy which includes administering the immunotherapy or cell therapy (e.g. T cell therapy, including engineered cells, such as CAR-T cell therapy) and the BCL2 inhibitor, is administered to a subject or patient having a cancer (e.g. CLL or SLL) or at risk for cancer.
  • the methods treat, e.g., ameliorate one or more symptom of, the disease or condition, such as by lessening tumor burden in a cancer expressing an antigen recognized by the immunotherapy or cell therapy, e.g. recognized by an engineered T cell.
  • the disease or condition that is treated can be any in which expression of an antigen is associated with and/or involved in the etiology of a disease condition or disorder such as a cancer, e.g. causes, exacerbates or otherwise is involved in such disease, condition, or disorder.
  • a disease condition or disorder such as a cancer
  • exemplary diseases and conditions can include diseases or conditions associated with malignancy or transformation of cells e.g. cancer).
  • exemplary antigens which include antigens associated with various diseases and conditions that can be treated, include any of antigens described herein.
  • the recombinant receptor expressed on engineered cells of a combination therapy including a chimeric antigen receptor or transgenic TCR, specifically binds to an antigen associated with the cancer.
  • the antigen associated with the disease or disorder such as cancer is selected from the group consisting of ROR1, B cell maturation antigen (BCMA), tEGFR, Her2, LI- CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, erbB dimers, EGFR vIII, FBP, FCRL5, FCRH5, fetal acethycholine e receptor, GD2, GD3, G Protein Coupled Receptor 5D (GPCR5D), HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, LI -cell adhesion molecule, (LI -CAM), Melanoma-associated antigen (MAGE), MAGE, MAGE, M
  • the disease or condition is a cancer or proliferative disease.
  • the disease or condition is a cancer.
  • the disease or condition is a proliferative disease.
  • the cancer or proliferative disease is a tumor, such as a solid tumor, lymphoma, leukemia, blood tumor, metastatic tumor, or other cancer or tumor type.
  • the cancer is a hematologic malignancy.
  • the cancer is a B cell malignancy.
  • the cancer is a leukemia or a lymphoma.
  • the cancer is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), or a subtype of NHL, such as diffuse large B-cell lymphoma (DLBCL).
  • the cancer is a leukemia.
  • the cancer is chronic lymphocytic leukemia (CLL).
  • the cancer is a lymphoma.
  • the cancer is small lymphocytic lymphoma (SLL).
  • the Eastern Cooperative Oncology Group (ECOG) performance status indicator can be used to assess or select subjects for treatment, e.g., subjects who have had poor performance from prior therapies (see, e.g., Oken et al. (1982) Am J Clin Oncol. 5:649-655).
  • the ECOG Scale of Performance Status describes a patient’s level of functioning in terms of their ability to care for themselves, daily activity, and physical ability (e.g., walking, working, etc.).
  • an ECOG performance status of 0 indicates that a subject can perform normal activity.
  • subjects with an ECOG performance status of 1 exhibit some restriction in physical activity but the subject is fully ambulatory.
  • patients with an ECOG performance status of 2 is more than 50% ambulatory.
  • the subject with an ECOG performance status of 2 may also be capable of selfcare; see e.g., Sprensen et al., (1993) Br J Cancer 67(4) 773-775.
  • the criteria reflective of the ECOG performance status are described in Table 1 below:
  • Antigens targeted by the receptors include antigens associated with a B cell malignancy, such as any of a number of known B cell marker.
  • the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.
  • the antigen is CD19.
  • the subject has CLL. In some embodiments of the provided methods, the subject has SLL. In some embodiments of the provided methods, the subject has a high risk CLL or SLL. In some embodiments of the provided methods, the subjects have a high risk CLL or SLL. In some embodiments of the provided methods, the subjects have a high risk CLL. In some embodiments of the provided methods, the subject has a high risk CLL. In some embodiments of the provided methods, the subjects have a high risk SLL. In some embodiments of the provided methods, the subject has a high risk SLL. In some embodiments of the provided methods, the subject has a high risk SLL. In some embodiments of the provided methods, the subject has a high risk SLL.
  • the subjects are a heavily pretreated population of subjects with high-risk CLL (or SLL).
  • the subject has received one or more prior therapies including ibrutinib.
  • the subject has received one or more prior therapies including venetoclax.
  • the subject has received one or more prior therapies including ibrutinib and venetoclax.
  • subjects with CLL include those with CLL diagnosis with indication of treatment based on the International Workshop on Chronic Lymphocytic Leukemia (iwCLL) guidelines and clinical measurable disease (bone marrow involvement by > 30% lymphocytes, peripheral blood lymphocytosis > 5xl0 9 /L, and/or measurable lymph nodes and/or hepatic or splenomegaly.
  • iwCLL Chronic Lymphocytic Leukemia
  • subjects with SLL include those with SLL diagnosis is based on lymphadenopathy and/or splenomegaly and ⁇ 5xl0 9 CD19+ CD5+ clonal B lymphocytes/L [ ⁇ 5000/pL] in the peripheral blood at diagnosis with measurable disease defined as at least one lesion > 1.5 cm in the greatest transverse diameter, and that is biopsy-proven SLL.
  • subjects with SLL include those with SLL diagnosis is based on lymphadenopathy and/or splenomegaly and ⁇ 5xl0 9 CD19+ CD5+ clonal B lymphocytes/L [ ⁇ 5000/pL] in the peripheral blood at diagnosis with measurable disease defined as at least one lesion > 1.5 cm in the greatest transverse diameter, and that is biopsy-proven SLL.
  • existing treatment strategies for high risk and very high risk subjects may include fludarabine, cyclophosphamide, and rituximab (FCR), Bruton’s tyrosine kinase (BTK) inhibitors (e.g. ibrutinib), and/or allogeneic stem cell transplantation.
  • FCR fludarabine
  • cyclophosphamide cyclophosphamide
  • rituximab FCR
  • BTK tyrosine kinase
  • ibrutinib allogeneic stem cell transplantation.
  • Many of the existing therapies include oral- targeted drugs, which have, for some patients with CLL, improved treatment outcomes. Nonetheless, some patients prove intolerant or resistant to therapy and/or fail to achieve complete response with undetectable MRD (uMRD).
  • subjects who have progressive disease after treatment with available therapies have poor outcomes.
  • subjects treated for CLL exhibit poor long-term outcomes.
  • refractory (R/R) high-risk CLL subjects exhibit poor survival after ibrutinib discontinuation (Jain et al. (2015) Blood 125(13):2062-2067).
  • Chronic lymphocytic leukemia is a generally a variable disease. Some subjects with CLL may survive without treatment while others may require immediate intervention.
  • subjects with CLL may be classified into groups that may inform disease prognosis and/or recommended treatment strategy. In some cases, these groups may be “low risk,” “intermediate risk,” “high risk,” and/or “very high risk” and patients may be classified as such depending on a number of factors including, but not limited to, genetic abnormalities and/or morphological or physical characteristics.
  • subjects treated in accord with the method are classified or identified based on the risk of CLL. In some embodiments, the subject is one that has high risk CLL. In some embodiments, the subject is one that has very high risk CLL.
  • the provided methods and uses provide for or achieve improved or more durable responses or efficacy as compared to certain alternative methods, such as in particular groups of subjects treated, such as in patients with a leukemia, such as CLL or SLL, including those with high-risk disease.
  • the methods are advantageous by virtue of administering T cell therapy, such as a composition including cells for adoptive cell therapy, e.g., such as a CAR-expressing T cells, e.g. anti-CD19 CAR+ T cells, and a BCL2 inhibitor (e.g. venetoclax).
  • the methods also include, prior to administration of the T cell therapy, a lymphodepleting therapy, e.g.
  • the BCL2 inhibitor (e.g. venetoclax) bridging therapy is provided prior to administration of the lymphodepleting therapy. In some cases, the BCL2 inhibitor (e.g. venetoclax) bridging therapy is discontinued at about or at least about 24 hours before administration of the lymphodepleting therapy. In some cases, the BCL2 inhibitor (e.g. venetoclax) bridging therapy is discontinued about 24 hours before administration of the lymphodepleting therapy. In some cases, the BCL2 inhibitor (e.g. venetoclax) bridging therapy is discontinued at least about 24 hours before administration of the lymphodepleting therapy. In some cases, the interval between discontinuation of the BCL2 inhibitor bridging therapy and administration of the lymphodepleting therapy is known as the “washout” period. In some cases, the washout period is about 1 day (24 hours).
  • the treated subjects include subjects that have relapsed following initial remission on ibrutinib or who are refractory or intolerant to treatment with ibrutinib. In some embodiments, the subject has relapsed following initial remission on ibrutinib. In some embodiments, the subject is refractory or intolerant to treatment with ibrutinib. In particular embodiments, the treated subjects include subjects that have relapsed following remission or are refractory or intolerant to one or more further prior therapy in addition to ibrutinib, such as 1, 2, 3, 4, 5 or more prior therapies.
  • the subjects have relapsed or are refractory to both a prior treatment of ibrutinib and venetoclax. In some embodiments, the subject has relapsed or is refractory to both ibrutinib and venetoclax. In some embodiments, subjects that are refractory to such treatment have progressed following one or more prior therapy. In some embodiments, subjects treated, including those treated with one or more prior therapies (e.g. ibrutinib and/or venetoclax) include those with a high-risk cytogenetics, including TP53 mutation, complex karyotype (i.e. at least three chromosomal alterations) and dell7(p).
  • prior therapies e.g. ibrutinib and/or venetoclax
  • the subject has CLL or is suspected of having CLL.
  • the subject is identified or selected as having CLL.
  • the CLL is relapsed or refractory CLL.
  • CLL is generally considered to be incurable and patients often eventually relapse or become refractory to available therapies (Dighiero and Hamblin (2008) The Lancet, 371:1017-1029).
  • the subject has SLL or is suspected of having SLL. In some embodiments, the subject is identified or selected as having SLL. In some embodiments, the SLL is a relapsed or refractory SLL.
  • the subject prior to the administration of the dose of engineered T cells, the subject has been treated with one or more prior therapies for the CLL or SLL, other than the therapy, e.g. dose of cells expressing CAR, or a lymphodepleting therapy.
  • the subject prior to the administration of the dose of engineered T cells, the subject has been treated with one or more prior therapies for the CLL , other than the therapy, e.g. dose of cells expressing CAR, or a lymphodepleting therapy.
  • prior to the administration of the dose of engineered T cells the subject has been treated with one or more prior therapies for the SLL, other than the therapy, e.g.
  • the one or more prior therapy comprises at least two prior therapies, optionally three, four, five, six, seven, eight, nine or more.
  • the one or more prior therapy comprises at least two prior therapies.
  • the one or more prior therapy comprises at least three prior therapies.
  • the one or more prior therapy comprises at least two four prior therapies.
  • the one or more prior therapy comprises at least two five therapies.
  • prior to the administration of the immunotherapy or cell therapy e.g. CAR T cells
  • the subject has been treated with two or more prior therapies for the SLL, other than the therapy, e.g.
  • the subject prior to the administration of the immunotherapy or cell therapy (e.g. CAR T cells), the subject has failed two or more prior therapies for the SLL, other than the therapy, e.g. dose of cells expressing CAR, or a lymphodepleting therapy.
  • the subject at or immediately prior to the time of the administration of the dose of cells, the subject has relapsed following remission after treatment with, or become refractory to, failed and/or was intolerant to treatment with the one or more prior therapies for the CLL.
  • the subject has relapsed following remission after treatment with the one or more prior therapies for the CLL. In some embodiments, at or immediately prior to the time of the administration of the dose of cells, the subject has become refractory to, has failed and/or was intolerant to treatment with the one or more prior therapies for the CLL. In some embodiments, the subject has failed two or more prior therapies. In some embodiments, the subject has relapsed following remission after treatment with, or become refractory to, failed and/or was intolerant to treatment with two or more prior therapies.
  • the subject has relapsed following remission after treatment with, or become refractory to, failed and/or was intolerant to treatment with three or more prior therapies.
  • the prior therapies are selected from a kinase inhibitor, optionally an inhibitor of Bruton’s tyrosine kinase (BTK), optionally ibrutinib; venetoclax; a combination therapy comprising fludarabine and rituximab; radiation therapy; and hematopoietic stem cell transplantation (HSCT).
  • the prior therapies comprise ibrutinib and/or venetoclax.
  • the prior therapies comprise ibrutinib and venetoclax. In some embodiments, the prior therapy comprises ibrutinib. In some embodiments, the prior therapy comprises venetoclax. In some embodiments, the subject has failed two or more prior therapies, including BTK inhibition (e.g. ibrutinib). In some embodiments, the subject has failed two or more prior therapies, including BTK inhibition (e.g. ibrutinib) and venetoclax. In some embodiments, the subject has failed two or more prior therapies, including BTK inhibition (e.g. ibrutinib) and another prior therapy that is not venetoclax.
  • BTK inhibition e.g. ibrutinib
  • venetoclax e.g. ibrutinib
  • the subject has failed two or more prior therapies, including BTK inhibition (e.g. ibrutinib) and another prior therapy that is not venetoclax.
  • the subject has relapsed following remission after treatment with, become refractory to failed treatment with and/or is intolerant to ibrutinib and/or venetoclax. In some embodiments, the subject has relapsed following remission after treatment with, become refractory to failed treatment with and/or is intolerant to ibrutinib. In some embodiments, the subject has relapsed following remission after treatment with, become refractory to failed treatment with and/or is intolerant to venetoclax. In some embodiments, the subject has relapsed following remission after treatment with, become refractory to, failed treatment with and/or is intolerant to ibrutinib and venetoclax.
  • a subject that has been previously treated with a BCL2 inhibitor (e.g. venetoclax) prior to the provided methods is not intolerant to venetoclax (e.g. at the time of administration of the cell therapy).
  • a subject has been treated with a BCL2 inhibitor (e.g. venetoclax) within 6 months prior to the provided methods, e.g. prior to receiving a lymphodepleting therapy or prior to receiving administration of a cytotoxic therapy.
  • a subject has been treated with a BCL2 inhibitor (e.g. venetoclax) within 6 months prior to the provided methods and does not have progressive disease (PD).
  • PD progressive disease
  • the subject did not exhibit progressive disease during the previous treatment with the BCL2 inhibitor.
  • a subject has been treated with a BCL2 inhibitor (e.g. venetoclax) within 6 months prior to the provided methods and did not exhibit progressive disease (PD) during the treatment with the inhibitor.
  • PD progressive disease
  • the previous treatment with the BCL2 inhibitor e.g. venetoclax
  • a Best Overall Result BOR
  • SBOR Best Overall Result
  • the previous treatment with the BCL2 inhibitor e.g. venetoclax
  • the previous treatment with the BCL2 inhibitor resulted in a Best Overall Result (BOR) of stable disease (SD) or progressive disease within six months of discontinuing the previous treatment, more than 6 months have passed since the last dose of the previous treatment.
  • the subject is not intolerant to the BCL2 inhibitor (e.g. venetoclax) at a time immediately following collection of autologous cells from the subject and/or immediately before administration of a lymphodepleting therapy to the subject.
  • the subject is not intolerant to the BCL2 inhibitor (e.g. venetoclax) at the time of initiation of administration of the immunotherapy or cell therapy.
  • the subject was previously treated with the cell therapy (e.g. CAR T cells) and achieved a complete response.
  • the subject previously achieved a complete response to the cell therapy (e.g. CAR T cells) and subsequently progressed, including progression by Richter’s transformation.
  • Richter’s transformation is characterized by the sudden transformation of a CLL or SLL into a more aggressive form of large cell lymphoma.
  • the subject was previously treated with a CD19-targeted therapy and has CD19-positive disease.
  • the CD19-positive disease is confirmed by immunohistochemistry or flow cytometry for CD 19 expression since completion of the previous CD 19- targeted therapy.
  • a subject is screened for expression of BCL2 family proteins. In some embodiments, a subject is screened for expression of BCL2 family proteins prior to administration of a lymphodepleting therapy to the subject. In some embodiments, a subject that has been previously treated with a BCL2 inhibitor, e.g. venetoclax, is screened for BCL2 mutations. In some embodiments, a subject that has been previously treated with a BCL2 inhibitor, e.g. venetoclax, is screened for BCL2 mutations prior to administration of a lymphodepleting therapy to the subject. In some embodiments, a subject that has been previously treated with a BCL2 inhibitor, e.g. venetoclax, is screened for BCL2 mutations prior to administration of the cell therapy to the subject.
  • a BCL2 inhibitor e.g. venetoclax
  • the subject does not have a mutation in BCL2 at a time immediately following collection of autologous cells from the subject and/or immediately before administration of a lymphodepleting therapy to the subject. In some embodiments, the subject does not have a mutation in BCL2 at the time of initiation of administration of the immunotherapy or cell therapy. In some embodiments, the subject does not have a mutation in BCL2. In some embodiments, the subject does not have a mutation in BCL2 or a BCL2 family protein. In some embodiments, the subject has a mutation in BCL2 or a BCL2 family protein at a time immediately following collection of autologous cells from the subject and/or immediately before administration of a lymphodepleting therapy to the subject.
  • the subject has a mutation in BCL2 or a BCL2 family protein at a time immediately following collection of autologous cells from the subject. In some embodiments, the subject has a mutation in BCL2 or a BCL2 family protein at a time immediately before administration of a lymphodepleting therapy to the subject. In some embodiments, the subject has a mutation in BCL2 or a BCL2 family protein at the time of initiation of administration of the immunotherapy or cell therapy (e.g. CAR T cells). In some embodiments, the subject has a mutation in BCL2 or a BCL2 family protein. In some embodiments, a subject is not excluded from receiving treatment with the combination therapy on the basis of BCL2 mutation status.
  • the subject exhibits measurable disease or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen at a time immediately following collection of autologous cells from the subject and/or immediately before administration of a lymphodepleting therapy to the subject.
  • the subject exhibits measurable disease or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen.
  • the subject at the time of initiation of the immunotherapy or cell therapy (e.g. CAR T cells), the subject exhibits measurable disease or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen.
  • the measurable disease is or includes lymph nodes of greater than 1.0 centimeters in greatest transverse diameter (GTD). In some embodiments, the measurable disease is or includes lymph nodes of greater than 1.5 centimeters in greatest transverse diameter (GTD). In some embodiments, the measurable disease is or includes lymph nodes of greater than 2.0 centimeters in greatest transverse diameter (GTD). In some embodiments, a subject exhibits measureable disease (e.g. lymph nodes of greater than 1.5 centimeters in greatest transverse diameter (GTD)) or evaluable or measurable disease in PB, BM, liver or spleen, and evidence of disease in the blood by local testing (e.g. minimum residual disease (MRD) in peripheral blood of greater than or equal to 10 4 ).
  • MRD minimum residual disease
  • the subject exhibits minimum residual disease (MRD) in peripheral blood of greater than or equal to 10 4 .
  • the subject exhibits minimum residual disease (MRD) in peripheral blood of greater than or equal to 10 4 at a time immediately following collection of autologous cells from the subject and/or immediately before administration of a lymphodepleting therapy to the subject.
  • the subject exhibits minimum residual disease (MRD) in peripheral blood of greater than or equal to 10 4 at a time immediately following collection of autologous cells from the subject.
  • the subject exhibits minimum residual disease (MRD) in peripheral blood of greater than or equal to 10 4 at a time immediately before administration of a lymphodepleting therapy to the subject.
  • the subject at the time of initiation of the immunotherapy or cell therapy (e.g. CAR T cells), the subject exhibits minimum residual disease (MRD) in peripheral blood of greater than or equal to 10 4 .
  • the subject exhibits evidence of CLL in blood by local testing. In some embodiments, the subject exhibits evidence of CLL in blood by local testing at a time immediately following collection of autologous cells from the subject and/or immediately before administration of a lymphodepleting therapy to the subject. In some embodiments, the subject exhibits evidence of CLL in blood by local testing at a time immediately following collection of autologous cells from the subject. In some embodiments, the subject exhibits evidence of CLL in blood by local testing at a time immediately before administration of a lymphodepleting therapy to the subject. In some embodiments, at the time of initiation of the immunotherapy or cell therapy (e.g. CAR T cells), the subject exhibits evidence of CLL in blood by local testing.
  • the immunotherapy or cell therapy e.g. CAR T cells
  • the subject exhibits measurable disease or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen and minimum residual disease (MRD) in peripheral blood of greater than or equal to 10 4 .
  • the subject exhibits measurable disease or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen and minimum residual disease (MRD) in peripheral blood of greater than or equal to 10 4 , at a time immediately following collection of autologous cells from the subject and/or immediately before administration of a lymphodepleting therapy to the subject.
  • the subject exhibits measurable disease or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen and minimum residual disease (MRD) in peripheral blood of greater than or equal to 10 4 .
  • the measurable disease is or includes lymph nodes of greater than 1.0 centimeters in greatest transverse diameter (GTD). In some embodiments, the measurable disease is or includes lymph nodes of greater than 1.5 centimeters in greatest transverse diameter (GTD). In some embodiments, the measurable disease is or includes lymph nodes of greater than 2.0 centimeters in greatest transverse diameter (GTD).
  • the subject is or has been identified as having one or more cytogenetic abnormalities, optionally associated with high-risk CLL, optionally selected from among: complex karyotype or cytogenetic abnormalities, del 17p, unmutated IGVH gene, and TP53 mutation; the subject is or has been identified as having high-risk CLL.
  • the subject is or has been identified as having one or more cytogenetic abnormalities.
  • the one or more cytogenetic abnormalities are associated with high-risk CLL.
  • the one or more cytogenetic abnormalities selected from among complex karyotype or cytogenetic abnormalities, del 17p, unmutated IGVH gene, and TP53 mutation.
  • the subject is or has been identified as having high-risk CLL.
  • the subject is or has been identified as having an ECOG status of 0 or 1; and/or the subject does not have an ECOG status of >1. In some embodiments, the subject is or has been identified as having an ECOG status of 0 or 1. In some embodiments, the subject is or has been identified as having an ECOG status of 0. In some embodiments, the subject is or has been identified as having an ECOG status of 1. In some embodiments, at or immediately prior to the administration of the dose of engineered cells or the lymphodepleting therapy the subject does not have a Richter’s transformation of the CLL or SLL.
  • the methods involve treating a subject that has an Eastern Cooperative Oncology Group Performance Status (ECOG) of 0-1 or 0-2.
  • subjects have Eastern Cooperative Oncology Group (ECOG) scores of between 0 and 2.
  • subjects are not excluded based on an ECOG score of 2.
  • the subject is or has been identified as having an ECOG status of 2.
  • the methods treat a poor-prognosis population or of DLBCL patients or subject thereof that generally responds poorly to therapies or particular reference therapies, such as one having one or more, such as two or three, chromosomal translocations (such as so-called “double -hit” or “triple-hit” lymphoma; having translocations MYC/8q24 loci, usually in combination with the t(14; 18) (q32; q21) BCL-2 gene or/and BCL6/3q27 chromosomal translocation; see, e.g., Xu et al. (2013) Int J Clin Exp Pathol. 6(4): 788-794), and/or one having relapsed, such as relapsed within 12 months, following administration of an autologous stem cell transplant (ASCT), and/or one having been deemed chemorefractory.
  • ASCT autologous stem cell transplant
  • the subject has measurable disease before initiation of a lymphodepleting therapy, such as is described in Section I.D.
  • the subject must have adequate organ function before initiation of a lymphodepleting therapy, such as is described in Section I.D. In some embodiments, the subject has adequate organ function prior to administration of the cell therapy, the BCL-2 inhibitor, or both.
  • adequate organ function is defined as (i) serum creatinine ⁇ 1.5 x age-adjusted upper limit of normal (ULN) or calculated creatinine clearance of > 30 mL/min; (ii) alanine aminotransferase (ALT) ⁇ 5 x ULN and total bilirubin ⁇ 2.0 mg/dL (or ⁇ 3.0 mg/dL for subjects with Gilbert’s syndrome or leukemic infiltration of the liver); (iii) adequate pulmonary function; and (iv) adequate cardiac function.
  • adequate pulmonary function is defined as ⁇ Common Terminology Criteria for Adverse Events (CTCAE) Grade 1 dyspnea and saturated oxygen (SaO2) > 92% on room air.
  • CCAE Common Terminology Criteria for Adverse Events
  • SaO2 saturated oxygen
  • adequate cardiac function is defined as left ventricular ejection fraction (LVEF) > 40% as assessed by echocardiogram (ECHO) or multiple uptake gated acquisition (MUGA) scan performed within 30 days prior to determination of eligibility.
  • LVEF left ventricular ejection fraction
  • ECHO echocardiogram
  • MUGA multiple uptake gated acquisition
  • the subject has hemoglobin > 9 g/dL, absolute neutrophil count (ANC) > 500/mm3, and platelets > 75,000/mm 3 , unless the subject has cytopenias due to CLL infiltration of the bone marrow.
  • the subject does not have active central nervous system (CNS) involvement by malignancy.
  • the subject has not been previously treated with any gene therapy product.
  • the subject has been previously treated with a therapy or a therapeutic agent targeting the disease or condition (e.g. CLL or SLL) prior to administration of the therapy, e.g. cells expressing the recombinant receptor.
  • a therapy or a therapeutic agent targeting the disease or condition e.g. CLL or SLL
  • the subject has been previously treated with a hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT or autogeneic HSCT.
  • HSCT hematopoietic stem cell transplantation
  • the subject has had poor prognosis after treatment with standard therapy and/or has failed one or more lines of previous therapy.
  • the subject has been treated or has previously received at least or at least about or about 1, 2, 3, or 4 other therapies for treating the disease or disorder (e.g.
  • CLL or SLL other than a lymphodepleting therapy and/or the therapy, e.g. dose of cells expressing the antigen receptor.
  • the subject has been treated or has previously received a therapy that includes anthracycline, a CD20 targeted agent, and/or ibrutinib.
  • the subject has been previously treated with chemotherapy or radiation therapy.
  • the subject is refractory or non-responsive to the other therapy or therapeutic agent.
  • the subject has persistent or relapsed disease, e.g., following treatment with another therapy or therapeutic intervention, including chemotherapy or radiation.
  • the subject is one that is eligible for a transplant, such as is eligible for a hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT.
  • HSCT hematopoietic stem cell transplantation
  • the subject has not previously received a transplant, despite being eligible, prior to administration of the therapy, such as cell therapy containing engineered cells (e.g. CAR-T cells) or a composition containing the cells to the subject as provided herein.
  • the subject has previously received an allogeneic stem cell transplantation (SCT).
  • SCT allogeneic stem cell transplantation
  • subjects are not excluded based on prior allogeneic stem cell transplantation (SCT).
  • the subject is one that is not eligible for a transplant, such as is not eligible for a hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT.
  • HSCT hematopoietic stem cell transplantation
  • subjects are not required to have a minimum absolute lymphocyte count (ALC) for apheresis.
  • ALC absolute lymphocyte count
  • the subject is an adult. In some embodiments, the subject is 18 years of age or older.
  • the method includes administration of cells to a subject selected or identified as having chronic lymphocytic leukemia (CLL).
  • CLL chronic lymphocytic leukemia
  • the methods include administration of cells to a subject selected or identified as having small lymphocytic lymphoma (SLL).
  • SLL small lymphocytic lymphoma
  • the subject exhibits one or more cytogenetic abnormalities, such as associated with the CLL or SLL, such as a high-risk CLL or a high-risk SLL.
  • the subject is selected or identified based on having a disease or condition characterized or determined to be aggressive CLL or SLL.
  • the subject has poor performance status.
  • the population to be treated includes subjects having an Eastern Cooperative Oncology Group Performance Status (ECOG) that is anywhere from 0-2.
  • ECOG Eastern Cooperative Oncology Group Performance Status
  • the subjects to be treated included ECOG 0-1 or do not include ECOG 2 subjects.
  • the subjects to be treated have failed two or more prior therapies.
  • the subject has features that correlate with poor overall survival.
  • the subject has never achieved a complete response (CR), never received autologous stem cell transplant (ASCT), is refractory to 1 or more second line therapy, has primary refractory disease, and/or has an ECOG performance score of 2 or an ECOG score of between 0 and 1.
  • CR complete response
  • ASCT autologous stem cell transplant
  • the subject to be treated includes a group of subjects with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after failure of 2 or more lines of therapy.
  • the subject may have previously been treated with allogeneic stem cell transplantation (SCT).
  • SCT allogeneic stem cell transplantation
  • the subject is not selected for treatment or excluded from treatment, if the subject has a poor performance status (e.g. ECOG 2).
  • the subject is selected for treatment if the subject has chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after failure of 2 lines of therapy, and ECOG score of 0 or 1.
  • the cancer is characterized by overexpression or aberrant expression of one or more prosurvival BCL2 family proteins.
  • the cancer is characterized by overexpression or aberrant expression of BCL2.
  • overexpression of BCL2 may be caused by the (14; 18)(q32;q21) translocation.
  • overexpression of BCL2 may be caused by amplification of the gene encoding the BCL2 protein.
  • the cancer is characterized by overexpression or aberrant expression of BCLXL, MCL1, and/or BFL1.
  • the cancer is characterized by a mutation in one or more genes encoding for a prosurvival BCL2 family protein.
  • the cancer is characterized by a mutation in the gene encoding the BCL2 protein. In some cases, the mutation is (14; 18)(q32;q21) translocation. In some embodiments, the cancer is resistant to treatment with an immunotherapy or cell therapy. In some embodiments, the cancer is resistant to treatment with a cell therapy, such as a CAR-expressing T cell therapy. In some embodiments, the cancer is resistant to treatment with a CD19-targeting CAR T cell therapy. In some embodiments, the BCL2 inhibitor sensitizes a cancer to treatment with an immunotherapy or cell therapy. In some embodiments, the BCL2 inhibitor sensitizes a cancer to treatment with a cell therapy, such as a CAR-expressing T cell therapy. In some embodiments, the BCL2 inhibitor sensitizes a cancer to treatment with a CD19-targeting CAR T cell therapy.
  • the combination therapy provided herein is carried out in a subject that has been previously treated with a BCL2 inhibitor, such as venetoclax, but in the absence of administration of a T cell therapy (e.g. CAR+ T cells) or T cell-engaging therapy.
  • a T cell therapy e.g. CAR+ T cells
  • T cell-engaging therapy e.g. CAR+ T cells
  • the subject is refractory to and/or develops resistance to, has relapsed following remission, has not achieved a CR after receiving such previous treatment for at least 6 months and/or exhibits an aggressive disease and/or high-risk features of the cancer.
  • the provided combination therapy can be carried out in a subject that has previously received administration of a BCL2 inhibitor, such as venetoclax.
  • timing of administration of an inhibitor in the present disclosure refers to timing of its administration relative to the immunotherapy or immunotherapeutic agent, e.g. T cell therapy (e.g. CAR+ T cells) or T cell-engaging therapy, in accord with the provided combination therapy methods and does not exclude the possibility that the subject has additionally previously been administered an inhibitor of a BCL2 inhibitor, such as venetoclax.
  • T cell therapy e.g. CAR+ T cells
  • T cell-engaging therapy e.g. T cell-engaging therapy
  • the appropriate dosage of a BCL2 inhibitor and/or immunotherapy such as a T cell therapy e.g. CAR-expressing T cells) or a T cell-engaging therapy, may depend on the type of disease to be treated, the particular inhibitor, cells and/or recombinant receptors expressed on the cells, the severity and course of the disease, route of administration, whether the BCL2 inhibitor and/or the immunotherapy, e.g., T cell therapy, are administered for preventive or therapeutic purposes, previous therapy, frequency of administration, the subject’s clinical history and response to the cells, and the discretion of the attending physician.
  • the compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments. Exemplary dosage regimens and schedules for the provided combination therapy are described.
  • the immunotherapy e.g. T cell therapy, and the BCL2 inhibitor are administered as part of a further combination treatment, which can be administered simultaneously with or sequentially to, in any order, another therapeutic intervention.
  • the immunotherapy e.g. engineered T cells, such as CAR-expressing T cells
  • the cells are administered prior to the one or more additional therapeutic agents.
  • the immunotherapy e.g. engineered T cells, such as CAR-expressing T cells, are administered after the one or more additional therapeutic agents.
  • the combination therapy methods further include a lymphodepleting therapy, such as administration of a chemotherapeutic agent.
  • the combination therapy further comprises administering another therapeutic agent, such as an anti-cancer agent, a checkpoint inhibitor, or another immune modulating agent.
  • Uses include uses of the combination therapies in such methods and treatments, and uses of such compositions in the preparation of a medicament in order to carry out such combination therapy methods.
  • the methods and uses thereby treat the disease or condition or disorder, such as a cancer or proliferative disease, in the subject.
  • the immunotherapy, e.g. T cell therapy, and the BCL2 inhibitor are administered without any other combination treatment.
  • the immunotherapy, e.g. T cell therapy, and the BCL2 inhibitor are administered without any other combination treatment, such as ibrutinib and/or rituximab.
  • the biological activity of the immunotherapy e.g. the biological activity of the engineered cell populations
  • the biological activity of the immunotherapy is measured, e.g., by any of a number of known methods. Parameters to assess include the ability of the engineered cells to destroy target cells, persistence and other measures of T cell activity, such as measured using any suitable method known in the art, such as assays described further below in Section III below.
  • the biological activity of the cells is measured by assaying cytotoxic cell killing, expression and/or secretion of one or more cytokines, proliferation or expansion, such as upon restimulation with antigen.
  • the biological activity is measured by assessing the disease burden and/or clinical outcome, such as reduction in tumor burden or load.
  • the biological activity is measured by assessing the presence of neutropenia in a subject.
  • administration of one or both agents of the combination therapy and/or any repeated administration of the therapy can be determined based on the results of the assays before, during, during the course of or after administration of one or both agents of the combination therapy.
  • the combined effect of the BCL2 inhibitor in combination with the cell therapy can be synergistic compared to treatments involving only the inhibitor or monotherapy with the cell therapy.
  • the combined effect of a subtherapeutically effective amount of the BCL2 inhibitor in combination with the cell therapy can be synergistic compared to treatments involving only the inhibitor or monotherapy with the cell therapy.
  • the combined effect of a subtherapeutically effective amount of the BC12 inhibitor in combination with a subtherapeutically effective amount of the cell therapy can be synergistic compared to treatments involving only the inhibitor or monotherapy with the cell therapy.
  • the provided methods, compositions and articles of manufacture herein result in an increase or an improvement in a desired therapeutic effect, such as an increased or an improvement in the reduction or inhibition of one or more symptoms associated with cancer.
  • the BCL2 inhibitor increases the expansion, proliferation, or cytotoxicity of the engineered T cells, such as CAR T cells.
  • the increase in expansion, proliferation, or cytotoxicity is observed in vivo following administration of the BCL2 inhibitor to a subject.
  • the increase in the number of engineered T cells e.g. CAR- T cells, is increased by greater than or greater than about 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 10.0 fold or more.
  • the increase in the cytotoxicity of the engineered T cells e.g.
  • CAR-T cells, against cancer cells is increased by greater than or greater than about 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0- fold, 9.0-fold, 10.0 fold or more.
  • the provided combination therapy methods, combinations, kits and uses involve administration of an inhibitor of BCL2 protein (a BCL2 inhibitor, e.g. venetoclax), which can be administered subsequently to administration of the immunotherapy or cell therapy, e.g., CAR T cells.
  • a BCL2 inhibitor e.g. venetoclax
  • the BCL2 protein that has the ability to promote survival and/or to mitigate pro- apoptotic signaling.
  • the BCL2 protein is anti-apoptotic.
  • the BCL2 protein has the ability to promote the survival of cancer cells.
  • the BCL2 protein has the ability to dampen pro-apoptotic signaling of cancer cells.
  • the BCL2 inhibitor in the combination therapy is an inhibitor of a BCL2 (e.g. venetoclax), which, in some cases, are involved in the regulation and promulgation of anti- apoptotic (prosurvival) signaling in a cell, such as via a cell’s intrinsic apoptotic pathway.
  • a BCL2 e.g. venetoclax
  • prosurvival BCL2 family proteins e.g. BCL2
  • prosurvival BCL2 family proteins are involved in apoptotic signaling, including antiapoptotic (prosurvival) signaling via a cell’ s intrinsic apoptosis pathway, via mitochondria.
  • prosurvival BCL2 family proteins e.g.
  • BCL2 are involved in antiapoptotic (prosurvival) signaling via granzyme and/or perforin-mediated apoptosis in the mitochondria.
  • the BCL2 inhibitor e.g. venetoclax
  • the BCL2 inhibitor also inhibits one or more other prosurvival proteins of the BCL2 family, including B-cell lymphoma extra-large (BCLXL), BCL2 related protein Al (BFL1), BCL2-like protein 2 (BCLW), BCL2-like protein 10 (BCLB), and induced myeloid leukemia cell differentiation protein (MCL1).
  • BCLXL B-cell lymphoma extra-large
  • BFL1 BCL2 related protein Al
  • BCLW BCL2-like protein 2
  • BCLB BCL2-like protein 10
  • MCL1 induced myeloid leukemia cell differentiation protein
  • the BCL2 inhibitor interacts with a BCL2 homology (BH) domain.
  • the BCL2 inhibitor is a mimetic of BH3.
  • the BCL2 inhibitor is a BH3 mimetic that occupies a BH3-binding domain.
  • the BCL2 inhibitor is a BH3 mimetic that occupies a BH3 binding domain and/or displaces pro-apoptotic BH3-only proteins from BCL2.
  • the BCL2 inhibitor blocks or reduces the interaction between BCL2 and proteins having a BH3 domain.
  • the BCL2 inhibitor occupies a BH3 binding domain to block or reduce heterodimerization of a prosurvival BCL2 family protein, such as BCL2 or BCLXL, with a pro-apoptotic BCL2 family protein, such as BAD, BAX, or BAK.
  • a prosurvival BCL2 family protein such as BCL2 or BCLXL
  • a pro-apoptotic BCL2 family protein such as BAD, BAX, or BAK.
  • the BCL2 inhibitor reduces or blocks the phosphorylation of BCL2.
  • the BCL2 inhibitor reduces prosurvival (antiapoptotic) signaling.
  • the reduction of prosurvival signaling lowers the apoptotic threshold of a cell.
  • the apoptosis is achieved by the cell’s intrinsic, mitochondrial- mediated apoptosis pathway.
  • the reduction of prosurvival signaling and/or lowering of the apoptotic threshold sensitizes a cell to cell death and/ or increases cell death.
  • the reduction of prosurvival signaling and/or lowering of the apoptotic threshold sensitizes a cell to cell death by one or more other agents and/ or increases cell death by one or more other agents. In some cases, the reduction of prosurvival signaling and/or lowering of the apoptotic threshold is achieved by inducing BAX and/or BAK-dependent apoptosis. In some cases, the reduction of prosurvival signaling and/or lowering of the apoptotic threshold sensitizes a cell to cell death by a cytotoxic therapy, such as an immunotherapy or cell therapy (e.g. CAR-expressing T cell therapy).
  • a cytotoxic therapy such as an immunotherapy or cell therapy (e.g. CAR-expressing T cell therapy).
  • the reduction of prosurvival signaling and/or lowering of the apoptotic threshold sensitizes a cell to cell death pathways targeted CAR T cells, including perforin and granzyme-mediated pathways (Benmabarek et al. (2019) Inti. J. Mol. Sci. (20): 1283).
  • the BCL2 inhibitor (e.g. venetoclax) is a selective BCL2 inhibitor.
  • a selective BCL2 inhibitor is a compound or agent, such as an inhibitor of a prosurvival BCL2 family protein, that is capable of being provided at a dosing regimen (e.g. dose and/or duration) that reduces or blocks BCL2 activity and/or signaling to a greater extent than that of other prosurvival BCL2 family proteins (e.g. BCLXL, BCLW, BCLB, MCL1).
  • a selective BCL2 inhibitor reduces or blocks the activity of BCL2 signaling and/or activity when provided at a dosing regimen, but does not reduce or block the signaling and/or activity of other prosurvival BCL2 family proteins when provided at the same dosing regimen. In some cases, a selective BCL2 inhibitors exert minimal or no effects on the activity and/or signaling of other prosurvival BCL2 family proteins, when provided at a dosing regimen.
  • the BCL2 inhibitor is a nonselective BCL2 inhibitor.
  • a nonselective BCL2 inhibitor is a compound or agent, such as an inhibitor of a prosurvival BCL2 family protein, that reduces or blocks the activity of more than one prosurvival BCL2 family protein.
  • a nonselective BCL2 inhibitor is a compound or agent, such as an inhibitor of a prosurvival BCL2 family protein, that is capable of being provided at a dosing regimen (e.g. dose and/or duration) that reduces or blocks the activity and/or signaling of a prosurvival BCL2 family protein, e.g.
  • BCL2 and additionally reduces or blocks the activity and/or signaling of one or more other prosurvival BCL2 family proteins (e.g. BCLXL, BCLW, BCLB, MCL1).
  • a nonselective BCL2 inhibitor reduces or blocks the activity and/or signaling of a prosurvival BCL2 family protein (e.g. BCL2) when provided at a dosing regimen, and also reduces or blocks the signaling and/or activity of one or more other prosurvival BCL2 family proteins (e.g. BCLXL, BCLW, BCLB, MCL1) when provided at the same dosing regimen.
  • the inhibitor inhibits BCL2 with a half-maximal inhibitory concentration (IC50) of less than or less than about 1000 nM, less than or less than about 900 nM, less than or less than about 800 nM, less than or less than about 700 nM, less than or less than about 600 nM, less than or less than about 500 nM, less than or less than about 400 nM, less than or less than about 300 nM, less than or less than about 200 nM, less than or less than about 100 nM, less than or less than about 90 nM, less than or less than about 80 nM, less than or less than about 70 nM, less than or less than about 60 nM, less than or less than about 50 nM, less than or less than about 40 nM, less than or less than about 30 nM, less than or less than about 20 nM, less than or less than about 10 nM, less than or less than about 9 nM, less than
  • IC50 half-max
  • the inhibitor inhibits one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1 with a half-maximal inhibitory concentration (IC50) of less than or less than about 1000 nM, less than or less than about 900 nM, less than or less than about 800 nM, less than or less than about 700 nM, less than or less than about 600 nM, less than or less than about 500 nM, less than or less than about 400 nM, less than or less than about 300 nM, less than or less than about 200 nM, less than or less than about 100 nM, less than or less than about 90 nM, less than or less than about 80 nM, less than or less than about 70 nM, less than or less than about 60 nM, less than or less than about 50 nM, less than or less than about 40 nM, less than or less than about 30 nM, less than or less than about 20 nM, less
  • the inhibition constant (Ki) of the inhibitor for BCL2 is lower than the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1.
  • the inhibition constant (Ki) of the inhibitor for BCL2 is at least 10 times lower, at least 100 times lower, at least 1,000 times lower, at least 5,000 times lower, at least 10,000 times lower, or at least 20,000 times lower than the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1.
  • the inhibition constant (Ki) of the inhibitor for BCL2 is at least 1 ,000 times lower than the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is at least 5,000 times lower than the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1.
  • the inhibition constant (Ki) of the inhibitor for BCL2 is at least 10,000 times lower than the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1.
  • the inhibition constant (Ki) of the inhibitor for BCL2 is at least 20,000 times lower than the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCLL
  • the inhibition constant (Ki) of the inhibitor for BCL2 is at least 1,000 times lower than the inhibition constant (Ki) of the inhibitor for BCLXL.
  • the inhibition constant (Ki) of the inhibitor for BCL2 is at least 4,000 times lower than the inhibition constant (Ki) of the inhibitor for BCLXL. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is at least 20,000 times lower than the inhibition constant (Ki) of the inhibitor for BCLW.
  • the inhibition constant (Ki) of the inhibitor for BCL2 is less than about 10 pM. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is less than about 1 pM. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is less than about 0.1 pM. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is less than about 10 nM. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is less than about 1.0 nM. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is less than about 0.1 nM.
  • the inhibition constant (Ki) of the inhibitor for BCL2 is less than about 0.01 nM. In some embodiments, the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1 is less than about 10 pM. In some embodiments, the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1 is less than about 1 pM.
  • the inhibition constant (Ki) of the inhibitor one or more other prosurvival BCL2 family proteins is less than about 0.1 pM. In some embodiments, the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1 is less than about 10 nM. In some embodiments, the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1 is less than about 1.0 nM.
  • the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins is less than about 0.1 nM. In some embodiments, the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1 is less than about 0.01 nM.
  • the IC50, Kd and/or Ki is measured or determined using an in vitro assay.
  • Assays to assess or quantitate or measure activity of protein tyrosine kinase inhibitors as described are known in the art. Such assays can be conducted in vitro and include assays to assess the ability of an agent to inhibit a specific biological or biochemical function. In some embodiments.
  • kinase activity studies can be performed. Protein tyrosine kinases catalyze the transfer of the terminal phosphate group from adenosine triphosphate (ATP) to the hydroxyl group of a tyrosine residue of the kinase itself or another protein substrate.
  • ATP adenosine triphosphate
  • kinase activity can be measured by incubating the kinase with the substrate (e.g., inhibitor) in the presence of ATP.
  • measurement of the phosphorylated substrate by a specific kinase can be assessed by several reporter systems including colorimetric, radioactive, and fluorometric detection. (Johnson, S.A. & T. Hunter (2005) Nat. Methods 2:17.)
  • inhibitors can be assessed for their affinity for a particular kinase or kinases, such as by using competition ligand binding assays (Ma et al., Expert Opin Drug Discov.
  • IC50 half-maximal inhibitory concentration
  • Ki ICso/(l+L/Kd), where L is the concentration of the inhibitor (Biochem Pharmacol 22: 3099-3108, 1973). Ki is the concentration of unlabeled inhibitor that would cause occupancy of 50 % of the binding sites present in the absence of ligand or other competitors.
  • the inhibitor is a small molecule.
  • the inhibitor is an inhibitor of a prosurvival BCL2 family protein, including but not limited to those described in US Patent No. 9,174,982, US Patent No. 8,546,399, US Patent No. 7,030,115, US Patent No. 7,390,799, US Patent No. 7,709,467, US Patent No. 8,624,027, US Patent No. 7,906,505, US Patent No. 6,720,338, published PCT application WO 13/096060, published PCT application WO 02/097053, published US application US 2016/0220573, US Patent No. 7,354,928, published US application 2015/0056186, and published PCT application WO 05/049594, which are each incorporated by reference in their entireties.
  • a prosurvival BCL2 family protein including but not limited to those described in US Patent No. 9,174,982, US Patent No. 8,546,399, US Patent No. 7,030,115, US Patent No. 7,390,799, US Patent No. 7,709,467,
  • the inhibitor inhibits BCL2, BCLXL, and BCLW, such as navitoclax. In some embodiments, the inhibitor inhibits BCL2, such as venetoclax.
  • the inhibitor inhibits or reduces the activity of BCL2, BCLXL, and
  • the inhibitor is navitoclax. In some cases, the inhibitor has the
  • the inhibitor inhibits or reduces the activity of BCL2, such as venetoclax.
  • the inhibitor is venetoclax.
  • the inhibitor has the structure
  • Exemplary prosurvival BCL2 family protein inhibitors include, but are not limited to venetoclax (ABT-199), navitoclax (ABT-263), ABT-737, AT-101/GDC-0199 (Gossypol), apogossypol, TW-37, G3139 (Genasense), GX15-070 (obatoclax), sabutoclax, HA14-1, antimycin A, BH3I-1, YC137, maritoclax (marinopyyrole A), clitocine, UMI-77, WEHI-539, and 544563.
  • the combination therapy can be administered in one or more compositions, e.g., a pharmaceutical composition containing a BCL2 inhibitor (e.g. venetoclax), and/or the immunotherapy or cell therapy, e.g., T cell therapy.
  • a pharmaceutical composition containing a BCL2 inhibitor e.g., venetoclax
  • the composition can include carriers such as a diluent, adjuvant, excipient, or vehicle with which a BCL2 inhibitor, e.g., venetoclax, and/or the cells are administered.
  • compositions will contain a therapeutically effective amount of a BCL2 inhibitor, e.g., venetoclax, generally in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • a BCL2 inhibitor e.g., venetoclax
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Saline solutions and aqueous dextrose and glycerol solutions also can be employed as liquid carriers, particularly for injectable solutions.
  • the pharmaceutical compositions can contain any one or more of a diluents(s), adjuvant(s), antiadherent(s), binder(s), coating(s), filler(s), flavor(s), color(s), lubricant(s), glidant(s), preservative(s), detergent(s), sorbent(s), emulsifying agent(s), pharmaceutical excipient(s), pH buffering agent(s), or sweetener(s) and a combination thereof.
  • the pharmaceutical composition can be liquid, solid, a lyophilized powder, in gel form, and/or combination thereof.
  • the choice of carrier is determined in part by the particular inhibitor and/or by the method of administration.
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine
  • compositions containing a pa BCL2 inhibitor can also be lyophilized.
  • the pharmaceutical compositions can be formulated for administration by any route known to those of skill in the art including intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, subcutaneous, intratumoral, epidural, nasal, oral, vaginal, rectal, topical, local, otic, inhalational, buccal e.g., sublingual), and transdermal administration or any route.
  • routes known to those of skill in the art including intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, subcutaneous, intratumoral, epidural, nasal, oral, vaginal, rectal, topical, local, otic, inhalational, buccal e.g., sublingual), and transdermal administration or any route.
  • other modes of administration also are contemplated.
  • the administration is by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon’s injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.
  • administration is by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • compositions also can be administered with other biologically active agents, either sequentially, intermittently or in the same composition.
  • administration also can include controlled release systems including controlled release formulations and device controlled release, such as by means of a pump.
  • the administration is oral.
  • a BCL2 inhibitor e.g., venetoclax
  • a BCL2 inhibitor is typically formulated and administered in unit dosage forms or multiple dosage forms.
  • Each unit dose contains a predetermined quantity of a therapeutically active BCL2 inhibitor, e.g., venetoclax, sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent.
  • unit dosage forms include, but are not limited to, tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil water emulsions containing suitable quantities of a BCL2 inhibitor, e.g., venetoclax.
  • Unit dose forms can be contained ampoules and syringes or individually packaged tablets or capsules. Unit dose forms can be administered in fractions or multiples thereof. In some embodiments, a multiple dose form is a plurality of identical unit dosage forms packaged in a single container to be administered in segregated unit dose form. Examples of multiple dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons.
  • the provided combination therapy method involves administering to the subject a a BCL2 inhibitor, e.g., venetoclax, and an immunotherapy or cell therapy (e.g. CAR- expressing T cells).
  • a BCL2 inhibitor e.g., venetoclax
  • an immunotherapy or cell therapy e.g. CAR- expressing T cells.
  • the provided combination therapy methods involve initiating administration of a BCL2 inhibitor, e.g., venetoclax, subsequent to the initiation of the immunotherapy or cell therapy, such as a T cell therapy e.g., CAR-expressing T cells).
  • the provided combination therapy methods involve initiating administration of a BCL2 inhibitor, e.g., venetoclax within about 2 days after initiation of administration of the immunotherapy or cell therapy, such as a T cell therapy (e.g., CAR-expressing T cells).
  • a T cell therapy e.g., CAR-expressing T cells
  • the method involves initiating administration of the BCL2 inhibitor, e.g., venetoclax, within about 1 day after initiation of administration of the immunotherapy or cell therapy (e.g. CAR T cell therapy).
  • the method involves initiating administration of the BCL2 inhibitor, e.g., venetoclax, at about 2 days after initiation of administration of the immunotherapy or cell therapy (e.g. CAR T cell therapy).
  • the method involves initiating administration of the BCL2 inhibitor, e.g.,venetoclax, at about 1 day after initiation of administration of the immunotherapy or cell therapy (e.g. CAR T cell therapy). In some embodiments, the method comprises initiating administration of the BCL2 inhibitor, e.g.,venetoclax, at about 1 day after initiation of administration of the cell therapy (e.g. CAR T cell therapy). In some embodiments, the provided methods include initiation of administration of the BCL2 inhibitor (e.g. venetoclax) after activation-induced cell death (AICD) of the cells of the immunotherapy or cell therapy (e.g. CAR T cells) has peaked. In some embodiments, the metof comprises initiating administration of the BCL2 inhibitor (e.g. venetoclax) after activation-induced cell death (AICD) of the cells of the cell therapy (e.g. CAR T cells) has peaked.
  • the metof comprises initiating administration of the BCL2 inhibitor (e.g. venetoclax
  • the provided combination therapy comprises (1) administering to a subject have a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds CD19 on Day 1; and (2) administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising administration of the BCL2 inhibitor within about 2 days (e.g. about 1 day) after initiation of administration of the cell therapy.
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • BCL2 inhibitor e.g. venetoclax
  • the provided combination therapy comprises (1) administering to a subject have a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds CD 19 on Day 1; and (2) administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising administration of the BCL2 inhibitor on Day 2.
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • BCL2 inhibitor e.g. venetoclax
  • the provided combination therapy comprises administering to a subject have a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor in a dosing regimen comprising administration of the BCL2 inhibitor (e.g. ventoclax) at or about 1 day after initiation of administration of a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds CD 19 on Day 1.
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • the provided combination therapy comprises administering to a subject have a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds CD19 on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen comprising initiation of the BCL2 inhibitor (e.g. venetoclax) at or about 1 day after initiation of administration of the cell therapy.
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • CAR chimeric antigen receptor
  • the method includes administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising (i) daily administration of a first dose for a first predetermined period and (ii) daily administration of at least one subsequent dose, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor.
  • each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor (e.g. venetoclax) compared to the preceding dose.
  • the at least one subsequent dose comprises a second dose, a third dose, a fourth dose, a fifth dose, a sixth dose, a seventh dose, an eighth dose, a ninth dose, and/or a tenth dose.
  • the at least one subsequent dose comprises a second dose.
  • the at least one subsequent dose comprises a second dose and a third dose.
  • the at least one subsequent dose comprises a second, third, and fourth dose.
  • the at least one subsequent dose comprises a second, third, fourth, and fifth dose.
  • initiation of administration of the BCL2 inhibitor begins on about Day 2.
  • the BCL2 inhibitor e.g. venetoclax
  • the at least one subsequent dose is or comprises a second dose.
  • the method includes administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising (i) daily administration of a first dose for a first predetermined period and (ii) daily administration of a second dose for a second predetermined period beginning after the first predetermined period, wherein the second dose is an increased amount of the BCL2 inhibitor compared to the first dose, and the BCL2 inhibitor is administered at no more than about 100 mg per day through Day 30.
  • a BCL2 inhibitor e.g. venetoclax
  • the at least one subsequent dose is or comprises a second dose and a third dose.
  • the method includes administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising (i) daily administration of a first dose for a first predetermined period; (ii) daily administration of a second dose for a second predertmined period beginning after the first predetermined period; and (iii) daily administration of a third dose for a third predetermined period beginning after the second predetermined period, wherein the second dose is an increased amount of the BCL2 inhibitor compared to the first dose, the third dose is an increased amount of the BCL2 inhibitor compared ot the first dose, and and the BCL2 inhibitor is administered at no more than about 100 mg per day through Day 30.
  • a BCL2 inhibitor e.g. venetoclax
  • the administration of the BCL2 inhibitor e.g., venetoclax
  • the combination therapy involves administration of the inhibitor (e.g. venetoclax) up to or about 3 months after initiation of administration of the immunotherapy or cell therapy, e.g.CAR T cells.
  • the combination therapy involves administration of the BCL2 inhibitor (e.g. venetoclax) for at least about 3 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells.
  • the combination therapy involves administration of the BCL2 inhibitor (e.g. venetoclax) for at least about 6 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells.
  • the combination therapy involves administration of the BCL2 inhibitor (e.g.
  • the combination therapy involves administration of the BCL2 inhibitor (e.g. venetoclax) for at least about 18 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells. In some cases, the combination therapy involves administration of the BCL2 inhibitor (e.g. venetoclax) for at least about 24 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells.
  • administration of the BCL2 inhibitor is discontinued about 3 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells, if the subject exhibits a desired response (e.g complete response). In some cases, administration of the BCL2 inhibitor is discontinued about 3 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells. In some cases, administration of the BCL2 inhibitor is discontinued about after Day 90. In some cases, administration of the BCL2 inhibitor is discontinued about 6 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells. In some cases, administration of the BCL2 inhibitor is discontinued about 12 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells. In some cases, administration of the BCL2 inhibitor is discontinued about 24 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells.
  • the outcome is a desired therapeutic response, as described below and in Section III.
  • the dose, frequency, duration, timing and/or order of administration of the BCL2 inhibitor e.g., venetoclax
  • the dose, frequency, duration, timing and/or order of administration of the BCL2 inhibitor is determined, based on particular thresholds or criteria of results of the screening step and/or assessment of treatment outcomes described herein, such as in Section III.
  • a subject if a subject exhibits minimum residual disease (MRD; greater than or equal to 10 4 in peripheral blood) at Day 90, the subject may continue treatment with the BCL2 inhibitor, e.g. venetoclax per standard of care, such as for a total treatment duration of 12 or 24 months.
  • the subject if a subject does not exhibit complete response (CR) at Day 90, the subject may continue treatment with the BCL2 inhibitor, e.g. venetoclax per standard of care, such as for a total treatment duration of 12 or 24 months.
  • Desired therapeutic results include but are not limited to a reduction of tumor size, bulk, metastasis, percentage of blasts in the bone marrow or molecularly detectable B cell malignancy and/or an improvement in prognosis or survival or other symptom associated with tumor burden. Desired therapeutic results, such as for the treatment of cancer (e.g. CLL or SLL), may additionally include an ability of the inhibitor to induce apoptosis in cancer cells and/or an increase in apoptosis of cancer cells. Methods for identifying and determining whether a desired therapeutic result for the treatment of cancer is achieved include but are not limited to any as described in Section III.
  • a therapeutically effective amount results in neutropenia, such as mild, moderate, or severe neutropenia.
  • mild neutropenia is defined as an absolute neutrophil count of 1,000 to l,5000/
  • moderate neutropenia is defined as an absolute neutrophil count of 500 to 1,000/ .L.
  • severe neutropenia is defined as an absolute neutrophil count of fewer than 500/
  • a subtherapeutically effective amount refers to an amount less effective, at dosages and/or for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment, as compared to a “therapeutically effective amount” of the same agent.
  • the subtherapeutically effective amount does not achieve or achieves less than a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment.
  • the desired therapeutic result is a reduction of tumor size, bulk, metastasis, percentage of blasts in the bone marrow or molecularly detectable B cell malignancy and/or an improvement in prognosis or survival or other symptom associated with tumor burden.
  • the desired therapeutic result is an ability of the inhibitor to induce apoptosis in cancer cells and/or an increase in apoptosis of cancer cells.
  • a subtherapeutically effective amount results in mild or moderate neutropenia
  • a subtherapeutically effective amount does not result in severe neutropenia.
  • mild neutropenia is defined as an absolute neutrophil count of 1,000 to l,5000/
  • moderate neutropenia is defined as an absolute neutrophil count of 500 to 1,000/ .L.
  • severe neutropenia is defined as an absolute neutrophil count of fewer than 500/
  • Methods for identifying and determining whether a desired therapeutic result for the treatment of cancer is achieved include but are not limited to any as described in Section III.
  • the subtherapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered.
  • the provided methods involve administering the cells and/or compositions at subeffective amounts, e.g., subtherapeutically effective amounts.
  • the provided methods involve administering a BCL2 inhibitor, e.g., venetoclax, engineered cells e.g. cell therapy), or compositions at subeffective amounts, e.g., subtherapeutically effective amounts.
  • a BCL2 inhibitor e.g. venetoclax
  • the dosing regimen e.g dose and/or duration
  • the BCL2 inhibitor dosing regimen comprises (i) daily administration of a first dose for a first predetermined period and (ii) daily administration of a second dose for a second predetermined period beginning after the first predetermined period, wherein the second dose is an increased amount of the BCL2 inhibitor compared to the first dose.
  • the first predetermined period is from about Day 2 to about Day 7. In some embodiments, the first predetermined period is from about Day 2 to about Day 30.
  • the first dose is between about 20 mg and about 200 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 20 mg, about 50 mg, about 100 mg, or about 200 mg of the BCL2 inhibitor. In some embodiments, the first dose is between about 20 mg and about 100 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 20 mg, about 50 mg, or about 100 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 20 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the first dose is no more than or no more than about 100 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 200 mg of the BCL2 inhibitor.
  • the first predetermined period is from about Day 2 to about Day 7, and the first dose is about 20 mg of the BCL2 inhibitor. In some embodiments, the first predetermined period is from about Day 2 to about Day 7, and the first dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the first predetermined period is from about Day 2 to about Day 30, and the first dose is about 20 mg of the BCL2 inhibitor. In some embodiments, the first predetermined period is from about Day 2 to about Day 30, and the first dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the first predetermined period is from about Day 2 to about Day 30, and the first dose is about 100 mg of the BCL2 inhibitor.
  • the BCL2 inhibitor dosing regimen comprises (i) daily administration of a first dose of the BCL2 inhibitor for a first predetermined period and (ii) daily administration of at least one subsequent dose of the BCL2 inhibitor for a predetermined period of time beginning after a preceding predetermined period of time, wherein each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose.
  • the at least one subsequent dose comprises a second dose, a third dose, a fourth dose, a fifth dose, a sixth dose, a seventh dose, an eighth dose, a ninth dose, and/or a tenth dose.
  • the at least one subsequent dose is or comprises a second dose. In some embodiments, the at least one subsequent dose is a second dose. In some embodiments, the at least one subsequent dose is or comprises a second dose and a third dose. In some embodiments, the at least one subsequent dose is a second dose and a third dose. In some embodiments, the at least one subsequent dose is or comprises a second, third, and fourth dose. In some embodiments, the at least one subsequent dose is a second, third, and fourth dose. In some embodiments, the at least one subsequent dose is or comprises a second, third, fourth, and fifth dose. In some embodiments, the at least one subsequent dose is a second, third, fourth, and fifth dose.
  • the at least one subsequent dose is or comprises a second dose.
  • the BCL2 inhibitor dosing regimen comprises (i) daily administration of a first dose of the BCL2 inhibitor for a first predetermined period; and (ii) daily administration of a second dose of the BCL2 inhibitor for a second predetermined period of time beginning after the first predetermined period of time, wherein the second dose is an increased amount of the BCL2 inhibitor compared to the first dose.
  • the second predetermined period is from about Day 8 to about Day 30. In some embodiments, the second predetermined period is from about Day 31 to about Day 37. In some embodiments, the second predetermined period is from about Day 31 to about Day 90.
  • the second dose is between about 50 mg and about 200 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 50 mg, about 100, about 200 mg, or about 400 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 200 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 400 mg of the BCL2 inhibitor.
  • the second predetermined period is from about Day 8 to about Day 30, and the second dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 8 to about Day 30, and the second dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 to about Day 37, and the second dose is about 200 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 to about Day 90, and the second dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 and Day 90, and the second dose is about 200 mg of the BCL2 inhibitor.
  • the dosing regimen comprises daily administration of about 20 mg the BCL2 inhibitor to the subject from about Day 2 to about Day 7; and daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 8 to about Day 30. In some embodiments, the dosing regimen comprises daily administration of about 50 mg per day of the BCL2 inhibitor to the subject from about Day 2 to about Day 7; and daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 8 to about Day 30. In some embodiments, the dosing regimen comprises daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 30; and daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 90.
  • the dosing regimen comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject between about Day 2 and about Day 30; and daily administration of about 200 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 90. In some embodiments, the dosing regimen comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 30; and daily administration of about 200 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 37.
  • the at least one subsequent dose is or comprises a second dose and a third dose.
  • the BCL2 inhibitor dosing regimen comprises (i) daily administration of a first dose of the BCL2 inhibitor for a first predetermined period; (ii) daily administration of a second dose of the BCL2 inhibitor for a second predetermined period of time beginning after the first predetermined period of time; and (iii) daily administration fo a third dose of the BCL2 inhibitor for a third predetermined period of time beginning after the second predetermined period of time, wherein the second dose is an increased amount of the BCL2 inhibitor compared to the first dose and the third dose is an increased amount of the BCL2 inhibitor compared to the second dose.
  • the third predetermined period is from about Day 31 to about Day 90. In some embodiments, the third predetermined period is from about Day 38 to about Day 90.
  • the third dose is between about 100 mg and about 400 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 100 mg, about 200 mg, or about 400 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 200 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 400 mg of the BCL2 inhibitor.
  • the third predetermined period is from about Day 31 to about Day 90, and the third dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the third predetermined period is from about Day 31 to about Day 90, and the third dose is about 200 mg of the BCL2 inhibitor. In some embodiments, the third predetermined period is from about Day 38 to about Day 90, and the third dose is about 400 mg of the BCL2 inhibitor.
  • the dosing regimen comprises (i) daily administration of about 20 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 7; (ii) daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 8 to about Day 30; and (iii) daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 90.
  • the dosing regimen comprises (i) daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 7; (ii) daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 8 to about Day 30; and (iii) daily administration of about 200 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 90.
  • the dosing regimen comprises (i) daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 2 and about Day 30; (ii) daily administration of about 200 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 37; and (iii) daily administration of about 400 mg of the BCL2 inhibitor to the subject from about Day 38 to about Day 90.
  • a subject is dosed in a dosing cohort (i.e. a dosing regimen).
  • a subject dosed in a dosing cohort is administered the BCL2 inhibitor in a dosing regimen comprising (i) daily administration of a first dose for a first predetermined period; and (ii) daily administration of at least one subsequent dose for a predetermined period of time beginning after a predetermined period of a preceding dose, wherein each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose.
  • a subject is dosed in a dosing cohort (i.e. a dosing regimen), wherein the at least one subsequent dose is or comprises a first dose and a second dose.
  • the BCL2 inhibitor dosing regimen comprises (i) daily administration of a first dose for a first predetermined period; and (ii) daily administration of a second dose for a second predetermined period following the first predetermined period, wherein the first dose is about 50 mg of the BCL2 inhibitor and the first predetermined period is from about Day 2 to about Day 30; and the second dose is about 100 mg of the BCL2 inhibitor and the second predetermined period is from about Day 31 to about Day 90.
  • a subject dosed in a dosing cohort is administered about 50 mg of the BCL2 inhibitor per day from about Day 2 to about Day 30, and about 100 mg of the BCL2 inhibitor per day from about Day 31 to about Day 90.
  • the BCL2 inhibitor dosing regimen comprises administration of a first dose and a second dose, wherein the first dose is about 100 mg of the BCL2 inhibitor and the first predetermined period is from about Day 2 to about Day 30; and the second dose is about 200 mg of the BCL2 inhibitor and the second predetermined period is from about Day 31 to about Day 90.
  • a subject dosed in a dosing cohort i.e. a dosing regimen
  • a subject is dosed in a dosing cohort (i.e. a dosing regimen), wherein the at least one subsequent dose is or comprises a second dose and a third dose.
  • a dosing cohort i.e. a dosing regimen
  • the dosing regimen comprises (i) daily administration of a first dose for a first predetermined period; (ii) daily administration of a second dose for a second predetermined period; and (iii) daily administration of a third dose, wherein the first dose is about 20 mg of the BCL2 inhibitor and the first predetermined period is from about Day 2 to about Day 7 ; the second dose is about 50 mg of the BCL2 inhibitor and the second predetermined period is from about Day 8 to about Day 30; and the third dose is about 100 mg of the BCL2 inhibitor and the third predetermined period is from about Day 31 to about Day 90.
  • a subject dosed in a dosing cohort i.e.
  • a dosing regimen is administered about 20 mg of the BCL2 inhibitor per day from about Day 2 to about Day 7, about 50 mg of the BCL2 inhibitor per day from about Day 8 to about Day 30, and about 100 mg of the BCL2 inhibitor per day from about Day 31 to Day 90.
  • the BCL2 inhibitor dosing regimen comprises (i) daily administration of a first dose for a first predetermined period; (ii) daily adminsitere of a second dose for a second predetermined period; and (iii) daily administration of a third dose for a third predetermined period, wherein the first dose is about 50 mg of the BCL2 and the first predetermined period is from about Day 2 to about Day 7; the second dose is about 100 mg and the second predetermined period is from about Day 8 to about Day 30; and the third dose is about 200 mg and the third predetermined period is from about Day 31 to about Day 90.
  • a subject dosed in a dosing cohort i.e.
  • a dosing regimen is administered about 50 mg of the BCL2 inhibitor per day from about Day 2 to about Day 8, about 100 mg of the BCL2 inhibitor per day from about Day 8 to about Day 30, and about 200 mg of the BCL2 inhibitor per day from about Day 31 to Day 90.
  • the BCL2 inhibitor dosing regimen comprises (i) daily administration of a first dose for a first predetermined period; (ii) daily adminsitere of a second dose for a second predetermined period; and (iii) daily administration of a third dose for a third predetermined period, wherein the first dose is about 100 mg of the BCL2 inhibitor and the first predetermined period is from about Day 2 to about Day 30; the second dose is about 200 mg of the BCL2 inhibitor and the second predetermined period is from about Day 31 to about Day 37 ; and the third dose is about 400 mg of the BCL2 inhibitor and the third predetermined period is from about Day 38 to about Day 90.
  • a subject dosed in a dosing cohort is administered about 100 mg of the BCL2 inhibitor per day from about Day 2 to about Day 30, about 200 mg of the BCL2 inhibitor per day from about Day 31 to about Day 37, about 400 mg of the BCL2 inhibitor per day from about Day 38 to about Day 90.
  • the dosing regimen does not result in a toxicity (e.g. a dose-limiting toxicity) in one or more of the subjects.
  • a toxicity e.g. a dose-limiting toxicity
  • the dosing regimen may be reduced or de-escalated.
  • the dosing regimen comprises (i) daily administration of a first dose of about 50 mg of the BCL2 inhibitor for a first predetermined period of from about Day 2 to about Day 30, and (ii) daily administration of a second dose of about 100 mg of the BCL2 inhibitor for a second predetermined period from about Day 31 to about Day 90.
  • the dosing regimen comprises (i) daily administration of a first dose of about 20 mg of the BCL2 inhibitor for a first predetermined period of from Day 2 to about Day 7,(ii) daily administration of a second dose of about 50 mg of the BCL2 inhibitor for a second predetermined period from about Day 8 to about Day 30, and (iii) daily administration of a third dose of about 100 mg of the BCL2 inhibitor for a third predetermined period from about Day 31 to about Day 90.
  • the dosing regimen comprises (i) daily administration of a first dose of about 100 mg of the BCL2 inhibitor for a first predetermined period of from about Day 2 to about Day 30, and (ii) daily administration of a second dose of about 200 mg of the BCL2 inhibitor for a second prefetermined period of from about Day 31 to about Day 90.
  • the dosing regimen comprises (i) daily administration of a first dose of about 50 mg of the BCL2 inhibitor for a first predetermined period of from about Day 2 to about Day 7, (ii) daily administration of a second dose level of about 100 mg of the BCL2 inhibitor for a second predertermined period of from about Day 8 to about Day 30, and (iii) daily administration of a third dose level of about 200 mg of the BCL2 inhibitor for a third predetermined period of from about Day 31 to about Day 90.
  • the dosing regimen does not result in a toxicity (e.g. a dose-limiting toxicity) in one or more of the subjects. In some embodiments, if the dosing regimen does not result in a DLT in one or more of the subjects among a plurality of subjects treated, the dosing regimen may be increased or escalated.
  • a toxicity e.g. a dose-limiting toxicity
  • the dosing regimen comprises (i) daily administration of a first dose of about 50 mg of the BCL2 inhibitor for a first predetermined period of from about Day 2 to about Day 30, and (ii) daily administration of a second dose of about 100 mg of the BCL2 inhibitor for a second predetermined period of from about Day 31 to about Day 90.
  • the dosing regimen comprises (i) daily administration of a first dose of about 100 mg of the BCL2 inhibitor for a first predetermined period of from about Day 2 to about Day 30, and (ii) daily administration of a second dose of about 200 mg of the BCL2 inhibitor for a second predetermined period of from about Day 31 to about Day 90.
  • the dosing regimen comprises (i) daily administration of a first dose of about 100 mg of the BCL2 inhibitor for a first predetermined period from about Day 2 to about Day 30, and (ii) daily administration of a second dose of about 200 mg of the BCL2 inhibitor for a second predetermined period of from about Day 31 to about Day 90.
  • the dosing regimen comprises (i) daily administration fo a first dose of about 100 mg of the BCL2 inhibitor for a first predetermined period from about Day 2 to about Day 30, (ii) daily administration of a second dose level of about 200 mg of the BCL2 inhibitor for a second predetermined period of from about Day 31 to about Day 37, and (iii) daily administration of a third dose level of about 400 mg of the BCL2 inhibitor for a third predetermined period of from about Day 38 to Day 90.
  • toxicity is determined by the pharmacokinetics (PK) of the cells of the cell therapy (eg. CAR T cells) and/or response, as determined by standard critera including any of those described in Section III. A and/or assessment of minimum residual disease (MRD).
  • PK pharmacokinetics
  • MRD minimum residual disease
  • CAR T cells administered to the subject shows that the cells did not persist and/or expand at satisfactory levels, response of the subject is also assessed. In some embodiments, if the cells of the cell therapy did not persist and/or expand at satisfactory levels, response of the subject is also assessed.
  • the dosing regimen is not de-escalated. In some cases, if the cells of the cell therapy did not persist and/or expand at satisfactory levels, and the subject demonstrates a response to the combination treatment, the dosing regimen is not de-escalated. In some cases, if the cells of the cell therapy did not persist and/or expand at satisfactory levels, and the subject demonstrates a response to the combination treatment, the dosing regimen is not de-escalated. In some cases, if the PK of the cells of the cell therapy (e.g. CAR T cells) administered to the subject shows that the cells did not persist and/or expand at satisfactory levels, and the subject demonstrates a response to the combination treatment, the dosing regimen is escalated. In some cases, if the cells of the cell therapy did not persist and/or expand at satisfactory levels, and the subject demonstrates a response to the combination treatment, the dosing regimen is escalated.
  • the dosing regimen is escalated.
  • the dosing regimen is de-escalated. In some cases, if the cells of the cell therapy did not persist and/or expand at satisfactory levels, and the subject does not demonstrate a satisfactory response to the combination treatment, the dosing regimen is de-escalated. In some cases, if the cells of the cell therapy did not persist and/or expand at satisfactory levels, and the subject does not demonstrate a satisfactory response to the combination treatment, the dosing regimen is de-escalated. In some cases, if the PK of the cells of the cell therapy (e.g. CAR T cells) administered to the subject shows that the cells did not persist and/or expand at satisfactory levels, and the subject does not demonstrate a satisfactory response to the combination treatment, the dosing regimen is de-escalated. In some cases, if the PK of the cells of the cell therapy (e.g.
  • CAR T cells administered to the subject shows that the cells did not persist and/or expand at satisfactory levels, and the subject does not demonstrate a satisfactory response to the combination treatment, this outcome is regarded as a doselimiting toxicity (DLT).
  • DLT doselimiting toxicity
  • the subject is identified as having a dose-limiting toxicity (DLT).
  • the dosing regimen is escalated. In some cases, if the cells of the cell therapy did persist and/or expand at satisfactory levels, and the subject demonstrates a satisfactory response to the combination treatment, the dosing regimen is escalated.
  • the precise dose of the BCL2 inhibitor and the timing thereof may depend on the particular therapeutic preparation, the regimen and dosing schedule, the route of administration, the seriousness of the disease, degree of toxicity, and/or one or more outcomes.
  • the combination therapy includes administering to a subject a therapy, e.g. an immunotherapy or cell therapy.
  • a therapy e.g. an immunotherapy or cell therapy.
  • the therapy is a T cell therapy (e.g. CAR-expressing T cells) or a T cell-engaging therapy.
  • Such therapies can be administered prior to administration of one or more BCL2 inhibitor as described.
  • the immunotherapy or cell therapy is or comprises a T cell-engaging therapy that is or comprises a binding molecule capable of binding to a surface molecule expressed on a T cell.
  • the surface molecule is an activating component of a T cell, such as a component of the T cell receptor complex.
  • the surface molecule is CD3 or is CD2.
  • the surface molecule is CD3.
  • the T cell-engaging therapy is or comprises an antibody or antigen-binding fragment.
  • the T cell-engaging therapy is a bispecific antibody containing at least one antigen-binding domain binding to an activating component of the T cell (e.g. a T cell surface molecule, e.g. CD3 or CD2) and at least one antigen-binding domain binding to a surface antigen on a target cell, such as a surface antigen on a tumor or cancer cell, for example any of the listed antigens as described herein, e.g. CD19.
  • the simultaneous or near simultaneous binding of such an antibody to both of its targets can result in a temporary interaction between the target cell and T cell, thereby resulting in activation, e.g. cytotoxic activity, of the T cell and subsequent lysis of the target cell.
  • bi-specific T cell engagers are used in connection with the provided methods, uses, articles of manufacture.
  • bi-specific T cell engagers have specificity toward two particular antigens (or markers or ligands).
  • the antigens are expressed on the surface of a particular type of cell.
  • the first antigen is associated with an immune cell or an engineered immune cell
  • the second antigen is associated with a target cell of the particular disease or condition, such as a cancer.
  • bi-specific T cell engagers Numerous methods of producing bi-specific T cell engagers are known, including fusion of two different hybridomas (Milstein and Cuello, Nature 1983;305:537-540), and chemical tethering though heterobifunctional cross linkers (Staerz et al. Nature 1985; 314:628-631).
  • exemplary bi- specific antibody T cell-engaging molecules are those which contain tandem scFv molecules fused by a flexible linker (see e.g. Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011); tandem scFv molecules fused to each other via, e.g.
  • a flexible linker and that further contain an Fc domain composed of a first and a second subunit capable of stable association
  • diabodies and derivatives thereof including tandem diabodies (Holliger et al, Prot Eng 9, 299-305 (1996); Kipriyanov et al, J Mol Biol 293, 41-66 (1999)); dual affinity retargeting (DART) molecules that can include the diabody format with a C-terminal disulfide bridge; or triomabs that include whole hybrid mouse/rat IgG molecules (Seimetz et al, Cancer Treat Rev 36, 458-467 (2010).
  • the bi-specific T cell engager is a molecule encoded by a polypeptide construct.
  • the polypeptide construct contains a first component comprising an antigen-binding domain binding to an activating portion of an immune cell or engineered immune cell, and a second component comprising an antigen-binding domain binding to a surface antigen (e.g. target or tumor associated antigen (TAA)) associated with a particular disease or condition (e.g. cancer).
  • TAA tumor associated antigen
  • the first and second components are coupled by a linker.
  • the first component is coupled to a leader sequence encoding a CD33 signal peptide.
  • the polypeptide is a construct containing from N-terminus to C- terminus: a first component comprising an antigen-binding domain binding to an activating portion of the T cell, a peptide linker, and a second component comprising an antigen-binding domain binding to a surface antigen (e.g. target or tumor associated antigen (TAA)) associated with a disease or condition (e.g. cancer).
  • a surface antigen e.g. target or tumor associated antigen (TAA)
  • TAA tumor associated antigen
  • an activating component of the T cell I a T cell surface molecule, such as CD3 or CD2.
  • the surface antigen of the target cell is a tumor associated antigen (TAA).
  • TAA tumor associated antigen
  • the TAA contains one or more epitopes.
  • the peptide linker is or comprises a cleavable peptide linker.
  • the antigen binding domain of the first component of the bi-specific T cell engager engages a receptor on an endogenous immune cell in the periphery of the tumor.
  • the endogenous immune cell is a T cell.
  • the engagement of the endogenous T cell receptor redirects the endogenous T cells to the tumor.
  • the engagement of the endogenous T cell receptor recruits tumor infiltrating lymphocytes (TILs) to the tumor.
  • TILs tumor infiltrating lymphocytes
  • the engagement of the endogenous T cell receptor activates the endogenous immune repertoire.
  • the simultaneous or near simultaneous binding of the bi-specific T cell engager to both of its targets can result in a temporary interaction between the target cell and T cell, thereby resulting in activation e.g. cytotoxic activity, cytokine release), of the T cell and subsequent lysis of the target cell.
  • the first component of the bi-specific T cell engager is or comprises an antigen binding domain that binds to an activating component of a T cell.
  • the activating component of the T cell is a surface molecule.
  • the surface molecule is or comprises a T-cell antigen.
  • Exemplary T-cell antigens include but are not limited to CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD44, CD45, CD69 and CD90.
  • the binding of the bispecific T cell engaging molecule with the T cell antigen stimulates and/or activates the T cell.
  • the anti-T cell binding domain includes an antibody or an antigenbinding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, an scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.
  • the T cell binding domain on the bi-specific T cell engager is an anti- CD3 domain.
  • the anti-CD3 domain is an scFv.
  • the anti-CD3 domain of the bi-specific T cell engager binds to a subunit of the CD3 complex on a receptor on a T cell.
  • the receptor is on an endogenous T cell.
  • the receptor is on an engineered immune cell further expressing a recombinant receptor.
  • the effects of CD3 engagement of T cells is well known in the art, and include but are not limited to T cell activation and other downstream cell signaling. Any of such bi-specific T cell engagers can be used in the provided disclosure herein.
  • the second component of the bi-specific T cell engager comprising an antigen-binding domain binding to a surface antigen associated with a disease or condition is a tumor or cancer antigen.
  • among the antigens targeted by the bi-specific T cell engager are those expressed in the context of a disease, condition, or cell type to be targeted via the adoptive cell therapy.
  • diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and/or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas.
  • cancers and tumors including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and/or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas.
  • the antigen includes av[36 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancertestis antigen, cancer/testis antigen IB (CT AG, also known as NY-ESO-1 and LAGE -2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), type III epidermal growth factor receptor mutation (EGFR
  • Antigens targeted by the receptors include antigens associated with a B cell malignancy, such as any of a number of known B cell marker.
  • the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.
  • the antigen is CD19.
  • both antigen binding domains comprising the first antigen binding domain and the second antigen binding domain, comprise an antibody or an antigen-binding fragment.
  • antibody herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab’)2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rlgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv) or fragments.
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • antibody should be understood to encompass functional antibody fragments thereof.
  • the term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
  • the antigen-binding proteins, antibodies and antigen binding fragments thereof specifically recognize an antigen of a full-length antibody.
  • the heavy and light chains of an antibody can be full-length or can be an antigen-binding portion (a Fab, F(ab’)2, Fv or a single chain Fv fragment (scFv)).
  • the antibody heavy chain constant region is chosen from, e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE, particularly chosen from, e.g., IgGl, IgG2, IgG3, and IgG4, more particularly, IgGl e.g., human IgGl).
  • the antibody light chain constant region is chosen from, e.g., kappa or lambda, particularly kappa.
  • antibody fragments refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; variable heavy chain (VH) regions, singlechain antibody molecules such as scFvs and single-domain VH single antibodies; and multispecific antibodies formed from antibody fragments.
  • the antibodies are single -chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs.
  • FRs conserved framework regions
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single -domain antibody is a human single -domain antibody.
  • the bi-specific T cell engager comprises an antibody heavy chain domain that specifically binds the antigen, such as a cancer marker or cell surface antigen of a cell or disease to be targeted, such as a tumor cell or a cancer cell, such as any of the target antigens described herein or known.
  • Exemplary single -domain antibodies include sdFv, nanobody, VHH or VNAR-
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells.
  • the antibodies are recombinantly produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody.
  • the antibody fragments are scFvs.
  • a “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs.
  • a humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • a non-human antibody e.g., the antibody from which the CDR residues are derived
  • the antigen binding domains are single chain variable fragments (scFv).
  • the scFv is a tandem scFv containing a heavy and a light chain.
  • the heavy and light chains are connected by peptide linkers.
  • the linker is composed primarily of serines and glycines.
  • the linkage of the heavy chain and the light chain forms a single polypeptide antigen binding domain.
  • the first antigen binding domain of the bi-specific T cell engager is an anti-CD3 scFv.
  • the second antigen binding domain of the bi-specific T cell engager is an anti-CD19 scFv.
  • the first antigen binding domain of the bi-specific T cell engager is an anti-CD3 scFv
  • the second antigen binding domain of the bi-specific T cell engager is an anti-CD19 scFv.
  • the bi-specific T cell engager polypeptide constructs contain a linker that joins the first component comprising the antigen-binding domain that binds to an activating portion of the T cell, to the second component comprising an antigen-binding domain binding to a surface antigen (e.g. target or tumor associated antigen (TAA)) associated with a particular disease or condition.
  • a surface antigen e.g. target or tumor associated antigen (TAA)
  • TAA tumor associated antigen
  • the linker is a peptide linker which is cleavable.
  • the cleavable linker includes a sequence that is a substrate for a protease.
  • the sequence comprises a bond that can be broken under in vivo conditions.
  • the linker sequence is selectively cleaved by a protease present in a physiological environment.
  • the environment is separate from the tumor microenvironment.
  • the protease is found in the periphery of the tumor.
  • the selectively cleavable linker is cleaved by a protease produced by cells that do not co-localize with the tumor. In some embodiments, the selectively cleavable linker is not cleaved by proteases that are in the proximity of the tumor microenvironment. In some embodiments, the cleavage of the linker by the protease renders the bi-specific T cell engaging molecule inactive. In some embodiments, the protease is found in the circulating blood of a subject. In some embodiments, the protease is a part of the intrinsic or extrinsic coagulation pathway. In some aspects, the protease is a serine protease. In some aspects, the protease comprises but is not limited to a thrombin, factor X, factor XI, factor XII, and plasmin.
  • bispecific antibody T cell-engagers are bispecific T cell engager (BiTE) molecules, which contain tandem scFv molecules fused by a flexible linker (see e.g. Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011); tandem scFv molecules fused to each other via, e.g.
  • the T-cell engaging therapy is blinatumomab or AMG 330. Any of such T cell-engagers can be used in used in the provided methods.
  • the immune system stimulator and/or the T cell engaging therapy can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon’s injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.
  • the immunotherapy is administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration.
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intrathoracic, intracranial, or subcutaneous administration.
  • one or more doses of a T cell engaging therapy are administered.
  • between or between about 0.001 pg and about 5,000 pg, inclusive, of the T cell engaging therapy is administered.
  • between or between about 0.001 pg and 1,000 pg, 0.001 pg tol pg, 0.01 pg tol pg, 0.1 pg tolO pg, 0.01 pg tol pg, 0.1 pg and 5 pg, 0.1 pg and 50 pg, 1 pg and 100 pg, 10 pg and 100 pg, 50 pg and 500 pg, 100 pg and 1,000 pg, 1,000 pg and 2,000 pg, or 2,000 pg and 5,000 pg of the T cell engaging therapy is administered.
  • the dose of the T cell engaging therapy is or includes between or between about 0.01 pg/kg and 100 mg/kg, 0.1 pg/kg and 10 pg/kg, 10 pg/kg and 50 pg/kg, 50 pg/kg and 100 pg/kg, 0.1 mg/kg and 1 mg/kg, 1 mg/kg and 10 mg/kg, 10 mg/kg and 100 mg/kg, 100 mg/kg and 500 mg/kg, 200 mg/kg and 300 mg/kg, 100 mg/kg and 250 mg/kg, 200 mg/kg and 400 mg/kg, 250 mg/kg and 500 mg/kg, 250 mg/kg and 750 mg/kg, 50 mg/kg and 750 mg/kg, 1 mg/kg and 10 mg/kg, or 100 mg/kg and 1,000 mg/kg, each inclusive.
  • the dose of the T cell engaging therapy is at least or at least about or is or is about 0.1 pg/kg, 0.5 pg/kg, 1 pg/kg, 5 pg/kg, 10 pg/kg, 20 pg/kg, 30 pg/kg, 40 pg/kg, 50 pg/kg, 60 pg/kg, 70 pg/kg, 80 pg/kg, 90 pg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg,
  • the therapy e.g. immunotherapy or cell therapy
  • the cell therapy is a tumor infiltrating lymphocytic (TIL) therapy, a natural kill (NK) cell therapy, a transgenic TCR therapy, or a recombinant-receptor expressing cell therapy, which optionally is a T cell therapy, which optionally is a chimeric antigen receptor(CAR)-expressing cell therapy.
  • TIL tumor infiltrating lymphocytic
  • NK natural kill
  • CAR chimeric antigen receptor
  • the T cell therapy includes administering T cells engineered to express a chimeric antigen receptor (CAR).
  • the T cell therapy is an adoptive T cell therapy comprising T cells that specifically recognize and/or target an antigen associated with the cancer, such as an antigen associated with a B cell malignancy, e.g. a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL).
  • the T cell therapy is an adoptive T cell therapy comprising T cells that specifically recognize and/or target an antigen associated with the cancer, such as an antigen associated with a B cell malignancy, e.g. a chronic lymphocytic leukemia (CLL).
  • CLL chronic lymphocytic leukemia
  • CLL small lymphocytic lymphoma
  • the T cell therapy is an adoptive T cell therapy comprising T cells that specifically recognize and/or target an antigen associated with the cancer, such as an antigen associated with a B cell malignancy, e.g. a small lymphocytic lymphoma (SLL).
  • the T cell therapy comprises T cells engineered with a chimeric antigen receptor (CAR) comprising an antigen binding domain that binds, such as specifically binds, to the antigen.
  • the antigen targeted by the T cell therapy is CD19.
  • the immune cells express a T cell receptor (TCR) or other antigenbinding receptor.
  • TCR T cell receptor
  • the immune cells express a recombinant receptor, such as a transgenic TCR or a chimeric antigen receptor (CAR).
  • the cells are autologous to the subject.
  • the cells are allogeneic to the subject. Exemplary of such cell therapies, e.g. T cell therapies, for use in the provided methods are described below.
  • the provided cells express and/or are engineered to express receptors, such as recombinant receptors, including those containing ligand-binding domains or binding fragments thereof, and T cell receptors (TCRs) and components thereof, and/or functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs).
  • receptors such as recombinant receptors, including those containing ligand-binding domains or binding fragments thereof, and T cell receptors (TCRs) and components thereof, and/or functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs).
  • the recombinant receptor contains an extracellular ligand-binding domain that specifically binds to an antigen.
  • the recombinant receptor is a CAR that contains an extracellular antigen-recognition domain that specifically binds to an antigen.
  • the ligand such as an antigen
  • the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which, like a TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule.
  • MHC major histocompatibility complex
  • the cells for use in or administered in connection with the provided methods contain or are engineered to contain an engineered receptor, e.g., an engineered antigen receptor, such as a chimeric antigen receptor (CAR), or a T cell receptor (TCR).
  • an engineered receptor e.g., an engineered antigen receptor, such as a chimeric antigen receptor (CAR), or a T cell receptor (TCR).
  • an engineered antigen receptor such as a chimeric antigen receptor (CAR), or a T cell receptor (TCR).
  • compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy.
  • therapeutic methods for administering the cells and compositions to subjects e.g., patients, in accord with the provided methods, and/or with the provided articles of manufacture or compositions.
  • engineered cells including engineered cells containing recombinant receptors
  • Exemplary recombinant receptors including CARs and recombinant TCRs, as well as methods for engineering and introducing the receptors into cells, include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, W02013/123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S.
  • the genetically engineered antigen receptors include a CAR as described in U.S. Patent No.: 7,446,190, and those described in International Patent Application Publication No.: WO/2014055668 Al.
  • the cell therapy e.g., adoptive T cell therapy
  • the cell therapy is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject.
  • the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.
  • the cell therapy e.g., adoptive T cell therapy
  • the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject.
  • the cells then are administered to a different subject, e.g., a second subject, of the same species.
  • the first and second subjects are genetically identical.
  • the first and second subjects are genetically similar.
  • the second subject expresses the same HLA class or supertype as the first subject.
  • the cells of the T cell therapy can be administered in a composition formulated for administration, or alternatively, in more than one composition (e.g., two compositions) formulated for separate administration.
  • the dose(s) of the cells may include a particular number or relative number of cells or of the engineered cells, and/or a defined ratio or compositions of two or more sub-types within the composition, such as CD4 vs.CD8 T cells.
  • the cells can be administered by any suitable means.
  • the cells are administered in a dosing regimen to achieve a therapeutic effect, such as a reduction in tumor burden.
  • Dosing and administration may depend in part on the schedule of administration of the BCL2 inhibitor, which can be administered subsequent to initiation of administration of the cell therapy, such as T cell therapy, e.g. CAR T cell therapy.
  • Various dosing schedules of the cell therapy include but are not limited to single or multiple administrations over various time -points, bolus administration, and pulse infusion.
  • the dose of cells of the cell therapy such as a T cell therapy comprising cells engineered with a recombinant antigen receptor, e.g. CAR or TCR
  • a composition or formulation such as a pharmaceutical composition or formulation.
  • Such compositions can be used in accord with the provided methods and/or with the provided articles of manufacture or compositions, such as in the treatment of a B cell malignancy (e.g. CLL or SLL).
  • pharmaceutical formulation refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • the cell therapy such as engineered T cells e.g. CAR T cells
  • a pharmaceutically acceptable carrier are formulated with a pharmaceutically acceptable carrier.
  • the choice of carrier is determined in part by the particular cell or agent and/or by the method of administration. Accordingly, there are a variety of suitable formulations.
  • the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition.
  • Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
  • Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such
  • Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
  • the formulations can include aqueous solutions.
  • the formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells or agents, where the respective activities do not adversely affect one another.
  • active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
  • the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
  • the pharmaceutical composition in some embodiments contains cells in amounts effective to treat the disease or condition, such as a therapeutically effective or prophylactically effective amount.
  • Therapeutic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs.
  • other dosage regimens may be useful and can be determined.
  • the desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
  • the cells may be administered using standard administration techniques, formulations, and/or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. With respect to cells, administration can be autologous or heterologous.
  • immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject.
  • Peripheral blood derived immunoresponsive cells or their progeny e.g., in vivo, ex vivo or in vitro derived
  • a therapeutic composition e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell
  • it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
  • Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration.
  • the agent or cell populations are administered parenterally.
  • parenteral includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration.
  • the agent or cell populations are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
  • compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH.
  • sterile liquid preparations e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH.
  • Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues.
  • Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
  • carriers can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
  • Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.
  • a suitable carrier such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.
  • formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.
  • the cells can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon’s injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.
  • injection e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon’s injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery.
  • injection e.g., intravenous or subcutaneous injection
  • Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
  • a given dose is administered by a single bolus administration of the cells. In some embodiments, it is administered by multiple bolus administrations of the cells, for example, over a period of no more than 3 days, or by continuous infusion administration of the cells.
  • administration of the cell dose or any additional therapies, e.g., the lymphodepleting therapy, intervention therapy and/or combination therapy is carried out via outpatient delivery.
  • the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, previous therapy, the subject’s clinical history and response to the cells, and the discretion of the attending physician.
  • the compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.
  • a dose of cells is administered to subjects in accord with the provided methods, and/or with the provided articles of manufacture or compositions.
  • the size or timing of the doses is determined as a function of the particular disease or condition (e.g., cancer, e.g., B cell malignancy) in the subject. In some cases, the size or timing of the doses for a particular disease in view of the provided description may be empirically determined.
  • the dose of cells comprises between at or about 2 x 10 5 of the cells/kg and at or about 2 x 10 6 of the cells/kg, such as between at or about 4 x 10 5 of the cells/kg and at or about 1 x 10 6 of the cells/kg or between at or about 6 x 10 5 of the cells/kg and at or about 8 x 10 5 of the cells/kg.
  • the dose of cells comprises no more than 2 x 10 5 of the cells (e.g.
  • antigen-expressing such as CAR-expressing cells
  • CAR-expressing cells per kilogram body weight of the subject (cells/kg), such as no more than at or about 3 x 10 5 cells/kg, no more than at or about 4 x 10 5 cells/kg, no more than at or about 5 x 10 5 cells/kg, no more than at or about 6 x 10 5 cells/kg, no more than at or about 7 x 10 5 cells/kg, no more than at or about 8 x 10 5 cells/kg, no more than at or about 9 x 10 5 cells/kg, no more than at or about 1 x 10 6 cells/kg, or no more than at or about 2 x 10 6 cells/kg.
  • the dose of cells comprises at least or at least about or at or about 2 x 10 5 of the cells e.g. antigenexpressing, such as CAR-expressing cells) per kilogram body weight of the subject (cells/kg), such as at least or at least about or at or about 3 x 10 5 cells/kg, at least or at least about or at or about 4 x 10 5 cells/kg, at least or at least about or at or about 5 x 10 5 cells/kg, at least or at least about or at or about 6 x 10 5 cells/kg, at least or at least about or at or about 7 x 10 5 cells/kg, at least or at least about or at or about 8 x 10 5 cells/kg, at least or at least about or at or about 9 x 10 5 cells/kg, at least or at least about or at or about 1 x 10 6 cells/kg, or at least or at least about or at or about 2 x 10 6 cells/kg.
  • antigenexpressing such as CAR-expressing cells
  • the cells, or individual populations of sub-types of cells are administered to the subject at a range of at or about one million to at or about 100 billion cells and/or that amount of cells per kilogram of body weight, such as, e.g. , 1 million to at or about 50 billion cells (e.g.
  • the dose of cells comprises from at or about 1 x 10 5 to at or about 5 x 10 8 total CAR-expressing T cells, from at or about 1 x 10 5 to at or about 2.5 x 10 8 total CAR-expressing T cells, from at or about 1 x 10 5 to at or about 1 x 10 8 total CAR-expressing T cells, from at or about 1 x 10 5 to at or about 5 x 10 7 total CAR-expressing T cells, from at or about 1 x 10 5 to at or about 2.5 x 10 7 total CAR-expressing T cells, from at or about 1 x 10 5 to at or about 1 x 10 7 total CAR-expressing T cells, from at or about 1 x 10 5 to at or about 5 x 10 6 total CAR-expressing T cells, from at or about 1 x
  • the dose of cells comprises at least or at least about 1 x 10 5 CAR- expressing cells, at least or at least about 2.5 x 10 5 CAR-expressing cells, at least or at least about 5 x 10 5 CAR-expressing cells, at least or at least about 1 x 10 6 CAR-expressing cells, at least or at least about 2.5 x 10 6 CAR-expressing cells, at least or at least about 5 x 10 6 CAR-expressing cells, at least or at least about 1 x 10 7 CAR-expressing cells, at least or at least about 2.5 x 10 7 CAR-expressing cells, at least or at least about 5 x 10 7 CAR-expressing cells, at least or at least about 1 x 10 8 CAR-expressing cells, at least or at least about 2.5 x 10 8 CAR-expressing cells, or at least or at least about 5 x 10 8 CAR-expressing cells.
  • the dose of cells is a flat dose of cells or fixed dose of cells such that the dose of cells is not tied to or based on the body surface area or weight of a subject.
  • the dose includes fewer than at or about 5 x 10 8 total recombinant receptor (e.g., CAR)-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), e.g., in the range of at or about 1 x 10 6 to at or about 5 x 10 8 such cells, such as at or about 2 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , 1 x 10 8 2 x 10 8 , 3 x 10 8 , 4 x 10 8 or 5 x 10 8 total such cells, or the range between any two of the foregoing values.
  • CAR total recombinant receptor
  • PBMCs peripheral blood mononuclear cells
  • the dose includes between at or about 1 x 10 6 and at or 3 x 10 8 total recombinant receptor (e.g., CAR)-expressing cells, e.g., in the range of at or about 1 x 10 7 to at or about 2 x 10 8 such cells, such as at or about 1 x 10 7 , 5 x 10 7 , 1 x 10 8 or 1.5 x 10 8 total such cells, or the range between any two of the foregoing values.
  • the patient is administered multiple doses, and each of the doses or the total dose can be within any of the foregoing values.
  • the dose of cells comprises the administration of from at or about 1 x 10 5 to at or about 5 x 10 8 total recombinant receptor (e.g. CAR)-expressing T cells or total T cells, from at or about 1 x 10 5 to at or about 1 x 10 8 total recombinant receptor e.g. CAR)-expressing T cells or total T cells, from at or about 5 x 10 5 to at or about 1 x 10 7 total recombinant receptor (e.g. CAR)-expressing T cells or total T cells, or from at or about 1 x 10 6 to at or about 1 x 10 7 total recombinant receptor (e.g. CAR)-expressing T cells or total T cells, each inclusive.
  • CAR total recombinant receptor
  • the dose of cells comprises the administration of from at or about 2.5 x 10 7 total recombinant receptor (e.g. CAR)-expressing T cells. In some embodiments, the dose of cells comprises the administration of from at or about 1 x 10 8 total recombinant receptor (e.g. CAR)- expressing T cells.
  • the T cells of the dose include CD4+ T cells, CD8+ T cells or CD4+ and CD8+ T cells. In some embodiments, the T cells of the dose include CD4+ T cells. In some embodiments, the T cells of the dose include CD8+ T cells. In some embodiments, the T cells of the dose include CD4+ and CD8+ T cells.
  • the CD8+ T cells of the dose includes between at or about 1 x 10 6 and at or about 1 x 10 8 total recombinant receptor (e.g., CAR)-expressing CD8+cells, e.g., in the range of at or about 5 x 10 6 to at or about 1 x 10 8 such cells, such cells at or about 1 x 10 7 , 2.5 x 10 7 , 5 x 10 7 , 7.5 x 10 7 , 1 x 10 8 , 1.5 x 10 8 , or 5 x 10 8 total such cells, or the range between any two of the foregoing values.
  • CAR total recombinant receptor
  • the patient is administered multiple doses, and each of the doses or the total dose can be within any of the foregoing values.
  • the dose of cells comprises the administration of from at or about 1 x 10 7 to at or about 0.75 x 10 8 total recombinant receptor-expressing CD8+ T cells, from at or about 1 x 10 7 to at or about 2.5 x 10 7 total recombinant receptor-expressing CD8+ T cells, from at or about 1 x 10 7 to at or about 0.75 x 10 8 total recombinant receptor-expressing CD8+ T cells, each inclusive.
  • the dose of cells comprises the administration of at or about 1 x 10 7 , 2.5 x 10 7 , 5 x 10 7 , 7.5 x 10 7 , 1 x 10 8 , 1.5 x 10 8 , or 5 x 10 8 total recombinant receptorexpressing CD8+ T cells.
  • the CD4+ T cells of the dose includes between at or about 1 x 10 6 and at or about 1 x 10 8 total recombinant receptor (e.g., CAR)-expressing CD4+cells, e.g., in the range of at or about 5 x 10 6 to 1 x 10 8 such cells, such at or about 1 x 10 7 , 2.5 x 10 7 , 5 x 10 7 , 7.5 x 10 7 , 1 x 10 8 , 1.5 x 10 8 , or 5 x 10 8 total such cells, or the range between any two of the foregoing values.
  • CAR total recombinant receptor
  • the patient is administered multiple doses, and each of the doses or the total dose can be within any of the foregoing values.
  • the dose of cells comprises the administration of from at or about 1 x 10 7 to at or about 0.75 x 10 8 total recombinant receptor-expressing CD4+ T cells, from at or about 1 x 10 7 to at or about 2.5 x 10 7 total recombinant receptor-expressing CD4+ T cells, from at or about 1 x 10 7 to at or about 0.75 x 10 8 total recombinant receptor-expressing CD4+ T cells, each inclusive.
  • the dose of cells comprises the administration of at or about 1 x 10 7 , 2.5 x 10 7 , 5 x 10 7 7.5 x 10 7 , 1 x 10 8 , 1.5 x 10 8 , or 5 x 10 8 total recombinant receptor-expressing CD4+ T cells.
  • the dose of cells e.g., recombinant receptor-expressing T cells
  • administration of a given “dose” encompasses administration of the given amount or number of cells as a single composition and/or single uninterrupted administration, e.g., as a single injection or continuous infusion, and also encompasses administration of the given amount or number of cells as a split dose or as a plurality of compositions, provided in multiple individual compositions or infusions, over a specified period of time, such as over no more than 3 days.
  • the dose is a single or continuous administration of the specified number of cells, given or initiated at a single point in time.
  • the dose is administered in multiple injections or infusions over a period of no more than three days, such as once a day for three days or for two days or by multiple infusions over a single day period.
  • the cells of the dose are administered in a single pharmaceutical composition.
  • the cells of the dose are administered in a plurality of compositions, collectively containing the cells of the dose.
  • the term “split dose” refers to a dose that is split so that it is administered over more than one day. This type of dosing is encompassed by the present methods and is considered to be a single dose.
  • the dose of cells may be administered as a split dose, e.g., a split dose administered over time.
  • the dose may be administered to the subject over 2 days or over 3 days.
  • Exemplary methods for split dosing include administering 25% of the dose on the first day and administering the remaining 75% of the dose on the second day. In other embodiments, 33% of the dose may be administered on the first day and the remaining 67% administered on the second day. In some aspects, 10% of the dose is administered on the first day, 30% of the dose is administered on the second day, and 60% of the dose is administered on the third day. In some embodiments, the split dose is not spread over more than 3 days.
  • cells of the dose may be administered by administration of a plurality of compositions or solutions, such as a first and a second, optionally more, each containing some cells of the dose.
  • the plurality of compositions, each containing a different population and/or sub-types of cells are administered separately or independently, optionally within a certain period of time.
  • the populations or sub-types of cells can include CD8 + and CD4 + T cells, respectively, and/or CD8+ and CD4+-enriched populations, respectively, e.g., CD4+ and/or CD8+ T cells each individually including cells genetically engineered to express the recombinant receptor.
  • the administration of the dose comprises administration of a first composition comprising a dose of CD8+ T cells or a dose of CD4+ T cells and administration of a second composition comprising the other of the dose of CD4+ T cells and the CD8+ T cells.
  • the administration of the composition or dose involves administration of the cell compositions separately.
  • the separate administrations are carried out simultaneously, or sequentially, in any order.
  • the dose comprises a first composition and a second composition, and the first composition and second composition are administered from at or about 0 to at or about 12 hours apart, from at or about 0 to at or about 6 hours apart or from at or about 0 to at or about 2 hours apart.
  • the initiation of administration of the first composition and the initiation of administration of the second composition are carried out no more than at or about 2 hours, no more than at or about 1 hour, or no more than at or about 30 minutes apart, no more than at or about 15 minutes, no more than at or about 10 minutes or no more than at or about 5 minutes apart.
  • the initiation and/or completion of administration of the first composition and the completion and/or initiation of administration of the second composition are carried out no more than at or about 2 hours, no more than at or about 1 hour, or no more than at or about 30 minutes apart, no more than at or about 15 minutes, no more than at or about 10 minutes or no more than at or about 5 minutes apart.
  • the first composition and the second composition are mixed prior to the administration into the subject. In some embodiments, the first composition and the second composition are mixed shortly e.g., within at or about 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1.5 hours, 1 hour, or 0.5 hour) before the administration, In some embodiments, the first composition and the second composition are mixed immediately before the administration.
  • the first composition e.g., first composition of the dose
  • the first composition comprises CD4+ T cells.
  • the first composition e.g., first composition of the dose
  • the first composition is administered prior to the second composition.
  • the dose or composition of cells includes a defined or target ratio of CD4+ cells expressing a recombinant receptor to CD8+ cells expressing a recombinant receptor and/or of CD4+ cells to CD8+ cells, which ratio optionally is approximately 1:1 or is between approximately 1:3 and approximately 3:1, such as approximately 1:1.
  • the dose or composition of cells includes a defined or target ratio of CD4+ cells expressing a recombinant receptor to CD8+ cells expressing a recombinant receptor and/or of CD4+ cells to CD8+ cells, which ratio optionally is approximately 1:1.
  • the dose or composition of cells includes a defined or target ratio of CD4+ cells expressing a recombinant receptor to CD8+ cells expressing a recombinant receptor and/or of CD4+ cells to CD8+ cells, which ratio optionally is between approximately 1:3 and approximately 3:1.
  • the administration of a composition or dose with the target or desired ratio of different cell populations involves the administration of a cell composition containing one of the populations and then administration of a separate cell composition comprising the other of the populations, where the administration is at or approximately at the target or desired ratio.
  • administration of a dose or composition of cells at a defined ratio leads to improved expansion, persistence and/or antitumor activity of the T cell therapy.
  • the subject receives multiple doses, e.g., two or more doses or multiple consecutive doses, of the cells.
  • two doses are administered to a subject.
  • the subject receives the consecutive dose, e.g., second dose, approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days after the first dose.
  • multiple consecutive doses are administered following the first dose, such that an additional dose or doses are administered following administration of the consecutive dose.
  • the number of cells administered to the subject in the additional dose is the same as or similar to the first dose and/or consecutive dose.
  • the additional dose or doses are larger than prior doses.
  • the size of the first and/or consecutive dose is determined based on one or more criteria such as response of the subject to prior treatment, e.g. chemotherapy, disease burden in the subject, such as tumor load, bulk, size, or degree, extent, or type of metastasis, stage, and/or likelihood or incidence of the subject developing toxic outcomes, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or a host immune response against the cells and/or recombinant receptors being administered.
  • a host immune response against the cells and/or recombinant receptors being administered e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or a host immune response against the cells and/or recombinant receptors being administered.
  • the time between the administration of the first dose and the administration of the consecutive dose is about 9 to about 35 days, about 14 to about 28 days, or 15 to 27 days. In some embodiments, the administration of the consecutive dose is at a time point more than about 14 days after and less than about 28 days after the administration of the first dose. In some aspects, the time between the first and consecutive dose is about 21 days. In some embodiments, an additional dose or doses, e.g. consecutive doses, are administered following administration of the consecutive dose. In some aspects, the additional consecutive dose or doses are administered at least about 14 and less than about 28 days following administration of a prior dose.
  • the additional dose is administered less than about 14 days following the prior dose, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days after the prior dose. In some embodiments, no dose is administered less than about 14 days following the prior dose and/or no dose is administered more than about 28 days after the prior dose.
  • the dose of cells comprises two doses e.g., a double dose), comprising a first dose of the T cells and a consecutive dose of the T cells, wherein one or both of the first dose and the second dose comprises administration of the split dose of T cells.
  • the dose of cells is generally large enough to be effective in reducing disease burden.
  • the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type(s) and/or a desired ratio of cell types.
  • the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+ to CD8+ ratio.
  • the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types.
  • the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.
  • the populations or sub-types of cells are administered at or within a tolerated difference of a desired dose of total cells, such as a desired dose of T cells.
  • the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg.
  • the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight.
  • the individual populations or sub-types are present at or near a desired output ratio (such as CD4 + to CD8 + ratio), e.g., within a certain tolerated difference or error of such a ratio.
  • a desired output ratio such as CD4 + to CD8 + ratio
  • the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or sub-types of cells, such as a desired dose of CD4+ cells and/or a desired dose of CD8+ cells.
  • the desired dose is a desired number of cells of the sub-type or population, or a desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg.
  • the desired dose is at or above a minimum number of cells of the population or sub-type, or minimum number of cells of the population or sub-type per unit of body weight.
  • the dosage is based on a desired fixed dose of total cells and a desired ratio, and/or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or sub-populations.
  • the dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4 + to CD8 + cells, and/or is based on a desired fixed or minimum dose of CD4 + and/or CD8 + cells.
  • the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4+ and CD8+ cells or sub-types.
  • the desired ratio can be a specific ratio or can be a range of ratios, for example, in some embodiments, the desired ratio (e.g., ratio of CD4 + to CD8 + cells) is between at or about 5:1 and at or about 5:1 (or greater than about 1:5 and less than about 5:1), or between at or about 1:3 and at or about 3:1 (or greater than about 1:3 and less than about 3:1), such as between at or about 2:1 and at or about 1:5 (or greater than about 1:5 and less than about 2:1, such as at or about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9: 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5.
  • the desired ratio
  • the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges.
  • the numbers and/or concentrations of cells refer to the number of recombinant receptor (e.g., CAR)-expressing cells. In other embodiments, the numbers and/or concentrations of cells refer to the number or concentration of all cells, T cells, or peripheral blood mononuclear cells (PBMCs) administered.
  • CAR recombinant receptor
  • PBMCs peripheral blood mononuclear cells
  • the size of the dose is determined based on one or more criteria such as response of the subject to prior treatment, e.g. chemotherapy, disease burden in the subject, such as tumor load, bulk, size, or degree, extent, or type of metastasis, stage, and/or likelihood or incidence of the subject developing toxic outcomes, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or a host immune response against the cells and/or recombinant receptors being administered.
  • toxic outcomes e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or a host immune response against the cells and/or recombinant receptors being administered.
  • the methods also include administering one or more additional doses of cells expressing a chimeric antigen receptor (CAR) and/or lymphodepleting therapy, and/or one or more steps of the methods are repeated.
  • the one or more additional dose is the same as the initial dose.
  • the one or more additional dose is different from the initial dose, e.g., higher, such as 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold or more higher than the initial dose, or lower, such as e.g., higher, such as 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold or 10-fold or more lower than the initial dose.
  • administration of one or more additional doses is determined based on response of the subject to the initial treatment or any prior treatment, disease burden in the subject, such as tumor load, bulk, size, or degree, extent, or type of metastasis, stage, and/or likelihood or incidence of the subject developing toxic outcomes, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or a host immune response against the cells and/or recombinant receptors being administered.
  • toxic outcomes e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or a host immune response against the cells and/or recombinant receptors being administered.
  • the biological activity of the engineered cell populations in some embodiments is measured, e.g., by any of a number of known methods.
  • Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry.
  • the ability of the engineered cells to destroy target cells can be measured using any suitable known methods, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004).
  • the biological activity of the cells is measured by assaying expression and/or secretion of one or more cytokines, such as CD107a, IFNy, IL -2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
  • cytokines such as CD107a, IFNy, IL -2, and TNF.
  • the BCL2 inhibitor e.g. venetoclax
  • a bridging therapy prior to administration of the immunotherapy or cell therapy, such as a T cell therapy (e.g. CAR T cells).
  • the bridging therapy is administered to a subject after collection of autologous cells from a subject to be treated.
  • the bridging therapy is administered to a subject prior to administration of a lymphodepleting chemotherapy, as described in Subsection D below.
  • the bridging therapy is administered after collection of autologous cells from the subject and prior to administration of a lymphodepleting chemotherapy to the subject.
  • the bridging therapy is ceased at least or at least about 1 day prior to administration of the lymphodepleting chemotherapy. In some aspects, the bridging therapy is ceased at or at about 1 day prior to administration of the lymphodepleting chemotherapy. In some aspects, this interval is known as a “washout” period, such that there is a washout period of at least about 1 day between cessation of the bridging therapy and initiation of administration of the lymphodepleting chemotherapy.
  • the bridging therapy comprises administration of a BCL2 inhibitor in weekly escalating doses.
  • the inhibitor is administered to the subject in escalating doses until a maximum dose of 100 mg daily is reached. In some embodiments, the inhibitor is administered to the subject in escalating doses until a maximum dose of 200 mg daily is reached. In some embodiments, the inhibitor is administered to the subject in escalating doses until a maximum dose of 400 mg daily is reached.
  • the bridging therapy comprises daily administration of between about 20 mg and 400 mg of the BCL2 inhibitor. In some embodiments, the bridging therapy comprises daily administration of a first dose of the BCL2 inhibitor for a first week, daily administration of a second dose of the BCL2 inhibitor for a second week, and daily administration of a third dose of the BCL2 inhibitor for a third week. In some embodiments, the second dose is an increased amount of BCL2 inhibitor compared to the first dose, and the third dose is an increased amount of BCL2 inhibitor compared to the second dose.
  • a subject is administered 20 mg per day of a BCL2 inhibitor for a first week. In some embodiments, a subject is administered 50 mg per day of a BCL2 inhibitor for a second week. In some embodiments, a subject is administered 100 mg per day of a BCL2 inhibitor for a third week. Thus, in some embodiments, a subject is administered 20 mg per day of a BCL2 inhibitor for a first week, 50 mg per day of a BCL2 inhibitor for a second week, and 100 mg per day of a BCL2 inhibitor for a third week.
  • bridging therapy comprises administration of 20 mg per day of a BCL2 inhibitor for a first week, 50 mg per day of a BCL2 inhibitor for a second week, and 100 mg per day of a BCL2 inhibitor for a third week. In some embodiments, the bridging therapy ceases after the third week of treatment.
  • the bridging therapy further comprises administration of an anti- CD20 antibody. In some embodiments, the bridging therapy further comprises administration of an anti- CD20 antibody if the subject has been previously treated with a BCL2 inhibitor (e.g. venetoclax). In some embodiments, if the subject has not been previously treated with a BCL2 inhibitor (e.g. venetoclax), the bridging therapy does not further comprise administration of the inhibitor.
  • a BCL2 inhibitor e.g. venetoclax
  • the bridging therapy further comprises administration of a BTK inhibitor (e.g. ibrutinib.)
  • the bridging therapy further comprises administration of the BTK inhibitor (e.g. ibrutinib) if the subject was receiving treatment with ibrutinib prior to treatment with the combination therapy.
  • the subject is administered a BTK inhibitor (e.g. ibrutinib) until autologous cells are collected from the subject and again during the bridging therapy.
  • the BCL2 inhibitor provided in the bridging therapy is venetoclax.
  • the BCL2 inhibitor provided in the bridging therapy is the same BCL2 inhibitor provided in the dosing regimen following administration of the cell therapy, e.g. CAR T cells, to the subject.
  • a subject receives BCL2 inhibitor bridging therapy for about three weeks prior to administration of CAR T cells, and a BCL2 inhibitor dosing regimen after administration of CAR T cells.
  • the BCL2 inhibitor administered in the bridging therapy and the BCL2 inhibitor administered in the dosing regimen are the same BCL2 inhibitor. In some cases, the BCL2 inhibitor administered in the bridging therapy and the BCL2 inhibitor administered in the dosing regimen are different BCL2 inhibitors. In some embodiments, the BCL2 inhibitor is venetoclax or navitoclax. In some embodiments, the BCL2 inhibitor is venetoclax. In some embodiments, the BCL2 inhibitor administered in the bridging therapy is venetoclax. In some embodiments, the BCL2 inhibitor administered in the bridging therapy is navitoclax.
  • a subject undergoes collection of autologous cells (e.g. leukapheresis), bridging therapy with the BCL2 inhibitor (e.g. venetoclax) for about three weeks, a washout period of about 1 day, lymphodepletion, initiation of administration of CAR T cells, and initiation of administration of the dosing regmen of the BCL2 inhibitor (e.g., venetoclax) about 1 day after initiation of administration of the CAR T cells, in that order.
  • autologous cells e.g. leukapheresis
  • the BCL2 inhibitor e.g. venetoclax
  • the provided methods can further include administering one or more lymphodepleting therapies, such as prior to or simultaneous with initiation of administration of the immunotherapy or cell thearpy, such as a T cell therapy (e.g. CAR-expressing T cells).
  • a T cell therapy e.g. CAR-expressing T cells.
  • the lymphodepleting therapy comprises administration of a phosphamide, such as cyclophosphamide.
  • the lymphodepleting therapy can include administration of fludarabine.
  • preconditioning subjects with immunodepleting can improve the effects of adoptive cell therapy (ACT).
  • ACT adoptive cell therapy
  • lymphodepleting agents including combinations of cyclosporine and fludarabine, have been effective in improving the efficacy of transferred tumor infiltrating lymphocyte (TIL) cells in cell therapy, including to improve response and/or persistence of the transferred cells.
  • TIL tumor infiltrating lymphocyte
  • lymphodepleting agents most commonly cyclophosphamide, fludarabine, bendamustine, or combinations thereof, sometimes accompanied by low-dose irradiation. See Han et al. Journal of Hematology & Oncology, 6:47 (2013); Kochenderfer et al., Blood, 119: 2709-2720 (2012); Kalos et al., Sci Transl Med, 3(95):95ra73 (2011); Clinical Trial Study Record Nos.: NCT02315612; NCT01822652.
  • Such preconditioning can be carried out with the goal of reducing the risk of one or more of various outcomes that could dampen efficacy of the therapy.
  • These include the phenomenon known as “cytokine sink,” by which T cells, B cells, NK cells compete with TILs for homeostatic and activating cytokines, such as IL -2, IL-7, and/or IL-15; suppression of TILs by regulatory T cells, NK cells, or other cells of the immune system; impact of negative regulators in the tumor microenvironment.
  • cytokine sink by which T cells, B cells, NK cells compete with TILs for homeostatic and activating cytokines, such as IL -2, IL-7, and/or IL-15
  • suppression of TILs by regulatory T cells, NK cells, or other cells of the immune system
  • impact of negative regulators in the tumor microenvironment Muranski et al., Nat Clin Pract Oncol. December; 3(12): 668-681 (2006).
  • the provided method further involves administering a lymphodepleting therapy to the subject.
  • the method involves administering the lymphodepleting therapy to the subject prior to the initiation of the administration of the dose of cells.
  • the lymphodepleting therapy contains a chemotherapeutic agent such as fludarabine and/or cyclophosphamide.
  • the administration of the cells and/or the lymphodepleting therapy is carried out via outpatient delivery.
  • the methods include administering a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, to a subject prior to the initiation of the administration of the dose of cells.
  • a preconditioning agent such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof
  • the subject may be administered a preconditioning agent at least 2 days prior, such as at least 3, 4, 5, 6, or 7 days prior, to the first or subsequent dose.
  • the subject is administered a preconditioning agent no more than 7 days prior, such as no more than 6, 5, 4, 3, or 2 days prior, to the initiation of administration of the dose of cells.
  • the subject is administered a preconditioning agent between 2 and 7, inclusive, such as at 2, 3, 4, 5, 6, or 7, days prior to the initiation of the administration of the dose of cells.
  • the subject is preconditioned with cyclophosphamide at a dose between or between about 20 mg/kg and 100 mg/kg, such as between or between about 40 mg/kg and 80 mg/kg. In some aspects, the subject is preconditioned with or with about 60 mg/kg of cyclophosphamide.
  • the cyclophosphamide can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some embodiments, the cyclophosphamide is administered once daily for one or two days.
  • the subject is administered cyclophosphamide at a dose between or between about 100 mg/m 2 and 500 mg/m 2 , such as between or between about 200 mg/m 2 and 400 mg/m 2 , or 250 mg/m 2 and 350 mg/m 2 , inclusive. In some instances, the subject is administered about 300 mg/m 2 of cyclophosphamide. In some embodiments, the cyclophosphamide can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days.
  • cyclophosphamide is administered daily, such as for 1-5 days, for example, for 3 to 5 days. In some instances, the subject is administered about 300 mg/m 2 of cyclophosphamide, daily for 3 days, prior to initiation of the cell therapy.
  • the subject is administered fludarabine at a dose between or between about 1 mg/m 2 and 100 mg/m 2 , such as between or between about 10 mg/m 2 and 75 mg/m 2 , 15 mg/m 2 and 50 mg/m 2 , 20 mg/m 2 and 40 mg/m 2 ” 24 mg/m 2 and 35 mg/m 2 , 20 mg/m 2 and 30 mg/m 2 , or 24 mg/m 2 and 26 mg/m 2 .
  • the subject is administered 25 mg/m 2 of fludarabine.
  • the subject is administered about 30 mg/m 2 of fludarabine.
  • the fludarabine can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some embodiments, fludarabine is administered daily, such as for 1-5 days, for example, for 3 to 5 days. In some instances, the subject is administered about 30 mg/m 2 of fludarabine, daily for 3 days, prior to initiation of the cell therapy.
  • the lymphodepleting agent comprises a combination of agents, such as a combination of cyclophosphamide and fludarabine.
  • the combination of agents may include cyclophosphamide at any dose or administration schedule, such as those described above, and fludarabine at any dose or administration schedule, such as those described above.
  • the subject is administered 60 mg/kg ( ⁇ 2 g/m 2 ) of cyclophosphamide and 3 to 5 doses of 25 mg/m 2 fludarabine prior to the dose of cells.
  • the subject is administered about 300 mg/m 2 cyclophosphamide and about 30 mg/m 2 fludarabine each daily for 3 days.
  • the preconditioning administration schedule ends between 2 and 7, inclusive, such as at 2, 3, 4, 5, 6, or 7, days prior to the initiation of the administration of the dose of cells.
  • a lymphodepleting preconditioning chemotherapy of cyclophosphamide and fludarabine (CY/FLU)
  • CY/FLU lymphodepleting preconditioning chemotherapy of cyclophosphamide and fludarabine
  • a subject is treated with BCL2 inhibitor (e.g. venetoclax) bridging therapy prior to receiving a lymphodepleting preconditioning chemotherapy of cyclophosphamide and fludarabine (CY/FLU), wherein the bridging therapy is ceased at least about one day before the subject receives the lymphodepleting therapy.
  • this interval is known as a “washout” period.
  • washout period of one day between conclusion of the bridging therapy and initiation of administration of the lymphodepleting preconditioning chemotherapy.
  • subjects are administered the dose of CAR-expressing T cells as described above.
  • the administration of the preconditioning agent prior to infusion of the dose of cells improves an outcome of the treatment.
  • preconditioning improves the efficacy of treatment with the dose or increases the persistence of the recombinant receptorexpressing cells (e.g., CAR-expressing cells, such as CAR-expressing T cells) in the subject.
  • preconditioning treatment increases disease-free survival, such as the percent of subjects that are alive and exhibit no minimal residual or molecularly detectable disease after a given period of time following the dose of cells. In some embodiments, the time to median disease-free survival is increased.
  • the biological activity of the engineered cell populations in some aspects is measured by any of a number of known methods.
  • Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry.
  • the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009) , and Herman et al. J.
  • the biological activity of the cells also can be measured by assaying expression and/or secretion of certain cytokines, such as CD 107a, IFNy, IL-2, and TNF.
  • the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
  • toxic outcomes, persistence and/or expansion of the cells, and/or presence or absence of a host immune response are assessed.
  • the administration of the preconditioning agent prior to infusion of the dose of cells improves an outcome of the treatment such as by improving the efficacy of treatment with the dose or increases the persistence of the recombinant receptor-expressing cells (e.g., CAR- expressing cells, such as CAR-expressing T cells) in the subject. Therefore, in some embodiments, the dose of preconditioning agent given in the method which is a combination therapy with the BCL2 inhibitor and cell therapy is higher or lower than the dose given in the method without the inhibitor. In some embodiments, the dose of preconditioning agent given in the method which is a combination therapy with the BCL2 inhibitor and cell therapy is higher than the dose given in the method without the inhibitor. In some embodiments, the dose of preconditioning agent given in the method which is a combination therapy with the BCL2 inhibitor and cell therapy is lower than the dose given in the method without the inhibitor.
  • the dose of preconditioning agent given in the method which is a combination therapy with the BCL2 inhibitor and cell therapy is higher than the dose given in the method without the inhibitor
  • the cells contain or are engineered to contain an engineered receptor, e.g., an engineered antigen receptor, such as a chimeric antigen receptor (CAR), or a T cell receptor (TCR).
  • an engineered receptor e.g., an engineered antigen receptor, such as a chimeric antigen receptor (CAR), or a T cell receptor (TCR).
  • populations of such cells compositions containing such cells and/or enriched for such cells, such as in which cells of a certain type such as T cells or CD8+ or CD4+ cells are enriched or selected.
  • pharmaceutical compositions and formulations for administration such as for adoptive cell therapy.
  • therapeutic methods for administering the cells and compositions to subjects e.g., patients.
  • the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids.
  • gene transfer is accomplished by first stimulating the cells, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.
  • the cell therapy for use in accord with the provided combination therapy methods includes administering engineered cells expressing recombinant receptors designed to recognize and/or specifically bind to molecules associated with the disease or condition, such as a cancer (e.g., a CLL or SLL), and result in a response, such as an immune response against such molecules upon binding to such molecules.
  • the receptors may include chimeric receptors, e.g., chimeric antigen receptors (CARs), and other transgenic antigen receptors including transgenic T cell receptors (TCRs).
  • the engineered cells such as T cells express a chimeric receptor, such as a chimeric antigen receptor (CAR), that contains one or more domains that combine a ligand-binding domain (e.g. antibody or antibody fragment) that provides specificity for a desired antigen (e.g., tumor antigen) with intracellular signaling domains.
  • a ligand-binding domain e.g. antibody or antibody fragment
  • the intracellular signaling domain is an activating intracellular domain portion, such as a T cell activating domain, providing a primary activation signal.
  • the intracellular signaling domain contains or additionally contains a costimulatory signaling domain to facilitate effector functions.
  • the receptor Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immunostimulatory signal, such as an IT AM-transduced signal, into the cell, thereby promoting an immune response targeted to the disease or condition.
  • an immunostimulatory signal such as an IT AM-transduced signal
  • chimeric receptors when genetically engineered into immune cells can modulate T cell activity, and, in some cases, can modulate T cell differentiation or homeostasis, thereby resulting in genetically engineered cells with improved longevity, survival and/or persistence in vivo, such as for use in adoptive cell therapy methods.
  • Exemplary antigen receptors including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, W02013/123061, U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S.
  • the antigen receptors include a CAR as described in U.S. Patent No.: 7,446,190, and those described in International Patent Application Publication No.: WO/2014055668 Al.
  • the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, U.S. Patent No.: 7,446,190, US Patent No.: 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, U.S. Patent No.: 7,446,190, and US Patent No.: 8,389,282.
  • the engineered cells express a recombinant receptor such as a chimeric antigen receptor (CAR) with specificity for a particular antigen (or marker or ligand), such as an antigen expressed on the surface of a particular cell type.
  • a recombinant receptor such as a chimeric antigen receptor (CAR) with specificity for a particular antigen (or marker or ligand), such as an antigen expressed on the surface of a particular cell type.
  • the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule.
  • the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on the engineered cells.
  • the chimeric receptors such as CARs, generally include an extracellular antigen binding domain that is an antigen-binding portion or portions of an antibody molecule.
  • the antigen-binding domain is a portion of an antibody molecule, generally a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment.
  • the antigen-binding domain is a single domain antibody (sdAb), such as sdFv, nanobody, VHH and VNAR.
  • an antigen-binding fragment comprises antibody variable regions joined by a flexible linker.
  • the chimeric receptors such as CARs, generally include an extracellular antigen binding domain, such as a portion of an antibody molecule, generally a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment.
  • the CAR contains an antibody or an antigen-binding fragment e.g. scFv) that specifically recognizes an antigen, such as an intact antigen, expressed on the surface of a cell.
  • the antigen receptors are a CAR containing an extracellular ntigen binding domain, such as antibody or antigen-binding fragment, that exhibits TCR-like specificity directed against peptide- MHC complexes, which also may be referred to as a TCR-like CAR.
  • the extracellular antigen binding domain specific for an MHC-peptide complex of a TCR-like CAR is linked to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s).
  • such molecules can typically mimic or approximate a signal through a natural antigen receptor, such as a TCR, and, optionally, a signal through such a receptor in combination with a costimulatory receptor.
  • MHC Major histocompatibility complex
  • a protein generally a glycoprotein, that contains a polymorphic peptide binding site or binding groove that can, in some cases, complex with peptide antigens of polypeptides, including peptide antigens processed by the cell machinery.
  • MHC molecules can be displayed or expressed on the cell surface, including as a complex with peptide, i.e. MHC-peptide complex, for presentation of an antigen in a conformation recognizable by an antigen receptor on T cells, such as a TCRs or TCR-like antibody.
  • MHC class I molecules are heterodimers having a membrane spanning a chain, in some cases with three a domains, and a non-covalently associated [32 microglobulin.
  • MHC class II molecules are composed of two transmembrane glycoproteins, a and [3, both of which typically span the membrane.
  • An MHC molecule can include an effective portion of an MHC that contains an antigen binding site or sites for binding a peptide and the sequences necessary for recognition by the appropriate antigen receptor.
  • MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where a MHC-peptide complex is recognized by T cells, such as generally CD8 + T cells, but in some cases CD4+ T cells.
  • MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are typically recognized by CD4 + T cells.
  • MHC molecules are encoded by a group of linked loci, which are collectively termed H-2 in the mouse and human leukocyte antigen (HLA) in humans.
  • HLA human leukocyte antigen
  • typically human MHC can also be referred to as human leukocyte antigen (HLA).
  • HLA human leukocyte antigen
  • MHC -peptide complex or “peptide -MHC complex” or variations thereof, refers to a complex or association of a peptide antigen and an MHC molecule, such as, generally, by non- covalent interactions of the peptide in the binding groove or cleft of the MHC molecule.
  • the MHC -peptide complex is present or displayed on the surface of cells.
  • the MHC -peptide complex can be specifically recognized by an antigen receptor, such as a TCR, TCR-like CAR or antigen-binding portions thereof.
  • a peptide, such as a peptide antigen or epitope, of a polypeptide can associate with an MHC molecule, such as for recognition by an antigen receptor.
  • the peptide is derived from or based on a fragment of a longer biological molecule, such as a polypeptide or protein.
  • the peptide typically is about 8 to about 24 amino acids in length.
  • a peptide has a length of from or from about 9 to 22 amino acids for recognition in the MHC Class II complex.
  • a peptide has a length of from or from about 8 to 13 amino acids for recognition in the MHC Class I complex.
  • the antigen receptor upon recognition of the peptide in the context of an MHC molecule, such as MHC -peptide complex, the antigen receptor, such as TCR or TCR-like CAR, produces or triggers an activation signal to the T cell that induces a T cell response, such as T cell proliferation, cytokine production, a cytotoxic T cell response or other response.
  • a TCR-like antibody or antigen-binding portion are known or can be produced by known methods (see e.g. US Published Application Nos. US 2002/0150914; US 2003/0223994; US 2004/0191260; US 2006/0034850; US 2007/00992530; US20090226474; US20090304679; and International PCT Publication No. WO 03/068201).
  • an antibody or antigen-binding portion thereof that specifically binds to a MHC-peptide complex can be produced by immunizing a host with an effective amount of an immunogen containing a specific MHC-peptide complex.
  • the peptide of the MHC-peptide complex is an epitope of antigen capable of binding to the MHC, such as a tumor antigen, for example a universal tumor antigen, or other antigen as described below.
  • an effective amount of the immunogen is then administered to a host for eliciting an immune response, wherein the immunogen retains a three-dimensional form thereof for a period of time sufficient to elicit an immune response against the three-dimensional presentation of the peptide in the binding groove of the MHC molecule.
  • Serum collected from the host is then assayed to determine if desired antibodies that recognize a three-dimensional presentation of the peptide in the binding groove of the MHC molecule is being produced.
  • the produced antibodies can be assessed to confirm that the antibody can differentiate the MHC-peptide complex from the MHC molecule alone, the peptide of interest alone, and a complex of MHC and irrelevant peptide. The desired antibodies can then be isolated.
  • an antibody or antigen-binding portion thereof that specifically binds to an MHC-peptide complex can be produced by employing antibody library display methods, such as phage antibody libraries.
  • phage display libraries of mutant Fab, scFv or other antibody forms can be generated, for example, in which members of the library are mutated at one or more residues of a CDR or CDRs. See e.g. US published application No. US20020150914, US2014/0294841; and Cohen CJ. et al. (2003) J Mol. Recogn. 16:324-332.
  • antibody herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab’)2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rlgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.
  • Fab fragment antigen binding
  • rlgG fragment antigen binding
  • VH variable heavy chain
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • antibody should be understood to encompass functional antibody fragments thereof.
  • the term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
  • the antigen-binding proteins, antibodies and antigen binding fragments thereof specifically recognize an antigen of a full-length antibody.
  • the heavy and light chains of an antibody can be full-length or can be an antigen-binding portion (a Fab, F(ab’)2, Fv or a single chain Fv fragment (scFv)).
  • the antibody heavy chain constant region is chosen from, e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE, particularly chosen from, e.g., IgGl, IgG2, IgG3, and IgG4, more particularly, IgGl e.g., human IgGl).
  • the antibody light chain constant region is chosen from, e.g., kappa or lambda, particularly kappa.
  • antibody fragments refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; variable heavy chain (VH) regions, singlechain antibody molecules such as scFvs and single-domain VH single antibodies; and multispecific antibodies formed from antibody fragments.
  • the antibodies are single -chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
  • CDR complementarity determining region
  • HVR hypervariable region
  • CDR-H1, CDR-H2, CDR-H3 three CDRs in each heavy chain variable region
  • CDR-L1, CDR-L2, CDR-L3 three CDRs in each light chain variable region
  • “Framework regions” and “FR” are known, in some cases, to refer to the non-CDR portions of the variable regions of the heavy and light chains.
  • FR-H1, FR- H2, FR-H3, and FR-H4 there are four FRs in each full-length heavy chain variable region (FR-H1, FR- H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).
  • the boundaries of a given CDR or FR may vary depending on the scheme used for identification.
  • the Kabat scheme is based on structural alignments
  • the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering.
  • the Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme.
  • the AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular’s AbM antibody modeling software.
  • Table 2 lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively.
  • residue numbering is listed using both the Kabat and Chothia numbering schemes.
  • FRs are located between CDRs, for example, with FR-L1 located before CDR-L1, FR-L2 located between CDR- L1 and CDR-L2, FR-L3 located between CDR-L2 and CDR-L3 and so forth.
  • CDR complementary determining region
  • individual specified CDRs e.g., CDR-H1, CDR-H2, CDR-H3
  • CDR-H1, CDR-H2, CDR-H3 individual specified CDRs
  • a particular CDR e.g., a CDR-H3
  • a CDR-H3 contains the amino acid sequence of a corresponding CDR in a given VH or VL region amino acid sequence
  • a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the aforementioned schemes, or other known schemes.
  • specific CDR sequences are specified. Exemplary CDR sequences of provided antibodies are described using various numbering schemes, although it is understood that a provided antibody can include CDRs as described according to any of the other aforementioned numbering schemes or other numbering schemes known to a skilled artisan.
  • FR or individual specified FR(s) e.g., FR-H1, FR- H2, FR-H3, FR-H4
  • FR-H1, FR- H2, FR-H3, FR-H4 should be understood to encompass a (or the specific) framework region as defined by any of the known schemes.
  • the scheme for identification of a particular CDR, FR, or FRs or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, AbM or Contact method, or other known schemes.
  • the particular amino acid sequence of a CDR or FR is given.
  • variable region refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs.
  • FRs conserved framework regions
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
  • Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody.
  • a single-domain antibody is a human single-domain antibody.
  • the CAR comprises an antibody heavy chain domain that specifically binds the antigen, such as a cancer marker or cell surface antigen of a cell or disease to be targeted, such as a tumor cell or a cancer cell, such as any of the target antigens described herein or known.
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells.
  • the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody.
  • the antibody fragments are scFvs.
  • a “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs.
  • a humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody.
  • a “humanized form” of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody.
  • some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
  • the recombinant receptor such as a chimeric receptor (e.g. CAR)
  • a chimeric receptor e.g. CAR
  • an extracellular antigen binding domain such as an antibody or antigen-binding fragment (e.g. scFv), that binds, such as specifically binds, to an antigen (or a ligand).
  • an antigen binding domain such as an antibody or antigen-binding fragment (e.g. scFv)
  • binds such as specifically binds, to an antigen (or a ligand).
  • the antigens targeted by the chimeric receptors are those expressed in the context of a disease, condition, or cell type to be targeted via the adoptive cell therapy.
  • diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and/or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas.
  • cancers and tumors including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and/or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas.
  • the antigen targeted by the receptor is or comprises selected from among av(36 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer/testis antigen IB (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial
  • Antigens targeted by the receptors include antigens associated with a B cell malignancy, such as any of a number of known B cell marker.
  • the antigen targeted by the receptor is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.
  • the antigen targeted by the receptor is CD19.
  • the disease or condition is a B cell malignancy, and the antigen is CD19.
  • the disease or condition is chronic lymphocytic leukemia (CLL), and the antigen is CD 19.
  • the disease or condition is small lymphocytic lymphoma (SLL), and the antigen is CD19.
  • Antigens targeted by the receptors include antigens associated with a B cell malignancy, such as any of a number of known B cell marker.
  • the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.
  • the antigen is CD19.
  • any of such antigens are antigens expressed on human B cells.
  • the antibody or an antigen-binding fragment specifically recognizes an antigen, such as CD19.
  • the antibody or antigen-binding fragment is derived from, or is a variant of, antibodies or antigen-binding fragment that specifically binds to CD19.
  • the antigen is CD19.
  • the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD 19.
  • the antibody or antibody fragment that binds CD 19 is a mouse derived antibody such as FMC63 and SJ25C1.
  • the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US 2016/0152723.
  • the antigen-binding domain includes a VH and/or VL derived from FMC63, which, in some aspects, can be an scFv.
  • FMC63 generally refers to a mouse monoclonal IgGl antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, N. R., et al. (1987). Leucocyte typing HI. 302).
  • the FMC63 antibody comprises CDR-H1 and CDR-H2 set forth in SEQ ID NO: 38 and 39, respectively, and CDR-H3 set forth in SEQ ID NO: 40 or 54 and CDR-L1 set forth in SEQ ID NO: 35 and CDR-L2 set forth in SEQ ID NO: 36 or 55 and CDR-L3 sequences set forth in SEQ ID NO: 37 or 56.
  • the FMC63 antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 41 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 42.
  • the scFv comprises a variable light chain containing the CDR-L1 sequence of SEQ ID NO:35, a CDR-L2 sequence of SEQ ID NO:36, and a CDR-L3 sequence of SEQ ID NO:37 and/or a variable heavy chain containing a CDR-H1 sequence of SEQ ID NO:38, a CDR-H2 sequence of SEQ ID NO:39, and a CDR-H3 sequence of SEQ ID NO:40, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
  • the scFv comprises a variable heavy chain region of FMC63 set forth in SEQ ID NO:41 and a variable light chain region of FMC63 set forth in SEQ ID NO:42, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
  • the variable heavy and variable light chains are connected by a linker.
  • the linker is set forth in SEQ ID NO:59.
  • the scFv comprises, in order, a VH, a linker, and a VL- In some embodiments, the scFv comprises, in order, a VL, a linker, and a VH- In some embodiments, the scFv is encoded by a sequence of nucleotides set forth in SEQ ID NO:57 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:57.
  • the scFv comprises the sequence of amino acids set forth in SEQ ID NO:43 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:43.
  • the antigen-binding domain includes a VH and/or VL derived from SJ25C1, which, in some aspects, can be an scFv.
  • SJ25C1 is a mouse monoclonal IgGl antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, N. R., et al. (1987). Leucocyte typing III. 302).
  • the SJ25C1 antibody comprises CDR-H1, CDR-H2 and CDR- H3 set forth in SEQ ID NOS: 47-49, respectively, and CDR-L1, CDR-L2 and CDR-L3 sequences set forth in SEQ ID NOS: 44-46, respectively.
  • the SJ25C1 antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 50 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 51.
  • the scFv comprises a variable light chain containing a CDR-L1 sequence of SEQ ID NO:44, a CDR-L2 sequence of SEQ ID NO: 45, and a CDR-L3 sequence of SEQ ID NO:46 and/or a variable heavy chain containing a CDR-H1 sequence of SEQ ID NO:47, a CDR-H2 sequence of SEQ ID NO:48, and a CDR-H3 sequence of SEQ ID NO:49, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
  • the scFv comprises a variable heavy chain region of SJ25C1 set forth in SEQ ID NO:50 and a variable light chain region of SJ25C1 set forth in SEQ ID NO:51, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
  • the variable heavy and variable light chains are connected by a linker.
  • the linker is set forth in SEQ ID NO:52.
  • the scFv comprises, in order, a VH, a linker, and a VL- In some embodiments, the scFv comprises, in order, a VL, a linker, and a VH- In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 53 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 53.
  • the recombinant receptor e.g., a chimeric antigen receptor
  • the recombinant receptor includes an extracellular portion containing one or more ligand- (e.g., antigen-) binding domains, such as an antibody or fragment thereof, and one or more intracellular signaling region or domain (also interchangeably called a cytoplasmic signaling domain or region).
  • the antibody or fragment includes an scFv.
  • the chimeric antigen receptor includes an extracellular portion containing an antibody or fragment and an intracellular signaling region.
  • the intracellular signaling region comprises an intracellular signaling domain.
  • the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain that is capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and/or a signaling domain comprising an immunoreceptor tyrosine-based activation motif (IT AM).
  • the recombinant receptor e.g., CAR
  • the spacer and/or transmembrane domain can link the extracellular portion containing the ligand- (e.g., antigen-) binding domain and the intracellular signaling region(s) or domain(s)
  • the recombinant receptor such as the CAR, further includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region, e.g., an IgG4 hinge region, and/or a CH1/CL and/or Fc region.
  • the recombinant receptor further comprises a spacer and/or a hinge region.
  • the constant region or portion is of a human IgG, such as IgG4 or IgGl.
  • the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain.
  • the spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. In some examples, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length.
  • Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges.
  • a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less.
  • Exemplary spacers include IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain.
  • Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, Hudecek et al. (2015) Cancer Immunol Res. 3(2): 125-135 or international patent application publication number WO2014031687, U.S. Patent No. 8,822,647 or published app. No. US2014/0271635.
  • the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgGl, such as the hinge only spacer set forth in SEQ ID NO: 1, and encoded by the sequence set forth in SEQ ID NO: 2.
  • the spacer is an Ig hinge, e.g., and IgG4 hinge, linked to a CH2 and/or CH3 domains.
  • the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ ID NO:4.
  • the spacer the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only, such as set forth in SEQ ID NO: 3.
  • the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers.
  • the constant region or portion is of IgD.
  • the spacer has the sequence set forth in SEQ ID NO: 5.
  • the spacer has a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 1, 3, 4 and 5.
  • the spacer has a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity SEQ ID NO: 1.
  • the spacer comprises the sequence set forth in SEQ ID NO: 1.
  • the spacer is a polypeptide spacer that (a) comprises or consists of all or a portion of an immunoglobulin hinge or a modified version thereof or comprises about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region, (b) comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof and/or comprises about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region, or (c) is at or about 12 amino acids in length and/or comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4, or a modified version thereof; or (d) consists or comprises the sequence of amino acids set forth in SEQ ID NOS: 1, 3-5, 27-34 or 58, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 9
  • the antigen receptor comprises an intracellular domain linked directly or indirectly to the extracellular domain.
  • the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain.
  • the intracellular signaling domain comprises an IT AM.
  • the antigen recognition domain e.g. extracellular domain
  • the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g. scFv) and intracellular signaling domain.
  • the antigen-binding component e.g., antibody
  • the antigen-binding component is linked to one or more transmembrane and intracellular signaling domains.
  • a transmembrane domain that naturally is associated with one of the domains in the receptor e.g., CAR
  • the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • the transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 (4-1BB), or CD154. Alternatively the transmembrane domain in some embodiments is synthetic.
  • the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • the linkage is by linkers, spacers, and/or transmembrane domain(s).
  • the transmembrane domain contains a transmembrane portion of CD28 or a variant thereof.
  • the extracellular domain and transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein.
  • the transmembrane domain of the receptor e.g., the CAR is a transmembrane domain of human CD28 or variant thereof, e.g., a 27-amino acid transmembrane domain of a human CD28 (Accession No.: P10747.1), or is a transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 8 or a sequence of amino acids that exhibits at least or at least about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8.
  • the transmembrane-domain containing portion of the recombinant receptor comprises the sequence of amino acids set forth in SEQ ID NO: 9 or a sequence of amino acids having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
  • the recombinant receptor e.g., CAR
  • the recombinant receptor includes at least one intracellular signaling component or components, such as an intracellular signaling region or domain.
  • T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences).
  • the CAR includes one or both of such signaling components.
  • a short oligo- or polypeptide linker for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
  • the cytoplasmic domain or intracellular signaling region of the CAR activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR.
  • the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors.
  • a truncated portion of an intracellular signaling region of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal.
  • the intracellular signaling regions include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capability.
  • the intracellular signaling regions include the cytoplasmic sequences of a region or domain that is involved in providing costimulatory signal.
  • the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex.
  • Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine -based activation motifs or IT AMs.
  • IT AM containing primary cytoplasmic signaling sequences include those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon.
  • cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
  • the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain.
  • the antigen-binding portion is linked to one or more cell signaling modules.
  • cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains.
  • the receptor e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor y, CD8alpha, CD8beta, CD4, CD25, or CD16.
  • the CAR or other chimeric receptor includes a chimeric molecule between CD3-zeta (CD3-Q or Fc receptor y and CD8alpha, CD8beta, CD4, CD25 or CD 16.
  • the intracellular (or cytoplasmic) signaling region comprises a human CD3 chain, optionally a CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD3 ⁇ (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Patent No.: 7,446,190 or U.S. Patent No. 8,911,993.
  • the intracellular signaling region comprises the sequence of amino acids set forth in SEQ ID NO: 13, 14 or 15 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, 14 or 15.
  • the intracellular signaling region comprises the sequence of amino acids set forth in SEQ ID NO: 13 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13. In some embodiments, the intracellular signaling region comprises the sequence of amino acids set forth in SEQ ID NO: 13.
  • full activation In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal.
  • a component for generating secondary or co-stimulatory signal is also included in the CAR.
  • the CAR does not include a component for generating a costimulatory signal.
  • an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.
  • the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule.
  • the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, 0X40 (CD134), CD27, DAP10, DAP12, ICOS and/or other costimulatory receptors.
  • the CAR includes a costimulatory region or domain of CD28 or 4-1BB, such as of human CD28 or human 4-1BB.
  • the intracellular signaling region or domain comprises an intracellular costimulatory signaling domain of human CD28 or functional variant or portion thereof, such as a 41 amino acid domain thereof and/or such a domain with an LL to GG substitution at positions 186-187 of a native CD28 protein.
  • the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 10 or 11 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10 or 11.
  • the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 10 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10.
  • the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 11 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11.
  • the intracellular region comprises an intracellular costimulatory signaling domain of 4- IBB or functional variant or portion thereof, such as a 42-amino acid cytoplasmic domain of a human 4-1BB (Accession No.
  • the intracellular region comprises an intracellular costimulatory signaling domain comprising the sequence of amino acids set forth in SEQ ID NO: 12.
  • the same CAR includes both the primary (or activating) cytoplasmic signaling regions and costimulatory signaling components.
  • the activating domain is included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen.
  • the CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014/055668).
  • the cells include one or more stimulatory or activating CAR and/or a costimulatory CAR.
  • the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sei. Transl.
  • the two receptors induce, respectively, an activating and an inhibitory signal to the cell, such that ligation of one of the receptor to its antigen activates the cell or induces a response, but ligation of the second inhibitory receptor to its antigen induces a signal that suppresses or dampens that response.
  • activating CARs and inhibitory CARs iCARs
  • Such a strategy may be used, for example, to reduce the likelihood of off-target effects in the context in which the activating CAR binds an antigen expressed in a disease or condition but which is also expressed on normal cells, and the inhibitory receptor binds to a separate antigen which is expressed on the normal cells but not cells of the disease or condition.
  • the chimeric receptor is or includes an inhibitory CAR (e.g. iCAR) and includes intracellular components that dampen or suppress an immune response, such as an ITAM- and/or co stimulatory-promoted response in the cell.
  • an immune response such as an ITAM- and/or co stimulatory-promoted response in the cell.
  • intracellular signaling components are those found on immune checkpoint molecules, including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptors, EP2/4 Adenosine receptors including A2AR.
  • the engineered cell includes an inhibitory CAR including a signaling domain of or derived from such an inhibitory molecule, such that it serves to dampen the response of the cell, for example, that induced by an activating and/or costimulatory CAR.
  • CARs are referred to as first, second, and/or third generation CARs.
  • a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding;
  • a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD 137;
  • a third generation CAR in some aspects is one that includes multiple costimulatory domains of different costimulatory receptors.
  • the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion.
  • exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.
  • the antigen receptor further includes a marker and/or cells expressing the CAR or other antigen receptor further includes a surrogate marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor.
  • a surrogate marker such as a cell surface marker
  • the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor, such as truncated version of such a cell surface receptor (e.g., tEGFR).
  • the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A.
  • a linker sequence such as a cleavable linker sequence, e.g., T2A.
  • a marker, and optionally a linker sequence can be any as disclosed in published patent application No. WO2014031687.
  • the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence.
  • An exemplary polypeptide for a truncated EGFR comprises the sequence of amino acids set forth in SEQ ID NO: 7 or 16 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7 or 16.
  • An exemplary T2A linker sequence comprises the sequence of amino acids set forth in SEQ ID NO: 6 or 17 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 6 or 17.
  • the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof.
  • the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self’ by the immune system of the host into which the cells will be adoptively transferred.
  • the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered.
  • the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.
  • the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof.
  • the CAR contains an antibody, e.g., antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4-1BB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof.
  • the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a hinge -only spacer.
  • an Ig molecule such as a human Ig molecule
  • an Ig hinge e.g. an IgG4 hinge, such as a hinge -only spacer.
  • the transmembrane domain of the recombinant receptor is or includes a transmembrane domain of human CD28 (e.g. Accession No. P01747.1) or variant thereof, such as a transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 8 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8; in some embodiments, the transmembrane -domain containing portion of the recombinant receptor comprises the sequence of amino acids set forth in SEQ ID NO: 9 or a sequence of amino acids having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
  • the transmembrane domain comprises the sequence of amino acids set forth in SEQ ID NO: 8 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8. In some embodiments, the transmembrane domain comprises the sequence of amino acids set forth in SEQ ID NO: 8.
  • the transmembrane domain comprises the sequence of amino acids set forth in SEQ ID NO: 9 or a sequence of amino acids having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the transmembrane domain comprises the sequence of amino acids set forth in SEQ ID NO: 9.
  • the intracellular signaling component(s) of the recombinant receptor e.g. the CAR
  • the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 10 or 11 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10 or 11.
  • the intracellular domain comprises an intracellular costimulatory signaling domain of 4-1BB (e.g. (Accession No. Q07011.1) or functional variant or portion thereof, such as the sequence of amino acids set forth in SEQ ID NO: 12 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.
  • 4-1BB e.g. (Accession No. Q07011.1
  • functional variant or portion thereof such as the sequence of amino acids set forth in SEQ ID NO: 12 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.
  • the intracellular domain comprises an intracellular costimulatory signaling domain comrpising the sequence of amino acids set forth in SEQ ID NO: 12 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.
  • the intracellular domain comprises an intracellular costimulatory signaling domain comrpising the sequence of amino acids set forth in SEQ ID NO: 12.
  • the intracellular signaling domain of the recombinant receptor comprises a human CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD3 ⁇ (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Patent No.: 7,446,190 or U.S. Patent No. 8,911,993.
  • a human CD3 zeta stimulatory signaling domain or functional variant thereof such as an 112 AA cytoplasmic domain of isoform 3 of human CD3 ⁇ (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Patent No.: 7,446,190 or U.S. Patent No. 8,911,993.
  • the intracellular signaling domain comprises the sequence of amino acids as set forth in SEQ ID NO: 13, 14 or 15 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, 14 or 15.
  • the intracellular signaling domain comprises the sequence of amino acids as set forth in SEQ ID NO: 13 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13. In some embodiments, the intracellular signaling domain comprises the sequence of amino acids as set forth in SEQ ID NO: 13.
  • the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgGl, such as the hinge only spacer set forth in SEQ ID NO: 1, and encoded by the sequence set forth in SEQ ID NO: 2.
  • the spacer is an Ig hinge, e.g., and IgG4 hinge, linked to a CH2 and/or CH3 domains.
  • the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ ID NO: 4.
  • the spacer the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only, such as set forth in SEQ ID NO: 3.
  • the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers.
  • the constant region or portion is of IgD.
  • the spacer has the sequence set forth in SEQ ID NO: 5.
  • the spacer has a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 1, 3, 4 and 5.
  • the CAR includes an antibody such as an antibody fragment, including scFvs, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3 zeta signaling domain.
  • an antibody such as an antibody fragment, including scFvs
  • a spacer such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain
  • the CAR includes an antibody or fragment, such as scFv, a spacer such as any of the Ig-hinge containing spacers, a CD28-derived transmembrane domain, a 4-lBB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.
  • nucleic acid molecules encoding such CAR constructs further includes a sequence encoding a T2A ribosomal skip element and/or a tEGFR sequence, e.g., downstream of the sequence encoding the CAR.
  • the sequence encodes a T2A ribosomal skip element set forth in SEQ ID NO: 6 or 17, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 6 or 17.
  • T cells expressing an antigen receptor e.g.
  • CAR can also be generated to express a truncated EGFR (EGFRt) as a non-immunogenic selection epitope (e.g. by introduction of a construct encoding the CAR and EGFRt separated by a T2A ribosome switch to express two proteins from the same construct), which then can be used as a marker to detect such cells (see e.g. U.S. Patent No. 8,802,374).
  • EGFRt truncated EGFR
  • the sequence encodes an tEGFR sequence set forth in SEQ ID NO: 7 or 16, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7 or 16.
  • the peptide such as T2A, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream (see, for example, de Felipe. Genetic Vaccines and Ther.
  • 2A sequences that can be used in the methods and nucleic acids disclosed herein, without limitation, 2A sequences from the foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 21), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 20), Thosea asigna virus (T2A, e.g., SEQ ID NO: 6 or 17), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 18 or 19) as described in U.S. Patent Publication No.
  • F2A foot-and-mouth disease virus
  • E2A equine rhinitis A virus
  • T2A e.g., SEQ ID NO: 6 or 17
  • P2A porcine teschovirus-1
  • the CAR comprises, in order, an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is or comprises a 4- IBB, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is or comprises a CD3zeta signaling domain and optionally further includes a spacer between the transmembrane domain and the scFv;
  • the CAR includes, in order, an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is or comprises a 4- IBB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is a CD3zeta signaling domain.
  • the CAR comprises or consists of, in order, an scFv specific for the antigen, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is or comprises a CD3zeta signaling domain.
  • the spacer is a polypeptide spacer that (a) comprises or consists of all or a portion of an immunoglobulin hinge or a modified version thereof or comprises about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region, (b) comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof and/or comprises about 15 amino acids or less, and does not comprise a CD28 extracellular
  • the spacer comprises or consists of SEQ ID NO: 1
  • the costimulatory domain comprises SEQ ID NO: 12 or variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto
  • the transmembrane domain is of CD28 or comprises SEQ ID NO: 9 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto
  • the scFv contains the binding domain of or CDRs of or VH and VL of FMC63
  • the primary signaling domain contains SEQ ID NO: 13, 14, or 15, and/or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%
  • the spacer comprises or consists of SEQ ID NO: 30, the costimulatory domain comprises SEQ ID NO: 12 or variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto,
  • the transmembrane domain is of CD28 or comprises SEQ ID NO: 9 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto
  • the scFv contains the binding domain of or CDRs of or VH and VL of FMC63
  • the primary signaling domain contains SEQ ID NO: 13, 14, or 15, and/or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%
  • the spacer comprises or consists of SEQ ID NO: 31, the costimulatory domain comprises SEQ ID NO: 12 or variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto,
  • the transmembrane domain is of CD28 or comprises SEQ ID NO: 9 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto
  • the scFv contains the binding domain of or CDRs of or VH and VL of FMC63
  • the primary signaling domain contains SEQ ID NO: 13, 14, or 15, and/or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%
  • the spacer comprises or consists of SEQ ID NO: 33
  • the costimulatory domain comprises SEQ ID NO: 12 or variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto
  • the transmembrane domain is of CD28 or comprises SEQ ID NO: 9 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto
  • the scFv contains the binding domain of or CDRs of or VH and VL of FMC63
  • the primary signaling domain contains SEQ ID NO: 13, 14, or 15, and/or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9
  • the spacer comprises or consists of SEQ ID NO: 34
  • the costimulatory domain comprises SEQ ID NO: 12 or variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto
  • the transmembrane domain is of CD28 or comprises SEQ ID NO: 9 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto
  • the scFv contains the binding domain of or CDRs of or VH and VL of FMC63
  • the primary signaling domain contains SEQ ID NO: 13, 14, or 15, and/or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 9
  • the recombinant receptors, such as CARs, expressed by the cells administered to the subject generally recognize or specifically bind to a molecule that is expressed in, associated with, and/or specific for the disease or condition or cells thereof being treated.
  • the receptor Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immunostimulatory signal, such as an ITAM-transduced signal, into the cell, thereby promoting an immune response targeted to the disease or condition.
  • the cells express a CAR that specifically binds to an antigen expressed by a cell or tissue of the disease or condition or associated with the disease or condition.
  • engineered cells such as T cells, used in connection with the provided methods, uses, articles of manufacture or compositions are cells that express a T cell receptor (TCR) or antigen-binding portion thereof that recognizes an peptide epitope or T cell epitope of a target polypeptide, such as an antigen of a tumor, viral or autoimmune protein.
  • TCR T cell receptor
  • a “T cell receptor” or “TCR” is a molecule that contains a variable a and (3 chains (also known as TCRa and TCR , respectively) or a variable y and 5 chains (also known as TCRa and TCR , respectively), or antigen-binding portions thereof, and which is capable of specifically binding to a peptide bound to an MHC molecule.
  • the TCR is in the a form.
  • TCRs that exist in aP and y5 forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions.
  • a TCR can be found on the surface of a cell or in soluble form.
  • a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the term “TCR” should be understood to encompass full TCRs as well as antigen-binding portions or antigen-binding fragments thereof.
  • the TCR is an intact or full-length TCR, including TCRs in the a[3 form or y5 form.
  • the TCR is an antigen-binding portion that is less than a full-length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC -peptide complex.
  • an antigen-binding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as MHC-peptide complex, to which the full TCR binds.
  • an antigen-binding portion contains the variable domains of a TCR, such as variable a chain and variable [3 chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex.
  • the variable chains of a TCR contain complementarity determining regions involved in recognition of the peptide, MHC and/or MHC-peptide complex.
  • variable domains of the TCR contain hypervariable loops, or complementarity determining regions (CDRs), which generally are the primary contributors to antigen recognition and binding capabilities and specificity.
  • CDRs complementarity determining regions
  • a CDR of a TCR or combination thereof forms all or substantially all of the antigen-binding site of a given TCR molecule.
  • the various CDRs within a variable region of a TCR chain generally are separated by framework regions (FRs), which generally display less variability among TCR molecules as compared to the CDRs (see, e.g., Jores et al., Proc. Nat’l Acad. Sci. U.S.A.
  • CDR3 is the main CDR responsible for antigen binding or specificity, or is the most important among the three CDRs on a given TCR variable region for antigen recognition, and/or for interaction with the processed peptide portion of the peptide -MHC complex.
  • the CDR1 of the alpha chain can interact with the N- terminal part of certain antigenic peptides.
  • CDR1 of the beta chain can interact with the C-terminal part of the peptide.
  • CDR2 contributes most strongly to or is the primary CDR responsible for the interaction with or recognition of the MHC portion of the MHC-peptide complex.
  • the variable region of the [3-chain can contain a further hypervariable region (CDR4 or HVR4), which generally is involved in superantigen binding and not antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).
  • a TCR also can contain a constant domain, a transmembrane domain and/or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997).
  • each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end.
  • a TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction.
  • a TCR chain contains one or more constant domain.
  • the extracellular portion of a given TCR chain e.g., a-chain or [3-chain
  • a constant domain e.g., a-chain constant domain or Ca, typically positions 117 to 259 of the chain based on Kabat numbering or
  • the extracellular portion of the TCR formed by the two chains contains two membrane -proximal constant domains, and two membrane -distal variable domains, which variable domains each contain CDRs.
  • the constant domain of the TCR may contain short connecting sequences in which a cysteine residue forms a disulfide bond, thereby linking the two chains of the TCR.
  • a TCR may have an additional cysteine residue in each of the a and [3 chains, such that the TCR contains two disulfide bonds in the constant domains.
  • the TCR chains contain a transmembrane domain.
  • the transmembrane domain is positively charged.
  • the TCR chain contains a cytoplasmic tail.
  • the structure allows the TCR to associate with other molecules like CD3 and subunits thereof.
  • a TCR containing constant domains with a transmembrane region may anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex.
  • the intracellular tails of CD3 signaling subunits e.g. CD3y, CD35, CD3s and CD3 ⁇ chains
  • the TCR may be a heterodimer of two chains a and [3 (or optionally y and 5) or it may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (a and [3 chains or y and 5 chains) that are linked, such as by a disulfide bond or disulfide bonds.
  • the TCR can be generated from a known TCR sequence(s), such as sequences of Va,[3 chains, for which a substantially full-length coding sequence is readily available. Methods for obtaining full-length TCR sequences, including V chain sequences, from cell sources are well known.
  • nucleic acids encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of TCR-encoding nucleic acids within or isolated from a given cell or cells, or synthesis of publicly available TCR DNA sequences.
  • PCR polymerase chain reaction
  • the TCR is obtained from a biological source, such as from cells such as from a T cell (e.g.
  • the T-cells can be obtained from in vivo isolated cells.
  • the TCR is a thymically selected TCR.
  • the TCR is a neoepitope -restricted TCR.
  • the T- cells can be a cultured T-cell hybridoma or clone.
  • the TCR or antigen-binding portion thereof or antigen-binding fragment thereof can be synthetically generated from knowledge of the sequence of the TCR.
  • the TCR is generated from a TCR identified or selected from screening a library of candidate TCRs against a target polypeptide antigen, or target T cell epitope thereof.
  • TCR libraries can be generated by amplification of the repertoire of Va and V(3 from T cells isolated from a subject, including cells present in PBMCs, spleen or other lymphoid organ.
  • T cells can be amplified from tumor-infiltrating lymphocytes (TILs).
  • TCR libraries can be generated from CD4 + or CD8 + cells.
  • the TCRs can be amplified from a T cell source of a normal of healthy subject, i.e. normal TCR libraries.
  • the TCRs can be amplified from a T cell source of a diseased subject, i.e. diseased TCR libraries.
  • degenerate primers are used to amplify the gene repertoire of Va and V(3, such as by RT- PCR in samples, such as T cells, obtained from humans.
  • scTv libraries can be assembled from naive Va and V(3 libraries in which the amplified products are cloned or assembled to be separated by a linker.
  • the libraries can be HLA allelespecific.
  • TCR libraries can be generated by mutagenesis or diversification of a parent or scaffold TCR molecule.
  • the TCRs are subjected to directed evolution, such as by mutagenesis, e.g., of the a or (3 chain. In some aspects, particular residues within CDRs of the TCR are altered. In some embodiments, selected TCRs can be modified by affinity maturation. In some embodiments, antigen-specific T cells may be selected, such as by screening to assess CTL activity against the peptide. In some aspects, TCRs, e.g. present on the antigen-specific T cells, may be selected, such as by binding activity, e.g., particular affinity or avidity for the antigen.
  • the TCR or antigen-binding portion thereof is one that has been modified or engineered.
  • directed evolution methods are used to generate TCRs with altered properties, such as with higher affinity for a specific MHC -peptide complex.
  • directed evolution is achieved by display methods including, but not limited to, yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci U S A, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), or T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84).
  • display approaches involve engineering, or modifying, a known, parent or reference TCR.
  • a wild-type TCR can be used as a template for producing mutagenized TCRs in which in one or more residues of the CDRs are mutated, and mutants with an desired altered property, such as higher affinity for a desired target antigen, are selected.
  • peptides of a target polypeptide for use in producing or generating a TCR of interest are known or can be readily identified.
  • peptides suitable for use in generating TCRs or antigen-binding portions can be determined based on the presence of an HLA- restricted motif in a target polypeptide of interest, such as a target polypeptide described below.
  • peptides are identified using available computer prediction models.
  • models include, but are not limited to, ProPredl (Singh and Raghava (2001) Bioinformatics 17(12): 1236-1237, and SYFPEITHI (see Schuler et al.
  • the MHC -restricted epitope is HLA-A0201, which is expressed in approximately 39-46% of all Caucasians and therefore, represents a suitable choice of MHC antigen for use preparing a TCR or other MHC-peptide binding molecule.
  • HLA-A0201 -binding motifs and the cleavage sites for proteasomes and immune - proteasomes using computer prediction models are known.
  • such models include, but are not limited to, ProPredl (described in more detail in Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17(12): 1236-1237 2001), and SYFPEITHI (see Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction, in Immunoinformatics Methods in Molecular Biology, vol 409(1): 75-93 2007)
  • the TCR or antigen binding portion thereof may be a recombinantly produced natural protein or mutated form thereof in which one or more property, such as binding characteristic, has been altered.
  • a TCR may be derived from one of various animal species, such as human, mouse, rat, or other mammal.
  • a TCR may be cell-bound or in soluble form.
  • the TCR is in cell-bound form expressed on the surface of a cell.
  • the TCR is a full-length TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single -chain TCR (sc-TCR). In some embodiments, a dTCR or scTCR have the structures as described in WO 03/020763, WO 04/033685, WO2011/044186.
  • the TCR contains a sequence corresponding to the transmembrane sequence. In some embodiments, the TCR does contain a sequence corresponding to cytoplasmic sequences. In some embodiments, the TCR is capable of forming a TCR complex with CD3. In some embodiments, any of the TCRs, including a dTCR or scTCR, can be linked to signaling domains that yield an active TCR on the surface of a T cell. In some embodiments, the TCR is expressed on the surface of cells.
  • a dTCR contains a first polypeptide wherein a sequence corresponding to a TCR a chain variable region sequence is fused to the N terminus of a sequence corresponding to a TCR a chain constant region extracellular sequence, and a second polypeptide wherein a sequence corresponding to a TCR P chain variable region sequence is fused to the N terminus a sequence corresponding to a TCR chain constant region extracellular sequence, the first and second polypeptides being linked by a disulfide bond.
  • the bond can correspond to the native interchain disulfide bond present in native dimeric aP TCRs. In some embodiments, the interchain disulfide bonds are not present in a native TCR.
  • one or more cysteines can be incorporated into the constant region extracellular sequences of dTCR polypeptide pair.
  • both a native and a non-native disulfide bond may be desirable.
  • the TCR contains a transmembrane sequence to anchor to the membrane.
  • a dTCR contains a TCR a chain containing a variable a domain, a constant a domain and a first dimerization motif attached to the C-terminus of the constant a domain, and a TCR P chain comprising a variable P domain, a constant P domain and a first dimerization motif attached to the C-terminus of the constant P domain, wherein the first and second dimerization motifs easily interact to form a covalent bond between an amino acid in the first dimerization motif and an amino acid in the second dimerization motif linking the TCR a chain and TCR P chain together.
  • the TCR is a scTCR.
  • a scTCR can be generated using methods known, See e.g., Soo Hoo, W. F. et al. PNAS (USA) 89, 4759 (1992); Wiilfing, C. and Pliickthun, A., J. Mol. Biol. 242, 655 (1994); Kurucz, I. et al. PNAS (USA) 90 3830 (1993); International published PCT Nos. WO 96/13593, WO 96/18105, W099/60120, WO99/18129, WO 03/020763, WO2011/044186; and Schlueter, C. J. et al. J.
  • a scTCR contains an introduced non-native disulfide interchain bond to facilitate the association of the TCR chains (see e.g. International published PCT No. WO 03/020763).
  • a scTCR is a non-disulfide linked truncated TCR in which heterologous leucine zippers fused to the C-termini thereof facilitate chain association (see e.g. International published PCT No. W099/60120).
  • a scTCR contain a TCRa variable domain covalently linked to a TCRP variable domain via a peptide linker (see e.g., International published PCT No. WO99/18129).
  • a scTCR contains a first segment constituted by an amino acid sequence corresponding to a TCR a chain variable region, a second segment constituted by an amino acid sequence corresponding to a TCR P chain variable region sequence fused to the N terminus of an amino acid sequence corresponding to a TCR P chain constant domain extracellular sequence, and a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
  • a scTCR contains a first segment constituted by an a chain variable region sequence fused to the N terminus of an a chain extracellular constant domain sequence, and a second segment constituted by a P chain variable region sequence fused to the N terminus of a sequence P chain extracellular constant and transmembrane sequence, and, optionally, a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
  • a scTCR contains a first segment constituted by a TCR chain variable region sequence fused to the N terminus of a P chain extracellular constant domain sequence, and a second segment constituted by an a chain variable region sequence fused to the N terminus of a sequence a chain extracellular constant and transmembrane sequence, and, optionally, a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
  • the linker of a scTCRs that links the first and second TCR segments can be any linker capable of forming a single polypeptide strand, while retaining TCR binding specificity.
  • the linker sequence may, for example, have the formula -P-AA-P- wherein P is proline and AA represents an amino acid sequence wherein the amino acids are glycine and serine.
  • the first and second segments are paired so that the variable region sequences thereof are orientated for such binding.
  • the linker has a sufficient length to span the distance between the C terminus of the first segment and the N terminus of the second segment, or vice versa, but is not too long to block or reduces bonding of the scTCR to the target ligand.
  • the linker can contain from or from about 10 to 45 amino acids, such as 10 to 30 amino acids or 26 to 41 amino acids residues, for example 29, 30, 31 or 32 amino acids.
  • the linker has the formula -PGGG-(SGGGG)5-P- wherein P is proline, G is glycine and S is serine (SEQ ID NO:22).
  • the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO:23)
  • the scTCR contains a covalent disulfide bond linking a residue of the immunoglobulin region of the constant domain of the a chain to a residue of the immunoglobulin region of the constant domain of the P chain.
  • the interchain disulfide bond in a native TCR is not present.
  • one or more cysteines can be incorporated into the constant region extracellular sequences of the first and second segments of the scTCR polypeptide. In some cases, both a native and a non-native disulfide bond may be desirable.
  • the native disulfide bonds are not present.
  • the one or more of the native cysteines forming a native interchain disulfide bonds are substituted to another residue, such as to a serine or alanine.
  • an introduced disulfide bond can be formed by mutating non-cysteine residues on the first and second segments to cysteine. Exemplary non-native disulfide bonds of a TCR are described in published International PCT No. W02006/000830.
  • the TCR or antigen-binding fragment thereof exhibits an affinity with an equilibrium binding constant for a target antigen of between or between about 10-5 and 10-12 M and all individual values and ranges therein.
  • the target antigen is an MHC-peptide complex or ligand.
  • nucleic acid or nucleic acids encoding a TCR such as a and [3 chains, can be amplified by PCR, cloning or other suitable means and cloned into a suitable expression vector or vectors.
  • the expression vector can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses.
  • the vector can a vector of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), or the pEX series (Clontech, Palo Alto, Calif.).
  • bacteriophage vectors such as G I 0. XGT11, XZapII (Stratagene), XEMBL4, and XNM1149, also can be used.
  • plant expression vectors can be used and include pBIOl, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech).
  • animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech).
  • a viral vector is used, such as a retroviral vector.
  • the recombinant expression vectors can be prepared using standard recombinant DNA techniques.
  • vectors can contain regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA-based.
  • the vector can contain a nonnative promoter operably linked to the nucleotide sequence encoding the TCR or antigenbinding portion (or other MHC -peptide binding molecule).
  • the promoter can be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus.
  • CMV cytomegalovirus
  • SV40 SV40 promoter
  • RSV RSV promoter
  • promoter found in the long-terminal repeat of the murine stem cell virus a promoter found in the long-terminal repeat of the murine stem cell virus.
  • Other known promoters also are contemplated.
  • the a and [3 chains are PCR amplified from total cDNA isolated from a T cell clone expressing the TCR of interest and cloned into an expression vector.
  • the a and [3 chains are cloned into the same vector.
  • the a and [3 chains are cloned into different vectors.
  • the generated a and [3 chains are incorporated into a retroviral, e.g. lentiviral, vector.
  • the provided methods involve administering to a subject having a disease or condition (e.g. a cancer such as CLL or SLL) cells expressing a recombinant antigen receptor.
  • a disease or condition e.g. a cancer such as CLL or SLL
  • recombinant receptors e.g., CARs or TCRs
  • Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.
  • the cells expressing the receptors and administered by the provided methods are engineered cells.
  • the genetic engineering generally involves introduction of a nucleic acid encoding the recombinant or engineered component into a composition containing the cells, such as by retroviral transduction, transfection, or transformation.
  • the engineered cells are produced by a process that generates an output composition of enriched T cells from one or more input compositions and/or from a single biological sample.
  • the output composition contains cells that express a recombinant receptor, e.g., a CAR, such as an anti-CD19 CAR.
  • the cells of the output compositions are suitable for administration to a subject as a therapy, e.g., an autologous cell therapy.
  • the output composition is a composition of enriched CD4+ or CD8+ T cells.
  • the output composition is a composition of enriched CD4+ and CD8+ T cells.
  • the process for generating or producing engineered cells is by a process that includes some or all of the steps of: collecting or obtaining a biological sample; isolating, selecting, or enriching input cells from the biological sample; cry opreserving and storing the input cells; thawing and/or incubating the input cells under stimulating conditions; engineering the stimulated cells to express or contain a recombinant polynucleotide, e.g., a polynucleotide encoding a recombinant receptor such as a CAR; cultivating the engineered cells, e.g.
  • the process is performed with two or more input compositions of enriched T cells, such as a separate CD4+ composition and a separate CD8+ composition, that are separately processed and engineered from the same starting or initial biological sample and re -infused back into the subject at a defined ratio, e.g. 1:1 ratio of CD4+ to CD8+ T cells.
  • the enriched T cells are or include engineered T cells, e.g., T cells transduced to express a recombinant receptor.
  • an output composition of engineered cells expressing a recombinant receptor is produced from an initial and/or input composition of cells.
  • the input composition is a composition of enriched CD3+ T cells, enriched CD4+ T cells, and/or enriched CD8+ T cells (herein after also referred to as compositions of enriched T cells, compositions of enriched CD4+ T cells, and compositions of enriched CD8+ T cells, respectively).
  • a composition enriched in CD4+ T cells contains at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% CD4+ T cells.
  • the composition of enriched CD4+ T cells contains about 100% CD4+ T cells.
  • the composition of enriched CD4+T cells includes or contains less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and/or contains no CD8+ T cells, and/or is free or substantially free of CD8+ T cells.
  • the populations of enriched CD4+T cells consist essentially of CD4+ T cells.
  • a composition enriched in CD8+ T cells contains at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% CD8+ T cells, or contains or contains about 100% CD8+ T cells.
  • the composition of enriched CD8+ T cells includes or contains less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and/or contains no CD4+ T cells, and/or is free or substantially free of CD4+ T cells.
  • the populations of enriched CD8+T cells consist essentially of CD8+ T cells.
  • a composition enriched in CD3+ T cells contains at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% CD3+ T cells.
  • the composition of enriched CD3+ T cells contains about 100% CD3+ T cells.
  • the composition of enriched CD3+T cells includes CD4+ and CD8+ T cells that are at a ratio of CD4+ T cells to CD8+ T cells of between approximately 1:3 and approximately 3:1, such as approximately 1:1. In some embodiments, the ratio is between approximately 1:3 and approximately 3:1. In some embodiments, the ratio is approximately 1:1.
  • the process for producing engineered cells further can include one or more of: activating and/or stimulating a cells, e.g., cells of an input composition; genetically engineering the activated and/or stimulated cells, e.g., to introduce a polynucleotide encoding a recombinant protein by transduction or transfection; and/or cultivating the engineered cells, e.g., under conditions that promote proliferation and/or expansion.
  • the provided methods may be used in connection with harvesting, collecting, and/or formulating output compositions produced after the cells have been incubated, activated, stimulated, engineered, transduced, transfected, and/or cultivated.
  • engineered cells such as those that express an anti-CD19 CAR, used in accord with the provided methods are produced or generated by a process for selecting, isolating, activating, stimulating, expanding, cultivating, and/or formulating cells. In some embodiments, such methods include any as described.
  • At least one separate composition of enriched CD4+ T cells and at least one separate composition of enriched CD8+ T cells are isolated, selected, enriched, or obtained from a single biological sample, e.g., a sample of PBMCs or other white blood cells from the same donor such as a patient or healthy individual.
  • a separate composition of enriched CD4+ T cells and a separate composition of enriched CD8+ T cells originated, e.g., are initially isolated, selected, and/or enriched, from the same biological sample, such as a single biological sample obtained, collected, and/or taken from a single subject.
  • a biological sample is first subjected to selection of CD4+ T cells, where both the negative and positive fractions are retained, and the negative fraction is further subjected to selection of CD8+ T cells.
  • a biological sample is first subjected to selection of CD8+ T cells, where both the negative and positive fractions are retained, and the negative fraction is further subjected to selection of CD4+ T cells.
  • methods of selection are carried out as described in International PCT publication No. WO2015/164675.
  • a biological sample is first positively selected for CD8+ T cells to generate at least one composition of enriched CD8+ T cells, and the negative fraction is then positively selected for CD4+ T cells to generate at least one composition of enriched CD4+ T cells, such that the at least one composition of enriched CD8+ T cells and the at least one composition of enriched CD4+ T cells are separate compositions from the same biological sample, e.g., from the same donor patient or healthy individual.
  • two or more separate compositions of enriched T cells are separately frozen, e.g., cryoprotected or cryopreserved in a cryopreservation media.
  • two or more separate compositions of enriched T cells are activated and/or stimulated by contacting with a stimulatory reagent (e.g., by incubation with CD3/CD28 conjugated magnetic beads for T cell activation).
  • a stimulatory reagent e.g., by incubation with CD3/CD28 conjugated magnetic beads for T cell activation.
  • each of the activated/stimulated cell composition is engineered, transduced, and/or transfected, e.g., using a viral vector encoding a recombinant protein (e.g.
  • the method comprises removing the stimulatory reagent, e.g., magnetic beads, from the cell composition.
  • a cell composition containing engineered CD4+ T cells and a cell compostion containing engineered CD8+ T cells are separately cultivated, e.g., for separate expansion of the CD4+ T cell and CD8+ T cell populations therein.
  • a cell composition from the cultivation is harvested and/or collected and/or formulated, e.g., by washing the cell composition in a formulation buffer.
  • a formulated cell composition comprising CD4+ T cells and a formulated cell composition comprising CD8+ T cells is frozen, e.g., cryoprotected or cryopreserved in a cryopreservation media.
  • engineered CD4+ T cells and CD8+ T cells in each formulation originate from the same donor or biological sample and express the same recombination protein (e.g., CAR, such as anti-CD19 CAR).
  • a separate engineered CD4+ formulation and a separate engineered CD8+ formulation are administered at a defined ratio, e.g. 1:1, to a subject in need thereof such as the same donor.
  • two or more separate compositions of enriched T cells e.g., at least one being a composition of enriched CD4+ T cells and at least one being a separate composition of enriched CD8+ T cells from the same biological sample, selected from a sample from a subject and then are combined at a defined ratio, e.g. 1:1.
  • the combined composition enriched in CD4+ and CD8+ T cells are activated and/or stimulated by contacting with a stimulatory reagent (e.g., by incubation with CD3/CD28 conjugated magnetic beads for T cell activation).
  • the activated/stimulated cell composition is engineered, transduced, and/or transfected, e.g., using a viral vector encoding a recombinant protein (e.g. CAR), to express the recombinant protein in the CD4+ T cells and CD8+ T cells of the cell composition.
  • the method comprises removing the stimulatory reagent, e.g., magnetic beads, from the cell composition.
  • the cell composition containing engineered CD4+ T cells and engineered CD8+ T cells are cultivated, e.g., for expansion of the CD4+ T cell and CD8+ T cell populations therein.
  • a cell composition from the cultivation is harvested and/or collected and/or formulated, e.g., by washing the cell composition in a formulation buffer.
  • a formulated cell composition comprising recombinant receptor (e.g. CAR) engineered CD4+ T cells and CD8+ T cells is frozen, e.g., cryoprotected or cryopreserved in a cryopreservation media.
  • engineered CD4+ T cells and CD8+ T cells in the formulation originate from the same donor or biological sample and express the same recombinant protein (e.g., CAR, such as anti-CD19 CAR).
  • a composition of enriched CD3+ T cells is selected from a sample from a subject.
  • the composition enriched in CD3+ T cells is activated and/or stimulated by contacting with a stimulatory reagent (e.g., by incubation with CD3/CD28 conjugated magnetic beads for T cell activation).
  • the activated/stimulated cell composition is engineered, transduced, and/or transfected, e.g., using a viral vector encoding a recombinant protein (e.g. CAR), to express the recombinant protein in the T cells of the cell composition.
  • the method comprises removing the stimulatory reagent, e.g., magnetic beads, from the cell composition.
  • the cell composition containing engineered CD3+ T cells are cultivated, e.g., for expansion of the T cells populations therein.
  • a cell composition from the cultivation is harvested and/or collected and/or formulated, e.g., by washing the cell composition in a formulation buffer.
  • a formulated cell composition comprising recombinant receptor (e.g. CAR) engineered CD3+ T cells is frozen, e.g., cryoprotected or cryopreserved in a cryopreservation media.
  • engineered CD3+ T cells in the formulation express a CAR, such as anti-CD19 CAR.
  • cells such as T cells, used in connection with the provided methods, uses, articles of manufacture or compositions are cells have been genetically engineered to express a recombinant receptor, e.g., a CAR or a TCR described herein.
  • the engineered cells are used in the context of cell therapy, e.g., adoptive cell therapy.
  • the engineered cells are immune cells.
  • the engineered cells are T cells, such as CD4+ and CD8+ T cells, CD4+ T cells, or CD8+ T cells.
  • the nucleic acids such as nucleic acids encoding a recombinant receptor
  • the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.
  • the cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells.
  • the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells.
  • Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs).
  • the cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen.
  • the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4 + cells, CD8 + cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation.
  • the cells may be allogeneic and/or autologous.
  • the methods include off-the-shelf methods.
  • the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs).
  • the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, and re-introducing them into the same subject, before or after cry opreservation.
  • T cells and/or of CD4 + and/or of CD8 + T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
  • TN naive T
  • TSCM stem cell memory T
  • TCM central memory T
  • TEM effector memory T
  • TIL tumor-infiltrating lymphocyte
  • the cells are natural killer (NK) cells.
  • the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
  • the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids.
  • the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived.
  • the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.
  • preparation of the engineered cells includes one or more culture and/or preparation steps.
  • the cells for introduction of the nucleic acid encoding the transgenic receptor such as the CAR may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject.
  • the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered.
  • the subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
  • the cells in some embodiments are primary cells, e.g., primary human cells.
  • the samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation.
  • the biological sample can be a sample obtained directly from a biological source or a sample that is processed.
  • Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
  • the sample from which the cells are derived or isolated is blood or a blood- derived sample, or is or is derived from an apheresis or leukapheresis product.
  • exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom.
  • PBMCs peripheral blood mononuclear cells
  • sample from which the cells are derived or isolated is an apheresis product. In some aspects, the sample from which the cells are derived or isolated is a leukapheresis product. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
  • the cells are derived from cell lines, e.g., T cell lines.
  • the cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig.
  • isolation of the cells includes one or more preparation and/or nonaffinity based cell separation steps.
  • cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents.
  • cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
  • cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis.
  • the samples contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
  • the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and 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/or magnesium and/or many or all divalent cations.
  • a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer’s instructions.
  • a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer’s instructions.
  • the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca ++ /Mg ++ free PBS.
  • components of a blood cell sample are removed and the cells directly resuspended in culture media.
  • the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
  • the selection step includes incubation of cells with a selection reagent.
  • the incubation with a selection reagent or reagents e.g., as part of selection methods which may be performed using one or more selection reagents for selection of one or more different cell types based on the expression or presence in or on the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid.
  • surface markers e.g., surface proteins, intracellular markers, or nucleic acid.
  • any known method using a selection reagent or reagents for separation based on such markers may be used.
  • the selection reagent or reagents result in a separation that is affinity- or immunoaffinity-based separation.
  • the selection in some aspects includes incubation with a reagent or reagents for separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
  • a reagent or reagents for separation of cells and cell populations based on the cells’ expression or expression level of one or more markers typically cell surface markers
  • an antibody or binding partner that specifically binds to such markers
  • a volume of cells is mixed with an amount of a desired affinity-based selection reagent.
  • the immunoaffinity-based selection can be carried out using any system or method that results in a favorable energetic interaction between the cells being separated and the molecule specifically binding to the marker on the cell, e.g., the antibody or other binding partner on the solid surface, e.g., particle.
  • methods are carried out using particles such as beads, e.g. magnetic beads, that are coated with a selection agent (e.g. antibody) specific to the marker of the cells.
  • the particles e.g.
  • beads can be incubated or mixed with cells in a container, such as a tube or bag, while shaking or mixing, with a constant cell density-to-particle (e.g., bead) ratio to aid in promoting energetically favored interactions.
  • the methods include selection of cells in which all or a portion of the selection is carried out in the internal cavity of a centrifugal chamber, for example, under centrifugal rotation.
  • incubation of cells with selection reagents, such as immunoaffinity-based selection reagents is performed in a centrifugal chamber.
  • the isolation or separation is carried out using a system, device, or apparatus described in International Patent Application, Publication Number W02009/072003, or US 20110003380 Al.
  • the system is a system as described in International Publication Number W02016/073602.
  • the user by conducting such selection steps or portions thereof (e.g., incubation with antibody-coated particles, e.g., magnetic beads) in the cavity of a centrifugal chamber, the user is able to control certain parameters, such as volume of various solutions, addition of solution during processing and timing thereof, which can provide advantages compared to other available methods.
  • certain parameters such as volume of various solutions, addition of solution during processing and timing thereof, which can provide advantages compared to other available methods.
  • the ability to decrease the liquid volume in the cavity during the incubation can increase the concentration of the particles (e.g. bead reagent) used in the selection, and thus the chemical potential of the solution, without affecting the total number of cells in the cavity. This in turn can enhance the pairwise interactions between the cells being processed and the particles used for selection.
  • carrying out the incubation step in the chamber permits the user to effect agitation of the solution at desired time(s) during the incubation, which also can improve the interaction.
  • At least a portion of the selection step is performed in a centrifugal chamber, which includes incubation of cells with a selection reagent.
  • a volume of cells is mixed with an amount of a desired affinity-based selection reagent that is far less than is normally employed when performing similar selections in a tube or container for selection of the same number of cells and/or volume of cells according to manufacturer’s instructions.
  • an amount of selection reagent or reagents that is/are no more than 5%, no more than 10%, no more than 15%, no more than 20%, no more than 25%, no more than 50%, no more than 60%, no more than 70% or no more than 80% of the amount of the same selection reagent(s) employed for selection of cells in a tube or container-based incubation for the same number of cells and/or the same volume of cells according to manufacturer’s instructions is employed.
  • the cells are incubated in the cavity of the chamber in a composition that also contains the selection buffer with a selection reagent, such as a molecule that specifically binds to a surface marker on a cell that it desired to enrich and/or deplete, but not on other cells in the composition, such as an antibody, which optionally is coupled to a scaffold such as a polymer or surface, e.g., bead, e.g., magnetic bead, such as magnetic beads coupled to monoclonal antibodies specific for CD3, CD4 and/or CD8.
  • a selection reagent such as a molecule that specifically binds to a surface marker on a cell that it desired to enrich and/or deplete, but not on other cells in the composition, such as an antibody, which optionally is coupled to a scaffold such as a polymer or surface, e.g., bead, e.g., magnetic bead, such as magnetic beads coupled to monoclonal antibodies specific for CD3, CD4 and/or CD8.
  • the selection reagent is added to cells in the cavity of the chamber in an amount that is substantially less than (e.g. is no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the amount) as compared to the amount of the selection reagent that is typically used or would be necessary to achieve about the same or similar efficiency of selection of the same number of cells or the same volume of cells when selection is performed in a tube with shaking or rotation.
  • the incubation is performed with the addition of a selection buffer to the cells and selection reagent to achieve a target volume with incubation of the reagent of, for example, 10 mL to 200 mL, such as at least or at least about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL or 200 mL.
  • the selection buffer and selection reagent are pre -mixed before addition to the cells.
  • the selection buffer and selection reagent are separately added to the cells.
  • the selection incubation is carried out with periodic gentle mixing condition, which can aid in promoting energetically favored interactions and thereby permit the use of less overall selection reagent while achieving a high selection efficiency.
  • the total duration of the incubation with the selection reagent is from or from about 5 minutes to 6 hours, such as 30 minutes to 3 hours, for example, at least or at least about 30 minutes, 60 minutes, 120 minutes or 180 minutes.
  • the incubation generally is carried out under mixing conditions, such as in the presence of spinning, generally at relatively low force or speed, such as speed lower than that used to pellet the cells, such as from or from about 600 rpm to 1700 rpm (e.g. at or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm), such as at an RCF at the sample or wall of the chamber or other container of from or from about 80g to 100g (e.g. at or about or at least 80 g, 85 g, 90 g, 95 g, or 100 g).
  • the spin is carried out using repeated intervals of a spin at such low speed followed by a rest period, such as a spin and/or rest for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, such as a spin at approximately 1 or 2 seconds followed by a rest for approximately 5, 6, 7, or 8 seconds.
  • a rest period such as a spin and/or rest for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, such as a spin at approximately 1 or 2 seconds followed by a rest for approximately 5, 6, 7, or 8 seconds.
  • such process is carried out within the entirely closed system to which the chamber is integral.
  • this process (and in some aspects also one or more additional step, such as a previous wash step washing a sample containing the cells, such as an apheresis sample) is carried out in an automated fashion, such that the cells, reagent, and other components are drawn into and pushed out of the chamber at appropriate times and centrifugation effected, so as to complete the wash and binding step in a single closed system using an automated program.
  • the incubated cells are subjected to a separation to select for cells based on the presence or absence of the particular reagent or reagents.
  • the separation is performed in the same closed system in which the incubation of cells with the selection reagent was performed.
  • incubated cells, including cells in which the selection reagent has bound are transferred into a system for immunoaffinity-based separation of the cells.
  • the system for immunoaffinity-based separation is or contains a magnetic separation column.
  • the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation.
  • the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
  • Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
  • the separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker.
  • positive selection of or enrichment for cells of a particular type refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker.
  • negative selection, removal, or depletion of cells of a particular type refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
  • multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection.
  • a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection.
  • multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
  • T cells such as cells positive or expressing high levels of one or more surface markers, e.g., CD28 + , CD62L + , CCR7 + , CD27 + , CD127 + , CD4 + , CD8 + , CD45RA + , and/or CD45RO + T cells, are isolated by positive or negative selection techniques.
  • surface markers e.g., CD28 + , CD62L + , CCR7 + , CD27 + , CD127 + , CD4 + , CD8 + , CD45RA + , and/or CD45RO + T cells.
  • isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection.
  • positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker + ) at a relatively higher level (marker hlgh ) on the positively or negatively selected cells, respectively.
  • a biological sample e.g., a sample of PBMCs or other white blood cells
  • CD4+ T cells are subjected to selection of CD4+ T cells, where both the negative and positive fractions are retained.
  • CD8+ T cells are selected from the negative fraction.
  • a biological sample is subjected to selection of CD8+ T cells, where both the negative and positive fractions are retained.
  • CD4+ T cells are selected from the negative fraction.
  • T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14.
  • a CD4 + or CD8 + selection step is used to separate CD4 + helper and CD8 + cytotoxic T cells.
  • Such CD4 + and CD8 + populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
  • CD8 + cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation.
  • enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve longterm survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood.1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701.
  • combining TcM-enriched CD8 + T cells and CD4 + T cells further enhances efficacy.
  • memory T cells are present in both CD62L + and CD62L subsets of CD8 + peripheral blood lymphocytes.
  • PBMC can be enriched for or depleted of CD62L CD8 + and/or CD62L + CD8 + fractions, such as using anti-CD8 and anti-CD62L antibodies.
  • the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B.
  • isolation of a CD8 + population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L.
  • enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L.
  • Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order.
  • the same CD4 expression-based selection step used in preparing the CD8 + cell population or subpopulation also is used to generate the CD4 + cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
  • a sample of PBMCs or other white blood cell sample is subjected to selection of CD4 + cells, where both the negative and positive fractions are retained.
  • the negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.
  • CD4 + T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens.
  • CD4 + lymphocytes can be obtained by standard methods.
  • naive CD4 + T lymphocytes are CD45RO , CD45RA + , CD62L + , CD4 + T cells.
  • central memory CD4 + cells are CD62L + and CD45RO + .
  • effector CD4 + cells are CD62L and CD45RO .
  • a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8.
  • the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection.
  • the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher ⁇ Humana Press Inc., Totowa, NJ).
  • the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as DynalbeadsTM or MACS® beads).
  • the magnetically responsive material, e.g., particle generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.
  • a binding partner e.g., an antibody
  • the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner.
  • a specific binding member such as an antibody or other binding partner.
  • Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference.
  • Colloidal sized particles such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.
  • the incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
  • the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
  • the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells.
  • positive selection cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained.
  • negative selection cells that are not attracted (unlabeled cells) are retained.
  • a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.
  • the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin.
  • the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers.
  • the cells, rather than the beads are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added.
  • streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
  • the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient.
  • the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.
  • the affinity-based selection is via magnetic-activated cell sorting (MACS®) (Miltenyi Biotec, Auburn, CA). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto.
  • MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered.
  • the non-target cells are labelled and depleted from the heterogeneous population of cells.
  • the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods.
  • the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination.
  • the system is a system as described in International Patent Application, Publication Number W02009/072003, or US 20110003380 Al.
  • the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion.
  • the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.
  • the separation and/or other steps is carried out using CliniMACS® system (Miltenyi Biotec), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system.
  • Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves.
  • the integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence.
  • the magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column.
  • the peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.
  • the CliniMACS® system in some aspects uses antibody -coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution.
  • the cells after labelling of cells with magnetic particles the cells are washed to remove excess particles.
  • a cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag.
  • the tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps.
  • the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag. [0565] In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy® system (Miltenyi Biotec). The CliniMACS Prodigy® system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation.
  • the CliniMACS Prodigy® system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into erythrocytes, white blood cells and plasma layers.
  • the CliniMACS Prodigy® system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immuno ther. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and Wang et al. (2012) J Immuno ther. 35(9):689-701.
  • a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream.
  • a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting.
  • a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. l(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.
  • MEMS microelectromechanical systems
  • the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection.
  • separation may be based on binding to fluorescently labeled antibodies.
  • separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flowcytometric detection system.
  • FACS fluorescence-activated cell sorting
  • MEMS microelectromechanical systems
  • the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering.
  • the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population.
  • the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used.
  • a freezing solution e.g., following a washing step to remove plasma and platelets.
  • Any of a variety of known freezing solutions and parameters in some aspects may be used.
  • PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively.
  • the cells are generally then frozen to -80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank
  • the isolation and/or selection results in one or more input compositions of enriched T cells, e.g., CD3+ T cells, CD4+ T cells, and/or CD8+ T cells.
  • two or more separate input composition are isolated, selected, enriched, or obtained from a single biological sample.
  • separate input compositions are isolated, selected, enriched, and/or obtained from separate biological samples collected, taken, and/or obtained from the same subject.
  • the one or more input compositions is or includes a composition of enriched T cells that includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or at about 100% CD3+ T cells.
  • the input composition of enriched T cells consists essentially of CD3+ T cells.
  • the one or more input compositions is or includes a composition of enriched CD4+ T cells that includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or at about 100% CD4+ T cells.
  • the input composition of CD4+ T cells includes less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and/or contains no CD8+ T cells, and/or is free or substantially free of CD8+ T cells.
  • the composition of enriched T cells consists essentially of CD4+ T cells.
  • the one or more compositions is or includes a composition of CD8+ T cells that is or includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or at about 100% CD8+ T cells.
  • the composition of CD8+ T cells contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and/or contains no CD4+ T cells, and/or is free of or substantially free of CD4+ T cells.
  • the composition of enriched T cells consists essentially of CD8+ T cells.
  • the cells are incubated and/or cultured prior to or in connection with genetic engineering.
  • the incubation steps can include culture, cultivation, stimulation, activation, and/or propagation.
  • the incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells.
  • the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
  • the conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
  • agents e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
  • the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of stimulating or activating an intracellular signaling domain of a TCR complex.
  • the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell.
  • agents can include antibodies, such as those specific for a TCR, e.g. anti-CD3.
  • the stimulating conditions include one or more agent, e.g. ligand, which is capable of stimulating a costimulatory receptor, e.g., anti-CD28.
  • agents and/or ligands may be, bound to solid support such as a bead, and/or one or more cytokines.
  • the expansion method may further comprise the step of adding anti-CD3 and/or anti-CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml).
  • the stimulating agents include IL-2, IL- 15 and/or IL-7.
  • the IL -2 concentration is at least about 10 units/mL.
  • the stimulating conditions can include incubation using anti-CD3/anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
  • anti-CD3/anti-CD28 conjugated magnetic beads e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander.
  • incubation is carried out in accordance with techniques such as those described in US Patent No. 6,040,177 to Riddell et al., Klebanoff et al.(2012) J Immunother. 35(9): 651— 660, Terakura et al. (2012) Blood.1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.
  • the T cells are expanded by adding to a culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells).
  • PBMC peripheral blood mononuclear cells
  • the non-dividing feeder cells can comprise gammairradiated PBMC feeder cells.
  • the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division.
  • the feeder cells are added to culture medium prior to the addition of the populations of T cells.
  • the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees Celsius, and generally at or about 37 degrees Celsius.
  • the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells.
  • LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads.
  • the LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.
  • antigen-specific T cells such as antigen-specific CD4 + and/or CD8 + T cells
  • antigenspecific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.
  • At least a portion of the incubation in the presence of one or more stimulating conditions or a stimulatory agents is carried out in the internal cavity of a centrifugal chamber, for example, under centrifugal rotation, such as described in International Publication Number W02016/073602.
  • at least a portion of the incubation performed in a centrifugal chamber includes mixing with a reagent or reagents to induce stimulation and/or activation.
  • cells, such as selected cells are mixed with a stimulating condition or stimulatory agent in the centrifugal chamber.
  • a volume of cells is mixed with an amount of one or more stimulating conditions or agents that is far less than is normally employed when performing similar stimulations in a cell culture plate or other system.
  • the stimulating agent is added to cells in the cavity of the chamber in an amount that is substantially less than (e.g. is no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the amount) as compared to the amount of the stimulating agent that is typically used or would be necessary to achieve about the same or similar efficiency of selection of the same number of cells or the same volume of cells when selection is performed without mixing in a centrifugal chamber, e.g. in a tube or bag with periodic shaking or rotation.
  • the incubation is performed with the addition of an incubation buffer to the cells and stimulating agent to achieve a target volume with incubation of the reagent of, for example, 10 mL to 200 mL, such as at least or at least about or about or 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL or 200 mL.
  • the incubation buffer and stimulating agent are pre-mixed before addition to the cells.
  • the incubation buffer and stimulating agent are separately added to the cells.
  • the stimulating incubation is carried out with periodic gentle mixing condition, which can aid in promoting energetically favored interactions and thereby permit the use of less overall stimulating agent while achieving stimulating and activation of cells.
  • the incubation generally is carried out under mixing conditions, such as in the presence of spinning, generally at relatively low force or speed, such as speed lower than that used to pellet the cells, such as from or from about 600 rpm to 1700 rpm (e.g. at or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm), such as at an RCF at the sample or wall of the chamber or other container of from or from about 80g to 100g (e.g. at or about or at least 80 g, 85 g, 90 g, 95 g, or 100 g).
  • the spin is carried out using repeated intervals of a spin at such low speed followed by a rest period, such as a spin and/or rest for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, such as a spin at approximately 1 or 2 seconds followed by a rest for approximately 5, 6, 7, or 8 seconds.
  • a rest period such as a spin and/or rest for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, such as a spin at approximately 1 or 2 seconds followed by a rest for approximately 5, 6, 7, or 8 seconds.
  • the total duration of the incubation is between or between about 1 hour and 96 hours, 1 hour and 72 hours, 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours or 12 hours and 24 hours, such as at least or at least about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours or 72 hours.
  • the further incubation is for a time between or about between 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours or 12 hours and 24 hours, inclusive.
  • the stimulating conditions include incubating, culturing, and/or cultivating a composition of enriched T cells with and/or in the presence of one or more cytokines.
  • the one or more cytokines are recombinant cytokines.
  • the one or more cytokines are human recombinant cytokines.
  • the one or more cytokines bind to and/or are capable of binding to receptors that are expressed by and/or are endogenous to T cells.
  • the one or more cytokines is or includes a member of the 4-alpha- helix bundle family of cytokines.
  • members of the 4-alpha-helix bundle family of cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin 12 (IL- 12), interleukin 15 (IL- 15), granulocyte colony-stimulating factor (G-CSF), and granulocyte -macrophage colony-stimulating factor (GM-CSF).
  • IL-2 interleukin-2
  • IL-4 interleukin-4
  • IL-7 interleukin-9
  • IL- 12 interleukin 12
  • IL- 15 interleukin 15
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte -macrophage colony-stimulating factor
  • the stimulation results in activation and/or proliferation of the cells, for example, prior to transduction.
  • engineered cells such as T cells, used in connection with the provided methods, uses, articles of manufacture or compositions are cells have been genetically engineered to express a recombinant receptor, e.g., a CAR or a TCR described herein.
  • the cells are engineered by introduction, delivery or transfer of nucleic acid sequences that encode the recombinant receptor and/or other molecules.
  • methods for producing engineered cells includes the introduction of a polynucleotide encoding a recombinant receptor (e.g. anti-CD19 CAR) into a cell, e.g., such as a stimulated or activated cell.
  • a recombinant receptor e.g. anti-CD19 CAR
  • the recombinant proteins are recombinant receptors, such as any described.
  • Introduction of the nucleic acid molecules encoding the recombinant protein, such as recombinant receptor, in the cell may be carried out using any of a number of known vectors.
  • Such vectors include viral and non-viral systems, including lentiviral and gammaretroviral systems, as well as transposon-based systems such as PiggyBac or Sleeping Beauty-based gene transfer systems.
  • Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.
  • the engineering produces one or more engineered compositions of enriched T cells.
  • the one or more compositions of stimulated T cells are or include two separate stimulated compositions of enriched T cells.
  • two separate compositions of enriched T cells are separately engineered.
  • the two separate compositions include a composition of enriched CD4+ T cells.
  • the two separate compositions include a composition of enriched CD8+ T cells.
  • two separate compositions of enriched CD4+ T cells and enriched CD8+ T cells are genetically engineered separately.
  • gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.
  • the gene transfer is accomplished by first incubating the cells under stimulating conditions, such as by any of the methods described.
  • methods for genetic engineering are carried out by contacting one or more cells of a composition with a nucleic acid molecule encoding the recombinant protein, e.g. recombinant receptor.
  • the contacting can be effected with centrifugation, such as spinoculation (e.g. centrifugal inoculation).
  • centrifugation such as spinoculation (e.g. centrifugal inoculation).
  • spinoculation e.g. centrifugal inoculation
  • Exemplary centrifugal chambers include those produced and sold by Biosafe SA, including those for use with the Sepax® and Sepax® 2 system, including an A-200/F and A-200 centrifugal chambers and various kits for use with such systems.
  • Exemplary chambers, systems, and processing instrumentation and cabinets are described, for example, in US Patent No. 6,123,655, US Patent No. 6,733,433 and Published U.S. Patent Application, Publication No.: US 2008/0171951, and published international patent application, publication no. WO 00/38762, the contents of each of which are incorporated herein by reference in their entirety.
  • Exemplary kits for use with such systems include, but are not limited to, single -use kits sold by BioSafe SA under product names CS-430.1, CS-490.1, CS-600.1 or CS-900.2.
  • the contacting can be effected with centrifugation, such as spinoculation (e.g., centrifugal inoculation).
  • the composition containing cells, the vector, e.g., viral particles and reagent can be rotated, generally at relatively low force or speed, such as speed lower than that used to pellet the cells, such as from or from about 600 rpm to 1700 rpm (e.g., at or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm).
  • the rotation is carried at a force, e.g., a relative centrifugal force, of from or from about 100 g to 3200 g (e.g., at or about or at least at or about 100 g, 200 g, 300 g, 400 g, 500 g, 1000 g, 1500 g, 2000 g, 2500 g, 3000 g or 3200 g), as measured for example at an internal or external wall of the chamber or cavity.
  • a force e.g., a relative centrifugal force, of from or from about 100 g to 3200 g (e.g., at or about or at least at or about 100 g, 200 g, 300 g, 400 g, 500 g, 1000 g, 1500 g, 2000 g, 2500 g, 3000 g or 3200 g), as measured for example at an internal or external wall of the chamber or cavity.
  • RCF relative centrifugal force
  • an object or substance such as a cell, sample, or pellet and/or a point in the chamber or other container being rotated
  • the value may be determined using well-known formulas, taking into account the gravitational force, rotation speed and the radius of rotation (distance from the axis of rotation and the object, substance, or particle at which RCF is being measured).
  • the system is included with and/or placed into association with other instrumentation, including instrumentation to operate, automate, control and/or monitor aspects of the transduction step and one or more various other processing steps performed in the system, e.g. one or more processing steps that can be carried out with or in connection with the centrifugal chamber system as described herein or in International Publication Number W02016/073602.
  • This instrumentation in some embodiments is contained within a cabinet.
  • the instrumentation includes a cabinet, which includes a housing containing control circuitry, a centrifuge, a cover, motors, pumps, sensors, displays, and a user interface.
  • An exemplary device is described in US Patent No. 6,123,655, US Patent No. 6,733,433 and US 2008/0171951.
  • the system comprises a series of containers, e.g., bags, tubing, stopcocks, clamps, connectors, and a centrifuge chamber.
  • the containers, such as bags include one or more containers, such as bags, containing the cells to be transduced and the viral vector particles, in the same container or separate containers, such as the same bag or separate bags.
  • the system further includes one or more containers, such as bags, containing medium, such as diluent and/or wash solution, which is pulled into the chamber and/or other components to dilute, resuspend, and/or wash components and/or compositions during the methods.
  • the containers can be connected at one or more positions in the system, such as at a position corresponding to an input line, diluent line, wash line, waste line and/or output line.
  • the chamber is associated with a centrifuge, which is capable of effecting rotation of the chamber, such as around its axis of rotation. Rotation may occur before, during, and/or after the incubation in connection with transduction of the cells and/or in one or more of the other processing steps. Thus, in some embodiments, one or more of the various processing steps is carried out under rotation, e.g., at a particular force.
  • the chamber is typically capable of vertical or generally vertical rotation, such that the chamber sits vertically during centrifugation and the side wall and axis are vertical or generally vertical, with the end wall(s) horizontal or generally horizontal.
  • the cells are transferred to a bioreactor bag assembly for culture of the genetically engineered cells, such as for cultivation or expansion of the cells.
  • recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV).
  • recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038/gt.2014.25; Carlens et al.
  • the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV) or spleen focus forming virus (SFFV).
  • LTR long terminal repeat sequence
  • MoMLV Moloney murine leukemia virus
  • MPSV myeloproliferative sarcoma virus
  • MSV murine embryonic stem cell virus
  • MSCV murine stem cell virus
  • SFFV spleen focus forming virus
  • retroviral vectors are derived from murine retroviruses.
  • the retroviruses include those derived from any avian or mammalian cell source.
  • the retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans.
  • the gene to be expressed replaces the retroviral gag, pol and/or env sequences.
  • retroviral systems e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
  • the viral vector particles contain a genome derived from a retroviral genome based vector, such as derived from a lentiviral genome based vector.
  • the heterologous nucleic acid encoding a recombinant receptor, such as an antigen receptor, such as a CAR is contained and/or located between the 5' LTR and 3' LTR sequences of the vector genome.
  • the viral vector genome is a lentivirus genome, such as an HIV-1 genome or an SIV genome.
  • lentiviral vectors have been generated by multiply attenuating virulence genes, for example, the genes env, vif, vpu and nef can be deleted, making the vector safer for therapeutic purposes. Lentiviral vectors are known. See Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136).
  • these viral vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection, and for transfer of the nucleic acid into a host cell.
  • Known lentiviruses can be readily obtained from depositories or collections such as the American Type Culture Collection (“ATCC”; 10801 University Boulevard., Manassas, Va. 20110-2209), or isolated from known sources using commonly available techniques.
  • Non-limiting examples of lentiviral vectors include those derived from a lentivirus, such as Human Immunodeficiency Virus 1 (HIV-1), HIV-2, an Simian Immunodeficiency Virus (SIV), Human T-lymphotropic virus 1 (HTLV-1), HTLV-2 or equine infection anemia virus (E1AV).
  • lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted, making the vector safer for therapeutic purposes.
  • Lentiviral vectors are known in the art, see Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136).
  • these viral vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection, and for transfer of the nucleic acid into a host cell.
  • Known lentiviruses can be readily obtained from depositories or collections such as the American Type Culture Collection (“ATCC”; 10801 University Boulevard., Manassas, Va. 20110-2209), or isolated from known sources using commonly available techniques.
  • ATCC American Type Culture Collection
  • the viral genome vector can contain sequences of the 5' and 3' LTRs of a retrovirus, such as a lentivirus.
  • the viral genome construct may contain sequences from the 5' and 3' LTRs of a lentivirus, and in particular can contain the R and U5 sequences from the 5' LTR of a lentivirus and an inactivated or self-inactivating 3' LTR from a lentivirus.
  • the LTR sequences can be LTR sequences from any lentivirus from any species. For example, they may be LTR sequences from HIV, SIV, FIV or BIV. Typically, the LTR sequences are HIV LTR sequences.
  • the nucleic acid of a viral vector lacks additional transcriptional units.
  • the vector genome can contain an inactivated or self-inactivating 3' LTR (Zufferey et al. J Virol 72: 9873, 1998; Miyoshi et al., J Virol 72:8150, 1998).
  • deletion in the U3 region of the 3' LTR of the nucleic acid used to produce the viral vector RNA can be used to generate self-inactivating (SIN) vectors. This deletion can then be transferred to the 5' LTR of the proviral DNA during reverse transcription.
  • a self-inactivating vector generally has a deletion of the enhancer and promoter sequences from the 3' long terminal repeat (LTR), which is copied over into the 5' LTR during vector integration.
  • LTR long terminal repeat
  • enough sequence can be eliminated, including the removal of a TATA box, to abolish the transcriptional activity of the LTR. This can prevent production of full-length vector RNA in transduced cells.
  • the U3 element of the 3' LTR contains a deletion of its enhancer sequence, the TATA box, Spl, and NF-kappa B sites.
  • the self-inactivating 3' LTR can be constructed by any method known in the art. In some embodiments, this does not affect vector titers or the in vitro or in vivo properties of the vector.
  • the U3 sequence from the lentiviral 5' LTR can be replaced with a promoter sequence in the viral construct, such as a heterologous promoter sequence.
  • a promoter sequence in the viral construct such as a heterologous promoter sequence.
  • An enhancer sequence can also be included. Any enhancer/promoter combination that increases expression of the viral RNA genome in the packaging cell line may be used.
  • the CMV enhancer/promoter sequence is used (U.S. Pat. No.
  • the risk of insertional mutagenesis can be minimized by constructing the retroviral vector genome, such as lentiviral vector genome, to be integration defective.
  • retroviral vector genome such as lentiviral vector genome
  • a variety of approaches can be pursued to produce a non-integrating vector genome.
  • a mutation(s) can be engineered into the integrase enzyme component of the pol gene, such that it encodes a protein with an inactive integrase.
  • the vector genome itself can be modified to prevent integration by, for example, mutating or deleting one or both attachment sites, or making the 3' LTR-proximal polypurine tract (PPT) non-functional through deletion or modification.
  • PPT 3' LTR-proximal polypurine tract
  • non-genetic approaches are available; these include pharmacological agents that inhibit one or more functions of integrase.
  • the approaches are not mutually exclusive; that is, more than one of them can be used at a time.
  • both the integrase and attachment sites can be nonfunctional, or the integrase and PPT site can be non-functional, or the attachment sites and PPT site can be non-functional, or all of them can be non-functional.
  • Such methods and viral vector genomes are known and available (see Philpott and Thrasher, Human Gene Therapy 18:483, 2007; Engelman et al.
  • the vector contains sequences for propagation in a host cell, such as a prokaryotic host cell.
  • the nucleic acid of the viral vector contains one or more origins of replication for propagation in a prokaryotic cell, such as a bacterial cell.
  • vectors that include a prokaryotic origin of replication also may contain a gene whose expression confers a detectable or selectable marker such as drug resistance.
  • the viral vector genome is typically constructed in a plasmid form that can be transfected into a packaging or producer cell line. Any of a variety of known methods can be used to produce retroviral particles whose genome contains an RNA copy of the viral vector genome.
  • at least two components are involved in making a virus-based gene delivery system: first, packaging plasmids, encompassing the structural proteins as well as the enzymes necessary to generate a viral vector particle, and second, the viral vector itself, i.e., the genetic material to be transferred. Biosafety safeguards can be introduced in the design of one or both of these components.
  • the packaging plasmid can contain all retroviral, such as HIV-1, proteins other than envelope proteins (Naldini et al., 1998).
  • viral vectors can lack additional viral genes, such as those that are associated with virulence, e.g., vpr, vif, vpu and nef, and/or Tat, a primary transactivator of HIV.
  • lentiviral vectors such as HIV-based lentiviral vectors, comprise only three genes of the parental virus: gag, pol and rev, which reduces or eliminates the possibility of reconstitution of a wild-type virus through recombination.
  • the viral vector genome is introduced into a packaging cell line that contains all the components necessary to package viral genomic RNA, transcribed from the viral vector genome, into viral particles.
  • the viral vector genome may comprise one or more genes encoding viral components in addition to the one or more sequences, e.g., recombinant nucleic acids, of interest.
  • endogenous viral genes required for replication are removed and provided separately in the packaging cell line.
  • a packaging cell line is transfected with one or more plasmid vectors containing the components necessary to generate the particles.
  • a packaging cell line is transfected with a plasmid containing the viral vector genome, including the LTRs, the cis-acting packaging sequence and the sequence of interest, i.e. a nucleic acid encoding an antigen receptor, such as a CAR; and one or more helper plasmids encoding the virus enzymatic and/or structural components, such as Gag, pol and/or rev.
  • multiple vectors are utilized to separate the various genetic components that generate the retroviral vector particles.
  • providing separate vectors to the packaging cell reduces the chance of recombination events that might otherwise generate replication competent viruses.
  • a single plasmid vector having all of the retroviral components can be used.
  • the retroviral vector particle such as lentiviral vector particle
  • a retroviral vector particle such as a lentiviral vector particle
  • a packaging cell line is transfected with a plasmid or polynucleotide encoding a nonnative envelope glycoprotein, such as to include xenotropic, polytropic or amphotropic envelopes, such as Sindbis virus envelope, GALV or VSV-G.
  • the packaging cell line provides the components, including viral regulatory and structural proteins, that are required in trans for the packaging of the viral genomic RNA into lentiviral vector particles.
  • the packaging cell line may be any cell line that is capable of expressing lentiviral proteins and producing functional lentiviral vector particles.
  • suitable packaging cell lines include 293 (ATCC CCL X), 293T, HeLA (ATCC CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL-10) and Cf2Th (ATCC CRL 1430) cells.
  • the packaging cell line stably expresses the viral protein(s).
  • a packaging cell line containing the gag, pol, rev and/or other structural genes but without the LTR and packaging components can be constructed.
  • a packaging cell line can be transiently transfected with nucleic acid molecules encoding one or more viral proteins along with the viral vector genome containing a nucleic acid molecule encoding a heterologous protein, and/or a nucleic acid encoding an envelope glycoprotein.
  • the viral vectors and the packaging and/or helper plasmids are introduced via transfection or infection into the packaging cell line.
  • the packaging cell line produces viral vector particles that contain the viral vector genome. Methods for transfection or infection are well known. Non-limiting examples include calcium phosphate, DEAE-dextran and lipofection methods, electroporation and microinjection.
  • the packaging sequences may permit the RNA transcript of the recombinant plasmid to be packaged into viral particles, which then may be secreted into the culture media.
  • the media containing the recombinant retroviruses in some embodiments is then collected, optionally concentrated, and used for gene transfer.
  • the viral vector particles are recovered from the culture media and titered by standard methods used by those of skill in the art.
  • a retroviral vector such as a lentiviral vector
  • a packaging cell line such as an exemplary HEK 293T cell line, by introduction of plasmids to allow generation of lentiviral particles.
  • a packaging cell is transfected and/or contains a polynucleotide encoding gag and pol, and a polynucleotide encoding a recombinant receptor, such as an antigen receptor, for example, a CAR.
  • the packaging cell line is optionally and/or additionally transfected with and/or contains a polynucleotide encoding a rev protein.
  • the packaging cell line is optionally and/or additionally transfected with and/or contains a polynucleotide encoding a non-native envelope glycoprotein, such as VSV-G.
  • a non-native envelope glycoprotein such as VSV-G.
  • the cell supernatant contains recombinant lentiviral vectors, which can be recovered and titered.
  • Recovered and/or produced retroviral vector particles can be used to transduce target cells using the methods as described. Once in the target cells, the viral RNA is reverse-transcribed, imported into the nucleus and stably integrated into the host genome. One or two days after the integration of the viral RNA, the expression of the recombinant protein, e.g., antigen receptor, such as CAR, can be detected.
  • the recombinant protein e.g., antigen receptor, such as CAR
  • the provided methods involve methods of transducing cells by contacting, e.g., incubating, a cell composition comprising a plurality of cells with a viral particle.
  • the cells to be transfected or transduced are or comprise primary cells obtained from a subject, such as cells enriched and/or selected from a subject.
  • the concentration of cells to be transduced of the composition is from or from about 1.0 x 10 5 cells/mL to 1.0 x 10 8 cells/mL, such as at least or at least about or about 1.0 x 10 5 cells/mL, 5 x 10 5 cells/mL, 1 x 10 6 cells/mL, 5 x 10 6 cells/mL, 1 x 10 7 cells/mL, 5 x 10 7 cells/mL or 1 x 10 8 cells/mL.
  • the viral particles are provided at a certain ratio of copies of the viral vector particles or infectious units (IU) thereof, per total number of cells to be transduced (lU/cell).
  • the viral particles are present during the contacting at or about or at least at or about 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or 60 IU of the viral vector particles per one of the cells.
  • the titer of viral vector particles is between or between about 1 x 10 6 lU/mL and 1 x 10 8 lU/mL, such as between or between about 5 x 10 6 lU/mL and 5 x 10 7 lU/mL, such as at least 6 x 10 6 lU/mL, 7 x 10 6 lU/mL, 8 x 10 6 lU/mL, 9 x 10 6 lU/mL, 1 x 10 7 lU/mL, 2 x 10 7 lU/mL, 3 x 10 7 lU/mL, 4 x 10 7 lU/mL, or 5 xlO 7 lU/mL.
  • transduction can be achieved at a multiplicity of infection (MOI) of less than 100, such as generally less than 60, 50, 40, 30, 20, 10, 5 or less.
  • MOI multiplicity of infection
  • the method involves contacting or incubating, the cells with the viral particles.
  • the contacting is for 30 minutes to 72 hours, such as 30 minute to 48 hours, 30 minutes to 24 hours or 1 hour to 24 hours, such as at least or at least about 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours or more.
  • contacting is performed in solution.
  • the cells and viral particles are contacted in a volume of from or from about 0.5 mL to 500 mL, such as from or from about 0.5 mL to 200 mL, 0.5 mL to 100 mL, 0.5 mL to 50 mL, 0.5 mL to 10 mL, 0.5 mL to 5 mL, 5 mL to 500 mL, 5 mL to 200 mL, 5 mL to 100 mL, 5 mL to 50 mL, 5 mL to 10 mL, 10 mL to 500 mL, 10 mL to 200 mL, 10 mL to 100 mL, 10 mL to 50 mL, 50 mL to 500 mL, 50 mL to 200 mL, 50 mL to 100 mL, 100 mL to 500 mL, 100 mL to 200 mL or 200 mL to
  • the input cells are treated, incubated, or contacted with particles that comprise binding molecules that bind to or recognize the recombinant receptor that is encoded by the viral DNA.
  • the incubation of the cells with the viral vector particles results in or produces an output composition comprising cells transduced with the viral vector particles.
  • recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437).
  • recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115- 126).
  • the cells may be transfected either during or after expansion e.g. with a T cell receptor (TCR) or a chimeric antigen receptor (CAR).
  • TCR T cell receptor
  • CAR chimeric antigen receptor
  • This transfection for the introduction of the gene of the desired receptor can be carried out with any suitable retroviral vector, for example.
  • the genetically modified cell population can then be liberated from the initial stimulus (the anti-CD3/anti-CD28 stimulus, for example) and subsequently be stimulated with a second type of stimulus e.g. via a de novo introduced receptor).
  • This second type of stimulus may include an antigenic stimulus in form of a peptide/MHC molecule, the cognate (cross-linking) ligand of the genetically introduced receptor (e.g.
  • a vector may be used that does not require that the cells, e.g., T cells, are activated.
  • the cells may be selected and/or transduced prior to activation.
  • the cells may be engineered prior to, or subsequent to culturing of the cells, and in some cases at the same time as or during at least a portion of the culturing.
  • genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319- 338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al.
  • the methods for generating the engineered cells include one or more steps for cultivating cells, e.g., cultivating cells under conditions that promote proliferation and/or expansion.
  • cells are cultivated under conditions that promote proliferation and/or expansion subsequent to a step of genetically engineering, e.g., introducing a recombinant polypeptide to the cells by transduction or transfection.
  • the cells are cultivated after the cells have been incubated under stimulating conditions and transduced or transfected with a recombinant polynucleotide, e.g., a polynucleotide encoding a recombinant receptor.
  • a composition of CAR-positive T cells that has been engineered by transduction or transfection with a recombinant polynucleotide encoding the CAR, is cultivated under conditions that promote proliferation and/or expansion.
  • the one or more compositions of engineered T cells are or include two separate compositions of enriched T cells, such as two separate compositions of enriched T cells that have been engineered with a polynucleotide encoding a recombinant receptor, e.g. a CAR.
  • two separate compositions of enriched T cells e.g., two separate compositions of enriched T cells selected, isolated, and/or enriched from the same biological sample, are separately cultivated under stimulating conditions , such as subsequent to a step of genetically engineering, e.g., introducing a recombinant polypeptide to the cells by transduction or transfection.
  • the two separate compositions include a composition of enriched CD4+ T cells, such as a composition of enriched CD4+ T cells that have been engineered with a polynucleotide encoding a recombinant receptor, e.g. a CAR.
  • the two separate compositions include a composition of enriched CD8+ T cells, such as a composition of enriched CD4+ T cells that have been engineered with a polynucleotide encoding a recombinant receptor, e.g. a CAR.
  • two separate compositions of enriched CD4+ T cells and enriched CD8+ T cells are separately cultivated, e.g., under conditions that promote proliferation and/or expansion.
  • cultivation is carried out under conditions that promote proliferation and/or expansion.
  • such conditions may be designed to induce proliferation, expansion, activation, and/or survival of cells in the population.
  • the stimulating conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to promote growth, division, and/or expansion of the cells.
  • the cells are cultivated in the presence of one or more cytokines.
  • the one or more cytokines are recombinant cytokines.
  • the one or more cytokines are human recombinant cytokines.
  • the one or more cytokines bind to and/or are capable of binding to receptors that are expressed by and/or are endogenous to T cells.
  • the one or more cytokines, e.g. a recombinant cytokine is or includes a member of the 4-alpha-helix bundle family of cytokines.
  • members of the 4-alpha-helix bundle family of cytokines include, but are not limited to, interleukin-2 (IL -2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin 12 (IL-12), interleukin 15 (IL- 15), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).
  • the one or more recombinant cytokine includes IL -2, IL-7 and/or IL-15.
  • the cells are cultivated in the presence of a cytokine, e.g., a recombinant human cytokine, at a concentration of between 1 lU/mL and 2,000 lU/mL, between 10 lU/mL and 100 lU/mL, between 50 lU/mL and 200 lU/mL, between 100 lU/mL and 500 lU/mL, between 100 lU/mL and 1,000 lU/mL, between 500 lU/mL and 2,000 lU/mL, or between 100 lU/mL and 1 ,500 lU/mL.
  • a cytokine e.g., a recombinant human cytokine
  • the cultivation is performed under conditions that generally include a temperature suitable for the growth of primary immune cells, such as human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees Celsius, and generally at or about 37 degrees Celsius.
  • the composition of enriched T cells is incubated at a temperature of 25 to 38°C, such as 30 to 37°C, for example at or about 37 °C ⁇ 2 °C.
  • the incubation is carried out for a time period until the culture, e.g. cultivation or expansion, results in a desired or threshold density, number or dose of cells.
  • the incubation is greater than or greater than about or is for about or 24 hours, 48 hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days or more.
  • the cultivation is performed in a closed system.
  • the cultivation is performed in a closed system under sterile conditions.
  • the cultivation is performed in the same closed system as one or more steps of the provided systems.
  • the composition of enriched T cells is removed from a closed system and placed in and/or connected to a bioreactor for the cultivation.
  • suitable bioreactors for the cultivation include, but are not limited to, GE Xuri W25, GE Xuri W5, Sartorius BioSTAT® RM 20
  • the bioreactor is used to perfuse and/or mix the cells during at least a portion of the cultivation step.
  • the mixing is or includes rocking and/or motioning.
  • the bioreactor can be subject to motioning or rocking, which, in some aspects, can increase oxygen transfer.
  • Motioning the bioreactor may include, but is not limited to rotating along a horizontal axis, rotating along a vertical axis, a rocking motion along a tilted or inclined horizontal axis of the bioreactor or any combination thereof.
  • at least a portion of the incubation is carried out with rocking. The rocking speed and rocking angle may be adjusted to achieve a desired agitation.
  • the rock angle is 20°, 19°, 18°, 17°, 16°, 15°, 14°, 13°, 12°, 11°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2° or 1°.
  • the rock angle is between 6-16°.
  • the rock angle is between 7-16°.
  • the rock angle is between 8-12°.
  • the rock rate is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 rpm.
  • the rock rate is between 4 and 12 rpm, such as between 4 and 6 rpm, inclusive.
  • the bioreactor maintains the temperature at or near 37°C and CO2 levels at or near 5% with a steady air flow at, at about, or at least 0.01 L/min, 0.05 L/min, 0.1 L/min, 0.2 L/min, 0.3 L/min, 0.4 L/min, 0.5 L/min, 1.0 L/min, 1.5 L/min, or 2.0 L/min or greater than 2.0 L/min.
  • At least a portion of the cultivation is performed with perfusion, such as with a rate of 290 ml/day, 580 ml/day, and/or 1160 ml/day, e.g., depending on the timing in relation to the start of the cultivation and/or density of the cultivated cells.
  • at least a portion of the cell culture expansion is performed with a rocking motion, such as at an angle of between 5° and 10°, such as 6°, at a constant rocking speed, such as a speed of between 5 and 15 RPM, such as 6 RPM or 10 RPM.
  • the methods for manufacturing, generating or producing a cell therapy and/or engineered cells may include formulation of cells, such as formulation of genetically engineered cells resulting from the processing steps prior to or after the incubating, engineering, and cultivating, and/or one or more other processing steps as described.
  • one or more of the processing steps, including formulation of cells can be carried out in a closed system.
  • the cells are processed in one or more steps (e.g.
  • the centrifugal chamber and/or closed system for manufacturing, generating or producing a cell therapy and/or engineered cells may include formulation of cells, such as formulation of genetically engineered cells resulting from the transduction processing steps prior to or after the culturing, e.g. cultivation and expansion, and/or one or more other processing steps as described.
  • the genetically engineered cells are formulated as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof.
  • the dose of cells comprising cells engineered with a recombinant antigen receptor is provided as a composition or formulation, such as a pharmaceutical composition or formulation.
  • a composition or formulation such as a pharmaceutical composition or formulation.
  • Such compositions can be used in accord with the provided methods, such as in the treatment of diseases, conditions, and disorders, or in detection, diagnostic, and prognostic methods, and uses and articles of manufacture.
  • the cells can be formulated in an amount for dosage administration, such as for a single unit dosage administration or multiple dosage administration.
  • the cells can be formulated into a container, such as a bag or vial.
  • the vial may be an infusion vial.
  • the vial is formulated with a single unit dose of the engineered cells, such as including the number of cells for administration in a given dose or fraction thereof.
  • the cells are formulated in a pharmaceutically acceptable buffer, which may, in some aspects, include a pharmaceutically acceptable carrier or excipient.
  • the processing includes exchange of a medium into a medium or formulation buffer that is pharmaceutically acceptable or desired for administration to a subject.
  • the processing steps can involve washing the transduced and/or expanded cells to replace the cells in a pharmaceutically acceptable buffer that can include one or more optional pharmaceutically acceptable carriers or excipients.
  • a pharmaceutically acceptable buffer can include one or more optional pharmaceutically acceptable carriers or excipients.
  • Exemplary of such pharmaceutical forms, including pharmaceutically acceptable carriers or excipients, can be any described below in conjunction with forms acceptable for administering the cells and compositions to a subject.
  • the pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount.
  • the formulation buffer contains a cryopreservative.
  • the cell are formulated with a cyropreservative solution that contains 1.0% to 30% DMSO solution, such as a 5% to 20% DMSO solution or a 5% to 10% DMSO solution.
  • the cryopreservation solution is or contains, for example, PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media.
  • the cryopreservative solution is or contains, for example, at least or about 7.5% DMSO.
  • the processing steps can involve washing the transduced and/or expanded cells to replace the cells in a cryopreservative solution.
  • the cells are frozen, e.g., cryoprotected or cryopreserved, in media and/or solution with a final concentration of or of about 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5.0% DMSO, or between 1% and 15%, between 6% and 12%, between 5% and 10%, or between 6% and 8% DMSO.
  • the cells are frozen, e.g., cryoprotected or cryopreserved, in media and/or solution with a final concentration of or of about 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5%, or 0.25% HSA, or between 0.1% and 5%, between 0.25% and 4%, between 0.5% and 2%, or between 1% and 2% HSA.
  • the formulation is carried out using one or more processing step including washing, diluting or concentrating the cells, such as the cultured or expanded cells.
  • the processing can include dilution or concentration of the cells to a desired concentration or number, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof.
  • the processing steps can include a volumereduction to thereby increase the concentration of cells as desired.
  • the processing steps can include a volume-addition to thereby decrease the concentration of cells as desired.
  • the processing includes adding a volume of a formulation buffer to transduced and/or expanded cells.
  • the volume of formulation buffer is from or from about 10 mL to 1000 mL, such as at least or at least about or about or 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL or 1000 mL.
  • such processing steps for formulating a cell composition is carried out in a closed system.
  • Exemplary of such processing steps can be performed using a centrifugal chamber in conjunction with one or more systems or kits associated with a cell processing system, such as a centrifugal chamber produced and sold by Biosafe SA, including those for use with the Sepax® or Sepax 2® cell processing systems.
  • a centrifugal chamber produced and sold by Biosafe SA, including those for use with the Sepax® or Sepax 2® cell processing systems.
  • An exemplary system and process is described in International Publication Number WO2016/073602.
  • the method includes effecting expression from the internal cavity of the centrifugal chamber a formulated composition, which is the resulting composition of cells formulated in a formulation buffer, such as pharmaceutically acceptable buffer, in any of the above embodiments as described.
  • the expression of the formulated composition is to a container, such as the vials of the biomedical material vessels described herein, that is operably linked as part of a closed system with the centrifugal chamber.
  • the biomedical material vessels are configured for integration and or operable connection and/or is integrated or operably connected, to a closed system or device that carries out one or more processing steps.
  • the biomedical material vessel is connected to a system at an output line or output position.
  • the closed system is connected to the vial of the biomedical material vessel at the inlet tube.
  • Exemplary close systems for use with the biomedical material vessels described herein include the Sepax® and Sepax® 2 system.
  • the closed system such as associated with a centrifugal chamber or cell processing system, includes a multi-port output kit containing a multi-way tubing manifold associated at each end of a tubing line with a port to which one or a plurality of containers can be connected for expression of the formulated composition.
  • a desired number or plurality of vials can be sterilely connected to one or more, generally two or more, such as at least 3, 4, 5, 6, 7, 8 or more of the ports of the multi-port output.
  • one or more containers e.g., biomedical material vessels, can be attached to the ports, or to fewer than all of the ports.
  • the system can effect expression of the output composition into a plurality of vials of the biomedical material vessels.
  • cells can be expressed to the one or more of the plurality of output containers, e.g., vials, in an amount for dosage administration, such as for a single unit dosage administration or multiple dosage administration.
  • the vials may each contain the number of cells for administration in a given dose or fraction thereof.
  • each vial in some aspects, may contain a single unit dose for administration or may contain a fraction of a desired dose such that more than one of the plurality of vials, such as two of the vials, or 3 of the vials, together constitute a dose for administration. In some embodiments, 4 vials together constitute a dose for administration.
  • the containers e.g.
  • bags or vials generally contain the cells to be administered, e.g., one or more unit doses thereof.
  • the unit dose may be an amount or number of the cells to be administered to the subject or twice the number (or more) of the cells to be administered. It may be the lowest dose or lowest possible dose of the cells that would be administered to the subject.
  • the provided articles of manufacture includes one or more of the plurality of output containers.
  • each of the containers individually comprises a unit dose of the cells.
  • each of the containers comprises the same or approximately or substantially the same number of cells.
  • each unit dose contains at or about or at least or at least about 1 x 10 6 , 2 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , or 1 x 10 8 engineered cells, total cells, T cells, or PBMCs.
  • each unit dose contains at or about or at least or at least about 1 x 10 6 , 2 x 10 6 , 5 x 10 6 , 1 x 10 7 , 5 x 10 7 , or 1 x 10 8 CAR+ T cells that are CD3+, such as CD4+ or CD8+, or a viable subset thereof.
  • the volume of the formulated cell composition in each container e.g. bag or vial, is between at or about 10 mL and at or about 100 mL, such as at or about or at least or at least about 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL or 100 mL.
  • the volume of the formulated cell composition in each container is between at or about 1 mL and at or about 10 mL, such as between at or about 1 mL and at or about 5 mL. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is between at or about 4 mL and at or about 5 mL. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is or is about 4.4 mL. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is or is about 4.5 mL.
  • the volume of the formulated cell composition in each container, e.g. bag or vial is or is about 4.6 mL. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is or is about 4.7 mL. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is or is about 4.8 mL. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is or is about 4.9 mL. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is or is about 5.0 mL.
  • the formulated cell composition has a concentration of greater than at or about 0.5 x 10 6 recombinant receptor-expressing (e.g. CAR + )/CD3+ cells or such viable cells per mL, greater than at or about 1.0 x 10 6 recombinant receptor-expressing (e.g. CAR + )/CD3+ cells or such viable cells per mL, greater than at or about 1.5 x 10 6 recombinant receptor-expressing (e.g. CAR + )/CD3+ cells or such viable cells per mL, greater than at or about 2.0 x 10 6 recombinant receptor-expressing (e.g. CAR + )/CD3+ cells or such viable cells per mL.
  • CAR + x 10 6 recombinant receptor-expressing
  • CAR + )/CD3+ cells or such viable cells per mL greater than at or about 2.9 x 10 6 recombinant receptor-expressing (e.g. CAR + )/CD3+ cells or such viable cells per mL greater than at or about 3.0 x 10 6 recombinant receptor-expressing (e.g. CAR + )/CD3+ cells or such viable cells per mL, greater than at or about 3.5 x 10 6 recombinant receptorexpressing (e.g. CAR + )/CD3+ cells or such viable cells per mL, greater than at or about 4.0 x 10 6 recombinant receptor-expressing (e.g.
  • the CD3+ cells are CD4+ T cells.
  • the CD3+ cells are CD8+ T cells.
  • the CD3+ T dels are CD4+ and CD8+ T cells.
  • the cells in the container e.g. bag or vials
  • the container e.g. vials
  • the container can be stored in liquid nitrogen until further use.
  • such cells produced by the method, or a composition comprising such cells are administered to a subject for beating a disease or condition, for example, in accord with the methods, uses and articles of manufacture described herein.
  • the provided combination therapy results in one or more treatment outcomes, such as a feature associated with any one or more of the parameters associated with the therapy or treatment, as described below.
  • the method includes assessment of the cytotoxicity of the T cells toward cancer cells, e.g., T cells administered for the T cell based therapy.
  • the method includes assessment of the exposure, persistence and proliferation of the T cells, e.g., T cells administered for the T cell based therapy.
  • the exposure, or prolonged expansion and/or persistence of the cells, and/or changes in cell phenotypes or functional activity of the cells, e.g., cells administered for immunotherapy, e.g. T cell therapy, in the methods provided herein can be measured by assessing the characteristics of the T cells in vitro or ex vivo.
  • such assays can be used to determine or confirm the function of the T cells, e.g. T cell therapy, before, during, or after administering the combination therapy provided herein.
  • the combination therapy can further include one or more screening steps to identify subjects for treatment with the combination therapy and/or continuing the combination therapy, and/or a step for assessment of heatment outcomes and/or monitoring treatment outcomes.
  • the step for assessment of heatment outcomes can include steps to evaluate and/or to monitor heatment and/or to identify subjects for administration of further or remaining steps of the therapy and/or for repeat therapy.
  • the screening step and/or assessment of treatment outcomes can be used to determine the dose, frequency, duration, timing and/or order of the combination therapy provided herein.
  • any of the screening steps and/or assessment of treatment of outcomes described herein can be used prior to, during, during the course of, or subsequent to administration of one or more steps of the provided combination therapy, e.g., administration of the immunotherapy or cell therapy e.g. CAR-expressing T cells), and/or a BCL2 inhibitor, e.g. venetoclax.
  • assessment is made prior to, during, during the course of, or after performing any of the methods provided herein.
  • the assessment is made prior to performing the methods provided herein.
  • assessment is made after performing one or more steps of the methods provided herein.
  • the assessment is performed prior to administration of one or more steps of the provided combination therapy, for example, to screen and identify patients suitable and/or susceptible to receive the combination therapy. In some embodiments, the assessment is performed during, during the course of, or subsequent to administration of one or more steps of the provided combination therapy, for example, to assess the intermediate or final treatment outcome, e.g., to determine the efficacy of the treatment and/or to determine whether to continue or repeat the treatments and/or to determine whether to administer the remaining steps of the combination therapy.
  • treatment of outcomes includes improved immune function, e.g., immune function of the T cells administered for cell based therapy and/or of the endogenous T cells in the body.
  • exemplary treatment outcomes include, but are not limited to, enhanced T cell proliferation, enhanced T cell functional activity, changes in immune cell phenotypic marker expression, such as such features being associated with the engineered T cells, e.g. CAR-T cells, administered to the subject.
  • exemplary treatment outcomes include decreased disease burden, e.g., tumor burden, improved clinical outcomes and/or enhanced efficacy of therapy.
  • the screening step and/or assessment of treatment of outcomes includes assessing the survival and/or function of the T cells administered for cell based therapy. In some embodiments, the screening step and/or assessment of treatment of outcomes includes assessing the levels of cytokines or growth factors. In some embodiments, the screening step and/or assessment of treatment of outcomes includes assessing disease burden and/or improvements, e.g., assessing tumor burden and/or clinical outcomes.
  • either of the screening step and/or assessment of treatment of outcomes can include any of the assessment methods and/or assays described herein and/or known in the art, and can be performed one or more times, e.g., prior to, during, during the course of, or subsequently to administration of one or more steps of the combination therapy.
  • Exemplary sets of parameters associated with a treatment outcome which can be assessed in some embodiments of the methods provided herein, include peripheral blood immune cell population profile and/or tumor burden.
  • the methods affect efficacy of the cell therapy in the subject.
  • the cytotoxicity of recombinant receptor-expressing, e.g., CAR-expressing, cells in the subject following administration of the dose of cells in the method with a BCL2 inhibitor, e.g., venetoclax is greater as compared to that achieved via a method without the administration of the inhibitor.
  • the cytotoxicity of recombinant receptor-expressing, e.g., CAR-expressing, cells in the subject following administration of the dose of cells in the method with a subtherapeutically effective amount of a BCL2 inhibitor, e.g., venetoclax is greater as compared to that achieved via a method without the administration of the inhibitor.
  • cytotoxicity in the subject of the administered T cell therapy e.g., CAR-expressing T cells is assessed as compared to a method in which the T cell therapy is administered to the subject in the absence of a BCL2 inhibitor, e.g., venetoclax.
  • the methods result in the administered T cells exhibiting increased or prolonged cytotoxicity in the subject as compared to a method in which the T cell therapy is administered to the subject in the absence of the inhibitor.
  • the administration of a BCL2 inhibitor decreases disease burden, e.g., tumor burden, in the subject as compared to a method in which the dose of cells expressing the recombinant receptor is administered to the subject in the absence of the inihibitor.
  • the administration of a subtherapeutically effective amount of a BCL2 inhibitor, e.g., venetoclax decreases disease burden, e.g., tumor burden, in the subject as compared to a method in which the dose of cells expressing the recombinant receptor is administered to the subject in the absence the inhibitor.
  • the administration of a BCL2 inhibitor decreases blast marrow in the subject as compared to a method in which the dose of cells expressing the recombinant receptor is administered to the subject in the absence of the inhibitor.
  • the administration of a BCL2 inhibitor results in improved clinical outcomes, e.g., objective response rate (ORR), progression-free survival (PFS) and/or overall survival (OS), compared to a method in which the dose of cells expressing the recombinant receptor is administered to the subject in the absence of the inhibitor.
  • ORR objective response rate
  • PFS progression-free survival
  • OS overall survival
  • the subject can be screened prior to the administration of one or more steps of the combination therapy.
  • the subject can be screened for characteristics of the disease and/or disease burden, e.g., tumor burden, prior to administration of the combination therapy, to determine suitability, responsiveness and/or susceptibility to administering the combination therapy.
  • the subject can be screened for characteristics of the disease, e.g., overexpression or aberrant expression of a prosurvival or proapoptotic BCL2 family protein, prior to administration of the combination therapy, to determine suitability, responsiveness and/or susceptibility to administering the combination therapy.
  • the screening step and/or assessment of treatment outcomes can be used to determine the dose, frequency, duration, timing and/or order of the combination therapy provided herein.
  • the subject can be screened after administration of one of the steps of the combination therapy, to determine and identify subjects to receive the remaining steps of the combination therapy and/or to monitor efficacy of the therapy.
  • the number, level or amount of administered T cells and/or proliferation and/or activity of the administered T cells is assessed after administration of the engineered T cells and/or after administration of a BCL2 inhibitor, e.g., venetoclax.
  • a change and/or an alteration e.g., an increase, an elevation, a decrease or a reduction, in levels, values or measurements of a parameter or outcome compared to the levels, values or measurements of the same parameter or outcome in a different time point of assessment, a different condition, a reference point and/or a different subject is determined or assessed.
  • the levels, values or measurements of two or more parameters are determined, and relative levels are compared.
  • the determined levels, values or measurements of parameters are compared to the levels, values or measurements from a control sample or an untreated sample.
  • the determined levels, values or measurements of parameters are compared to the levels from a sample from the same subject but at a different time point.
  • the values obtained in the quantification of individual parameter can be combined for the purpose of disease assessment, e.g., by forming an arithmetical or logical operation on the levels, values or measurements of parameters by using multi-parametric analysis.
  • a ratio of two or more specific parameters can be calculated.
  • Assessment and determination of parameters associated with T cell health, function, activity, and/or outcomes, such as response, efficacy and/or toxicity outcomes, can be assessed at various time points.
  • the assessment can be performed multiple times, e.g., prior to, during, and/or after manufacturing of the cells, prior to, during, and/or after the initiation of administrationof the cell therapy, and/or prior to, during, and/or after the initiation of administration of the BCL2 inhibitor, e.g., venetoclax.
  • functional attributes of the administered cells and/or cell compositions include monitoring pharmacokinetic (PK) and pharmacodynamics parameters, expansion and persistence of the cells, cell functional assays (e.g., any described herein, such as cytotoxicity assay, cytokine secretion assay and in vivo assays), high-dimensional T cell signaling assessment, and assessment of exhaustion phenotypes and/or signatures of the T cells.
  • PK pharmacokinetic
  • cell functional assays e.g., any described herein, such as cytotoxicity assay, cytokine secretion assay and in vivo assays
  • MRD minimal residual disease
  • other attributes that can be assessed or monitored include pharmacodynamic parameters of the BCL2 inhibitor, e.g., venetoclax.
  • parameters associated with therapy or a treatment outcome which include parameters that can be assessed for the screening steps and/or assessment of treatment of outcomes and/or monitoring treatment outcomes, includes tumor or disease burden.
  • the administration of the therapy that is an immunotherapy or cell therapy such as a T cell therapy (e.g. CAR-expressing T cells) and/or a BCL2 inhibitor, e.g., venetoclax, can reduce or prevent the expansion or burden of the disease or condition in the subject.
  • the methods generally reduce tumor size, bulk, metastasis, percentage of blasts in the bone marrow or molecularly detectable B cell malignancy and/or improve prognosis or survival or other symptom associated with tumor burden.
  • the disaease or condition is a cancer, such as a CLL or SLL.
  • the administration in accord with the provided methods, and/or with the provided articles of manufacture or compositions generally reduces or prevents the expansion or burden of the disease or condition in the subject.
  • the methods generally reduce tumor size, bulk, metastasis, percentage of blasts in the bone marrow or molecularly detectable B cell malignancy and/or improve prognosis or survival or other symptom associated with tumor burden.
  • the provided methods result in a decreased tumor burden in treated subjects compared to alternative methods in which the therapy, such as a T cell therapy (e.g. CAR- expressing T cells) is given without administration of a BCL2 inhibitor, e.g., venetoclax.
  • the provided methods result in a decreased tumor burden in subjects treated with a subtherapeutically effective amount of a BCL2 inhibitor, e.g. venetoclax, compared to alternative methods in which the immunotherapy, such as a T cell therapy e.g. CAR-expressing T cells) is given without administration of the inhibitor.
  • tumor burden it is not necessary that the tumor burden actually be reduced in all subjects receiving the combination therapy, but that tumor burden is reduced on average in subjects treated, such as based on clinical data, in which a majority of subjects treated with such a combination therapy exhibit a reduced tumor burden, such as at least 50%, 60%, 70%, 80%, 90%, 95% or more of subjects treated with the combination therapy, exhibit a reduced tumor burden.
  • the provided methods result in increased cytotoxic activity of the immunotherapy or cell therapy as compared to alternative methods in which the therapy, such as a T cell therapy (e.g. CAR-expressing T cells) is given without administration of a BCL2 inhibitor, e.g., venetoclax.
  • the provided methods result in increased cytotoxicity in subjects treated with a subtherapeutically effective amount of a BCL2 inhibitor, e.g. venetoclax, compared to alternative methods in which the immunotherapy, such as a T cell therapy (e.g. CAR-expressing T cells) is given without administration of the inhibitor.
  • the provided methods result in an increased cytotoxicity of the immunotherapy or cell therapy, optionally via perforin- and/or granzyme- mediate apoptosis, of one or more cancer cells, compared to alternative methods in which the immunotherapy, such as a T cell therapy (e.g. CAR-expressing T cells) is given without administration of a BCL2 inhibitor, e.g., venetoclax.
  • a T cell therapy e.g. CAR-expressing T cells
  • a BCL2 inhibitor e.g., venetoclax.
  • cytotoxicity it is not necessary that the cytotoxicity actually be increased in all subjects receiving the combination therapy, but that cytotoxicity is increased on average in subjects treated, such as based on clinical data, in which a majority of subjects treated with such a combination therapy exhibit a reduced tumor burden, such as at least 50%, 60%, 70%, 80%, 90%, 95% or more of subjects treated with the combination therapy, exhibit a reduced tumor burden.
  • Disease burden can encompass a total number of cells of the disease in the subject or in an organ, tissue, or bodily fluid of the subject, such as the organ or tissue of the tumor or another location, e.g., which would indicate metastasis.
  • tumor cells may be detected and/or quantified in the blood, lymph or bone marrow in the context of certain hematological malignancies.
  • tumor cells may be detected and/or quantified in the peripheral blood (PB).
  • PB peripheral blood
  • tumor cells may be detected and/or quantified in the lymph nodes.
  • tumor cells may be detected and/or quantified in the bone marrow.
  • Disease burden can include, in some embodiments, the mass of a tumor, the number or extent of metastases and/or the percentage of blast cells present in the bone marrow.
  • the subject has a myeloma, a lymphoma or a leukemia.
  • the extent of disease burden can be determined by assessment of residual leukemia in blood or bone marrow.
  • the subject has a non-Hodgkin lymphoma (NHL), an acute lymphoblastic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a small lymphocytic lymphoma (SLL) a diffuse large B-cell lymphoma (DLBCL) or a myeloma, e.g., a multiple myeloma (MM).
  • NHL non-Hodgkin lymphoma
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • DLBCL diffuse large B-cell lymphoma
  • MM multiple myeloma
  • the subject has a leukemia or lymphoma.
  • the subject has a leukemia. In some cases, the leukemia is CLL. In some embodiments, the subject has a lymphoma. In some cases, the subject has a NHL, including DBCBL. In some cases, the lymphoma is SLL.
  • response rates in subjects are based on the Lugano criteria.
  • Lugano criteria Cheson et al., (2014) JCO., 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323- 338; Cheson, B.D. (2015) Chin. Clin. Oncol. 4(1):5).
  • response assessment utilizes any of clinical, hematologic, and/or molecular methods.
  • response assessed using the Lugano criteria involves the use of positron emission tomography (PET)-computed tomography (CT) and/or CT as appropriate.
  • PET positron emission tomography
  • CT computed tomography
  • PET-CT evaluations may further comprise the use of fluorodeoxyglucose (FDG) for FDG-avid lymphomas.
  • FDG fluorodeoxyglucose
  • a 5-point scale may be used.
  • the 5-point scale comprises the following criteria: 1, no uptake above background; 2, uptake ⁇ mediastinum; 3, uptake > mediastinum but ⁇ liver; 4, uptake moderately > liver; 5, uptake markedly higher than liver and/or new lesions; X, new areas of uptake unlikely to be related to lymphoma.
  • a complete response as described using the Lugano criteria involves a complete metabolic response and a complete radiologic response at various measureable sites.
  • these sites include lymph nodes and extralymphatic sites, wherein a CR is described as a score of 1, 2, or 3 with or without a residual mass on the 5-point scale, when PET -CT is used.
  • uptake may be greater than normal mediastinum and/or liver. In this circumstance, complete metabolic response may be inferred if uptake at sites of initial involvement is no greater than surrounding normal tissue even if the tissue has high physiologic uptake.
  • response is assessed in the lymph nodes using CT, wherein a CR is described as no extralymphatic sites of disease and target nodes/nodal masses must regress to ⁇ 1.5 cm in longest transverse diameter of a lesion (LDi).
  • Further sites of assessment include the bone marrow wherein PET-CT-based assessment should indicate a lack of evidence of FDG-avid disease in marrow and a CT-based assessment should indicate a normal morphology, which if indeterminate should be IHC negative. Further sites may include assessment of organ enlargement, which should regress to normal.
  • nonmeasured lesions and new lesions are assessed, which in the case of CR should be absent (Cheson et al., (2014) JCO., 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323-338; Cheson, B.D. (2015) Chin. Clin. Oncol. 4(1):5).
  • a partial response (PR) as described using the Lugano criteria involves a partial metabolic and/or radiological response at various measureable sites.
  • these sites include lymph nodes and extralymphatic sites, wherein a PR is described as a score of 4 or 5 with reduced uptake compared with baseline and residual mass(es) of any size, when PET-CT is used.
  • a PR is described as a score of 4 or 5 with reduced uptake compared with baseline and residual mass(es) of any size, when PET-CT is used.
  • findings can indicate responding disease.
  • At the end of treatment such findings can indicate residual disease.
  • response is assessed in the lymph nodes using CT, wherein a PR is described as >50% decrease in SPD of up to 6 target measureable nodes and extranodal sites.
  • 5 mm x 5 mm is assigned as the default value; if the lesion is no longer visible, the value is 0 mm x 0 mm; for a node >5 mm x 5 mm, but smaller than normal, actual measurements are used for calculation.
  • Further sites of assessment include the bone marrow wherein PET-CT-based assessment should indicate residual uptake higher than uptake in normal marrow but reduced compared with baseline (diffuse uptake compatible with reactive changes from chemotherapy allowed).
  • consideration should be given to further evaluation with MRI or biopsy, or an interval scan.
  • further sites may include assessment of organ enlargement, where the spleen must have regressed by >50% in length beyond normal.
  • nonmeasured lesions and new lesions are assessed, which in the case of PR should be absent/normal, regressed, but no increase.
  • No response/stable disease (SD) or progressive disease (PD) can also be measured using PET-CT and/or CT based assessments.
  • progression-free survival is described as the length of time during and after the treatment of a disease, such as a B cell malignancy, that a subject lives with the disease but it does not get worse.
  • objective response is described as a measurable response.
  • objective response rate is described as the proportion of patients who achieved CR or PR.
  • overall survival is described as the length of time from either the date of diagnosis or the start of treatment for a disease, such as a B cell malignancy, that subjects diagnosed with the disease are still alive.
  • event-free survival is described as the length of time after treatment for a B cell malignancy ends that the subject remains free of certain complications or events that the treatment was intended to prevent or delay. These events may include the return of the B cell malignancy or the onset of certain symptoms, such as bone pain from B cell malignancy that has spread to the bone, or death.
  • the measure of duration of response includes the time from documentation of tumor response to disease progression.
  • the parameter for assessing response can include durable response, e.g., response that persists after a period of time from initiation of therapy.
  • durable response is indicated by the response rate at approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18 or 24 months after initiation of therapy.
  • the response is durable for greater than 3 months or greater than 6 months.
  • the RECIST criteria is used to determine objective tumor response. (Eisenhauer et al., European Journal of Cancer 45 (2009) 228-247.) In some aspects, the RECIST criteria is used to determine objective tumor response for target lesions. In some respects, a complete response as determined using RECIST criteria is described as the disappearance of all target lesions and any pathological lymph nodes (whether target or non-target) must have reduction in short axis to ⁇ 10 mm. In other aspects, a partial response as determined using RECIST criteria is described as at least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters.
  • progressive disease is described as at least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm (in some aspects the appearance of one or more new lesions is also considered progression).
  • stable disease is described as neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study.
  • exemplary parameters to assess the extent of disease burden include such parameters as number of clonal plasma cells (e.g., >10% on bone marrow biopsy or in any quantity in a biopsy from other tissues; plasmacytoma), presence of monoclonal protein (paraprotein) in either serum or urine, evidence of end-organ damage felt related to the plasma cell disorder (e.g., hypercalcemia (corrected calcium >2.75 mmol/1); renal insufficiency attributable to myeloma; anemia (hemoglobin ⁇ 10 g/dl); and/or bone lesions (lytic lesions or osteoporosis with compression fractures)).
  • number of clonal plasma cells e.g., >10% on bone marrow biopsy or in any quantity in a biopsy from other tissues; plasmacytoma
  • paraprotein monoclonal protein
  • evidence of end-organ damage felt related to the plasma cell disorder e.g., hypercalcemia (corrected calcium >2.75 mmol/1); renal insufficiency
  • exemplary parameters to assess the extent of disease burden include such parameters as cellular morphology (e.g., centroblastic, immunobias tic, and anaplastic cells), gene expression, miRNA expression and protein expression (e.g., expression of BCL2, BCL6, MUM1, LM02, MYC, and p21).
  • cellular morphology e.g., centroblastic, immunobias tic, and anaplastic cells
  • miRNA expression and protein expression e.g., expression of BCL2, BCL6, MUM1, LM02, MYC, and p21.

Abstract

Provided are combination therapies involving immunotherapies and cell therapies, such as adoptive cell therapy, e.g. a T cell therapy, and the use of an inhibitor of BCL2 protein, for treating subjects having or suspected of having a cancer, and related methods, uses, and articles of manufacture. The T cell therapy includes cells that express recombinant receptors such as chimeric antigen receptors (CARs).

Description

COMBINATION THERAPY OF A CELL THERAPY AND A BCL2 INHIBITOR
Cross-Reference to Related Applications
[0001] This application claims priority to U.S. Provisional Applications Nos. 63/126,406, filed December 16, 2020, entitled “COMBINATION THERAPY OF A CELL THERAPY AND A BCL2 INHIBITOR” and 63/166,211, filed March 25, 2021, entitled “COMBINATION THERAPY OF A CELL THERAPY AND A BCL2 INHIBITOR,” the contents of which are incorporated by reference in their entirety for all purposes.
Incorporation by Reference of Sequence Listing
[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042024640SEQLIST.TXT, created December 7, 2021, which is 35,424 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.
Field
[0003] The present disclosure relates in some aspects to methods and uses of combination therapies involving a therapy such as an immunotherapy or a cell therapy, e.g., a T cell therapy, and the use of an inhibitor of BCL2 protein, for treating subjects with cancers such as leukemias and lymphomas, and related methods, uses, and articles of manufacture. The T cell therapy includes cells that express recombinant receptors such as chimeric antigen receptors (CARs).
Background
[0004] Various strategies are available for immunotherapy and cell therapy for treating cancers, for example, adoptive cell therapies, including those involving the administration of cells expressing chimeric receptors specific for a disease or disorder of interest, such as chimeric antigen receptors (CARs) and/or other recombinant antigen receptors, as well as other adoptive immune cell and adoptive T cell therapies. Subsets of cancers are resistant to or develop resistance to such therapies. Improved methods are therefore needed, for example, to overcome this resistance and increase the efficacy of such methods. Provided are methods and uses that meet such needs.
Summary
[0005] Provided herein is a method of treating cancer, the method including (1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1; and (2) administering to the subject a BCL2 inhibitor in a dosing regimen including: (i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy; and (ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
[0006] Also provided herein is a method of treating cancer, the method including administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the BCL2 inhibitor is administered in a dosing regimen including: (i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of a cell therapy, wherein the cell therapy includes a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) and was administered to the subject on Day 1; and (ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
[0007] Also provided herein is a method of treating cancer, the method including administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen including: (i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy; and (ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
[0008] In some embodiments, the daily administration of the first dose of the BCL2 inhibitor begins about 1 day after or within about 1 day after initiation of administration of the cell therapy. In some embodiments, the daily administration of the first dose of the BCL2 inhibitor begins about 1 day after initiation of administration of the cell therapy. In some embodiments, the daily administration of the first dose of the BCL2 inhibitor begins within about 1 day after initiation of administration of the cell therapy. In some embodiments, the daily administration of the first dose of the BCL2 inhibitor begins at or after a time when activation-induced cell death (AICD) of the cells of the cell therapy has peaked following initiation of administration of the cell therapy. In some embodiments, the daily administration of the first dose of the BCL2 inhibitor begins at a time when activation-induced cell death (AICD) of the cells of the cell therapy has peaked following initiation of administration of the cell therapy. In some embodiments, the daily administration of the first dose of the BCL2 inhibitor begins after a time when activation- induced cell death (AICD) of the cells of the cell therapy has peaked following initiation of administration of the cell therapy.
[0009] In some embodiments, the at least one subsequent dose includes a second dose, and the dosing regimen includes: (i) daily administration of a first dose of the BCL2 inhibitor for a first predetermined period; and (ii) daily administration of a second dose of the BCL2 inhibitor for a second predetermined period beginning after the first predetermined period.
[0010] In some embodiments, the at least one subsequent dose includes a second dose and a third dose, and the dosing regimen includes: (i) daily administration of a first dose of the BCL2 inhibitor for a first predetermined period; (ii) daily administration of a second dose of the BCL2 inhibitor for a second predetermined period beginning after the first predetermined period; and (iii) daily administration of a third dose of the BCL2 inhibitor for a third predetermined period beginning after the second predetermined period.
[0011] In some embodiments, the first predetermined period is from about Day 2 to about Day 30. In some embodiments, the first predetermined period is from about Day 2 to about Day 7.
[0012] In some embodiments, the second predetermined period is from about Day 31 to about Day 90. In some embodiments, the second predetermined period is from about Day 8 to about Day 30. In some embodiments, the second predetermined period is from about Day 31 to about Day 37.
[0013] In some embodiments, the third predetermined period is from about Day 31 to about Day 90. In some embodiments, the third predetermined period is from about Day 38 to about Day 90.
[0014] In some embodiments, the first dose is at least about 20 mg of the BCL2 inhibitor. In some embodiments, no dose is greater than about 400 mg of the BCL2 inhibitor. In some embodiments, no dose of the dosing regimen is greater than about 400 mg of the BCL2 inhibitor.
[0015] In some embodiments, the first dose is between about 20 mg and about 100 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 20 mg, about 50 mg, or about 100 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 20 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 100 mg of the BCL2 inhibitor.
[0016] In some embodiments, the second dose is between about 50 mg and about 200 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 50 mg, about 100 mg, or about 200 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 50 mg, about 100 mg, about 150 mg, or about 200 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 150 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 200 mg of the BCL2 inhibitor.
[00171 In some embodiments, the third dose is between about 100 mg and 400 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 100 mg, about 200 mg, or about 400 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 100 mg, about 200 mg, about 300 mg, or about 400 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 200 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 300 mg of the BCL2 inhibitor.In some embodiments, the third dose is about 400 mg of the BCL2 inhibitor.
[0018] In some embodiments, the BCL2 inhibitor is venetoclax or navitoclax. In some embodiments, the BCL2 inhibitor is navitoclax.In some embodiments, the BCL2 inhibitor is venetoclax.
[0019] Also provided herein are methods of treating a cancer, the method including: (1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and (2) administering to the subject a BCL2 inhibitor in a dosing regimen including: (i) daily administration of about 20 mg of the BCL2 inhibitor from about Day 2 to about Day 7 ; (ii) daily administration of about 50 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and (iii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
[0020| Also provided herein is a method of treating a cancer, the method including administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the subject has been administered a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, and wherein the BCL2 inhibitor is administered in a dosing regimen including: (i) daily administration of about 20 mg of the BCL2 inhibitor from about Day 2 to about Day 7 ; (ii) daily administration of about 50 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and (iii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
[0021] Also provided herein is a method of treating cancer, the method including administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen including: (i) daily administration of about 20 mg of the BCL2 inhibitor from about Day 2 to about Day 7; (ii) daily administration of about 50 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and (iii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day 90. [0022] Also provided herein is a method of treating cancer, the method including: (1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and (2) administering to the subject a BCL2 inhibitor in a dosing regimen including: (i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and (ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
[0023] Also provided herein is a method of treating cancer, the method including administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the subject has been administered a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1, and wherein the BCL2 inhibitor is administered in a dosing regimen including: (i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and (ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
[0024] Also provided herein is a method of treating cancer, the method including administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen including: (i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and (ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
[0025] Also provided herein is a method of treating cancer, the method including: (1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and (2) administering to the subject a BCL2 inhibitor in a dosing regimen including: (i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 7; (ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and (iii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
[0026] Also provided herein is a method of treating cancer, the method including administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the subject has been administered a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, and the BCL2 inhibitor is administered in a dosing regimen including: (i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 7 ; (ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and (iii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
[0027] Also provided herein is a method of treating cancer, the method including administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen including: (i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 7; (ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and (iii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
[0028] Also provided herein is a method of treating cancer, the method including: (1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and (2) administering to the subject a BCL2 inhibitor in a dosing regimen including: (i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and (ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
[0029] Also provided herein is a method of treating cancer, the method including administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the subject has been administered a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1, and wherein the BCL2 inhibitor is administered in a dosing regimen including: (i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and (ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
[0030] Also provided herein is a method of treating cancer, the method including administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen including (i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and (ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
[0031] Also provided herein is a method of treating cancer, the method including: (1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and (2) administering to the subject a BCL2 inhibitor in a dosing regimen including: (i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30; (ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 37; and (iii) daily administration of about 400 mg of the BCL2 inhibitor from about Day 38 to about Day 90.
[0032] Also provided herein is a method of treating cancer, the method including administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the subject has been administered a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1, and wherein the BCL2 inhibitor is administering in a dosing regimen including: (i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30; (ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 37 ; and (iii) daily administration of about 400 mg of the BCL2 inhibitor from about Day 38 to about Day 90.
[0033] Also provided herein is a method of treating cancer, the method including administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy containing a dose of engineered cells including T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is be administered a BCL2 inhibitor in a dosing regimen including: (i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30; (ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 37; and (iii) daily administration of about 400 mg of the BCL2 inhibitor from about Day 38 to about Day 90.
[0034] In some embodiments, the BCL2 inhibitor is administered for no more than about 24 months. In some embodiments, the BCL2 inhibitor is administered for no more than about 18 months. In some embodiments, the BCL2 inhibitor is administered for no more than about 12 months. In some embodiments, if the subject exhibits minimum residual disease (MRD) in peripheral blood (PB) at Day 90, the BCL2 inhibitor is administered for up to 12 months or for up to 24 months. In some embodiments, if the subject exhibits minimum residual disease (MRD) in peripheral blood (PB) at Day 90, the BCL2 inhibitor is administered for up to 12 months. In some embodiments, if the subject exhibits minimum residual disease (MRD) in peripheral blood (PB) at Day 90, the BCL2 inhibitor is administered for up to 18 months. In some embodiments, if the subject exhibits minimum residual disease (MRD) in peripheral blood (PB) at Day 90, the BCL2 inhibitor is administered for up to 24 months. In some embodiments, if the subject does not exhibit a complete response (CR) at Day 90, the BCL2 inhibitor is administered for up to 12 months or for up to 24 months. In some embodiments, if the subject does not exhibit a complete response (CR) at Day 90, the BCL2 inhibitor is administered for up to 12 months. In some embodiments, if the subject does not exhibit a complete response (CR) at Day 90, the BCL2 inhibitor is administered for up to 18 months. In some embodiments, if the subject does not exhibit a complete response (CR) at Day 90, the BCL2 inhibitor is administered for up to 24 months.
[0035] In some embodiments, daily administration of the BCL2 inhibitor is discontinued after or about after Day 90. In some embodiments, the BCL2 inhibitor is administered daily until at or about Day 90.
[0036] In some embodiments, the BCL2 inhibitor is venetoclax or navitoclax. In some embodiments, the BCL2 inhibitor is navitoclax.In some embodiments, the BCL2 inhibitor is venetoclax.
[0037| In some embodiments, the subject has a chronic lymphocytic leukemia (CLL). In some embodiments, the CLL is a relapsed or refractory (r/r) CLL. In some embodiments, the subject has a small lymphocytic lymphoma (SLL). In some embodiments, the SLL is a relapsed or refractory (r/r) SLL.
[0038] In some embodiments, the method further includes, prior to initiation of administration of the cell therapy, administering a lymphodepleting therapy to the subject. In some embodiments, the subject has been preconditioned with a lymphodepleting therapy prior to initiation of administration of the cell therapy. In some embodiments, the lymphodepleting therapy includes the administration of fludarabine and/or cyclophosphamide. In some embodiments, the lymphodepleting therapy includes the administration of fludarabine. In some embodiments, the lymphodepleting therapy includes the administration of cyclophosphamide. In some embodiments, the lymphodepleting therapy includes the administration of fludarabine and cyclophosphamide.
[0039] In some embodiments, the lymphodepleting therapy includes administration of cyclophosphamide at about 200-400 mg/m2, optionally at or about 300 mg/m2, inclusive, and/or fludarabine at about 20-40 mg/m2, optionally 30 mg/m2, daily for 2-4 days. In some embodiments, the lymphodepleting therapy includes administration of cyclophosphamide at about 200-400 mg/m2, optionally at or about 300 mg/m2, inclusive, and/or fludarabine at about 20-40 mg/m2, optionally 30 mg/m2, daily for 3 days. In some embodiments, the lymphodepleting therapy includes administration of cyclophosphamide at about 200-400 mg/m2 daily for 2-4 days. In some embodiments, the lymphodepleting therapy includes administration of cyclophosphamide at about 200-400 mg/m2 daily for 3 days. In some embodiments, the lymphodepleting therapy includes administration of cyclophosphamide at or about 300 mg/m2 daily for 2-4 days. In some embodiments, the lymphodepleting therapy includes administration of cyclophosphamide at or about 300 mg/m2 daily for 3 days. In some embodiments, the lymphodepleting therapy includes administration of fludarabine at about 20-40 mg/m2 daily for 2-4 days. In some embodiments, the lymphodepleting therapy includes administration of fludarabine at about 20-40 mg/m2 daily for 3 days. In some embodiments, the lymphodepleting therapy includes administration of fludarabine at or about 30 mg/m2 daily for 2-4 days. In some embodiments, the lymphodepleting therapy includes administration of fludarabine at or about 30 mg/m2 daily for 3 days. In some embodiments, the lymphodepleting therapy includes administration of cyclophosphamide at or about 300 mg/m2 and fludarabine at about 30 mg/m2 daily for 3 days. In some embodiments, the cell therapy is administered at least at or about 2-7 days after the lymphodepleting therapy. In some embodiments, the cell therapy is administered at least at or about 2-7 days after the initiation of the lymphodepleting therapy.
[0040] In some embodiments, the method further includes administering to the subject a bridging therapy including a BCL2 inhibitor. In some embodiments, the subject is administered a bridging therapy comprising a BCL2 inhibitor. In some embodiments, the bridging therapy is administered prior to initiation of administration of a lymphodepleting therapy. In some embodiments, the bridging therapy is administered at a time between collecting of autologous cells from the subject and prior to administering a lymphodepleting therapy to the subject. In some embodiments, the cells of the cell therapy are the autologous cells collected from the subject. In some embodiments, the collecting is by apheresis or leukapheresis.
[0041] In some embodiments, the bridging therapy includes daily administration of a first dose of the BCL2 inhibitor for a first week, daily administration of a second dose of the BCL2 inhibitor for a second week, and daily administration of a third dose of the BCL2 inhibitor for a third week. In some embodiments, the bridging therapy includes daily administration of between about 20 mg and about 400 mg of the BCL2 inhibitor. In some embodiments, the second dose of the bridging therapy is an increased amount of BCL2 inhibitor compared to the first dose of the bridging therapy, and the third dose of the bridging therapy is an increased amount of BCL2 inhibitor compared to the second dose of the bridging therapy. In some embodiments, the first dose of the bridging therapy is 20 mg of the BCL2 inhibitor, the second dose of the bridging therapy is 50 mg of the BCL2 inhibitor, and the third dose of the bridging therapy is 100 mg of the BCL2 inhibitor. In some embodiments, the first dose of the bridging therapy is 50 mg of the BCL2 inhibitor, the second dose of the bridging therapy is 100 mg of the BCL2 inhibitor, and the third dose of the bridging therapy is 200 mg of the BCL2 inhibitor. In some embodiments, the first dose of the bridging therapy is 100 mg of the BCL2 inhibitor, the second dose of the bridging therapy is 200 mg of the BCL2 inhibitor, and the third dose of the bridging therapy is 400 mg of the BCL2 inhibitor. In some embodiments, the bridging therapy is ceased about or at least about 1 day prior to initiation of administration of a lymphodepleting therapy.
[0042] In some embodiments, the BCL2 inhibitor is venetoclax or navitoclax. In some embodiments, the BCL2 inhibitor is navitoclax.In some embodiments, the BCL2 inhibitor is venetoclax.
[0043] In some embodiments, the BCL2 inhibitor administered in the bridging therapy and the BCL2 inhibitor administered in the dosing regimen are the same BCL2 inhibitor. In some embodiments, the BCL2 inhibitor administered in the bridging therapy and the BCL2 inhibitor administered in the dosing regimen is venetoclax. In some embodiments, the BCL2 inhibitor administered in the bridging therapy and the BCL2 inhibitor administered in the dosing regimen are different BCL2 inhibitors.
[0044] In some embodiments, the bridging therapy further includes administration of an anti-CD20 antibody to the subject. In some embodiments, if the subject has been previously treated with a BCL2 inhibitor, the bridging therapy further includes administration of an anti-CD20 antibody to the subject. In some embodiments, the bridging therapy further includes administration of a BTK inhibitor to the subject. In some embodiments, the BTK inhibitor is or comprises ibrutinib.
[0045] In some embodiments, the subject has failed at least one prior line of therapy, and at least one of the at least one prior lines of therapy is a Bruton’s tyrosine kinase (BTK) inhibitor, or the subject is ineligible for treatment with a BTK inhibitor. In some embodiments, the subject has failed at least one prior line of therapy, and at least one of the at least one prior lines of therapy is a Bruton’ s tyrosine kinase (BTK) inhibitor. In some embodiments, the subject has failed a BTK inhibitor. In some embodiments, the subject is ineligible for treatment with a BTK inhibitor.
[0046] In some embodiments, the subject has been previously treated with a Bruton’s tyrosine kinase (BTK) inhibitor. In some embodiments, the BTK inhibitor is or comprises ibrutinib.
[0047] In some embodiments, prior to the administration of the cell therapy, the subject has been treated with one or more prior therapies for the CLL or the SLL, other than another dose of cells expressing a CAR or a lymphodepleting therapy. In some embodiments, prior to the administration of the cell therapy, the subject has relapsed following remission after treatment with, or become refractory to, failed and/or was intolerant to treatment with one or more prior therapies. In some embodiments, prior to the administration of the cell therapy, the subject has relapsed following remission after treatment with one or more prior therapies. In some embodiments, prior to the administration of the cell therapy, the subject has become refractory to treatment with one or more prior therapies. In some embodiments, prior to the administration of the cell therapy, the subject has failed treatment with one or more prior therapies. In some embodiments, prior to the administration of the cell therapy, the subject is intolerant to treatment with one or more prior therapies.
[0048] In some embodiments, prior to the administration of the cell therapy, the subject has been treated with at least two prior therapies for the CLL or the SLL, other than another dose of cells expressing a CAR or a lymphodepleting therapy. In some embodiments, prior to the administration of the cell therapy, the subject has relapsed following remission after treatment with, or become refractory to, failed and/or was intolerant to treatment with two or more prior therapies. In some embodiments, prior to the administration of the cell therapy, the subject has relapsed following remission after treatment with two or more prior therapies. In some embodiments, prior to the administration of the cell therapy, the subject has become refractory to treatment with two or more prior therapies. In some embodiments, prior to the administration of the cell therapy, the subject has failed treatment with two or more prior therapies. In some embodiments, prior to the administration of the cell therapy, the subject is intolerant to treatment with two or more prior therapies.
[0049] In some embodiments, the one or more prior therapies are selected from a kinase inhibitor; venetoclax; a combination therapy comprising fludarabine and rituximab; radiation therapy; and hematopoietic stem cell transplantation (HSCT). In some embodiments, the kinase inhibitor is an inhibitor of Bruton’s tyrosine kinase (BTK). In some cases, the kinase inhibitor is ibrutinib. In some embodiments, the one or more prior therapies are selected from a Bruton’s tyrosine kinase inhibitor; venetoclax; a combination therapy comprising fludarabine and rituximab; radiation therapy; and hematopoietic stem cell transplantation (HSCT). In some embodiments, the one or more prior therapies are selected from ibrutinib; venetoclax; a combination therapy comprising fludarabine and rituximab; radiation therapy; and hematopoietic stem cell transplantation (HSCT).
[0050] In some embodiments, the one or more prior therapies includes an inhibitor of Bruton’s tyrosine kinase (BTK). In some embodiments, the BTK inhibitor is ibrutinib. In some embodiments, the one or more prior therapies includes ibrutinib.
[0051] In some embodiments, the subject has failed a BTK inhibitor or is ineligible to receive a BTK inhibitor. In some embodiments, the subject has failed a BTK inhibitor. In some embodiments, failure of a BTK inhibitor is defined as having stable disease or progressive disease (PD) as best response, or progression after previous response, or discontinuation due to intolerance. In some embodiments, intolerance is defined as failure to tolerate treatment due to unmanageable toxicity. In some embodiments, a subject is ineligible for treatment with a BTK inhibitor. In some embodiments, a subject is ineligible for treatment with a BTK inhibitor due to a requirement for full-dose anticoagulation or history of arrhythmia.
[0052] In some embodiments, the one or more prior therapies includes ibrutinib and a BCL2 inhibitor. In some embodiments, the BCL2 inhibitor is venetoclax. In some embodiments, the one or more prior therapies comprises ibrutinib and venetoclax.
[0053] In some embodiments, the subject has not been previously treated with venetoclax.
[0054] In some embodiments, the subject has been previously treated with venetoclax. In some embodiments, the subject is not intolerant to venetoclax at the time of initiation of administration of the cell therapy and/or at the time of initiation of administration of the BCL2 inhibitor. In some embodiments, the subject is not intolerant to venetoclax at the time of initiation of administration of the cell therapy. In some embodiments, the subject is not intolerant to venetoclax at the time of initiation of administration of the BCL2 inhibitor. In some embodiments, the subject is not intolerant to venetoclax at the time of initiation of administration of the cell therapy nor at the time of initiation of administration of the BCL2 inhibitor. In some embodiments, the subject is not intolerant to venetoclax at the time of initiation of administration of the cell therapy or at the time of initiation of administration of the BCL2 inhibitor.
[0055] In some embodiments, more than six months have passed since the last dose of the previous treatment with the venetoclax if the subject’s best overall response (BOR) during or within 6 months of discontinuing the previous treatment with venetoclax was stable disease (SD) or progressive disease (PD). In some embodiments, more than six months have passed since the last dose of the previous treatment with the venetoclax if the subject’s best overall response (BOR) during or within 6 months of discontinuing the previous treatment with venetoclax was stable disease (SD). In some embodiments, more than six months have passed since the last dose of the previous treatment with the venetoclax if the subject’s best overall response (BOR) during or within 6 months of discontinuing the previous treatment with venetoclax was progressive disease (PD). [0056] In some embodiments, prior to administration of the cell therapy and/or prior to administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes and/or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, prior to administration of the cell therapy, the subject has measurable disease in the lymph nodes. In some embodiments, prior to administration of the cell therapy the subject has evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, prior to administration of the cell therapy, the subject has measurable disease in the lymph nodes and evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, prior to administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes. In some embodiments, prior to administration of the BCL2 inhibitor, the subject has evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, prior to administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes and evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, prior to administration of the cell therapy and prior to administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes. In some embodiments, prior to administration of the cell therapy and prior to administration of the BCL2 inhibitor, the subject has evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, prior to administration of the cell therapy and prior to administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes and evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen.
[0057| In some embodiments, at the time of administration of the cell therapy and/or at the time of administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes and/or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, at the time of administration of the cell therapy, the subject has measurable disease in the lymph nodes. In some embodiments, at the time of administration of the cell therapy the subject has evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, at the time of administration of the cell therapy, the subject has measurable disease in the lymph nodes and evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, at the time of administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes. In some embodiments, at the time of administration of the BCL2 inhibitor, the subject has evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, at the time of administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes and evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, at the time of administration of the cell therapy and at the time of administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes. In some embodiments, at the time of administration of the cell therapy and at the time of administration of the BCL2 inhibitor, the subject has evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, at the time of administration of the cell therapy and at the time of administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes and evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen.
[0058] In some embodiments, prior to administration of the cell therapy and/or prior to administration of the BCL2 inhibitor, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 104. In some embodiments, prior to administration of the cell therapy, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 104. In some embodiments, prior to administration of the BCL2 inhibitor, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 104. In some embodiments, prior to administration of the cell therapy and prior to administration of the BCL2 inhibitor, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 104.
[0059] In some embodiments, at the time of administration of the cell therapy and/or at the time of administration of the BCL2 inhibitor, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 104. In some embodiments, at the time of administration of the cell therapy, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 104. In some embodiments, at the time of administration of the BCL2 inhibitor, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 104. In some embodiments, at the time of administration of the cell therapy and prior to administration of the BCL2 inhibitor, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 104.
[0060] In some embodiments, the subject has Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.
[0061] In some embodiments, the dose of engineered cells has a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR. In some embodiments, the ratio is between approximately 1:3 and approximately 3:1. In some embodiments, the dose of engineered cells has a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR. In some embodiments, the ratio is between approximately 1:2 and approximately 2:1. In some embodiments, the dose of engineered cells has a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR that is or is approximately 1:1.
[0062] In some embodiments, the dose of engineered cells includes between about 2.5 x 107 total CAR-expressing cells and about 1.0 x 108 total CAR-expressing cells. In some embodiments, the dose of engineered cells includes at or about 2.5 x 107 total CAR-expressing cells. In some embodiments, the dose of engineered cells includes at or about 5 x 107 total cells or total CAR-expressing cells. In some embodiments, the dose of engineered cells includes at or about 7.5 x 107 total cells or total CAR- expressing cells. In some embodiments, the dose of engineered cells includes at or about 1 x 108 total cells or total CAR-expressing cells.
[0063| In some embodiments, administration of the cell therapy includes administering a plurality of separate compositions, wherein the plurality of separate compositions includes a first composition of one of the CD4+ T cells and the CD8+ T cells and a second composition of the other of the CD4+ T cells and the CD8+ T cells. In some embodiments, the first composition includes the CD8+ T cells and the second composition includes the CD4+ T cells. In some embodiments, the first composition includes the CD4+ T cells and the second composition includes the CD8+ T cells.
[0064] In some embodiments, the first composition and the second composition are administered 0 to 12 hours apart, 0 to 6 hours apart, or 0 to 2 hours apart. In some embodiments, the first composition and the second composition are administered 0 to 12 hours apart. In some embodiments, the first composition and the second composition are administered 0 to 6 hours apart. In some embodiments, the first composition and the second composition are administered 0 to 2 hours apart. In some embodiments, the the administration of the first composition and the administration of the second composition are carried out on the same day, between about 0 and about 12 hours apart, between about 0 and about 6 hours apart, or between about 0 and 2 hours apart. In some embodiments, the the administration of the first composition and the administration of the second composition are carried out on the same day. In some embodiments, the the administration of the first composition and the administration of the second composition are carried out on between about 0 and about 12 hours apart. In some embodiments, the the administration of the first composition and the administration of the second composition are carried out between about 0 and about 6 hours apart. In some embodiments, the the administration of the first composition and the administration of the second composition are carried out between about 0 and 2 hours apart. In some embodiments, the initiation of administration of the first composition and the initiation of administration of the second composition are carried out between about 1 minute and about 1 hour apart, or between about 5 minutes and about 30 minutes apart. In some embodiments, the initiation of administration of the first composition and the initiation of administration of the second composition are carried out between about 1 minute and about 1 hour apart. In some embodiments, the initiation of administration of the first composition and the initiation of administration of the second composition are carried out between about 5 minutes and about 30 minutes apart. In some embodiments, the first composition and the second composition are administered no more than 2 hours, no more than 1 hour, no more than 30 minutes, no more than 15 minutes, no more than 10 minutes, or no more than 5 minutes apart. In some embodiments, the first composition and the second composition are administered no more than 2 hours apart. In some embodiments, the first composition and the second composition are administered no more than 1 hour apart. In some embodiments, the first composition and the second composition are administered no more than 30 minutes apart. In some embodiments, the first composition and the second composition are administered no more than 15 minutes apart. In some embodiments, the first composition and the second composition are administered no more than 10 minutes apart. In some embodiments, the first composition and the second composition are administered no more than 5 minutes apart.
[0065] In some embodiments, the first composition is administered prior to the second composition. In some embodiments, the first composition is enriched for CD8+ cells. In some embodiments, the first composition is enriched for CD4+ cells. In some embodiments, the first composition is essentially free of CD4+ cells. In some embodiments, the first composition is essentially free of CD8+ cells. In some embodiments, the second composition is essentially free of CD4+ cells. In some embodiments, the second composition is essentially free of CD8+ cells.
[0066] In some embodiments, the CAR comprised by the CD4+ T cells and the CAR comprised by the CD8+ T cells comprises a CAR that is the same and/or wherein the CD4+ T cells and the CD8+ T cells are genetically engineered to express a CAR that is the same. In some embodiments, the CAR comprised by the CD4+ T cells and the CAR comprised by the CD8+ T cells is the same. In some embodiments, the CD4+ T cells and the CD8+ T cells are genetically engineered to express a CAR that is the same.
[0067] In some embodiments, the CAR contains an extracellular antigen-binding domain specific for CD19, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule. In some embodiments, the CAR contains an extracellular antigen-binding domain specific for CD19, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4- IBB, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is a CD3zeta. In some embodiments, the cytoplasmic signaling domain is derived from 4-1BB. In some embodiments, the cytoplasmic signaling domain is derived from CD3zeta. In some embodiments, the CAR has an extracellular antigen-binding domain specific for CD19, a transmembrane domain, a cytoplasmic signaling domain derived from a 4-1BB, and a cytoplasmic signaling domain derived from a CD3zeta.
[0068] In some embodiments, the CAR contains, in order, an extracellular antigen-binding domain specific for CD19, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, and a cytoplasmic signaling domain derived from a primary signaling ITAM- containing molecule.
[0069] In some embodiments, the antigen-binding domain is an scFv. In some embodiments, the scFv contains a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37). In some embodiments, the scFv contains a CDRH1 sequence of DYGVS (SEQ ID NO: 38), a CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and a CDRH3 sequence of YAMDYWG (SEQ ID NO: 40). In some embodiments, the scFv contains a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37), a CDRH1 sequence of DYGVS (SEQ ID NO: 38), a CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and a CDRH3 sequence of YAMDYWG (SEQ ID NO: 40).
[0070| In some embodiments, the scFv contains a variable heavy chain region of FMC63 and a variable light chain region of FMC63. In some embodiments, the scFv contains a CDRL1 sequence of FMC63, a CDRL2 sequence of FMC63, a CDRL3 sequence of FMC63, a CDRH1 sequence of FMC63, a CDRH2 sequence of FMC63, and a CDRH3 sequence of FMC63. In some embodiments, the scFv binds to the same epitope as or competes for binding with any of the foregoing.
[0071] In some embodiments, the scFv comprises a VH set forth in SEQ ID NO: 41 and a VL set forth in SEQ ID NO: 42. In some embodiments, the VH and VL are separated by a flexible linker. In some embodiments, the flexible linker is or contains the sequence set forth in SEQ ID NO: 59. In some embodiments, the scFv is or contains the sequence set forth in SEQ ID NO:43.
[0072] In some embodiments, the costimulatory signaling domain is a signaling domain of CD28 or 4-1BB. In some embodiments, the costimulatory signaling domain is a signaling domain of 4-1BB. In some embodiments, the costimulatory signaling domain is a signaling domain of CD28. In some embodiments, the costimulatory signaling domain contains the sequence set forth in SEQ ID NO: 12 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the costimulatory signaling domain contains a variant of the sequence set forth in SEQ ID NO: 12 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the costimulatory signaling domain contains the sequence set forth in SEQ ID NO:
12 .
[0073] In some embodiments, the primary signaling domain is a CD3zeta signaling domain. In some embodiments, the primary signaling domain contains the sequence set forth in SEQ ID NO: 13 or 14 or 15, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the primary signaling domain is a variant of the sequence set forth in SEQ ID NO: 13 or 14 or 15 having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the primary signaling domain is a CD3zeta signaling domain. In some embodiments, the primary signaling domain contains the sequence set forth in SEQ ID NO: 13 or 14 or 15. In some embodiments, the primary signaling domain contains the sequence set forth in SEQ ID NO:
13 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the primary signaling domain contains the sequence set forth in SEQ ID NO: 13.
[0074] In some embodiments, the CAR further contains a spacer between the transmembrane domain and the scFv. In some embodiments, the spacer is a polypeptide spacer that contains or consists of all or a portion of an immunoglobulin hinge or a modified version thereof. In some embodiments, the spacer is a polypeptide spacer that contains or consists of all or a portion of an IgG4 hinge, or a modified version thereof. In some embodiments, the spacer is about 15 amino acids or less. In some embodiments, the spacer does not contain a CD28 extracellular region. In some embodiments, the spacer does not contain a CD8 extracellular region. In some embodiments, the spacer does not contain a CD28 extracellular region or a CD8 extracellular region. In some embodiments, the spacer is about 15 amino acids or less and does not contain a CD28 extracellular region or a CD8 extracellular region. In some embodiments, the spacer is at or about 12 amino acids in length. In some embodiments, the spacer contains or consists of the sequence of SEQ ID NO: 1, a sequence encoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the spacer contains or consists of the sequence of SEQ ID NO: 1. In some embodiments, the spacer contains the sequence of SEQ ID NO: 1. In some embodiments, the spacer consists of the sequence of SEQ ID NO: 1. In some embodiments, the spacer contains or consists of the formula X1PPX2P, where Xi is glycine, cysteine or arginine and X2 is cysteine or threonine.
[0075] In some embodiments, the scFv comprises, in order, a VH, a linker, and a VL- In some embodiments, the linker contains the sequence set forth in SEQ ID NO: 59. In some embodiments, the scFv comprises, in order, a VH, a linker containing the sequence set forth in SEQ ID NO:59, and a VL- In some embodiments, the antigen binding domain contains an scFv that contains a variable heavy chain region of FMC63 and a variable light chain region of FMC63; the spacer is a polypeptide spacer that contains the sequence set forth in SEQ ID NO: 1; the costimulatory domain contains the sequence set forth in SEQ ID NO: 12; and the primary signaling domain contains the sequence set forth in SEQ ID NO: 13, 14 or 15. In some embodiments, the antigen binding domain contains an scFv that contains a variable heavy chain region of FMC63 and a variable light chain region of FMC63; the spacer is a polypeptide spacer that contains the sequence set forth in SEQ ID NO: 1; the costimulatory domain contains the sequence set forth in SEQ ID NO: 12; and the primary signaling domain contains the sequence set forth in SEQ ID NO: 13.
[0076] In some embodiments, the engineered cells are primary T cells obtained from a subject. In some embodiments, the engineered cells are autologous to the subject.
[0077] In some embodiments, the subject is a human subject.
[0078] Also provided herein is an article of manufacture containing a composition for a cell therapy, or one of a plurality of compositions for a cell therapy, comprising T cells expressing a CAR that binds CD19 and an inhibitor of BCL2 protein, and instructions for administering the cell therapy and the BCL2 inhibitor to the subject, wherein the instructions specify administering the cell therapy and the BCL2 inhibitor according to any of the methods provided herein. In some embodiments, the BCL2 inhibitor is venetoclax or navitoclax. In some embodiments, the BCL2 inhibitor is venetoclax.
[0079] Also provided herein are uses of a cell therapy and a BCL2 inhibitor in accord with any of the methods provided herein.
[0080| Also provided herein is use of a cell therapy and a BCL2 inhibitor for the treatment of a subject having a CLL or a SLL. Also provided herein is use of a cell therapy and a BCL2 inhibitor for the treatment of a subject having a CLL. Also provided herein is use of a cell therapy and a BCL2 inhibitor for the treatment of a subject having a CLL. Also provided herein is a cell therapy and a BCL2 inhibitor for the treatment of a subject having a CLL or a SLL. Also provided herein is a cell therapy and a BCL2 inhibitor for the treatment of a subject having a CLL. Also provided herein is a cell therapy and a BCL2 inhibitor for the treatment of a subject having a SLL. Also provided herein is use of a cell therapy in the manufacture of a medicament for the treatment of a subject having a CLL or a SLL, wherein the medicament is for use with a BCL2 inhibitor. Also provided herein is use of a cell therapy in the manufacture of a medicament for the treatment of a subject having a CLL, wherein the medicament is for use with a BCL2 inhibitor. Also provided herein is use of a cell therapy in the manufacture of a medicament for the treatment of a subject having a SLL, wherein the medicament is for use with a BCL2 inhibitor. Also provided herein is use of a BCL2 inhibitor in the manufacture of a medicament for the treatment of a subject having a CLL or a SLL, wherein the medicament is for use with a cell therapy. Also provided herein is use of a BCL2 inhibitor in the manufacture of a medicament for the treatment of a subject having a CLL, wherein the medicament is for use with a cell therapy. Also provided herein is use of a BCL2 inhibitor in the manufacture of a medicament for the treatment of a subject having a SLL, wherein the medicament is for use with a cell therapy.
[0081] Also provided herein is a cell therapy including a dose of engineered cells containing T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) and a BCL2 inhibitor for use in a method of treating a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL), where the method comprises: (1) administering to the subject the cell therapy on Day 1; and (2) administering to the subject the BCL2 inhibitor in a dosing regimen including: (i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy; and (ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
[0082] Also provided herein is a BCL2 inhibitor for use in a method of treating a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) that has been treated with a cell therapy including a dose of engineered T cells containing T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19), wherein the method comprises administering the BCL2 inhibitor to the subject in dosing regimen including: (i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy, wherein the cell therapy was administered to the subject on Day 1; and (ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
[0083] Also provided herein is a cell therapy including a dose of engineered cells containing T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) for use in a method of treating a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL), wherein the method comprises administering the cell therapy to the subject on Day 1, and the subject is to be administered a BCL2 inhibitor in a dosing regimen including: (i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy; and (ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
[0084] Also provided herein is use of a BCL2 inhibitor in the manufacture of a medicament for treatment of a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) that has been treated with a cell therapy including a dose of engineered T cells containing T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19), wherein the medicament is to be administered to the subject in dosing regimen including: (i) daily administration of a first dose of the medicament for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy, wherein the cell therapy was administered to the subject on Day 1; and (ii) daily administration of at least one subsequent dose of the medicament, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the medicament, wherein: each of the at least one subsequent dose is an increased amount of the medicament compared to the preceding dose; and the medicament is administered at no more than or no more than about 100 mg per day through Day 30.
[0085] Also provided herein is use of a cell therapy including a dose of engineered cells containing T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) in the manufacture of a medicament for the treatment of a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) that is to be administered a BCL2 inhibitor in a dosing regimen including: (i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the medicament, wherein the medicament is administered to the subject on Day 1; and (ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
Brief Description of the Drawings
[0086] FIG. 1A shows the cell viability of human CD19-expressing lymphoma and leukemia target cell lines co-cultured with anti-CD19 CAR T cells at increasing ratios of effector cells to target cells (E:T).
[0087] FIG. IB shows the size of spheroids generated from the non-Hodgkin lymphoma (NHL) RL cell line over time, co-cultured with CD19-targeting CAR T cells at E:T ratios of 0.25:1, 0.5:1 and 1: 1.
[0088] FIG. 2 shows the fold change in cell count of human CD19-expressing leukemia and lymphoma target cell lines co-cultured for 120 hours with CD19-targeting CAR T cells at an E:T ratio of 2.5:1.
[0089] FIG. 3 shows the cell viability of anti-CD19 CAR-expressing T cells treated for 96 hours with an exemplary BCL2 inhibitor.
[0090] FIG. 4A shows the cell count of human CD19-expressing lymphoma cell lines cultured alone, with CD19-targeting CAR T cells at an E:T ratio of 2.5:1, with an exemplary BCL2 inhibitor, or with both.
[0091 | FIG. 4B shows the cell count of CD19-expressing Granta-519 lymphoma target cells cultured alone, with anti-CD19 CAR T cells at a suboptimal E:T ratio of 1:1, with an exemplary BCL2 inhibitor, or both.
[0092] FIG. 5A shows the cell count of RL cells cultured alone, with CD19-targeting CAR T cells at an E:T ratio of 1:1, with an exemplary BCL2 inhibitor, or with both.
[0093] FIG. SB shows the tumor volume of RL spheroids cultured alone, with CD19-targeting CAR T cells at an E:T ratio of 1:1, with an exemplary BCL2 inhibitor, or with both.
[0094] FIG. SC shows the tumor volume of RL spheroids cultured alone or with CD19-targeting CAR T cells for 9 days, in the presence of increasing concentrations of an exemplary BCL2 inhibitor.
[0095] FIG. A shows the number of RL cells cultured for 8 days alone or with anti-CD19 CAR T cells (E:T ratio of 1:1) that had been previously subjected to chronic stimulation by incubation with anti- idiotypic antibody-coated beads, in the presence or absence of an exemplary BCL2 inhibitor. [0096] FIG. 6B shows the tumor volume of RL spheroids cultured for 8 days alone or with anti- CD19 CAR T cells (E:T ratio of 1:1) that had been previously subjected to chronic stimulation by incubation with anti-idiotypic antibody-coated beads, in the presence or absence of an exemplary BCL2 inhibitor.
[0097] FIG. 7A shows the expression, indicated by mean fluorescence intensity (MFI), of BCL2 by anti-CD19 CAR-expressing T cell compositions generated from three healthy human donors, compared to a fluorescence minus one (FMO) control.
[0098] FIG. 7B shows the percent of CAR+caspase 3+ cells among CD 19 CAR T cell compositions generated from three healthy human donors (each dot represents an individual donor), following chronic stimulation by incubation with anti-idiotypic antibody-coated beads and exposure to increasing concentrations of an exemplary BCL2 inhibitor.
[0099] FIGS. 7C and 7D show cell viability and expansion kinetics of CD19-targeting CAR T cells, respectively, following chronic stimulation by incubation with anti-idiotypic antibody-coated beads and exposure to an exemplary BCL2 inhibitor.
[0100] FIG. 8 shows JeKo-1 mantle cell lymphoma (MCL) target cells cultured alone, with CD19- targeting CAR T cells, with an exemplary BCL2 inhibitor, or with both.
[0101] FIG. 9 shows the IC50 of an exemplary BCL2 inhibitor against anti-CD19 CAR T cells in culture with 5%, 10%, or 20% serum.
[0102] FIGS. 10A and 10B show the tumor burden and body weight, respectively, of NOD scid gamma (NSG) mice injected with JeKo-1 MCL cells at Day -7 and treated daily with an exemplary BCL2 inhibitor from Day 0 to Day 21.
[0103] FIGS. 11A to 11C show tumor burden (FIG. 11 A) and survival in NSG mice injected wth JeKo-1 MCL cells at Day -7 and treated with an exemplary BCL2 inhibitor daily from Day 0 to Day 21 (FIG. 1 IB), anti-CD19 CAR T cells on Day -1 and Day 0 (FIG. 11C), or both (FIG. 11C).
[0104] FIG. 12 shows the number of CD4+CAR+ and CD8+CAR+ cells in the blood of NSG mice injected with JeKo-1 MCL cells and treated with CD19-targeting CAR T cells, in the presence or absence of an exemplary BCL2 inhibitor, on Days 7, 13, and 19.
[0105] FIGS. 13A and 13B show the number of CD3+CAR+ T cells and RL target cells, respectively, following a 6-day co-culture. Various concentrations of an exemplary BCL2 inhibitor were provided in the co-culture at its initiation, or 24, 48, or 72 hours after the initiation of co-culture.
[0106] FIGS. 14A and 14B show the survival of NSG mice injected with Granta-519 cells at Day - 14 and treated with anti-CD19 CAR T cells on Day 0 and an exemplary BCL2 inhibitor at 25 mg/kg (FIG. 14A) or 100 mg/kg (FIG. 14B) daily, beginning at either Day -1 or Day 5.
[0107] FIG. 15 shows gene expression data of tumor biopsies from 36 DLBCL patients enrolled in a clinical trial for a CD19-targeting CAR T cell therapy. A hypothetical threshold was set assuming, at 3 months after admistration of the cell therapy, 30% of subjects (11/36) would not respond and 70% of subjects (25/36) would respond. Actual responses are shown, designated as complete reponse (CR), partial response (PR), progressive disease (PD), or status unavailable (Not Available).
[0108] FIG. 16 shows exemplary dosing regimens for beating a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) with a combination therapy of CAR-expressing T cells and the exemplary BCL2 inhibitor venetoclax (D: Day; DC1: Dosing cohort 1; DC2: Dosing cohort 2; DC3: Dosing cohort 3; LD: lymphodepleting therapy).
Detailed Description
[0109] Provided herein are combination therapies for treating a subject having a cancer involving administration of an immunotherapy or a cell therapy (e.g. a T cell therapy, such as CAR-T cell) for treating a cancer and an inhibitor of BCL2 protein (a BCL2 inhibitor). In some embodiments, the immunotherapy or cell therapy includes any such therapy that specifically binds to an antigen associated with, expressed by, or present on cells of the cancer. In particular embodiments, the immunotherapy or cell therapy is or involves a therapy that results in cytolytic effector-mediated killing of cancer cells, such as by target cell apoptosis (hereinafter “cytotoxic therapy”). Among the provided embodiments are combination therapies involving administration of an immunotherapy involving T cell function or activity, such as a T-cell engaging therapy or a T cell therapy (e.g., CAR-expressing T cells), and administration of a BCL2 inhibitor. In some embodiments, the cytotoxic therapy is a T cell therapy, such as CAR-T cells. In particular embodiments, the provided combination therapies and methods improve responses to the immunotherapy or cell therapy by activity of the BCL2 inhibitor to increase susceptibility of the cancer cells, such as tumor cells, to apoptosis. In some embodiments, the cancer cells are thereby rendered more sensitive to cytolytic effector-mediated killing.
[0110] Also provided are combinations and articles of manufacture, such as kits, that contain a composition comprising the therapy and/or a composition comprising a BCL2 inhibitor, e.g., venetoclax, and uses of such compositions and combinations to treat or prevent cancers, such as a B cell malignancy (e.g. CLL or SLL).
[0111] Cell therapies, such as T cell-based therapies, for example, adoptive T cell therapies (including those involving the administration of cells expressing chimeric receptors specific for a cancer of interest, such as chimeric antigen receptors (CARs) and/or other recombinant antigen receptors, as well as other adoptive immune cell and adoptive T cell therapies) can be effective in the treatment of diseases and disorders such as a B cell malignancies. The engineered expression of recombinant receptors, such as chimeric antigen receptors (CARs), on the surface of T cells enables the redirection of T cell specificity. In clinical studies, CAR-T cells, for example anti-CD19 CAR-T cells, have produced durable, complete responses in both leukemia and lymphoma patients (Porter et al. (2015) Sci Transl Med., 7:303ral39; Kochenderfer (2015) J. Clin. Oncol., 33: 540-9; Lee et al. (2015) Lancet, 385:517-28; Maude et al. (2014) N Engl J Med, 371:1507-17). 10112] In certain contexts, available approaches to adoptive cell therapy may not always be entirely satisfactory. In some contexts, optimal efficacy can depend on the ability of the administered cells to recognize and bind to a target, e.g., target antigen, and to exert various effector functions, including cytotoxic killing of cancer cells and secretion of various factors such as cytokines. In some cases, however, certain cancer cells exhibit resistance to certain therapies, such as immunotherapies and cell therapies. In particular, results herein demonstrate that certain cancers are resistant to CAR T cell- mediated killing while others are more sensitive.
[0113] In some aspects, the provided methods, combinations and uses provide for or achieve improved or more durable responses or efficacy as compared to alternative methods, such as alternative methods involving only the administration of the immunotherapy or cell therapy but not in combination with a BCL2 inhibitor (e.g. venetoclax). In some embodiments, the methods are advantageous by virtue of administering a BCL2 inhibitor (e.g. venetoclax) shortly after (e.g. within 1 or 2 days of) administration of an immunotherapy or a cell therapy (e.g. T cell therapy, such as CAR-T cell), thereby sensitizing the tumor and/or making the tumor less resistant to, or more susceptible to, treatment with the immunotherapy or cell therapy. In some embodiments of the provided methods, the immunotherapy or cell therapy is a cell therapy (e.g. T cell therapy, such as CAR-T cell), and it is further found that the advantageous effect of sensitizing the tumor and/or making the tumor less resistant to, or more suspectible to, treatment with the cell therapy can be achieved by initiating administration of the BCL2 inhibitor (e.g. venetoclax) in a window of time after initiation of administration of the cell therapy to minimize or avoid a detrimental effect of the inhibitor on cells of the cell therapy. In particular, observations herein demonstrate that a BCL2 inhibitor (e.g. venetoclax), particularly if given at too high of a dose, can exacerbate activation induced cell death (AICD) of cells of the cell therapy. However, delaying administration of the BCL2 inhibitor (e.g. venetoclax) until a time after AICD is at or has reached its peak, or has decreased after having peaked, can avoid detrimental effects on the cells of the cell therapy while substantially improving, e.g. synergistically increasing, T cell-mediated killing of the tumor by cells of the cell therapy (e.g. CAR-T cells).
[0114] In some embodiments of the provided methods, the immunotherapy or cell therapy is a cell therapy (e.g. T cell therapy, such as CAR-T cell), and it is further found that the advantageous effect of sensitizing the tumor and/or making the tumor less resistant to, or more suspectible to, treatment with the cell therapy can be achieved by initiating administration of the BCL2 inhibitor (e.g. venetoclax) in a window of time shortly after initiation of administration of the cell therapy, such as at a time before symptoms of cytokine release syndrome (CRS) have begun or peaked. In particular, administering the BCL2 inhibitor (e.g. venetoclax) after initiation of administration of the cell therapy (e.g. 1-2 days after, such as 1 day after) may avoid exacerbating CRS symptoms and/or may improve clinical outcomes. In some aspects, initiating administration of the BCL2 inhibitor (e.g. venetoclax) in a window of time shortly after initiation of administration of the cell therapy (e.g. 1-2 days after, such as 1 day after) minimizes the amount of time between a bridging therapy with a BCL2 inhibitor (e.g. venetoclax) and administration of the BCL2 inhibitor dosing regimen (venetoclax), and may thereby improve clinical outcomes.
[01151 The provided methods are based on observations that certain cancers that are resistant to CAR T cell-mediated killing exhibit high expression of the prosurvival tumor suppressor p53 and other genes involved in prosurvival cell cycle pathways. It is found herein that the presence of a BCL2 inhibitor, e.g. venetoclax, improves T cell-mediated killing of cancer cells by a T cell therapy (e.g. CAR T cells), particularly among cancers that exhibit resistance following exposure to the T cell therapy (e.g. CAR T cells) alone. Such results were observed with low doses of the BCL2 inhibitor, which did not exhibit activity against the tumor when administered alone. These results evidence that administration of a BCL2 inhibitor, including at subtherapeutic doses, may improve responses to certain effector-mediated immunotherapies, such as T cell engagers or T cell therapies.
[0116] It is additionally found herein that the presence of a BCL2 inhibitor, e.g. venetoclax, increases the susceptibility of cancer cells to T cell-mediated killing by a T cell therapy (e.g. CAR T cells), such as among cancers that exhibit resistance to T cell therapy-mediated cell death (e.g. CAR T cells) alone, even when initiation of administration of the inhibitor is after or subsequent to administration of the T cell therapy (e.g. CAR T cells). Thus, in some cases, the cytotoxic effects of a BCL2 inhibitor, e.g. venetoclax, and a T cell therapy (e.g. CAR T cells) may be synergistic to result in cell death of cells otherwise resistant to treatment with the T cell therapy alone. In some cases, administration of the inhibitor, e.g. venetoclax, may sensitize otherwise resistant cancer cells to CAR T cell-mediated killing when it is administered within or at about 1-2 days after administration of the cell therapy. For example, as demonstrated in Example 8, in the absence of venetoclax, tumor cells were relatively resistant to cell death mediated by CAR T cells. Although a therapeutic dose of venetoclax given concurrently or after 24 hours reduced CAR-T cell numbers and activity, a therapeutic dose of venetoclax administered 48 or 72 hours after initiation of co-culture of tumor cells and CAR T cells was able to sensitize the tumor cells to CAR T cell-mediated killing, without deleterious effects on the CAR T cells being observed. These results surprisingly indicated that there is a window of time after initiation of the cell therapy (e.g. CAR-T cells) at which deleterious effects on cells of the cell therapy by a BCL2 inhibitor, e.g. venetoclax, even at therapeutic doses, can be avoided or minimized while maintaining the ability of the inhibitor to increase susceptibility of the tumor to the T cell therapy. These results indicate that administration of a BCL2 inhibitor, e.g. venetoclax, such as within or at about 1-2 days following administration of a T cell therapy, including at therapeutic doses, may improve responses to T cell therapies without exerting significant deleterious effects on T cells.
[0117] Venetoclax (ABT-199) is a small molecule inhibitor (SMI) that blocks the activity of the B- cell lymphoma 2 (BCL2) protein. Venetoclax is approved for use (1) in chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL); and (2) in combination with azacitidine, decitabine, or low-dose cytarabine for newly diagnosed acute myeloid leukemia (AML) in adult who are at least 75 years of age, or who have comorbidities that preclude use of intensive induction chemotherapy. Navitoclax (ABT-263), another prosurvival BCL2 family protein inhibitor, is a SMI that blocks the activity of the BCL2, B-cell lymphoma extra-large (BCLXL), and BCL2-like protein 2 (BCLW) proteins. Other BCL2 inhibitors include, but are not limited to ABT-737, AT-101/GDC-0199 (Gossypol), apogossypol, TW-37, G3139 (Genasense), GX15-070 (obatoclax), sabutoclax, HA14-1, antimycin A, BH3I-1, YC137, maritoclax, clitocine, UMI-77, WEHI-539, and 544563.
[0118] Members of the BCL2 family include both pro-apoptotic and anti-apoptotic proteins, and the balance of signaling between these two groups of proteins can determine whether a cell is more sensitized to or more resistant to apoptosis. In some cases, the overexpression of one or more prosurvival BCL2 family proteins (e.g. BCL2) can result in increased anti-apoptotic signaling and resistance to cell death. For example, in some cases, overexpression of BCL2 may be caused by a (14;18)(q32;q21) translocation. In some cases, overexpression of BCL2 may be caused by amplification of the gene encoding the BCL2 protein. Resistance to cell death may, in some cases, occur when the signaling of prosurvival (anti-apoptotic) BCL2 proteins (e.g. BCL2, BCLXL, BCLB, BCLW, BFL1, MCL1) outweighs the signaling of pro-apoptotic BCL2 proteins (e.g. BAX, BAK, BIG, BIM, NOXA, PUMA), such as when one or more prosurvival BCL2 proteins are overexpressed. In certain aspects, overexpression of one or more prosurvival BCL2 family proteins can support and increase cancer cell survival. In some aspects, increased expression of prosurvival BCL2 family proteins blocks pro-apoptotic BCL2 family proteins and inhibits a cancer cell’s intrinsic (mitochondrial) apoptotic pathway.
[Oil 9| Among the provided embodiments, the methods involve combination therapy of a therapy that targets or is directed to killing of cells of a cancer, e.g. an immunotherapy or cell therapy, such as a CAR T cell therapy, and an inhibitor of BCL2 protein. In some aspects, the inhibitor inhibits activity of a BCL2 family protein that is a prosurvival (anti-apoptotic) BCL2 family protein such as BCL2, B-cell lymphoma extra-large (BCLXL), BCL2 related protein Al (BFL1), BCL2-like protein 2 (BCLW), BCL2-like protein 10 (BCLB), induced myeloid leukemia cell differentiation protein (MCL1), or combinations thereof. In some aspects, the cancer is one in which the prosurvival BCL2 family protein is overexpressed. In some aspects, the inhibitor does not inhibit or reduce the activity of a BCL2 family protein that is a proapoptopic BCL2 family protein such as BCL2 associated X (BAX), BCL2 antagonist/killer 1 (BAK), DIVA, BCLXS, BCL2 interacting killer (BIK), BCL2-like protein 11 (BIM), BCL2 associated agonist of cell death (BAD), or combinations thereof. In some aspects, the cancer is one in which the proapoptopic BCL2 family protein is underexpressed or its activity is inhibited.
[0120] In some aspects, overexpression of a prosurvival BCL2 family protein is implicated in a number of cancers, including bladder cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, head and neck cancers, lung cancer, ovarian cancer, pancreatic cancer, renal cancer, skin cancer, and hematologic malignancies, such as leukemias and lymphomas (see e.g., WO 2005/049593 and W02005/024636). In some cases, overexpression or aberrant expression of one or more prosurvival BCL2 family proteins is a mechanism underlying leukemias, lymphomas, and solid tumors, whereby overexpression dampens pro-apoptotic signaling to promote the survival of cancer cells (see e.g., Campbell, K.J. and Tait, S.W.G. (2018) Open Biol., 8:180002). In many cases, existing methods of employing BCL2 inhibitors, e.g. venetoclax, involve use of the inhibitors as therapeutics for treating a variety of cancers. For example, venetoclax is indicated for treatment of certain cancers, such as B cell lymphomas, with a dose of 400 to 800 milligrams per day after a ramp-up period and for a duration of time that can extend for months to years.
[0121] However, reports indicate that certain inhibitors of prosurvival BCL2 family proteins (e.g. BCL2) may have deleterious effects, including direct cytotoxicity, on T cells (Karlsson et al. (2013) Cancer Gene Ther., 20:386-93). Further, BCL2 is known to be involved in and regulate the survival of T cells (Wilson et al. (2010) Lancet Oncol., 11:70261-8). A study investigating the effect of navitoclax, an inhibitor of BCL2, BCLXL, and BCLW, on T cells found that exposure to navitoclax induced a relative and absolute reduction of CD3+CD4+ and CD3+CD8+ T cells in the peripheral blood of mice (Cippa et al., (2012) Cell Death and Disease, 3:e299). In a Phase I clinical trial of navitoclax ((NCT00406809), it was reported that patients in the trial showed a relatively rapid and substantial decrease in T cells (an average decrease of 241 CD3+ cclls/pL) after 14 days of drug exposure to navitoclax at a dose of 200 milligrams/day or more, which was maintained at the time of the patients’ final visits occurring on average 89 days after drug exposure (Wilson et al. (2010) Lancet Oncol., 11:70261-8).
[0122] These reports indicate that the combination of a T cell therapy and a BCL2 inhibitor may not be a viable therapeutic strategy, particularly at therapeutic doses of the BCL2 inhibtor. In particular, such observations indicate that the combination of a BCL2 inhibitor and CAR T cell therapy would result in a loss of CAR T cells. As the proliferation and persistence of CAR T cells in vivo represent an ongoing challenge in the field of T cell therapies, the addition of an agent that is cytotoxic to T cells, such as an inhibitor of thevprosurvival BCL2 protein, would be not be favored as it would diminish efficacy of a CAR T cell therapy by reducing proliferation and persistence of the CAR-expressing T cells.
[0123] In addition, CAR-expressing T cells therapy may undergo activation-induced cell death (AICD). Specifically, reports indicate that CAR T cells may be prone to upregulation of expression of Fas, FasL, DR5, and TRAIL, such as upon excessive T cell stimulation, thereby resulting in programmed cell death. (Tschumi et al., J. Immunother. Cancer (2018) 71:6). To this end, combinatorial treatment with a BCL2 inhibitor (e.g. venetoclax), which blocks anti-apoptotic signaling, could exacerbate the programmed cell death observed in CAR T cells. Despite this, the observations herein indicate that the combination of a therapy, e.g. an immunotherapy or a cell therapy, including a T cell therapy such as a CAR-T cell therapy is advantageous, even at therapeutic doses of the inhibitor capable of exerting deleterious effects on CAR T cells. When the inhibitor (e.g. venetoclax) is administered at a time when activation-induced cell death of the CAR T cells has peaked, not only were deleterious effects on CAR T cells not observed, but the CAR T cells demonstrated potent antitumor effects (see Example 8). In some aspects, the methods are advantageous by virtue of administering a BCL2 inhibitor at a therapeutic dose subsequent to administration of a T cell therapy (e.g. within or at about 1-2 days after administration of the T cell therapy), such as a dose in which the inhibitor would be expected to exert deleterious effects on T cells, including T cell viability. In some aspects, the provided methods and uses provide for or achieve improved or more durable responses or efficacy as compared to certain alternative methods.
[0124] Further, the observations herein indicate that the combination of a therapy, e.g. an immunotherapy or a cell therapy, including a T cell therapy such as a CAR-T cell therapy is advantageous, even at subtherapeutic or lower doses of the inhibitor. The results herein also show that certain doses of the inhibitor do not impact viability of T cells. In some aspects, the methods are advantageous by virtue of administering a BCL2 inhibitor at a subtherapeutic dose, such as a dose in which the inhibitor alone would not be expected to or would not reduce tumor burden in the subject. In some aspects, the provided methods and uses provide for or achieve improved or more durable responses or efficacy as compared to certain alternative methods. In some aspects, the provided methods enhance or modulate the cytotoxic activity, such as via perforin- and/or granzyme-mediated apoptosis, toward one or more cells of a cancer of T cells against cancer cells, such as associated with administration of a T cell engaging therapy or a T cell therapy (e.g. CAR-expressing T cells). In some embodiments, observations herein indicate that a BCL2 inhibitor, e.g. venetoclax, may improve CAR T cell-mediated cytotoxicity activation at therapeutic and/or subtherapeutic doses. The provided findings indicate that combination therapy of the inhibitor in methods involving T cells, such as involving administration of adoptive T cell therapy, achieves improved function of the T cell therapy. In some embodiments, combination of the cell therapy (e.g., administration of engineered T cells such as CAR T cells) with the BCL2 inhibitor improves or enhances one or more functions and/or effects of the T cell therapy, such as cytotoxicity and/or therapeutic outcomes, e.g., ability to kill or reduce the burden of tumor or other disease or target cell.
[0125] In some aspects, such effects are observed despite that the tumor or disease or target cell itself is insensitive, resistant and/or otherwise not sufficiently responsive to the therapy, e.g. immunotherapy or cell therapy, such as immunotherapy including T cell engaging therapy or T cell therapy (e.g. CAR T cells), or to the dose of the inhibitor when each is administered alone. In some embodiments, the cancer is insensitive to or has become resistant to treatment with a therapy for treating the cancer that is directed to or targets killing of the cancer, e.g. immunotherapy or cell therapy, including a T cell engaging therapy or a T cell therapy (e.g. CAR T cell therapy). In some embodiments, the cancer is insensitive to or has become resistant to such therapies by virtue of the cells of the cancer suppressing pro-apoptotic signaling. For example, in some embodiments, the cancer is insensitive to or has become resistant to CAR T cells targeting the cancer-associated antigen, e.g. CD19. In some embodiments, the provided combination therapy achieves synergistic effects and activity compared to a therapy involving only administration of the therapy, e.g. immunotherapy or cell therapy, or of the BCL2 inhibitor given at the same dosing regimen, e.g. dose and frequency.
[0126] In some embodiments, the provided methods, uses and combination therapies include administration of a BCL2 inhibitor, in combination with a therapy for treating the cancer that is directed to or targets killing of the cancer, e.g. immunotherapy or cell therapy, such as an immunotherapy, including a T cell engaging therapy or a T cell therapy (e.g. CAR T cell therapy) in a subject that has already been administered the BCL2 inhibitor or another prosurvival BCL2 family protein inhibitor. In some embodiments, the combination therapy, methods and uses include continued administration of the BCL2 inhibitor, e.g., venetoclax, in combination with a T cell therapy (e.g. CAR+ T cells) in a subject that has previously received administration of the inhibitor, e.g., venetoclax, but in the absence of (or not in combination with) a therapy for treating the cancer that is directed to or targets killing of the cancer, e.g. immunotherapy or cell therapy, such as an immunotherapy, including a T cell engaging therapy or a T cell therapy (e.g. CAR T cell therapy). In some embodiments, the combination therapy, methods, and uses include or involve administration of a lower dose of the BCL2 inhibitor, e.g., venetoclax, than the previous treatment.
[0127] In some embodiments, the methods and combinations result in improvements in T cell- mediated cytotoxicity against cancer cells. In some embodiments, the methods and combinations result in improvements in T cell-mediated cytotoxicity against cancer cells, optionally by increasing perforin- and/or granzyme-mediated apoptosis. In some embodiments, the methods and combinations result in improvements in T cell-mediated cytotoxicity against cancer cells by increasing perforin-mediated apoptosis. In some embodiments, the methods and combinations result in improvements in T cell- mediated cytotoxicity against cancer cells by increasing granzyme-mediated apoptosis. Such improvements in some aspects result without compromising, or without substantially compromising, one or more other desired properties of functionality, e.g., of CAR-T cell functionality, proliferation, and/or persistence. In some embodiments, the combination with the BCL2 inhibitor, while improving the cytotoxicity of the T cells, does not reduce the ability of the cells to become activated, secrete one or more desired cytokines, expand and/or persist, e.g., as measured in an in vitro assay as compared to such cells cultured under conditions otherwise the same but in the absence of the inhibitor.
[0128] In some embodiments, the provided embodiments involve initiating the administration of a BCL2 inhibitor, e.g., venetoclax, after administration of an immunotherapy or a cell therapy (e.g. CAR T cell therapy) in a dosing regimen. In some embodiments, the initiation of the administration of a BCL2 inhibitor, e.g., venetoclax, is after administration of the immunotherapy or cell therapy, such as between about 1 day after and about 2 days after (e.g. 1 day after) administration of the therapy for treating the cancer. In some embodiments, the initiation of the administration of a BCL2 inhibitor, e.g., venetoclax, is not until activation-induced cell death (AICD) of the cells of the immunotherapy or cell therapy has peaked. In some embodiments, the initiation of the administration of a BCL2 inhibitor, e.g., venetoclax, is not until after activation-induced cell death (AICD) of the cells of the immunotherapy or cell therapy has peaked and subsequently begun to decline. In some embodiments, administration of a prosurvival BCL2 family protein inhibitor, such as a BCL2 inhibitor, e.g., venetoclax, is not until about 1 day after administration of the immunotherapy or cell therapy. In some embodiments, administration of a prosurvival BCL2 family protein inhibitor, such as a BCL2 inhibitor, e.g., venetoclax, is not until about 1 day after administration of the cytotoxic therapy. In some cases, administration of the immunotherapy or cell therapy (e.g. CAR T cell therapy) occurs on Day 1, and administration of the BCL2 inhibitor begins on Day 2.
[0129] In some embodiments, the method includes administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising daily administration of a first dose for a first predetermined period and daily administration of at least one subsequent dose for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor. In some embodiments, each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor (e.g. venetoclax) compared to the preceding dose.
[0130] In some embodiments, the at least one subsequent dose comprises a second dose, a third dose, a fourth dose, a fifth dose, a sixth dose, a seventh dose, an eighth dose, a ninth dose, and/or a tenth dose. In some embodiments, the at least one subsequent dose comprises a second dose. In some embodiments, the at least one subsequent dose comprises a second dose and a third dose. In some embodiments, the at least one subsequent dose comprises a second, third, and fourth dose, n some embodiments, the at least one subsequent dose comprises a second, third, fourth, and fifth dose.
[01311 It is understood that in a transition from a preceding dose in the preceding predetermined period to a subsequent dose in a subsequent predetermined period, administration of the subsequent dose may begin immediately after the predetermined period of the preceding dose. For example, the subsequent dose (e.g. the second dose) in the dosing regimen is administered for a predetermined period, which begins immediately after the predetermined period of the preceding dose (e.g. the first dose). In other embodiments, administration of the BCL2 inhibitor may be paused for one or more days between one or more predetermined periods of any given dose.
[0132] In some cases, the method includes administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising (i) daily administration of a first dose for a first predetermined period and (ii) daily administration of a second dose for a second predetermined period beginning after the first predetermined period, wherein the second dose is an increased amount of the BCL2 inhibitor compared to the first dose.
[0133] In some cases, the method includes administering to the subject a BCL2 (e.g. venetoclax) inhibitor in a dosing regimen comprising (i) daily administration of a first dose for a first predetermined period, (ii) daily administration of a second dose for a second predetermined period beginning after the first predetermined period, and (iii) daily administration of a third dose for a third predetermined period beginning after the second predetermined period, wherein the second dose is an increased amount of the BCL2 inhibitor compared to the first dose and the third dose is an increased amount of the inhibitor compared to the third dose.
[0134| In some embodiments, the BCL2 inhibitor (e.g. venetoclax) is provided at no more than or no more than about 100 mg per day through Day 30. In some aspects, providing the BCL2 inhibitor (e.g. venetoclax) at no more than or no more than about 100 mg per day through Day 30 can minimize neutropenia and/or thrombocytopenia in the subject.
[0135] In some cases, the method includes administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising (i) daily administration of a first dose for a first predetermined period and (ii) daily administration of a second dose for a second predetermined period beginning after the first predetermined period, wherein the second dose comprises an increased amount of the BCL2 inhibitor compared to the first dose and the BCL2 inhibitor is provided at no more than or no more than about 100 mg per day through Day 30.
[0136] In some cases, the method includes administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising (i) daily administration of a first dose for a first predetermined period, (ii) daily administration of a second dose for a second predetermined period beginning after the first predetermined period, and (iii) daily administration of a third dose for a third predetermined period beginning after the second predetermined period, wherein the second dose comprises an increased amount of the BCL2 inhibitor compared to the first dose, the third dose comprises an increased amount of the BCL2 inhibitor compared to the second dose, and the BCL2 inhibitor is provided at no more than or no more than about 100 mg per day through Day 30.
[0137] In some embodiments, the first predetermined period is from about Day 2 to about Day 7. In some embodiments, the first predetermined period is from about Day 2 to about Day 30. In some embodiments, the first dose is about 20 mg, about 50 mg, or about 100 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 20 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 100 mg of the BCL2 inhibitor.
[0138] In some embodiments, the first predetermined period is from about Day 2 to about Day 7, and the first dose is about 20 mg of the BCL2 inhibitor. In some embodiments, the first predetermined period is from about Day 2 to about Day 7, and the first dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the first predetermined period is from about Day 2 to about Day 7, and the first dose is about 100 mg of the BCL2 inhibitor.
[0139] In some embodiments, the first dose predetermined period is from about Day 2 to about Day 30, and the first dose is about 20 mg of the BCL2 inhibitor. In some embodiments, the first predetermined period is from about Day 2 to about Day 30, and the first dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the first predetermined period is from about Day 2 to about Day 30, and the first dose is about 100 mg of the BCL2 inhibitor.
[0140] In some embodiments, the second predetermined period is from about Day 8 to about Day 30. In some embodiments, the second predetermined period is from about Day 31 to Day 37. In some embodiments, the second predetermined period is from about Day 31 to Day 90.
[0141] In some embodiments, the second dose is about 50 mg, about 100 mg, or about 200 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 200 mg of the BCL2 inhibitor.
[0142] In some embodiments, the second predetermined period is from about Day 8 to about Day 30, and the second dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 8 to about Day 30, and the second dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 8 to about Day 30, and the second dose is about 200 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 to Day 37, and the second dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 to Day 37, and the second dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 to Day 37, and the second dose is about 200 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 to Day 90, and the second dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 to Day 90, and the second dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 and Day 90, and the second dose is about 200 mg of the BCL2 inhibitor.
[0143] In some embodiments, administration of a first dose for a first predetermined period comprises daily administration of about 20 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 7 ; and administration of a second dose for a second predetermined period comprises daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 8 to about Day 30. In some embodiments, administration of a first dose for a first predetermined period comprises daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 7; and administration of a second dose for a second predetermined period comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 8 to about Day 30. In some embodiments, administration of a first dose for a first predetermined period comprises daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 30; and administration of a second dose for a second predetermined period comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 90. In some embodiments, administration of a first dose for a first predetermined period comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 30; and administration of a second dose for a second predetermined period comprises daily administration of about 200 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 90. In some embodiments, administration of a first dose for a first predetermined period comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 30; and administration of a second dose for a second predetermined period comprises daily administration of about 200 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 37.
[0144] In some embodiments, administration of a third dose for a third predetermined period comprises daily administration of the BCL2 inhibitor to the subject from about Day 31 to Day 90. In some embodiments, administration of a third dose for a third predetermined period comprises daily administration of the BCL2 inhibitor to the subject from about Day 38 to Day 90. In some embodiments, the third dose is about 100 mg, about 200 mg, or about 400 mg of the BCL inhibitor. In some embodiments, the third dose is about 100 mg of the BCL inhibitor. In some embodiments, the third dose is about 200 mg of the BCL inhibitor. In some embodiments, the third dose is about 400 mg of the BCL inhibitor.
[0145] In some embodiments, the third predetermined period is from about Day 31 to Day 90, and the third dose is about 100 mg of the BCL inhibitor. In some embodiments, the third predetermined period is from about Day 31 to Day 90, and the third dose is about 200 mg of the BCL inhibitor. In some embodiments, the third predetermined period is from about Day 38 to Day 90, and the third dose is about 400 mg of the BCL inhibitor.
[0146] In some embodiments, administration of a first dose for a first predetermined period comprises daily administration of about 20 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 7 ; administration of a second dose for a second predetermined period comprises daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 8 to about Day 30; and administration of a third dose for a third predetermined period comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 90. In some embodiments, administration of a first dose for a first predetermined period comprises daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 2 toabout Day 7; administration of a second dose for a second predetermined period comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 8 to about Day 30; and administration of a third dose for a third predetermined period comprises daily administration of about 200 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 90. In some embodiments, administration of a first dose for a first predetermined period comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 30; administration of a second dose for a second predetermined period comprises daily administration of about 200 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 37; and administration of a third dose for a third predetermined period comprises daily administration of about 400 mg of the BCL2 inhibitor to the subject from about Day 38 to about Day 90.
[0147] In some cases, the combination therapy involves administration of the inhibitor for up to or or about 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more (e.g. 24 months) after the subject has received administration of the therapy, e.g. CAR T cell therapy. In some cases, the combination therapy involves administration of the inhibitor up to or or about 3 months after the subject has received administration of the therapy, e.g. CAR T cell therapy. In some cases, the combination therapy involves administration of the inhibitor up to or or about 6 months after the subject has received administration of the therapy, e.g. CAR T cell therapy. In some cases, the combination therapy involves administration of the inhibitor up to or or about 9 months after the subject has received administration of the therapy, e.g. CAR T cell therapy. In some cases, the combination therapy involves administration of the inhibitor up to or or about 12 months after the subject has received administration of the therapy, e.g. CAR T cell therapy. In some cases, the combination therapy involves administration of the inhibitor up to or or about 15 months after the subject has received administration of the therapy, e.g. CAR T cell therapy. In some cases, the combination therapy involves administration of the inhibitor up to or or about 18 months after the subject has received administration of the therapy, e.g. CAR T cell therapy. In some cases, the combination therapy involves administration of the inhibitor up to or or about 21 months after the subject has received administration of the therapy, e.g. CAR T cell therapy. In some cases, the combination therapy involves administration of the inhibitor up to or or about 24 months after the subject has received administration of the therapy, e.g. CAR T cell therapy.
[0148| In some cases, if the subject does not exhibit a complete response (CR) at Day 90, the combination therapy involves administration of the inhibitor up to or or about 12 months after the subject has received administration of the therapy, e.g. CAR T cell therapy. In some cases, if the subject exhibit minimum residual disease in peripheral blood at Day 90, the combination therapy involves administration of the inhibitor up to or or about 12 months after the subject has received administration of the therapy, e.g. CAR T cell therapy.
[0149] In some cases, if the subject does not exhibit a complete response (CR) at Day 90, the combination therapy involves administration of the inhibitor up to or or about 18 months after the subject has received administration of the therapy, e.g. CAR T cell therapy. In some cases, if the subject exhibit minimum residual disease in peripheral blood at Day 90, the combination therapy involves administration of the inhibitor up to or or about 18 months after the subject has received administration of the therapy, e.g. CAR T cell therapy.
[0150] In some cases, if the subject does not exhibit a complete response (CR) at Day 90, the combination therapy involves administration of the inhibitor up to or or about 24 months after the subject has received administration of the therapy, e.g. CAR T cell therapy. In some cases, if the subject exhibit minimum residual disease in peripheral blood at Day 90, the combination therapy involves administration of the inhibitor up to or or about 24 months after the subject has received administration of the therapy, e.g. CAR T cell therapy.
[0151] In some aspects, a BCL2 inhibitor, e.g., venetoclax, is administered for no more than three months after administration of the therapy for treating the cancer that is directed to or targets killing of the cancer, e.g. immunotherapy or cell therapy, such as an immunotherapy, including a T cell engaging therapy or a T cell therapy (e.g. CAR T cell therapy). In some embodiments, inhibitor is administered in combination with a T cell therapy (e.g. CAR T cells) and administration of the inhibitor is continued in a dosing regimen that includes continued administration of the inhibitor until a time after which the cells of the T cell therapy have reached peak levels in the subject and/or are persisting in the subject. In some aspects, advantages of the provided embodiments also include the ability to modulate the dosing or administration of the inhibitor, e.g., venetoclax, or removing or discontinuing the administration of the inhibitor, e.g., venetoclax, depending on the tolerability in the subject. In some aspects, the provided embodiments, e.g., involving combination treatment with a BCL2 inhibitor, e.g., venetoclax, can help reduce tumor burden and/or mitigate cancer cell resistance to certain therapies, such as cell therapies, e.g. CAR T cell therapy.
[0152] In some embodiments, the provided methods can potentiate CAR-T cell therapy, which, in some aspects, can improve outcomes for treatment of subjects that have a cancer that is resistant or refractory to other therapies, is an aggressive or high-risk cancer, and/or that is or is likely to exhibit a relatively lower response rate to a CAR-T cell therapy when administered without the inhibitor. In some aspects, administering a BCL2 inhibitor, e.g., venetoclax, according to the provided methods could increase the activity of CAR-expressing cells for treating a cancer, e.g. B cell malignancy such as CLL or NHL, e.g. DLBCL or SLL, by increasing T cell cytotoxicity by reducing the resistance of cancer cells to the CAR T cell therapy, optionally by increasing perforin- and/or granzyme-mediated apoptosis of the cancer cells. In some aspects, anti-tumor activity of administered CAR+ T cells against human lymphoma cell is improved. In some aspects, anti-tumor activity of administered CAR+ T cells against CLL cells is improved. In some aspects, anti-tumor activity of administered CAR+ T cells against SLL cells is improved.
I. COMBINATION THERAPY
[0153] Provided herein are methods for combination therapy for treating a disease or condition, such as a proliferative disease (e.g. a cancer) that include administering to a subject a combination therapy of (1) an inhibitor of BCL2 protein (e.g. venetoclax) and (2) an immunotherapy or a cell therapy, e.g. a T cell therapy (e.g. CAR-expressing T cells).
[0154] In some embodiments, the immunotherapy or cell therapy, such as an adoptive immune cell therapy comprising T cells (e.g. CAR-expressing T cells) specifically recognizes and/or binds to an antigen associated with, expressed by or present on cells of the cancer. Also provided are combinations and articles of manufacture, such as kits, that contain a composition comprising the T cell therapy and/or a composition comprising the BCL2 inhibitor, and uses of such compositions and combinations to treat or prevent conditions or diseases such as cancers, including hematologic malignancies (e.g. CLL or SLL).
[0155] In some embodiments, methods can include administration of the BCL2 inhibitor (e.g. venetoclax) subsequently to the administration (e.g., 1 or 2 days after initiation of the administration) of the immunotherapy or cell therapy, wherein the therapy specifically binds to an antigen associated with, expressed by or present on cells of the cancer (e.g. CLL or SLL). In some embodiments, the methods include administering the BCL2 inhibitor in a dosing regimen comprising (i) daily admistration of a first dose for a first predetermined period and (ii) daily administration of at least one subsequent dose for a predetermined period beginning after a preceding predetermined period, wherein each of the at least one dose is a greater amount of the BCL2 inhibitor compared to the preceding dose. In some embodiments, no more than 100 mg per day of the BCL2 inhibitor is provided though Day 30, where the immunotherapy or cell therapy (e.g. CAR T cells) is administered on Day 1.
[0156] In some embodiments, methods can include administration of the BCL2 inhibitor (e.g. venetoclax) about 1 day after initiation of the administration of the immunotherapy or cell (e.g. CAR T cells), wherein the therapy specifically binds to an antigen associated with, expressed by or present on cells of the cancer (e.g. CLL or SLL), wherein the inhibitor is administered in a dosing regimen comprising (i) daily admistration of a first dose for a first predetermined period and (ii) daily administration of at least one subsequent dose for a predetermined period beginning after a preceding predetermined period, wherein each of the at least one dose is a greater amount of the BCL2 inhibitor compared to the preceding dose and no more than 100 mg per day of the BCL2 inhibitor is provided through Day 30, where the immunotherapy or cell therapy (e.g. CAR T cells) is administered on Day 1.
[0157] In some embodiments, the BCL2 inhibitor is administered to the subject as a single agent therapy (e.g. monotherapy) in combination with an immunotherapy or a cell therapy (e.g. CAR T celsl). In some embodiments administration as a monotherapy consists of a single type of treatment alone, to treat a disease or condition, except where otherwise provided. In some embodiments, an inhibitor of a prosurvival BCL2 family protein is provided as a monotherapy with an immunotherapy or a cell therapy, such that no other treatment is provided to treat a disease or condition beyond provision of (1) the BCL2 inhibitor and (2) the immunotherapy or the cell therapy.
[0158] In some embodiments, the subject is administered a BCL2 inhibitor (e.g. venetoclax) as a bridging therapy, prior to administration of the immunotherapy or cell therapy (e.g. CAR T cells). In some cases, the bridging therapy comprises administration a BCL2 inhibitor (e.g. venetoclax) at 20 mg per day for a first week, at 50 mg per day for a second week, and at 100 mg per day for a third week. In some embodiments, the bridging therapy further comprises administration of an anti-CD20 antibody. In some embodiments, the bridging therapy further comprises administration of an anti-CD20 antibody if the subject has been previously treated with a BCL2 inhibitor (e.g. venetoclax). In some embodiments, if the subject has not been previously treated with a BCL2 inhibitor (e.g. venetoclax), the bridging therapy does not further comprise administration of the BCL2 inhibitor. In some embodiments, the bridging therapy further comprises administration of a BTK inhibitor ( e.g. ibrutinib.) In some embodiments, the bridging therapy further comprises administration of the BTK inhibitor (e.g. ibrutinib) if the subject was receiving treatment with ibrutinib prior to treatment with the combination therapy. Thus, in some cases, the subject is administered a BTK inhibitor (e.g. ibrutinib) until autologous cells are collected from the subject and again during the bridging therapy.
[0159] In some embodiments, the immunotherapy or cell therapy is adoptive cell therapy. In some embodiments, the cell therapy is or comprises a transgenic TCR therapy or a recombinant-receptor expressing cell therapy (optionally T cell therapy), which optionally is a chimeric antigen receptor (CAR)-expressing cell therapy. In some embodiments, the therapy targets CD19 or is a B cell targeted therapy. In some cases, the cytotoxic therapy is a cell therapy comprising T cells expressing a chimeric antigen-receptor (CAR), wherein the antigen-binding domain of the CAR binds to CD 19. In some embodiments, the cells and dosage regimens for administering the cells can include any as described in the following Subsection A under “Administration of an Immunotherapy or Cell Therapy.”
[0160] In some embodiments, the immunotherapy or cell therapy is capable of mediating and/or inducing a cell’s intrinsic, mitochondrial-mediated apoptotic pathway. In some embodiments, the immunotherapy or cell therapy is capable of mediating and/or inducing a cell’s perforin- and or granzyme-mediated apoptotic pathway. In some embodiments, the cancer cells are resistant to the instrinsic apoptotic pathways. In some embodiments, the BCL2 inhibitor sensitizes cells to apoptosis via the intrinsic apoptotic pathways. In some embodiments, the BCL2 inhibitor sensitizes cells to apoptosis, as mediated or induced by the immunotherapy or cell therapy. In some ways, the BCL2 inhibitor lowers the apoptotic resistance of cells to the immunotherapy or cell therapy.
[0161] In some embodiments, the immunotherapy or cell therapy is an adoptive cell therapy (e.g. a T cell therapy). In some embodiments, the adoptive cell therapy comprises cells that are autologous to the subject. In some embodiments, the cells that are autologous to the subject are engineered to express a chimeric antigen receptor (CAR). In some embodiments, CAR-expressing autologous T cells are provided to the subject.
[0162] In some embodiments, the immunotherapy or cell therapy, such as a T cell therapy e.g. CAR-expressing T cells) or a T cell-engaging therapy, and the BCL2 inhibitor are provided as pharmaceutical compositions for administration to the subject. In some embodiments, the pharmaceutical compositions contain therapeutically effective amounts of one or both of the agents for combination therapy, e.g., T cells for adoptive cell therapy and a BCL2 inhibitor as described. In some embodiments, the pharmaceutical compositions contain therapeutically effective amounts of a BCL2 inhibitor. In some embodiments, the pharmaceutical compositions contain subtherapeutically effective amounts of one or both of the agents for combination therapy, e.g., T cells for adoptive cell therapy and a BCL2 inhibitor as described. In some embodiments, the pharmaceutical compositions contain subtherapeutically effective amounts of a BCL2 inhibitor. In some embodiments, the agents are formulated for administration in separate pharmaceutical compositions. In some embodiments, any of the pharmaceutical compositions provided herein can be formulated in dosage forms appropriate for each route of administration.
[0163] In some embodiments, the combination therapy, which includes administering the immunotherapy or cell therapy (e.g. T cell therapy, including engineered cells, such as CAR-T cell therapy) and the BCL2 inhibitor, is administered to a subject or patient having a cancer (e.g. CLL or SLL) or at risk for cancer. In some aspects, the methods treat, e.g., ameliorate one or more symptom of, the disease or condition, such as by lessening tumor burden in a cancer expressing an antigen recognized by the immunotherapy or cell therapy, e.g. recognized by an engineered T cell.
[0164] In some embodiments, the disease or condition that is treated can be any in which expression of an antigen is associated with and/or involved in the etiology of a disease condition or disorder such as a cancer, e.g. causes, exacerbates or otherwise is involved in such disease, condition, or disorder. Exemplary diseases and conditions can include diseases or conditions associated with malignancy or transformation of cells e.g. cancer). Exemplary antigens, which include antigens associated with various diseases and conditions that can be treated, include any of antigens described herein. In particular embodiments, the recombinant receptor expressed on engineered cells of a combination therapy, including a chimeric antigen receptor or transgenic TCR, specifically binds to an antigen associated with the cancer.
[0165| In some embodiments, the antigen associated with the disease or disorder such as cancer is selected from the group consisting of ROR1, B cell maturation antigen (BCMA), tEGFR, Her2, LI- CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, anti-folate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, erbB dimers, EGFR vIII, FBP, FCRL5, FCRH5, fetal acethycholine e receptor, GD2, GD3, G Protein Coupled Receptor 5D (GPCR5D), HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, LI -cell adhesion molecule, (LI -CAM), Melanoma-associated antigen (MAGE)-Al, MAGE- A3, MAGE- A6, Preferentially expressed antigen of melanoma (PRAME), survivin, EGP2, EGP40, TAG72, B7-H6, IL-13 receptor a2 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250/CAIX, HLA-AI MAGE Al, HLA-A2 NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptors, 5T4, Foetal AchR, NKG2D ligands, CD44v6, dual antigen, and an antigen associated with a universal tag, a cancer-testes antigen, mesothelin, MUC1, MUC16, PSCA, NKG2D Ligands, NY-ESO-1, MART-1, gplOO, G Protein Coupled Receptor 5D (GPCR5D), oncofetal antigen, ROR1, TAG72, VEGF- R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1, CD138, and a pathogen-specific antigen. In some embodiments, the antigen associated with the disease or disorder is CD19. In some embodiments, the antigen is CD19. In some embodiments, the antigen is associated with or is a universal tag.
[0166] In some embodiments, the disease or condition is a cancer or proliferative disease. In some embodiments, the disease or condition is a cancer. In some embodiments, the disease or condition is a proliferative disease. In some embodiments, the cancer or proliferative disease is a tumor, such as a solid tumor, lymphoma, leukemia, blood tumor, metastatic tumor, or other cancer or tumor type. In some embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is a B cell malignancy. In some embodiments, the cancer is a leukemia or a lymphoma. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), or a subtype of NHL, such as diffuse large B-cell lymphoma (DLBCL). In some embodiments, the cancer is a leukemia. In some embodiments, the cancer is chronic lymphocytic leukemia (CLL). In some embodiments, the cancer is a lymphoma. In some embodiments, the cancer is small lymphocytic lymphoma (SLL).
[0167] In some embodiments, the Eastern Cooperative Oncology Group (ECOG) performance status indicator can be used to assess or select subjects for treatment, e.g., subjects who have had poor performance from prior therapies (see, e.g., Oken et al. (1982) Am J Clin Oncol. 5:649-655). The ECOG Scale of Performance Status describes a patient’s level of functioning in terms of their ability to care for themselves, daily activity, and physical ability (e.g., walking, working, etc.). In some embodiments, an ECOG performance status of 0 indicates that a subject can perform normal activity. In some aspects, subjects with an ECOG performance status of 1 exhibit some restriction in physical activity but the subject is fully ambulatory. In some aspects, patients with an ECOG performance status of 2 is more than 50% ambulatory. In some cases, the subject with an ECOG performance status of 2 may also be capable of selfcare; see e.g., Sprensen et al., (1993) Br J Cancer 67(4) 773-775. The criteria reflective of the ECOG performance status are described in Table 1 below:
Figure imgf000040_0001
Figure imgf000041_0001
[0168] Antigens targeted by the receptors (e.g. CAR) in some embodiments include antigens associated with a B cell malignancy, such as any of a number of known B cell marker. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30. In some embodiments, the antigen is CD19.
[0169] In particular, among provided embodiments are methods of treating subjects with CLL or SLL. In some embodiments of the provided methods, the subject has CLL. In some embodiments of the provided methods, the subject has SLL. In some embodiments of the provided methods, the subject has a high risk CLL or SLL. In some embodiments of the provided methods, the subjects have a high risk CLL or SLL. In some embodiments of the provided methods, the subjects have a high risk CLL. In some embodiments of the provided methods, the subject has a high risk CLL. In some embodiments of the provided methods, the subjects have a high risk SLL. In some embodiments of the provided methods, the subject has a high risk SLL. In some embodiments, the subjects are a heavily pretreated population of subjects with high-risk CLL (or SLL). In some embodiments, the subject has received one or more prior therapies including ibrutinib. In some embodiments, the subject has received one or more prior therapies including venetoclax. In some embodiments, the subject has received one or more prior therapies including ibrutinib and venetoclax.
[0170] In some embodiments, subjects with CLL include those with CLL diagnosis with indication of treatment based on the International Workshop on Chronic Lymphocytic Leukemia (iwCLL) guidelines and clinical measurable disease (bone marrow involvement by > 30% lymphocytes, peripheral blood lymphocytosis > 5xl09/L, and/or measurable lymph nodes and/or hepatic or splenomegaly. In some embodiments, subjects with SLL include those with SLL diagnosis is based on lymphadenopathy and/or splenomegaly and < 5xl09 CD19+ CD5+ clonal B lymphocytes/L [< 5000/pL] in the peripheral blood at diagnosis with measurable disease defined as at least one lesion > 1.5 cm in the greatest transverse diameter, and that is biopsy-proven SLL. In some embodiments, subjects with SLL include those with SLL diagnosis is based on lymphadenopathy and/or splenomegaly and < 5xl09 CD19+ CD5+ clonal B lymphocytes/L [< 5000/pL] in the peripheral blood at diagnosis with measurable disease defined as at least one lesion > 1.5 cm in the greatest transverse diameter, and that is biopsy-proven SLL.
[0171] In some cases, existing treatment strategies for high risk and very high risk subjects may include fludarabine, cyclophosphamide, and rituximab (FCR), Bruton’s tyrosine kinase (BTK) inhibitors (e.g. ibrutinib), and/or allogeneic stem cell transplantation. (Puiggros et al., BioMed Research International, Volume 2014 (2014), Article ID 435983). Many of the existing therapies include oral- targeted drugs, which have, for some patients with CLL, improved treatment outcomes. Nonetheless, some patients prove intolerant or resistant to therapy and/or fail to achieve complete response with undetectable MRD (uMRD). In some aspects, subjects who have progressive disease after treatment with available therapies have poor outcomes. For instance, in some aspects, subjects treated for CLL exhibit poor long-term outcomes. For example, in some cases, refractory (R/R) high-risk CLL subjects exhibit poor survival after ibrutinib discontinuation (Jain et al. (2015) Blood 125(13):2062-2067). There is a need for improved methods of treating CLL, and in some aspects, for those appropriate for treating high and/or very high-risk CLL and/or subjects having relapsed or become refractory to multiple prior therapies.
[0172] Chronic lymphocytic leukemia (CLL) is a generally a variable disease. Some subjects with CLL may survive without treatment while others may require immediate intervention. In some cases, subjects with CLL may be classified into groups that may inform disease prognosis and/or recommended treatment strategy. In some cases, these groups may be “low risk,” “intermediate risk,” “high risk,” and/or “very high risk” and patients may be classified as such depending on a number of factors including, but not limited to, genetic abnormalities and/or morphological or physical characteristics. In some embodiments, subjects treated in accord with the method are classified or identified based on the risk of CLL. In some embodiments, the subject is one that has high risk CLL. In some embodiments, the subject is one that has very high risk CLL.
[0173] In some embodiments, the provided methods and uses provide for or achieve improved or more durable responses or efficacy as compared to certain alternative methods, such as in particular groups of subjects treated, such as in patients with a leukemia, such as CLL or SLL, including those with high-risk disease. In some embodiments, the methods are advantageous by virtue of administering T cell therapy, such as a composition including cells for adoptive cell therapy, e.g., such as a CAR-expressing T cells, e.g. anti-CD19 CAR+ T cells, and a BCL2 inhibitor (e.g. venetoclax). In some embodiments, the methods also include, prior to administration of the T cell therapy, a lymphodepleting therapy, e.g. such as cyclophosphamide, fludarabine, or combinations thereof. In some embodiments, the BCL2 inhibitor (e.g. venetoclax) bridging therapy is provided prior to administration of the lymphodepleting therapy. In some cases, the BCL2 inhibitor (e.g. venetoclax) bridging therapy is discontinued at about or at least about 24 hours before administration of the lymphodepleting therapy. In some cases, the BCL2 inhibitor (e.g. venetoclax) bridging therapy is discontinued about 24 hours before administration of the lymphodepleting therapy. In some cases, the BCL2 inhibitor (e.g. venetoclax) bridging therapy is discontinued at least about 24 hours before administration of the lymphodepleting therapy. In some cases, the interval between discontinuation of the BCL2 inhibitor bridging therapy and administration of the lymphodepleting therapy is known as the “washout” period. In some cases, the washout period is about 1 day (24 hours).
[0174] In some embodiments, the treated subjects include subjects that have relapsed following initial remission on ibrutinib or who are refractory or intolerant to treatment with ibrutinib. In some embodiments, the subject has relapsed following initial remission on ibrutinib. In some embodiments, the subject is refractory or intolerant to treatment with ibrutinib. In particular embodiments, the treated subjects include subjects that have relapsed following remission or are refractory or intolerant to one or more further prior therapy in addition to ibrutinib, such as 1, 2, 3, 4, 5 or more prior therapies. In some embodiments, the subjects have relapsed or are refractory to both a prior treatment of ibrutinib and venetoclax. In some embodiments, the subject has relapsed or is refractory to both ibrutinib and venetoclax. In some embodiments, subjects that are refractory to such treatment have progressed following one or more prior therapy. In some embodiments, subjects treated, including those treated with one or more prior therapies (e.g. ibrutinib and/or venetoclax) include those with a high-risk cytogenetics, including TP53 mutation, complex karyotype (i.e. at least three chromosomal alterations) and dell7(p).
[0175] In some embodiments of any of the provided methods, the subject has CLL or is suspected of having CLL. In some embodiments, the subject is identified or selected as having CLL. In some embodiments, the CLL is relapsed or refractory CLL. In particular, CLL is generally considered to be incurable and patients often eventually relapse or become refractory to available therapies (Dighiero and Hamblin (2008) The Lancet, 371:1017-1029).
[0176] In some embodiments, the subject has SLL or is suspected of having SLL. In some embodiments, the subject is identified or selected as having SLL. In some embodiments, the SLL is a relapsed or refractory SLL.
[0177] In some embodiments, prior to the administration of the dose of engineered T cells, the subject has been treated with one or more prior therapies for the CLL or SLL, other than the therapy, e.g. dose of cells expressing CAR, or a lymphodepleting therapy. In some embodiments, prior to the administration of the dose of engineered T cells, the subject has been treated with one or more prior therapies for the CLL , other than the therapy, e.g. dose of cells expressing CAR, or a lymphodepleting therapy. In some embodiments, prior to the administration of the dose of engineered T cells, the subject has been treated with one or more prior therapies for the SLL, other than the therapy, e.g. dose of cells expressing CAR, or a lymphodepleting therapy. In some embodiments, the one or more prior therapy comprises at least two prior therapies, optionally three, four, five, six, seven, eight, nine or more. In some embodiments, the one or more prior therapy comprises at least two prior therapies. In some embodiments, the one or more prior therapy comprises at least three prior therapies. In some embodiments, the one or more prior therapy comprises at least two four prior therapies. In some embodiments, the one or more prior therapy comprises at least two five therapies. In some embodiments, prior to the administration of the immunotherapy or cell therapy (e.g. CAR T cells), the subject has been treated with two or more prior therapies for the SLL, other than the therapy, e.g. dose of cells expressing CAR, or a lymphodepleting therapy. In some embodiments, prior to the administration of the immunotherapy or cell therapy (e.g. CAR T cells), the subject has failed two or more prior therapies for the SLL, other than the therapy, e.g. dose of cells expressing CAR, or a lymphodepleting therapy. [0178] In some embodiments, at or immediately prior to the time of the administration of the dose of cells, the subject has relapsed following remission after treatment with, or become refractory to, failed and/or was intolerant to treatment with the one or more prior therapies for the CLL. In some embodiments, at or immediately prior to the time of the administration of the dose of cells, the subject has relapsed following remission after treatment with the one or more prior therapies for the CLL. In some embodiments, at or immediately prior to the time of the administration of the dose of cells, the subject has become refractory to, has failed and/or was intolerant to treatment with the one or more prior therapies for the CLL. In some embodiments, the subject has failed two or more prior therapies. In some embodiments, the subject has relapsed following remission after treatment with, or become refractory to, failed and/or was intolerant to treatment with two or more prior therapies. In some embodiments, at or immediately prior to the time of the administration of the dose of cells, the subject has relapsed following remission after treatment with, or become refractory to, failed and/or was intolerant to treatment with three or more prior therapies. In some embodiments, the prior therapies are selected from a kinase inhibitor, optionally an inhibitor of Bruton’s tyrosine kinase (BTK), optionally ibrutinib; venetoclax; a combination therapy comprising fludarabine and rituximab; radiation therapy; and hematopoietic stem cell transplantation (HSCT). In some embodiments, the prior therapies comprise ibrutinib and/or venetoclax. In some embodiments, the prior therapies comprise ibrutinib and venetoclax. In some embodiments, the prior therapy comprises ibrutinib. In some embodiments, the prior therapy comprises venetoclax. In some embodiments, the subject has failed two or more prior therapies, including BTK inhibition (e.g. ibrutinib). In some embodiments, the subject has failed two or more prior therapies, including BTK inhibition (e.g. ibrutinib) and venetoclax. In some embodiments, the subject has failed two or more prior therapies, including BTK inhibition (e.g. ibrutinib) and another prior therapy that is not venetoclax.
[0179] In some embodiments, the subject has relapsed following remission after treatment with, become refractory to failed treatment with and/or is intolerant to ibrutinib and/or venetoclax. In some embodiments, the subject has relapsed following remission after treatment with, become refractory to failed treatment with and/or is intolerant to ibrutinib. In some embodiments, the subject has relapsed following remission after treatment with, become refractory to failed treatment with and/or is intolerant to venetoclax. In some embodiments, the subject has relapsed following remission after treatment with, become refractory to, failed treatment with and/or is intolerant to ibrutinib and venetoclax.
[0180] In some embodiments a subject that has been previously treated with a BCL2 inhibitor (e.g. venetoclax) prior to the provided methods is not intolerant to venetoclax (e.g. at the time of administration of the cell therapy). In some embodiments, a subject has been treated with a BCL2 inhibitor (e.g. venetoclax) within 6 months prior to the provided methods, e.g. prior to receiving a lymphodepleting therapy or prior to receiving administration of a cytotoxic therapy. In some embodiments a subject has been treated with a BCL2 inhibitor (e.g. venetoclax) within 6 months prior to the provided methods and does not have progressive disease (PD). In some embodiments, the subject did not exhibit progressive disease during the previous treatment with the BCL2 inhibitor. In some embodiments a subject has been treated with a BCL2 inhibitor (e.g. venetoclax) within 6 months prior to the provided methods and did not exhibit progressive disease (PD) during the treatment with the inhibitor. In some embodiments, if the previous treatment with the BCL2 inhibitor (e.g. venetoclax) resulted in a Best Overall Result (BOR) of stable disease (SD) or progressive disease during the previous BCL2 inhibitor (e.g. venetoclax) treatment, more than 6 months have passed since the last dose of the previous treatment. In some embodiments, if the previous treatment with the BCL2 inhibitor (e.g. venetoclax) resulted in a Best Overall Result (BOR) of stable disease (SD) or progressive disease within six months of discontinuing the previous treatment, more than 6 months have passed since the last dose of the previous treatment.
[0181] In some embodiments, the subject is not intolerant to the BCL2 inhibitor (e.g. venetoclax) at a time immediately following collection of autologous cells from the subject and/or immediately before administration of a lymphodepleting therapy to the subject. In some embodiments, the subject is not intolerant to the BCL2 inhibitor (e.g. venetoclax) at the time of initiation of administration of the immunotherapy or cell therapy.
[0182] In some embodiments, the subject was previously treated with the cell therapy (e.g. CAR T cells) and achieved a complete response. In some embodiments, the subject previously achieved a complete response to the cell therapy (e.g. CAR T cells) and subsequently progressed, including progression by Richter’s transformation. In some cases, Richter’s transformation is characterized by the sudden transformation of a CLL or SLL into a more aggressive form of large cell lymphoma.
[0183] In some embodiments, the subject was previously treated with a CD19-targeted therapy and has CD19-positive disease. In some embodiments, the CD19-positive disease is confirmed by immunohistochemistry or flow cytometry for CD 19 expression since completion of the previous CD 19- targeted therapy.
[0184] In some embodiments, a subject is screened for expression of BCL2 family proteins. In some embodiments, a subject is screened for expression of BCL2 family proteins prior to administration of a lymphodepleting therapy to the subject. In some embodiments, a subject that has been previously treated with a BCL2 inhibitor, e.g. venetoclax, is screened for BCL2 mutations. In some embodiments, a subject that has been previously treated with a BCL2 inhibitor, e.g. venetoclax, is screened for BCL2 mutations prior to administration of a lymphodepleting therapy to the subject. In some embodiments, a subject that has been previously treated with a BCL2 inhibitor, e.g. venetoclax, is screened for BCL2 mutations prior to administration of the cell therapy to the subject.
[0185] In some embodiments, the subject does not have a mutation in BCL2 at a time immediately following collection of autologous cells from the subject and/or immediately before administration of a lymphodepleting therapy to the subject. In some embodiments, the subject does not have a mutation in BCL2 at the time of initiation of administration of the immunotherapy or cell therapy. In some embodiments, the subject does not have a mutation in BCL2. In some embodiments, the subject does not have a mutation in BCL2 or a BCL2 family protein. In some embodiments, the subject has a mutation in BCL2 or a BCL2 family protein at a time immediately following collection of autologous cells from the subject and/or immediately before administration of a lymphodepleting therapy to the subject. In some embodiments, the subject has a mutation in BCL2 or a BCL2 family protein at a time immediately following collection of autologous cells from the subject. In some embodiments, the subject has a mutation in BCL2 or a BCL2 family protein at a time immediately before administration of a lymphodepleting therapy to the subject. In some embodiments, the subject has a mutation in BCL2 or a BCL2 family protein at the time of initiation of administration of the immunotherapy or cell therapy (e.g. CAR T cells). In some embodiments, the subject has a mutation in BCL2 or a BCL2 family protein. In some embodiments, a subject is not excluded from receiving treatment with the combination therapy on the basis of BCL2 mutation status.
[0186] In some embodiments, the subject exhibits measurable disease or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen at a time immediately following collection of autologous cells from the subject and/or immediately before administration of a lymphodepleting therapy to the subject. In some embodiments, the subject exhibits measurable disease or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, at the time of initiation of the immunotherapy or cell therapy (e.g. CAR T cells), the subject exhibits measurable disease or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen. In some embodiments, the measurable disease is or includes lymph nodes of greater than 1.0 centimeters in greatest transverse diameter (GTD). In some embodiments, the measurable disease is or includes lymph nodes of greater than 1.5 centimeters in greatest transverse diameter (GTD). In some embodiments, the measurable disease is or includes lymph nodes of greater than 2.0 centimeters in greatest transverse diameter (GTD). In some embodiments, a subject exhibits measureable disease (e.g. lymph nodes of greater than 1.5 centimeters in greatest transverse diameter (GTD)) or evaluable or measurable disease in PB, BM, liver or spleen, and evidence of disease in the blood by local testing (e.g. minimum residual disease (MRD) in peripheral blood of greater than or equal to 104).
[0187] In some embodiments, the subject exhibits minimum residual disease (MRD) in peripheral blood of greater than or equal to 104. In some embodiments, the subject exhibits minimum residual disease (MRD) in peripheral blood of greater than or equal to 104 at a time immediately following collection of autologous cells from the subject and/or immediately before administration of a lymphodepleting therapy to the subject. In some embodiments, the subject exhibits minimum residual disease (MRD) in peripheral blood of greater than or equal to 104 at a time immediately following collection of autologous cells from the subject. In some embodiments, the subject exhibits minimum residual disease (MRD) in peripheral blood of greater than or equal to 104 at a time immediately before administration of a lymphodepleting therapy to the subject. In some embodiments, at the time of initiation of the immunotherapy or cell therapy (e.g. CAR T cells), the subject exhibits minimum residual disease (MRD) in peripheral blood of greater than or equal to 104.
[0188] In some embodiments, the subject exhibits evidence of CLL in blood by local testing. In some embodiments, the subject exhibits evidence of CLL in blood by local testing at a time immediately following collection of autologous cells from the subject and/or immediately before administration of a lymphodepleting therapy to the subject. In some embodiments, the subject exhibits evidence of CLL in blood by local testing at a time immediately following collection of autologous cells from the subject. In some embodiments, the subject exhibits evidence of CLL in blood by local testing at a time immediately before administration of a lymphodepleting therapy to the subject. In some embodiments, at the time of initiation of the immunotherapy or cell therapy (e.g. CAR T cells), the subject exhibits evidence of CLL in blood by local testing.
[0189] In some embodiments, the subject exhibits measurable disease or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen and minimum residual disease (MRD) in peripheral blood of greater than or equal to 104. In some embodiments, the subject exhibits measurable disease or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen and minimum residual disease (MRD) in peripheral blood of greater than or equal to 104, at a time immediately following collection of autologous cells from the subject and/or immediately before administration of a lymphodepleting therapy to the subject. In some embodiments, at the time of initiation of administration of the cytotoxic therapy, the subject exhibits measurable disease or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen and minimum residual disease (MRD) in peripheral blood of greater than or equal to 104. In some embodiments, the measurable disease is or includes lymph nodes of greater than 1.0 centimeters in greatest transverse diameter (GTD). In some embodiments, the measurable disease is or includes lymph nodes of greater than 1.5 centimeters in greatest transverse diameter (GTD). In some embodiments, the measurable disease is or includes lymph nodes of greater than 2.0 centimeters in greatest transverse diameter (GTD).
[0190] In some embodiments, at or prior to the administration of the dose of cells, the subject is or has been identified as having one or more cytogenetic abnormalities, optionally associated with high-risk CLL, optionally selected from among: complex karyotype or cytogenetic abnormalities, del 17p, unmutated IGVH gene, and TP53 mutation; the subject is or has been identified as having high-risk CLL. In some embodiments, at or prior to initiation of administration of the cytotoxic therapy, the subject is or has been identified as having one or more cytogenetic abnormalities. In some embodiments, the one or more cytogenetic abnormalities are associated with high-risk CLL. In some embodiments, the one or more cytogenetic abnormalities selected from among complex karyotype or cytogenetic abnormalities, del 17p, unmutated IGVH gene, and TP53 mutation. In some embodiments, at or prior to initiation of administration of the cytotoxic therapy, the subject is or has been identified as having high-risk CLL.
[0191] In some embodiments, the subject is or has been identified as having an ECOG status of 0 or 1; and/or the subject does not have an ECOG status of >1. In some embodiments, the subject is or has been identified as having an ECOG status of 0 or 1. In some embodiments, the subject is or has been identified as having an ECOG status of 0. In some embodiments, the subject is or has been identified as having an ECOG status of 1. In some embodiments, at or immediately prior to the administration of the dose of engineered cells or the lymphodepleting therapy the subject does not have a Richter’s transformation of the CLL or SLL.
[0192] In some embodiments, the methods involve treating a subject that has an Eastern Cooperative Oncology Group Performance Status (ECOG) of 0-1 or 0-2. In some embodiments, subjects have Eastern Cooperative Oncology Group (ECOG) scores of between 0 and 2. In some embodiments, subjects are not excluded based on an ECOG score of 2. In some embodiments, the subject is or has been identified as having an ECOG status of 2. In some embodiments, the methods treat a poor-prognosis population or of DLBCL patients or subject thereof that generally responds poorly to therapies or particular reference therapies, such as one having one or more, such as two or three, chromosomal translocations (such as so-called “double -hit” or “triple-hit” lymphoma; having translocations MYC/8q24 loci, usually in combination with the t(14; 18) (q32; q21) BCL-2 gene or/and BCL6/3q27 chromosomal translocation; see, e.g., Xu et al. (2013) Int J Clin Exp Pathol. 6(4): 788-794), and/or one having relapsed, such as relapsed within 12 months, following administration of an autologous stem cell transplant (ASCT), and/or one having been deemed chemorefractory.
[0193| In some embodiments, the subject has measurable disease before initiation of a lymphodepleting therapy, such as is described in Section I.D.
[0194] In some embodiments, the subject must have adequate organ function before initiation of a lymphodepleting therapy, such as is described in Section I.D. In some embodiments, the subject has adequate organ function prior to administration of the cell therapy, the BCL-2 inhibitor, or both. In some embodiments, adequate organ function is defined as (i) serum creatinine < 1.5 x age-adjusted upper limit of normal (ULN) or calculated creatinine clearance of > 30 mL/min; (ii) alanine aminotransferase (ALT) < 5 x ULN and total bilirubin < 2.0 mg/dL (or < 3.0 mg/dL for subjects with Gilbert’s syndrome or leukemic infiltration of the liver); (iii) adequate pulmonary function; and (iv) adequate cardiac function. In some embodiments, adequate pulmonary function is defined as < Common Terminology Criteria for Adverse Events (CTCAE) Grade 1 dyspnea and saturated oxygen (SaO2) > 92% on room air. In some embodiments, adequate cardiac function is defined as left ventricular ejection fraction (LVEF) > 40% as assessed by echocardiogram (ECHO) or multiple uptake gated acquisition (MUGA) scan performed within 30 days prior to determination of eligibility. In some embodiments, the subject has hemoglobin > 9 g/dL, absolute neutrophil count (ANC) > 500/mm3, and platelets > 75,000/mm3, unless the subject has cytopenias due to CLL infiltration of the bone marrow. [0195] In some embodiments, the subject does not have active central nervous system (CNS) involvement by malignancy. In some embodiments, the subject has not been previously treated with any gene therapy product.
[0196| In some embodiments, the subject has been previously treated with a therapy or a therapeutic agent targeting the disease or condition (e.g. CLL or SLL) prior to administration of the therapy, e.g. cells expressing the recombinant receptor. In some embodiments, the subject has been previously treated with a hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT or autogeneic HSCT. In some embodiments, the subject has had poor prognosis after treatment with standard therapy and/or has failed one or more lines of previous therapy. In some embodiments, the subject has been treated or has previously received at least or at least about or about 1, 2, 3, or 4 other therapies for treating the disease or disorder (e.g. CLL or SLL), other than a lymphodepleting therapy and/or the therapy, e.g. dose of cells expressing the antigen receptor. In some embodiments, the subject has been treated or has previously received a therapy that includes anthracycline, a CD20 targeted agent, and/or ibrutinib.
[0197] In some embodiments, the subject has been previously treated with chemotherapy or radiation therapy. In some aspects, the subject is refractory or non-responsive to the other therapy or therapeutic agent. In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapy or therapeutic intervention, including chemotherapy or radiation.
[0198] In some embodiments, the subject is one that is eligible for a transplant, such as is eligible for a hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT. In some such embodiments, the subject has not previously received a transplant, despite being eligible, prior to administration of the therapy, such as cell therapy containing engineered cells (e.g. CAR-T cells) or a composition containing the cells to the subject as provided herein. In some such embodiments, the subject has previously received an allogeneic stem cell transplantation (SCT). In some such embodiments, the subject has not previously received an allogeneic stem cell transplantation (SCT). In some embodiments, subjects are not excluded based on prior allogeneic stem cell transplantation (SCT).
[0199] In some embodiments, the subject is one that is not eligible for a transplant, such as is not eligible for a hematopoietic stem cell transplantation (HSCT), e.g., allogeneic HSCT.
[0200] In some embodiments, subjects are not required to have a minimum absolute lymphocyte count (ALC) for apheresis.
[0201] In some embodiments, the subject is an adult. In some embodiments, the subject is 18 years of age or older.
[0202] In some embodiments, the method includes administration of cells to a subject selected or identified as having chronic lymphocytic leukemia (CLL). In some embodiments, the methods include administration of cells to a subject selected or identified as having small lymphocytic lymphoma (SLL). In some embodiments, the subject exhibits one or more cytogenetic abnormalities, such as associated with the CLL or SLL, such as a high-risk CLL or a high-risk SLL. In some embodiments, the subject is selected or identified based on having a disease or condition characterized or determined to be aggressive CLL or SLL.
[0203] In some embodiments, the subject has poor performance status. In some aspects, the population to be treated includes subjects having an Eastern Cooperative Oncology Group Performance Status (ECOG) that is anywhere from 0-2. In other aspects of any of the embodiments, the subjects to be treated included ECOG 0-1 or do not include ECOG 2 subjects. In some aspects of any of the embodiments, the subjects to be treated have failed two or more prior therapies. In some embodiments, the subject has features that correlate with poor overall survival. In some embodiments, the subject has never achieved a complete response (CR), never received autologous stem cell transplant (ASCT), is refractory to 1 or more second line therapy, has primary refractory disease, and/or has an ECOG performance score of 2 or an ECOG score of between 0 and 1.
[0204] In some embodiments, the subject to be treated includes a group of subjects with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after failure of 2 or more lines of therapy. Optionally, the subject may have previously been treated with allogeneic stem cell transplantation (SCT). In some embodiments, the subject is not selected for treatment or excluded from treatment, if the subject has a poor performance status (e.g. ECOG 2). Thus, in some embodiments, the subject is selected for treatment if the subject has chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) after failure of 2 lines of therapy, and ECOG score of 0 or 1.
[0205] In some embodiments, the cancer is characterized by overexpression or aberrant expression of one or more prosurvival BCL2 family proteins. In some embodiments, the cancer is characterized by overexpression or aberrant expression of BCL2. In some cases, overexpression of BCL2 may be caused by the (14; 18)(q32;q21) translocation. In some cases, overexpression of BCL2 may be caused by amplification of the gene encoding the BCL2 protein. In some embodiments, the cancer is characterized by overexpression or aberrant expression of BCLXL, MCL1, and/or BFL1. In some embodiments, the cancer is characterized by a mutation in one or more genes encoding for a prosurvival BCL2 family protein. In some embodiments, the cancer is characterized by a mutation in the gene encoding the BCL2 protein. In some cases, the mutation is (14; 18)(q32;q21) translocation. In some embodiments, the cancer is resistant to treatment with an immunotherapy or cell therapy. In some embodiments, the cancer is resistant to treatment with a cell therapy, such as a CAR-expressing T cell therapy. In some embodiments, the cancer is resistant to treatment with a CD19-targeting CAR T cell therapy. In some embodiments, the BCL2 inhibitor sensitizes a cancer to treatment with an immunotherapy or cell therapy. In some embodiments, the BCL2 inhibitor sensitizes a cancer to treatment with a cell therapy, such as a CAR-expressing T cell therapy. In some embodiments, the BCL2 inhibitor sensitizes a cancer to treatment with a CD19-targeting CAR T cell therapy.
[0206] In some embodiments, the combination therapy provided herein is carried out in a subject that has been previously treated with a BCL2 inhibitor, such as venetoclax, but in the absence of administration of a T cell therapy (e.g. CAR+ T cells) or T cell-engaging therapy. In some cases, after such previous treatment the subject is refractory to and/or develops resistance to, has relapsed following remission, has not achieved a CR after receiving such previous treatment for at least 6 months and/or exhibits an aggressive disease and/or high-risk features of the cancer. Thus, it is understood that the provided combination therapy can be carried out in a subject that has previously received administration of a BCL2 inhibitor, such as venetoclax. Reference to timing of administration of an inhibitor in the present disclosure refers to timing of its administration relative to the immunotherapy or immunotherapeutic agent, e.g. T cell therapy (e.g. CAR+ T cells) or T cell-engaging therapy, in accord with the provided combination therapy methods and does not exclude the possibility that the subject has additionally previously been administered an inhibitor of a BCL2 inhibitor, such as venetoclax.
[0207] For the prevention or treatment of disease, the appropriate dosage of a BCL2 inhibitor and/or immunotherapy, such as a T cell therapy e.g. CAR-expressing T cells) or a T cell-engaging therapy, may depend on the type of disease to be treated, the particular inhibitor, cells and/or recombinant receptors expressed on the cells, the severity and course of the disease, route of administration, whether the BCL2 inhibitor and/or the immunotherapy, e.g., T cell therapy, are administered for preventive or therapeutic purposes, previous therapy, frequency of administration, the subject’s clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments. Exemplary dosage regimens and schedules for the provided combination therapy are described.
[0208] In some embodiments, the immunotherapy, e.g. T cell therapy, and the BCL2 inhibitor are administered as part of a further combination treatment, which can be administered simultaneously with or sequentially to, in any order, another therapeutic intervention. In some contexts, the immunotherapy, e.g. engineered T cells, such as CAR-expressing T cells, are co-administered with another therapy sufficiently close in time such that the immunotherapy enhances the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered prior to the one or more additional therapeutic agents. In some embodiments, the immunotherapy, e.g. engineered T cells, such as CAR-expressing T cells, are administered after the one or more additional therapeutic agents. In some embodiments, the combination therapy methods further include a lymphodepleting therapy, such as administration of a chemotherapeutic agent. In some embodiments, the combination therapy further comprises administering another therapeutic agent, such as an anti-cancer agent, a checkpoint inhibitor, or another immune modulating agent. Uses include uses of the combination therapies in such methods and treatments, and uses of such compositions in the preparation of a medicament in order to carry out such combination therapy methods. In some embodiments, the methods and uses thereby treat the disease or condition or disorder, such as a cancer or proliferative disease, in the subject.
[0209] In some embodiments, the immunotherapy, e.g. T cell therapy, and the BCL2 inhibitor are administered without any other combination treatment. In some embodiments, the immunotherapy, e.g. T cell therapy, and the BCL2 inhibitor are administered without any other combination treatment, such as ibrutinib and/or rituximab.
[0210] Prior to, during or following administration of the immunotherapy (e.g. T cell therapy, such as CAR-T cell therapy) and/or an inhibitor of BCL2, the biological activity of the immunotherapy, e.g. the biological activity of the engineered cell populations, in some embodiments is measured, e.g., by any of a number of known methods. Parameters to assess include the ability of the engineered cells to destroy target cells, persistence and other measures of T cell activity, such as measured using any suitable method known in the art, such as assays described further below in Section III below. In some embodiments, the biological activity of the cells, e.g., T cells administered for the T cell based therapy, is measured by assaying cytotoxic cell killing, expression and/or secretion of one or more cytokines, proliferation or expansion, such as upon restimulation with antigen. In some aspects the biological activity is measured by assessing the disease burden and/or clinical outcome, such as reduction in tumor burden or load. In some aspects the biological activity is measured by assessing the presence of neutropenia in a subject. In some embodiments, administration of one or both agents of the combination therapy and/or any repeated administration of the therapy, can be determined based on the results of the assays before, during, during the course of or after administration of one or both agents of the combination therapy.
[0211] In some embodiments, the combined effect of the BCL2 inhibitor in combination with the cell therapy can be synergistic compared to treatments involving only the inhibitor or monotherapy with the cell therapy. In some embodiments, the combined effect of a subtherapeutically effective amount of the BCL2 inhibitor in combination with the cell therapy can be synergistic compared to treatments involving only the inhibitor or monotherapy with the cell therapy. In some embodiments, the combined effect of a subtherapeutically effective amount of the BC12 inhibitor in combination with a subtherapeutically effective amount of the cell therapy can be synergistic compared to treatments involving only the inhibitor or monotherapy with the cell therapy. For example, in some embodiments, the provided methods, compositions and articles of manufacture herein result in an increase or an improvement in a desired therapeutic effect, such as an increased or an improvement in the reduction or inhibition of one or more symptoms associated with cancer.
[0212] In some embodiments, the BCL2 inhibitor increases the expansion, proliferation, or cytotoxicity of the engineered T cells, such as CAR T cells. In some embodiments, the increase in expansion, proliferation, or cytotoxicity is observed in vivo following administration of the BCL2 inhibitor to a subject. In some embodiments, the increase in the number of engineered T cells, e.g. CAR- T cells, is increased by greater than or greater than about 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0-fold, 9.0-fold, 10.0 fold or more. In some embodiments, the increase in the cytotoxicity of the engineered T cells, e.g. CAR-T cells, against cancer cells is increased by greater than or greater than about 1.2-fold, 1.5-fold, 2.0-fold, 3.0-fold, 4.0-fold, 5.0-fold, 6.0-fold, 7.0-fold, 8.0- fold, 9.0-fold, 10.0 fold or more.
A. ADMINISTRATION OF AN INHIBITOR OF BCL2 PROTEIN
[0213] The provided combination therapy methods, combinations, kits and uses involve administration of an inhibitor of BCL2 protein (a BCL2 inhibitor, e.g. venetoclax), which can be administered subsequently to administration of the immunotherapy or cell therapy, e.g., CAR T cells. In some embodiments, the BCL2 protein that has the ability to promote survival and/or to mitigate pro- apoptotic signaling. In some embodiments, the BCL2 protein is anti-apoptotic. In some embodiments, the BCL2 protein has the ability to promote the survival of cancer cells. In some embodiments, the BCL2 protein has the ability to dampen pro-apoptotic signaling of cancer cells.
[0214] In some embodiments, the BCL2 inhibitor in the combination therapy is an inhibitor of a BCL2 (e.g. venetoclax), which, in some cases, are involved in the regulation and promulgation of anti- apoptotic (prosurvival) signaling in a cell, such as via a cell’s intrinsic apoptotic pathway. In some cases, prosurvival BCL2 family proteins (e.g. BCL2) are involved in apoptotic signaling, including antiapoptotic (prosurvival) signaling via a cell’ s intrinsic apoptosis pathway, via mitochondria. In some cases, prosurvival BCL2 family proteins (e.g. BCL2) are involved in antiapoptotic (prosurvival) signaling via granzyme and/or perforin-mediated apoptosis in the mitochondria. In some embodiments, the BCL2 inhibitor (e.g. venetoclax) also inhibits one or more other prosurvival proteins of the BCL2 family, including B-cell lymphoma extra-large (BCLXL), BCL2 related protein Al (BFL1), BCL2-like protein 2 (BCLW), BCL2-like protein 10 (BCLB), and induced myeloid leukemia cell differentiation protein (MCL1).
[0215] In some embodiments, the BCL2 inhibitor (e.g. venetoclax) interacts with a BCL2 homology (BH) domain. In some embodiments, the BCL2 inhibitor is a mimetic of BH3. In some embodiments, the BCL2 inhibitor is a BH3 mimetic that occupies a BH3-binding domain. In some embodiments, the BCL2 inhibitor is a BH3 mimetic that occupies a BH3 binding domain and/or displaces pro-apoptotic BH3-only proteins from BCL2. In some embodiments, the BCL2 inhibitor blocks or reduces the interaction between BCL2 and proteins having a BH3 domain. In some embodiments, the BCL2 inhibitor occupies a BH3 binding domain to block or reduce heterodimerization of a prosurvival BCL2 family protein, such as BCL2 or BCLXL, with a pro-apoptotic BCL2 family protein, such as BAD, BAX, or BAK. In some embodiments, the BCL2 inhibitor reduces or blocks the phosphorylation of BCL2.
[0216] In some embodiments, the BCL2 inhibitor (e.g. venetoclax) reduces prosurvival (antiapoptotic) signaling. In some cases, the reduction of prosurvival signaling lowers the apoptotic threshold of a cell. In some cases, the apoptosis is achieved by the cell’s intrinsic, mitochondrial- mediated apoptosis pathway. In some cases, the reduction of prosurvival signaling and/or lowering of the apoptotic threshold sensitizes a cell to cell death and/ or increases cell death. In some cases, the reduction of prosurvival signaling and/or lowering of the apoptotic threshold sensitizes a cell to cell death by one or more other agents and/ or increases cell death by one or more other agents. In some cases, the reduction of prosurvival signaling and/or lowering of the apoptotic threshold is achieved by inducing BAX and/or BAK-dependent apoptosis. In some cases, the reduction of prosurvival signaling and/or lowering of the apoptotic threshold sensitizes a cell to cell death by a cytotoxic therapy, such as an immunotherapy or cell therapy (e.g. CAR-expressing T cell therapy). In some cases, the reduction of prosurvival signaling and/or lowering of the apoptotic threshold sensitizes a cell to cell death pathways targeted CAR T cells, including perforin and granzyme-mediated pathways (Benmabarek et al. (2019) Inti. J. Mol. Sci. (20): 1283).
[0217] In some embodiments, the BCL2 inhibitor (e.g. venetoclax) is a selective BCL2 inhibitor. In some embodiments, a selective BCL2 inhibitor is a compound or agent, such as an inhibitor of a prosurvival BCL2 family protein, that is capable of being provided at a dosing regimen (e.g. dose and/or duration) that reduces or blocks BCL2 activity and/or signaling to a greater extent than that of other prosurvival BCL2 family proteins (e.g. BCLXL, BCLW, BCLB, MCL1). In some cases, a selective BCL2 inhibitor reduces or blocks the activity of BCL2 signaling and/or activity when provided at a dosing regimen, but does not reduce or block the signaling and/or activity of other prosurvival BCL2 family proteins when provided at the same dosing regimen. In some cases, a selective BCL2 inhibitors exert minimal or no effects on the activity and/or signaling of other prosurvival BCL2 family proteins, when provided at a dosing regimen.
[0218] In some embodiments, the BCL2 inhibitor is a nonselective BCL2 inhibitor. In some embodiments, a nonselective BCL2 inhibitor is a compound or agent, such as an inhibitor of a prosurvival BCL2 family protein, that reduces or blocks the activity of more than one prosurvival BCL2 family protein. In some cases, a nonselective BCL2 inhibitor is a compound or agent, such as an inhibitor of a prosurvival BCL2 family protein, that is capable of being provided at a dosing regimen (e.g. dose and/or duration) that reduces or blocks the activity and/or signaling of a prosurvival BCL2 family protein, e.g. BCL2 and additionally reduces or blocks the activity and/or signaling of one or more other prosurvival BCL2 family proteins (e.g. BCLXL, BCLW, BCLB, MCL1). In some cases, a nonselective BCL2 inhibitor reduces or blocks the activity and/or signaling of a prosurvival BCL2 family protein (e.g. BCL2) when provided at a dosing regimen, and also reduces or blocks the signaling and/or activity of one or more other prosurvival BCL2 family proteins (e.g. BCLXL, BCLW, BCLB, MCL1) when provided at the same dosing regimen.
[0219] In some embodiments, the inhibitor inhibits BCL2 with a half-maximal inhibitory concentration (IC50) of less than or less than about 1000 nM, less than or less than about 900 nM, less than or less than about 800 nM, less than or less than about 700 nM, less than or less than about 600 nM, less than or less than about 500 nM, less than or less than about 400 nM, less than or less than about 300 nM, less than or less than about 200 nM, less than or less than about 100 nM, less than or less than about 90 nM, less than or less than about 80 nM, less than or less than about 70 nM, less than or less than about 60 nM, less than or less than about 50 nM, less than or less than about 40 nM, less than or less than about 30 nM, less than or less than about 20 nM, less than or less than about 10 nM, less than or less than about 9 nM, less than or less than about 8 nM, less than or less than about 7 nM, less than or less than about 6 nM, less than or less than about 5 nM, less than or less than about 4 nM, less than or less than about 3 nM, less than or less than about 2 nM, less than or less than about 1 nM, less than or less than about 0.9 nM, less than or less than about 0.8 nM, less than or less than about 0.7 nM, less than or less than about 0.6 nM, less than or less than about 0.5 nM, less than or less than about 0.4 nM, less than or less than about 0.3 nM, less than or less than about 0.2 nM, less than or less than about 0.1 nM, or less than or less than about 0.01 nM. In some embodiments, the inhibitor inhibits one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1 with a half-maximal inhibitory concentration (IC50) of less than or less than about 1000 nM, less than or less than about 900 nM, less than or less than about 800 nM, less than or less than about 700 nM, less than or less than about 600 nM, less than or less than about 500 nM, less than or less than about 400 nM, less than or less than about 300 nM, less than or less than about 200 nM, less than or less than about 100 nM, less than or less than about 90 nM, less than or less than about 80 nM, less than or less than about 70 nM, less than or less than about 60 nM, less than or less than about 50 nM, less than or less than about 40 nM, less than or less than about 30 nM, less than or less than about 20 nM, less than or less than about 10 nM, less than or less than about 9 nM, less than or less than about 8 nM, less than or less than about 7 nM, less than or less than about 6 nM, less than or less than about 5 nM, less than or less than about 4 nM, less than or less than about 3 nM, less than or less than about 2 nM, less than or less than about 1 nM, less than or less than about 0.9 nM, less than or less than about 0.8 nM, less than or less than about 0.7 nM, less than or less than about 0.6 nM, less than or less than about 0.5 nM, less than or less than about 0.4 nM, less than or less than about 0.3 nM, less than or less than about 0.2 nM, less than or less than about 0.1 nM, or less than or less than about 0.01 nM.
[0220] In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is lower than the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is at least 10 times lower, at least 100 times lower, at least 1,000 times lower, at least 5,000 times lower, at least 10,000 times lower, or at least 20,000 times lower than the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is at least 1 ,000 times lower than the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is at least 5,000 times lower than the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is at least 10,000 times lower than the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is at least 20,000 times lower than the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCLL In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is at least 1,000 times lower than the inhibition constant (Ki) of the inhibitor for BCLXL. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is at least 4,000 times lower than the inhibition constant (Ki) of the inhibitor for BCLXL. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is at least 20,000 times lower than the inhibition constant (Ki) of the inhibitor for BCLW.
[0221] In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is less than about 10 pM. In In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is less than about 1 pM. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is less than about 0.1 pM. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is less than about 10 nM. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is less than about 1.0 nM. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is less than about 0.1 nM. In some embodiments, the inhibition constant (Ki) of the inhibitor for BCL2 is less than about 0.01 nM. In some embodiments, the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1 is less than about 10 pM. In In some embodiments, the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1 is less than about 1 pM. In some embodiments, the inhibition constant (Ki) of the inhibitor one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1 is less than about 0.1 pM. In some embodiments, the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1 is less than about 10 nM. In some embodiments, the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1 is less than about 1.0 nM. In some embodiments, the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1 is less than about 0.1 nM. In some embodiments, the inhibition constant (Ki) of the inhibitor for one or more other prosurvival BCL2 family proteins, such as BCLXL, BCLW, BCLB, and/or MCL1 is less than about 0.01 nM.
[0222] In some embodiments, the IC50, Kd and/or Ki is measured or determined using an in vitro assay. Assays to assess or quantitate or measure activity of protein tyrosine kinase inhibitors as described are known in the art. Such assays can be conducted in vitro and include assays to assess the ability of an agent to inhibit a specific biological or biochemical function. In some embodiments. In some embodiments, kinase activity studies can be performed. Protein tyrosine kinases catalyze the transfer of the terminal phosphate group from adenosine triphosphate (ATP) to the hydroxyl group of a tyrosine residue of the kinase itself or another protein substrate. In some embodiments, kinase activity can be measured by incubating the kinase with the substrate (e.g., inhibitor) in the presence of ATP. In some embodiments, measurement of the phosphorylated substrate by a specific kinase can be assessed by several reporter systems including colorimetric, radioactive, and fluorometric detection. (Johnson, S.A. & T. Hunter (2005) Nat. Methods 2:17.) In some embodiments, inhibitors can be assessed for their affinity for a particular kinase or kinases, such as by using competition ligand binding assays (Ma et al., Expert Opin Drug Discov. 2008 Jun; 3(6):607-621) From these assays, the half-maximal inhibitory concentration (IC50) can be calculated. IC50 is the concentration that reduces a biological or biochemical response or function by 50% of its maximum. In some cases, such as in kinase activity studies, IC50 is the concentration of the compound that is required to inhibit the target kinase activity by 50%. In some cases, the dissociation constant (Kd) and/or the inhibition constant (Ki values) can be determined additionally or alternatively. IC50 and Kd can be calculated by any number of means known in the art. The inhibition constant (Ki values) can be calculated from the IC50 and Kd values according to the Cheng-Prusoff equation: Ki = ICso/(l+L/Kd), where L is the concentration of the inhibitor (Biochem Pharmacol 22: 3099-3108, 1973). Ki is the concentration of unlabeled inhibitor that would cause occupancy of 50 % of the binding sites present in the absence of ligand or other competitors.
[0223] In some embodiments, the inhibitor is a small molecule.
[0224] In some embodiments, the inhibitor is an inhibitor of a prosurvival BCL2 family protein, including but not limited to those described in US Patent No. 9,174,982, US Patent No. 8,546,399, US Patent No. 7,030,115, US Patent No. 7,390,799, US Patent No. 7,709,467, US Patent No. 8,624,027, US Patent No. 7,906,505, US Patent No. 6,720,338, published PCT application WO 13/096060, published PCT application WO 02/097053, published US application US 2016/0220573, US Patent No. 7,354,928, published US application 2015/0056186, and published PCT application WO 05/049594, which are each incorporated by reference in their entireties.
[0225] In some embodiments, the inhibitor inhibits BCL2, BCLXL, and BCLW, such as navitoclax. In some embodiments, the inhibitor inhibits BCL2, such as venetoclax.
[0226] In some embodiments, the inhibitor inhibits or reduces the activity of BCL2, BCLXL, and
BCLW, such as navitoclax. In some cases, the inhibitor is navitoclax. In some cases, the inhibitor has the
Figure imgf000058_0001
structure , or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof, including and compositions thereof, for the treatment of subjects with cancer.
[0227] In some embodiments, the inhibitor inhibits or reduces the activity of BCL2, such as venetoclax. In some cases, the inhibitor is venetoclax. In some cases, the inhibitor has the structure
Figure imgf000058_0002
, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer or racemic mixtures thereof, including and compositions thereof.
[0228] Exemplary prosurvival BCL2 family protein inhibitors include, but are not limited to venetoclax (ABT-199), navitoclax (ABT-263), ABT-737, AT-101/GDC-0199 (Gossypol), apogossypol, TW-37, G3139 (Genasense), GX15-070 (obatoclax), sabutoclax, HA14-1, antimycin A, BH3I-1, YC137, maritoclax (marinopyyrole A), clitocine, UMI-77, WEHI-539, and 544563.
J. COMPOS/F/ONS AND FORMULA F/ONS
[0229] In some embodiments of the combination therapy methods, combinations, kits and uses provided herein, the combination therapy can be administered in one or more compositions, e.g., a pharmaceutical composition containing a BCL2 inhibitor (e.g. venetoclax), and/or the immunotherapy or cell therapy, e.g., T cell therapy. [0230] In some embodiments, the composition, e.g., a pharmaceutical composition containing a BCL2 inhibitor, e.g., venetoclax, can include carriers such as a diluent, adjuvant, excipient, or vehicle with which a BCL2 inhibitor, e.g., venetoclax, and/or the cells are administered. Examples of suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of a BCL2 inhibitor, e.g., venetoclax, generally in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Saline solutions and aqueous dextrose and glycerol solutions also can be employed as liquid carriers, particularly for injectable solutions. The pharmaceutical compositions can contain any one or more of a diluents(s), adjuvant(s), antiadherent(s), binder(s), coating(s), filler(s), flavor(s), color(s), lubricant(s), glidant(s), preservative(s), detergent(s), sorbent(s), emulsifying agent(s), pharmaceutical excipient(s), pH buffering agent(s), or sweetener(s) and a combination thereof. In some embodiments, the pharmaceutical composition can be liquid, solid, a lyophilized powder, in gel form, and/or combination thereof. In some aspects, the choice of carrier is determined in part by the particular inhibitor and/or by the method of administration.
[0231] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG), stabilizers and/or preservatives. The compositions containing a pa BCL2 inhibitor, e.g., venetoclax, can also be lyophilized.
[0232] In some embodiments, the pharmaceutical compositions can be formulated for administration by any route known to those of skill in the art including intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, subcutaneous, intratumoral, epidural, nasal, oral, vaginal, rectal, topical, local, otic, inhalational, buccal e.g., sublingual), and transdermal administration or any route. In some embodiments, other modes of administration also are contemplated. In some embodiments, the administration is by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon’s injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, administration is by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.
[0233] In some embodiments, compositions also can be administered with other biologically active agents, either sequentially, intermittently or in the same composition. In some embodiments, administration also can include controlled release systems including controlled release formulations and device controlled release, such as by means of a pump. In some embodiments, the administration is oral.
[0234] In some embodiments, a BCL2 inhibitor, e.g., venetoclax, is typically formulated and administered in unit dosage forms or multiple dosage forms. Each unit dose contains a predetermined quantity of a therapeutically active BCL2 inhibitor, e.g., venetoclax, sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. In some embodiments, unit dosage forms, include, but are not limited to, tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil water emulsions containing suitable quantities of a BCL2 inhibitor, e.g., venetoclax. Unit dose forms can be contained ampoules and syringes or individually packaged tablets or capsules. Unit dose forms can be administered in fractions or multiples thereof. In some embodiments, a multiple dose form is a plurality of identical unit dosage forms packaged in a single container to be administered in segregated unit dose form. Examples of multiple dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons.
2. DOSING
[0235| In some embodiments, the provided combination therapy method involves administering to the subject a a BCL2 inhibitor, e.g., venetoclax, and an immunotherapy or cell therapy (e.g. CAR- expressing T cells). In some embodiments, the provided combination therapy methods involve initiating administration of a BCL2 inhibitor, e.g., venetoclax, subsequent to the initiation of the immunotherapy or cell therapy, such as a T cell therapy e.g., CAR-expressing T cells).
[0236] In some embodiments, the provided combination therapy methods involve initiating administration of a BCL2 inhibitor, e.g., venetoclax within about 2 days after initiation of administration of the immunotherapy or cell therapy, such as a T cell therapy (e.g., CAR-expressing T cells). In some embodiments, the method involves initiating administration of the BCL2 inhibitor, e.g., venetoclax, within about 1 day after initiation of administration of the immunotherapy or cell therapy (e.g. CAR T cell therapy). In some embodiments, the method involves initiating administration of the BCL2 inhibitor, e.g., venetoclax, at about 2 days after initiation of administration of the immunotherapy or cell therapy (e.g. CAR T cell therapy). In some embodiments, the method involves initiating administration of the BCL2 inhibitor, e.g.,venetoclax, at about 1 day after initiation of administration of the immunotherapy or cell therapy (e.g. CAR T cell therapy). In some embodiments, the method comprises initiating administration of the BCL2 inhibitor, e.g.,venetoclax, at about 1 day after initiation of administration of the cell therapy (e.g. CAR T cell therapy). In some embodiments, the provided methods include initiation of administration of the BCL2 inhibitor (e.g. venetoclax) after activation-induced cell death (AICD) of the cells of the immunotherapy or cell therapy (e.g. CAR T cells) has peaked. In some embodiments, the metof comprises initiating administration of the BCL2 inhibitor (e.g. venetoclax) after activation-induced cell death (AICD) of the cells of the cell therapy (e.g. CAR T cells) has peaked.
[0237] In some embodiments, the provided combination therapy comprises (1) administering to a subject have a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds CD19 on Day 1; and (2) administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising administration of the BCL2 inhibitor within about 2 days (e.g. about 1 day) after initiation of administration of the cell therapy. In some embodiments, the provided combination therapy comprises (1) administering to a subject have a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds CD 19 on Day 1; and (2) administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising administration of the BCL2 inhibitor on Day 2.
[0238] In some embodiments, the provided combination therapy comprises administering to a subject have a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor in a dosing regimen comprising administration of the BCL2 inhibitor (e.g. ventoclax) at or about 1 day after initiation of administration of a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds CD 19 on Day 1.
|0239] In some embodiments, the provided combination therapy comprises administering to a subject have a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds CD19 on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen comprising initiation of the BCL2 inhibitor (e.g. venetoclax) at or about 1 day after initiation of administration of the cell therapy.
[0240] In some embodiments, the method includes administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising (i) daily administration of a first dose for a first predetermined period and (ii) daily administration of at least one subsequent dose, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor. In some embodiments, each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor (e.g. venetoclax) compared to the preceding dose. In some embodiments, the at least one subsequent dose comprises a second dose, a third dose, a fourth dose, a fifth dose, a sixth dose, a seventh dose, an eighth dose, a ninth dose, and/or a tenth dose. In some embodiments, the at least one subsequent dose comprises a second dose. In some embodiments, the at least one subsequent dose comprises a second dose and a third dose. In some embodiments, the at least one subsequent dose comprises a second, third, and fourth dose. In some embodiments, the at least one subsequent dose comprises a second, third, fourth, and fifth dose.
[0241] In some embodiments, initiation of administration of the BCL2 inhibitor (e.g. venetoclax) begins on about Day 2. In some embodiments, the BCL2 inhibitor (e.g. venetoclax) is administered at no more than about 100 mg per day through Day 30.
[0242] In some embodiments, the at least one subsequent dose is or comprises a second dose. In some embodiments, the method includes administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising (i) daily administration of a first dose for a first predetermined period and (ii) daily administration of a second dose for a second predetermined period beginning after the first predetermined period, wherein the second dose is an increased amount of the BCL2 inhibitor compared to the first dose, and the BCL2 inhibitor is administered at no more than about 100 mg per day through Day 30.
[0243] In some embodiments, the at least one subsequent dose is or comprises a second dose and a third dose. In some embodiments, the method includes administering to the subject a BCL2 inhibitor (e.g. venetoclax) in a dosing regimen comprising (i) daily administration of a first dose for a first predetermined period; (ii) daily administration of a second dose for a second predertmined period beginning after the first predetermined period; and (iii) daily administration of a third dose for a third predetermined period beginning after the second predetermined period, wherein the second dose is an increased amount of the BCL2 inhibitor compared to the first dose, the third dose is an increased amount of the BCL2 inhibitor compared ot the first dose, and and the BCL2 inhibitor is administered at no more than about 100 mg per day through Day 30.
[0244] In some embodiments, the administration of the BCL2 inhibitor, e.g., venetoclax, is continued and/or further administered over a period of time, e.g., until a determined time point or until a particular outcome is achieved.
[0245] In some cases, the combination therapy involves administration of the inhibitor (e.g. venetoclax) up to or about 3 months after initiation of administration of the immunotherapy or cell therapy, e.g.CAR T cells. In some cases, the combination therapy involves administration of the BCL2 inhibitor (e.g. venetoclax) for at least about 3 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells. In some cases, the combination therapy involves administration of the BCL2 inhibitor (e.g. venetoclax) for at least about 6 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells. In some cases, the combination therapy involves administration of the BCL2 inhibitor (e.g. venetoclax) for at least about 12 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells. In some cases, the combination therapy involves administration of the BCL2 inhibitor (e.g. venetoclax) for at least about 18 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells. In some cases, the combination therapy involves administration of the BCL2 inhibitor (e.g. venetoclax) for at least about 24 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells.
[0246] In some cases, administration of the BCL2 inhibitor is discontinued about 3 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells, if the subject exhibits a desired response (e.g complete response). In some cases, administration of the BCL2 inhibitor is discontinued about 3 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells. In some cases, administration of the BCL2 inhibitor is discontinued about after Day 90. In some cases, administration of the BCL2 inhibitor is discontinued about 6 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells. In some cases, administration of the BCL2 inhibitor is discontinued about 12 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells. In some cases, administration of the BCL2 inhibitor is discontinued about 24 months after initiation of administration of the immunotherapy or cell therapy, e.g. CAR T cells.
[0247] In some cases, the outcome is a desired therapeutic response, as described below and in Section III. In some embodiments, the dose, frequency, duration, timing and/or order of administration of the BCL2 inhibitor, e.g., venetoclax, is determined, based on particular thresholds or criteria of results of the screening step and/or assessment of treatment outcomes described herein, such as in Section III.
[0248| In some embodiments, if a subject exhibits minimum residual disease (MRD; greater than or equal to 104 in peripheral blood) at Day 90, the subject may continue treatment with the BCL2 inhibitor, e.g. venetoclax per standard of care, such as for a total treatment duration of 12 or 24 months. In some embodiments, if a subject does not exhibit complete response (CR) at Day 90, the subject may continue treatment with the BCL2 inhibitor, e.g. venetoclax per standard of care, such as for a total treatment duration of 12 or 24 months.
[0249] Desired therapeutic results, such as for the treatment of cancer (e.g. a CLL or SLL), include but are not limited to a reduction of tumor size, bulk, metastasis, percentage of blasts in the bone marrow or molecularly detectable B cell malignancy and/or an improvement in prognosis or survival or other symptom associated with tumor burden. Desired therapeutic results, such as for the treatment of cancer (e.g. CLL or SLL), may additionally include an ability of the inhibitor to induce apoptosis in cancer cells and/or an increase in apoptosis of cancer cells. Methods for identifying and determining whether a desired therapeutic result for the treatment of cancer is achieved include but are not limited to any as described in Section III. [0250] In some cases, a therapeutically effective amount results in neutropenia, such as mild, moderate, or severe neutropenia. In some cases, mild neutropenia is defined as an absolute neutrophil count of 1,000 to l,5000/|lL. In some cases, moderate neutropenia is defined as an absolute neutrophil count of 500 to 1,000/ .L. In some cases, severe neutropenia is defined as an absolute neutrophil count of fewer than 500/|lL. Methods for identifying and determining whether a desired therapeutic result for the treatment of cancer is achieved include but are not limited to any as described in Section III. In some cases, the inhibitor is administered at a subtherapeutically effective amount. In some cases, in the context of administration, a subtherapeutically effective amount refers to an amount less effective, at dosages and/or for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment, as compared to a “therapeutically effective amount” of the same agent. In some cases, the subtherapeutically effective amount does not achieve or achieves less than a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment. In some cases the desired therapeutic result is a reduction of tumor size, bulk, metastasis, percentage of blasts in the bone marrow or molecularly detectable B cell malignancy and/or an improvement in prognosis or survival or other symptom associated with tumor burden. In some cases the desired therapeutic result is an ability of the inhibitor to induce apoptosis in cancer cells and/or an increase in apoptosis of cancer cells. In some cases, a subtherapeutically effective amount results in mild or moderate neutropenia In some cases, a subtherapeutically effective amount does not result in severe neutropenia. In some cases, mild neutropenia is defined as an absolute neutrophil count of 1,000 to l,5000/|lL. In some cases, moderate neutropenia is defined as an absolute neutrophil count of 500 to 1,000/ .L. In some cases, severe neutropenia is defined as an absolute neutrophil count of fewer than 500/|lL.
[0251] Methods for identifying and determining whether a desired therapeutic result for the treatment of cancer is achieved include but are not limited to any as described in Section III. The subtherapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered.
[0252] In some embodiments, the provided methods involve administering the cells and/or compositions at subeffective amounts, e.g., subtherapeutically effective amounts. In some embodiments, the provided methods involve administering a BCL2 inhibitor, e.g., venetoclax, engineered cells e.g. cell therapy), or compositions at subeffective amounts, e.g., subtherapeutically effective amounts. In some cases, a BCL2 inhibitor, e.g. venetoclax, is administered at a subtherapeutic amount, i.e., the dosing regimen (e.g dose and/or duration) is less than dosing regimen commonly prescribed by clinicians to achieve a therapeutic or prophylactic result. A. EIBSTDOSE
[0253] In some of any of the provided embodiments, the BCL2 inhibitor dosing regimen comprises (i) daily administration of a first dose for a first predetermined period and (ii) daily administration of a second dose for a second predetermined period beginning after the first predetermined period, wherein the second dose is an increased amount of the BCL2 inhibitor compared to the first dose.
[0254] In some embodiments, the first predetermined period is from about Day 2 to about Day 7. In some embodiments, the first predetermined period is from about Day 2 to about Day 30.
[0255] In some embodiments, the first dose is between about 20 mg and about 200 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 20 mg, about 50 mg, about 100 mg, or about 200 mg of the BCL2 inhibitor. In some embodiments, the first dose is between about 20 mg and about 100 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 20 mg, about 50 mg, or about 100 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 20 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the first dose is no more than or no more than about 100 mg of the BCL2 inhibitor. In some embodiments, the first dose is about 200 mg of the BCL2 inhibitor.
[0256] In some embodiments, the first predetermined period is from about Day 2 to about Day 7, and the first dose is about 20 mg of the BCL2 inhibitor. In some embodiments, the first predetermined period is from about Day 2 to about Day 7, and the first dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the first predetermined period is from about Day 2 to about Day 30, and the first dose is about 20 mg of the BCL2 inhibitor. In some embodiments, the first predetermined period is from about Day 2 to about Day 30, and the first dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the first predetermined period is from about Day 2 to about Day 30, and the first dose is about 100 mg of the BCL2 inhibitor.
B. SUBSEQUENT DOSE(S)
[0257] In some of any of the provided embodiments, the BCL2 inhibitor dosing regimen comprises (i) daily administration of a first dose of the BCL2 inhibitor for a first predetermined period and (ii) daily administration of at least one subsequent dose of the BCL2 inhibitor for a predetermined period of time beginning after a preceding predetermined period of time, wherein each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose. In some embodiments, the at least one subsequent dose comprises a second dose, a third dose, a fourth dose, a fifth dose, a sixth dose, a seventh dose, an eighth dose, a ninth dose, and/or a tenth dose. In some embodiments, the at least one subsequent dose is or comprises a second dose. In some embodiments, the at least one subsequent dose is a second dose. In some embodiments, the at least one subsequent dose is or comprises a second dose and a third dose. In some embodiments, the at least one subsequent dose is a second dose and a third dose. In some embodiments, the at least one subsequent dose is or comprises a second, third, and fourth dose. In some embodiments, the at least one subsequent dose is a second, third, and fourth dose. In some embodiments, the at least one subsequent dose is or comprises a second, third, fourth, and fifth dose. In some embodiments, the at least one subsequent dose is a second, third, fourth, and fifth dose.
[0258] In some embodiments, the at least one subsequent dose is or comprises a second dose. In some of any of the provided embodiments, the BCL2 inhibitor dosing regimen comprises (i) daily administration of a first dose of the BCL2 inhibitor for a first predetermined period; and (ii) daily administration of a second dose of the BCL2 inhibitor for a second predetermined period of time beginning after the first predetermined period of time, wherein the second dose is an increased amount of the BCL2 inhibitor compared to the first dose.
[0259] In some embodiments, the second predetermined period is from about Day 8 to about Day 30. In some embodiments, the second predetermined period is from about Day 31 to about Day 37. In some embodiments, the second predetermined period is from about Day 31 to about Day 90.
[0260] In some embodiments, the second dose is between about 50 mg and about 200 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 50 mg, about 100, about 200 mg, or about 400 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 200 mg of the BCL2 inhibitor. In some embodiments, the second dose is about 400 mg of the BCL2 inhibitor.
[0261 | In some embodiments, the second predetermined period is from about Day 8 to about Day 30, and the second dose is about 50 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 8 to about about Day 30, and the second dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 to about Day 37, and the second dose is about 200 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 to about Day 90, and the second dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the second predetermined period is from about Day 31 and Day 90, and the second dose is about 200 mg of the BCL2 inhibitor.
[0262] In some embodiments, the dosing regimen comprises daily administration of about 20 mg the BCL2 inhibitor to the subject from about Day 2 to about Day 7; and daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 8 to about Day 30. In some embodiments, the dosing regimen comprises daily administration of about 50 mg per day of the BCL2 inhibitor to the subject from about Day 2 to about Day 7; and daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 8 to about Day 30. In some embodiments, the dosing regimen comprises daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 30; and daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 90. In some embodiments, the dosing regimen comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject between about Day 2 and about Day 30; and daily administration of about 200 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 90. In some embodiments, the dosing regimen comprises daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 30; and daily administration of about 200 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 37.
[0263] In some embodiments, the at least one subsequent dose is or comprises a second dose and a third dose. In some of any of the provided embodiments, the BCL2 inhibitor dosing regimen comprises (i) daily administration of a first dose of the BCL2 inhibitor for a first predetermined period; (ii) daily administration of a second dose of the BCL2 inhibitor for a second predetermined period of time beginning after the first predetermined period of time; and (iii) daily administration fo a third dose of the BCL2 inhibitor for a third predetermined period of time beginning after the second predetermined period of time, wherein the second dose is an increased amount of the BCL2 inhibitor compared to the first dose and the third dose is an increased amount of the BCL2 inhibitor compared to the second dose.
[0264] In some embodiments, the third predetermined period is from about Day 31 to about Day 90. In some embodiments, the third predetermined period is from about Day 38 to about Day 90.
[0265] In some embodiments, the third dose is between about 100 mg and about 400 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 100 mg, about 200 mg, or about 400 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 200 mg of the BCL2 inhibitor. In some embodiments, the third dose is about 400 mg of the BCL2 inhibitor.
[0266] In some embodiments, the third predetermined period is from about Day 31 to about Day 90, and the third dose is about 100 mg of the BCL2 inhibitor. In some embodiments, the third predetermined period is from about Day 31 to about Day 90, and the third dose is about 200 mg of the BCL2 inhibitor. In some embodiments, the third predetermined period is from about Day 38 to about Day 90, and the third dose is about 400 mg of the BCL2 inhibitor.
[0267] In some embodiments, the dosing regimen comprises (i) daily administration of about 20 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 7; (ii) daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 8 to about Day 30; and (iii) daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 90. In some embodiments, the dosing regimen comprises (i) daily administration of about 50 mg of the BCL2 inhibitor to the subject from about Day 2 to about Day 7; (ii) daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 8 to about Day 30; and (iii) daily administration of about 200 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 90. In some embodiments, the dosing regimen comprises (i) daily administration of about 100 mg of the BCL2 inhibitor to the subject from about Day 2 and about Day 30; (ii) daily administration of about 200 mg of the BCL2 inhibitor to the subject from about Day 31 to about Day 37; and (iii) daily administration of about 400 mg of the BCL2 inhibitor to the subject from about Day 38 to about Day 90.
C. DGSING REGIMENS
[0268] In some embodiments, a subject is dosed in a dosing cohort (i.e. a dosing regimen). In some embodiments, a subject dosed in a dosing cohort (i.e. a dosing regimen) is administered the BCL2 inhibitor in a dosing regimen comprising (i) daily administration of a first dose for a first predetermined period; and (ii) daily administration of at least one subsequent dose for a predetermined period of time beginning after a predetermined period of a preceding dose, wherein each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose.
[0269] In some embodiments, a subject is dosed in a dosing cohort (i.e. a dosing regimen), wherein the at least one subsequent dose is or comprises a first dose and a second dose. In some embodiments, the BCL2 inhibitor dosing regimen comprises (i) daily administration of a first dose for a first predetermined period; and (ii) daily administration of a second dose for a second predetermined period following the first predetermined period, wherein the first dose is about 50 mg of the BCL2 inhibitor and the first predetermined period is from about Day 2 to about Day 30; and the second dose is about 100 mg of the BCL2 inhibitor and the second predetermined period is from about Day 31 to about Day 90. Thus, in some embodiments, a subject dosed in a dosing cohort (i.e. a dosing regimen) is administered about 50 mg of the BCL2 inhibitor per day from about Day 2 to about Day 30, and about 100 mg of the BCL2 inhibitor per day from about Day 31 to about Day 90.
[0270] In some embodiments, the BCL2 inhibitor dosing regimen comprises administration of a first dose and a second dose, wherein the first dose is about 100 mg of the BCL2 inhibitor and the first predetermined period is from about Day 2 to about Day 30; and the second dose is about 200 mg of the BCL2 inhibitor and the second predetermined period is from about Day 31 to about Day 90. Thus, in some embodiments, a subject dosed in a dosing cohort (i.e. a dosing regimen) is administered about 100 mg of the BCL2 inhibitor per day from about Day 2 to about Day 30, and about 200 mg of the BCL2 inhibitor per day from about Day 31 to about Day 90.
[0271] In some embodiments, a subject is dosed in a dosing cohort (i.e. a dosing regimen), wherein the at least one subsequent dose is or comprises a second dose and a third dose.
[0272] In some embodiments, the dosing regimen comprises (i) daily administration of a first dose for a first predetermined period; (ii) daily administration of a second dose for a second predetermined period; and (iii) daily administration of a third dose, wherein the first dose is about 20 mg of the BCL2 inhibitor and the first predetermined period is from about Day 2 to about Day 7 ; the second dose is about 50 mg of the BCL2 inhibitor and the second predetermined period is from about Day 8 to about Day 30; and the third dose is about 100 mg of the BCL2 inhibitor and the third predetermined period is from about Day 31 to about Day 90. Thus, in some embodiments, a subject dosed in a dosing cohort (i.e. a dosing regimen) is administered about 20 mg of the BCL2 inhibitor per day from about Day 2 to about Day 7, about 50 mg of the BCL2 inhibitor per day from about Day 8 to about Day 30, and about 100 mg of the BCL2 inhibitor per day from about Day 31 to Day 90.
[02731 In some embodiments, the BCL2 inhibitor dosing regimen comprises (i) daily administration of a first dose for a first predetermined period; (ii) daily adminsitere of a second dose for a second predetermined period; and (iii) daily administration of a third dose for a third predetermined period, wherein the first dose is about 50 mg of the BCL2 and the first predetermined period is from about Day 2 to about Day 7; the second dose is about 100 mg and the second predetermined period is from about Day 8 to about Day 30; and the third dose is about 200 mg and the third predetermined period is from about Day 31 to about Day 90. Thus, in some embodiments, a subject dosed in a dosing cohort (i.e. a dosing regimen) is administered about 50 mg of the BCL2 inhibitor per day from about Day 2 to about Day 8, about 100 mg of the BCL2 inhibitor per day from about Day 8 to about Day 30, and about 200 mg of the BCL2 inhibitor per day from about Day 31 to Day 90.
[0274] In some embodiments, the BCL2 inhibitor dosing regimen comprises (i) daily administration of a first dose for a first predetermined period; (ii) daily adminsitere of a second dose for a second predetermined period; and (iii) daily administration of a third dose for a third predetermined period, wherein the first dose is about 100 mg of the BCL2 inhibitor and the first predetermined period is from about Day 2 to about Day 30; the second dose is about 200 mg of the BCL2 inhibitor and the second predetermined period is from about Day 31 to about Day 37 ; and the third dose is about 400 mg of the BCL2 inhibitor and the third predetermined period is from about Day 38 to about Day 90. Thus, in some embodiments, a subject dosed in a dosing cohort (i.e. a dosing regimen) is administered about 100 mg of the BCL2 inhibitor per day from about Day 2 to about Day 30, about 200 mg of the BCL2 inhibitor per day from about Day 31 to about Day 37, about 400 mg of the BCL2 inhibitor per day from about Day 38 to about Day 90.
[0275] In some embodiments, among a plurality of subjects treated, the dosing regimen does not result in a toxicity (e.g. a dose-limiting toxicity) in one or more of the subjects. In some embodiments, if the dosing regimen results in a DLT in one or more of the subjects among a plurality of subjects treated, a different dosing regimen is chosen that does not result in a toxicity (e.g. a DLT) in one or more of the subjects. In some embodiments, if a dosing regimen results in a DLT in one or more of the subjects among a plurality of subjects treated, the dosing regimen may be reduced or de-escalated.
[0276] In some embodiments, the dosing regimen comprises (i) daily administration of a first dose of about 50 mg of the BCL2 inhibitor for a first predetermined period of from about Day 2 to about Day 30, and (ii) daily administration of a second dose of about 100 mg of the BCL2 inhibitor for a second predetermined period from about Day 31 to about Day 90. In some embodiments, the dosing regimen comprises (i) daily administration of a first dose of about 20 mg of the BCL2 inhibitor for a first predetermined period of from Day 2 to about Day 7,(ii) daily administration of a second dose of about 50 mg of the BCL2 inhibitor for a second predetermined period from about Day 8 to about Day 30, and (iii) daily administration of a third dose of about 100 mg of the BCL2 inhibitor for a third predetermined period from about Day 31 to about Day 90.
[0277] In some embodiments, the dosing regimen comprises (i) daily administration of a first dose of about 100 mg of the BCL2 inhibitor for a first predetermined period of from about Day 2 to about Day 30, and (ii) daily administration of a second dose of about 200 mg of the BCL2 inhibitor for a second prefetermined period of from about Day 31 to about Day 90. In some embodiments, the dosing regimen comprises (i) daily administration of a first dose of about 50 mg of the BCL2 inhibitor for a first predetermined period of from about Day 2 to about Day 7, (ii) daily administration of a second dose level of about 100 mg of the BCL2 inhibitor for a second predertermined period of from about Day 8 to about Day 30, and (iii) daily administration of a third dose level of about 200 mg of the BCL2 inhibitor for a third predetermined period of from about Day 31 to about Day 90.
[0278] In some embodiments, among a plurality of subjects treated, the dosing regimen does not result in a toxicity (e.g. a dose-limiting toxicity) in one or more of the subjects. In some embodiments, if the dosing regimen does not result in a DLT in one or more of the subjects among a plurality of subjects treated, the dosing regimen may be increased or escalated.
[0279] In some embodiments, the dosing regimen comprises (i) daily administration of a first dose of about 50 mg of the BCL2 inhibitor for a first predetermined period of from about Day 2 to about Day 30, and (ii) daily administration of a second dose of about 100 mg of the BCL2 inhibitor for a second predetermined period of from about Day 31 to about Day 90. In some embodiments, the dosing regimen comprises (i) daily administration of a first dose of about 100 mg of the BCL2 inhibitor for a first predetermined period of from about Day 2 to about Day 30, and (ii) daily administration of a second dose of about 200 mg of the BCL2 inhibitor for a second predetermined period of from about Day 31 to about Day 90.
[0280] In some embodiments, the dosing regimen comprises (i) daily administration of a first dose of about 100 mg of the BCL2 inhibitor for a first predetermined period from about Day 2 to about Day 30, and (ii) daily administration of a second dose of about 200 mg of the BCL2 inhibitor for a second predetermined period of from about Day 31 to about Day 90. In some embodiments, the dosing regimen comprises (i) daily administration fo a first dose of about 100 mg of the BCL2 inhibitor for a first predetermined period from about Day 2 to about Day 30, (ii) daily administration of a second dose level of about 200 mg of the BCL2 inhibitor for a second predetermined period of from about Day 31 to about Day 37, and (iii) daily administration of a third dose level of about 400 mg of the BCL2 inhibitor for a third predetermined period of from about Day 38 to Day 90.
[0281] In some embodiments, toxicity is determined by the pharmacokinetics (PK) of the cells of the cell therapy (eg. CAR T cells) and/or response, as determined by standard critera including any of those described in Section III. A and/or assessment of minimum residual disease (MRD). [0282] In some embodiments, if a subject dosed in a dosing cohort (i.e. a dosing regimen) does not exhibit toxicity (e.g. a DLT), the PK of the cells of the cell therapy (e.g. CAR T cells) administered to the subject is assessed. In some embodiments, if the PK of the cells of the cell therapy (e.g. CAR T cells) administered to the subject shows that the cells did not persist and/or expand at satisfactory levels, response of the subject is also assessed. In some embodiments, if the cells of the cell therapy did not persist and/or expand at satisfactory levels, response of the subject is also assessed.
[0283] In some cases, if the PK of the cells of the cell therapy (e.g. CAR T cells) administered to the subject shows that the cells did not persist and/or expand at satisfactory levels, and the subject demonstrates a response to the combination treatment, the dosing regimen is not de-escalated. In some cases, if the cells of the cell therapy did not persist and/or expand at satisfactory levels, and the subject demonstrates a response to the combination treatment, the dosing regimen is not de-escalated. In some cases, if the PK of the cells of the cell therapy (e.g. CAR T cells) administered to the subject shows that the cells did not persist and/or expand at satisfactory levels, and the subject demonstrates a response to the combination treatment, the dosing regimen is escalated. In some cases, if the cells of the cell therapy did not persist and/or expand at satisfactory levels, and the subject demonstrates a response to the combination treatment, the dosing regimen is escalated.
[0284] In some cases, if the PK of the cells of the cell therapy (e.g. CAR T cells) administered to the subject shows that the cells did not persist and/or expand at satisfactory levels, and the subject does not demonstrate a satisfactory response to the combination treatment, the dosing regimen is de-escalated. In some cases, if the cells of the cell therapy did not persist and/or expand at satisfactory levels, and the subject does not demonstrate a satisfactory response to the combination treatment, the dosing regimen is de-escalated. In some cases, if the PK of the cells of the cell therapy (e.g. CAR T cells) administered to the subject shows that the cells did not persist and/or expand at satisfactory levels, and the subject does not demonstrate a satisfactory response to the combination treatment, this outcome is regarded as a doselimiting toxicity (DLT). In some cases, if the cells of the cell therapy did not persist and/or expand at satisfactory levels, and the subject does not demonstrate a satisfactory response to the combination treatment, the subject is identified as having a dose-limiting toxicity (DLT).
[0285] In some cases, if the PK of the cells of the cell therapy (e.g. CAR T cells) administered to subject in a dosing regimen shows that the cells did persist and/or expand at satisfactory levels, and the subject demonstrates a satisfactory response to the combination treatment, the dosing regimen is escalated. In some cases, if the cells of the cell therapy did persist and/or expand at satisfactory levels, and the subject demonstrates a satisfactory response to the combination treatment, the dosing regimen is escalated.
[0286] The precise dose of the BCL2 inhibitor and the timing thereof, which can each be independently determined empirically, may depend on the particular therapeutic preparation, the regimen and dosing schedule, the route of administration, the seriousness of the disease, degree of toxicity, and/or one or more outcomes.
B. ADMINISTRATION OF AN IMMUNOTHERAPY OR CELL THERAPY
[0287] In some embodiments of the methods, compositions, combinations, kits and uses provided herein, the combination therapy includes administering to a subject a therapy, e.g. an immunotherapy or cell therapy. In some embodiments, the therapy is a T cell therapy (e.g. CAR-expressing T cells) or a T cell-engaging therapy. Such therapies can be administered prior to administration of one or more BCL2 inhibitor as described.
J. / CELL-ENGA G/\G THERAPY
[0288] In some embodiments, the immunotherapy or cell therapy is or comprises a T cell-engaging therapy that is or comprises a binding molecule capable of binding to a surface molecule expressed on a T cell. In some embodiments, the surface molecule is an activating component of a T cell, such as a component of the T cell receptor complex. In some embodiments, the surface molecule is CD3 or is CD2. In some embodiments, the surface molecule is CD3. In some embodiments, the T cell-engaging therapy is or comprises an antibody or antigen-binding fragment.
[0289] In some embodiments, the T cell-engaging therapy is a bispecific antibody containing at least one antigen-binding domain binding to an activating component of the T cell (e.g. a T cell surface molecule, e.g. CD3 or CD2) and at least one antigen-binding domain binding to a surface antigen on a target cell, such as a surface antigen on a tumor or cancer cell, for example any of the listed antigens as described herein, e.g. CD19. In some embodiments, the simultaneous or near simultaneous binding of such an antibody to both of its targets can result in a temporary interaction between the target cell and T cell, thereby resulting in activation, e.g. cytotoxic activity, of the T cell and subsequent lysis of the target cell.
[0290] In some embodiments, bi-specific T cell engagers (BiTE) are used in connection with the provided methods, uses, articles of manufacture. In some embodiments, bi-specific T cell engagers have specificity toward two particular antigens (or markers or ligands). In some embodiments, the antigens are expressed on the surface of a particular type of cell. In particular embodiments, the first antigen is associated with an immune cell or an engineered immune cell, and the second antigen is associated with a target cell of the particular disease or condition, such as a cancer.
[0291] Numerous methods of producing bi-specific T cell engagers are known, including fusion of two different hybridomas (Milstein and Cuello, Nature 1983;305:537-540), and chemical tethering though heterobifunctional cross linkers (Staerz et al. Nature 1985; 314:628-631). Among exemplary bi- specific antibody T cell-engaging molecules are those which contain tandem scFv molecules fused by a flexible linker (see e.g. Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011); tandem scFv molecules fused to each other via, e.g. a flexible linker, and that further contain an Fc domain composed of a first and a second subunit capable of stable association (WO2013026837); diabodies and derivatives thereof, including tandem diabodies (Holliger et al, Prot Eng 9, 299-305 (1996); Kipriyanov et al, J Mol Biol 293, 41-66 (1999)); dual affinity retargeting (DART) molecules that can include the diabody format with a C-terminal disulfide bridge; or triomabs that include whole hybrid mouse/rat IgG molecules (Seimetz et al, Cancer Treat Rev 36, 458-467 (2010).
[0292] In certain embodiments, the bi-specific T cell engager is a molecule encoded by a polypeptide construct. In certain embodiments, the polypeptide construct contains a first component comprising an antigen-binding domain binding to an activating portion of an immune cell or engineered immune cell, and a second component comprising an antigen-binding domain binding to a surface antigen (e.g. target or tumor associated antigen (TAA)) associated with a particular disease or condition (e.g. cancer). In some embodiments, the first and second components are coupled by a linker. In some embodiments, the first component is coupled to a leader sequence encoding a CD33 signal peptide.
[0293] In some embodiments, the polypeptide is a construct containing from N-terminus to C- terminus: a first component comprising an antigen-binding domain binding to an activating portion of the T cell, a peptide linker, and a second component comprising an antigen-binding domain binding to a surface antigen (e.g. target or tumor associated antigen (TAA)) associated with a disease or condition (e.g. cancer).
[0294] In some aspects, an activating component of the T cell I a T cell surface molecule, such as CD3 or CD2. In some embodiments, the surface antigen of the target cell is a tumor associated antigen (TAA). In some aspects, the TAA contains one or more epitopes. In some embodiments, the peptide linker is or comprises a cleavable peptide linker.
[0295] In some embodiments, the antigen binding domain of the first component of the bi-specific T cell engager engages a receptor on an endogenous immune cell in the periphery of the tumor. In some embodiments, the endogenous immune cell is a T cell. In some aspects, the engagement of the endogenous T cell receptor redirects the endogenous T cells to the tumor. In some aspects, the engagement of the endogenous T cell receptor recruits tumor infiltrating lymphocytes (TILs) to the tumor. In some aspects, the engagement of the endogenous T cell receptor activates the endogenous immune repertoire.
[0296] In some embodiments, the simultaneous or near simultaneous binding of the bi-specific T cell engager to both of its targets (e.g. the immune cell and the TAA) can result in a temporary interaction between the target cell and T cell, thereby resulting in activation e.g. cytotoxic activity, cytokine release), of the T cell and subsequent lysis of the target cell.
[0297] In some embodiments, the first component of the bi-specific T cell engager is or comprises an antigen binding domain that binds to an activating component of a T cell. In some embodiments, the activating component of the T cell is a surface molecule. In some embodiments, the surface molecule is or comprises a T-cell antigen. Exemplary T-cell antigens include but are not limited to CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD44, CD45, CD69 and CD90. In some aspects, the binding of the bispecific T cell engaging molecule with the T cell antigen stimulates and/or activates the T cell.
[0298] In some embodiments, the anti-T cell binding domain includes an antibody or an antigenbinding fragment thereof selected from the group consisting of a Fab fragment, a F(ab')2 fragment, an Fv fragment, an scFv, a scAb, a dAb, a single domain heavy chain antibody, and a single domain light chain antibody.
[0299] In some embodiments, the T cell binding domain on the bi-specific T cell engager is an anti- CD3 domain. In some aspects, the anti-CD3 domain is an scFv. In some embodiments, the anti-CD3 domain of the bi-specific T cell engager binds to a subunit of the CD3 complex on a receptor on a T cell. In some aspects, the receptor is on an endogenous T cell. In some embodiments, the receptor is on an engineered immune cell further expressing a recombinant receptor. The effects of CD3 engagement of T cells is well known in the art, and include but are not limited to T cell activation and other downstream cell signaling. Any of such bi-specific T cell engagers can be used in the provided disclosure herein.
[0300] In some embodiments, the second component of the bi-specific T cell engager comprising an antigen-binding domain binding to a surface antigen associated with a disease or condition is a tumor or cancer antigen. In some embodiments, among the antigens targeted by the bi-specific T cell engager are those expressed in the context of a disease, condition, or cell type to be targeted via the adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and/or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas.
[0301] In some embodiments, the antigen includes av[36 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancertestis antigen, cancer/testis antigen IB (CT AG, also known as NY-ESO-1 and LAGE -2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrine receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gplOO), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPCR5D), Her2/neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight-melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen Al (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha(IL-22Ra), IL-13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, LI cell adhesion molecule (Ll-CAM), CE7 epitope of LI- CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-Al, MAGE- A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PS MA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome deltaisomer ase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal tag, and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens. Antigens targeted by the receptors in some embodiments include antigens associated with a B cell malignancy, such as any of a number of known B cell marker. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30. In some embodiments, the antigen is CD19.
[0302] In some embodiments, both antigen binding domains, including the first antigen binding domain and the second antigen binding domain, comprise an antibody or an antigen-binding fragment.
[0303| The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab’)2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rlgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv) or fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
[0304] In some embodiments, the antigen-binding proteins, antibodies and antigen binding fragments thereof specifically recognize an antigen of a full-length antibody. In some embodiments, the heavy and light chains of an antibody can be full-length or can be an antigen-binding portion (a Fab, F(ab’)2, Fv or a single chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is chosen from, e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE, particularly chosen from, e.g., IgGl, IgG2, IgG3, and IgG4, more particularly, IgGl e.g., human IgGl). In another embodiment, the antibody light chain constant region is chosen from, e.g., kappa or lambda, particularly kappa.
[0305] Among the provided antibodies are antibody fragments. An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; variable heavy chain (VH) regions, singlechain antibody molecules such as scFvs and single-domain VH single antibodies; and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single -chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
[0306] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
[0307] Single-domain antibodies (sdAb) are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single -domain antibody is a human single -domain antibody. In some embodiments, the bi-specific T cell engager comprises an antibody heavy chain domain that specifically binds the antigen, such as a cancer marker or cell surface antigen of a cell or disease to be targeted, such as a tumor cell or a cancer cell, such as any of the target antigens described herein or known. Exemplary single -domain antibodies include sdFv, nanobody, VHH or VNAR-
[0308] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some embodiments, the antibody fragments are scFvs. [0309] A “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
[0310] In certain embodiments, the antigen binding domains are single chain variable fragments (scFv). In some embodiments, the scFv is a tandem scFv containing a heavy and a light chain. In some embodiments, the heavy and light chains are connected by peptide linkers. In some embodiments, the linker is composed primarily of serines and glycines. In some aspects, the linkage of the heavy chain and the light chain forms a single polypeptide antigen binding domain.
[0311] In certain embodiments, the first antigen binding domain of the bi-specific T cell engager is an anti-CD3 scFv. In certain embodiments, the second antigen binding domain of the bi-specific T cell engager is an anti-CD19 scFv. In certain embodiments, the first antigen binding domain of the bi-specific T cell engager is an anti-CD3 scFv, and the second antigen binding domain of the bi-specific T cell engager is an anti-CD19 scFv.
[0312] In some aspects, the bi-specific T cell engager polypeptide constructs contain a linker that joins the first component comprising the antigen-binding domain that binds to an activating portion of the T cell, to the second component comprising an antigen-binding domain binding to a surface antigen (e.g. target or tumor associated antigen (TAA)) associated with a particular disease or condition. In some aspects, the linker is a short, medium or long linker.
[0313] In some embodiments, the linker is a peptide linker which is cleavable. In some aspects, the cleavable linker includes a sequence that is a substrate for a protease. In some embodiments, the sequence comprises a bond that can be broken under in vivo conditions. In some cases, the linker sequence is selectively cleaved by a protease present in a physiological environment. In some aspects, the environment is separate from the tumor microenvironment. In some embodiments, the protease is found in the periphery of the tumor.
[0314] In some embodiments, the selectively cleavable linker is cleaved by a protease produced by cells that do not co-localize with the tumor. In some embodiments, the selectively cleavable linker is not cleaved by proteases that are in the proximity of the tumor microenvironment. In some embodiments, the cleavage of the linker by the protease renders the bi-specific T cell engaging molecule inactive. In some embodiments, the protease is found in the circulating blood of a subject. In some embodiments, the protease is a part of the intrinsic or extrinsic coagulation pathway. In some aspects, the protease is a serine protease. In some aspects, the protease comprises but is not limited to a thrombin, factor X, factor XI, factor XII, and plasmin.
[0315] Among such exemplary bispecific antibody T cell-engagers are bispecific T cell engager (BiTE) molecules, which contain tandem scFv molecules fused by a flexible linker (see e.g. Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011); tandem scFv molecules fused to each other via, e.g. a flexible linker, and that further contain an Fc domain composed of a first and a second subunit capable of stable association (WO2013026837); diabodies and derivatives thereof, including tandem diabodies (Holliger et al, Prot Eng 9, 299-305 (1996); Kipriyanov et al, J Mol Biol 293, 41-66 (1999)); dual affinity retargeting (DART) molecules that can include the diabody format with a C-terminal disulfide bridge; or triomabs that include whole hybrid mouse/rat IgG molecules (Seimetz et al, Cancer Treat Rev 36, 458- 467 (2010). In some embodiments, the T-cell engaging therapy is blinatumomab or AMG 330. Any of such T cell-engagers can be used in used in the provided methods.
[0316] The immune system stimulator and/or the T cell engaging therapy can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, transseptal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon’s injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, the immunotherapy is administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intrathoracic, intracranial, or subcutaneous administration.
[0317] In certain embodiments, one or more doses of a T cell engaging therapy are administered. In particular embodiments, between or between about 0.001 pg and about 5,000 pg, inclusive, of the T cell engaging therapy is administered. In particular embodiments, between or between about 0.001 pg and 1,000 pg, 0.001 pg tol pg, 0.01 pg tol pg, 0.1 pg tolO pg, 0.01 pg tol pg, 0.1 pg and 5 pg, 0.1 pg and 50 pg, 1 pg and 100 pg, 10 pg and 100 pg, 50 pg and 500 pg, 100 pg and 1,000 pg, 1,000 pg and 2,000 pg, or 2,000 pg and 5,000 pg of the T cell engaging therapy is administered. In some embodiments, the dose of the T cell engaging therapy is or includes between or between about 0.01 pg/kg and 100 mg/kg, 0.1 pg/kg and 10 pg/kg, 10 pg/kg and 50 pg/kg, 50 pg/kg and 100 pg/kg, 0.1 mg/kg and 1 mg/kg, 1 mg/kg and 10 mg/kg, 10 mg/kg and 100 mg/kg, 100 mg/kg and 500 mg/kg, 200 mg/kg and 300 mg/kg, 100 mg/kg and 250 mg/kg, 200 mg/kg and 400 mg/kg, 250 mg/kg and 500 mg/kg, 250 mg/kg and 750 mg/kg, 50 mg/kg and 750 mg/kg, 1 mg/kg and 10 mg/kg, or 100 mg/kg and 1,000 mg/kg, each inclusive. In some embodiments, the dose of the T cell engaging therapy is at least or at least about or is or is about 0.1 pg/kg, 0.5 pg/kg, 1 pg/kg, 5 pg/kg, 10 pg/kg, 20 pg/kg, 30 pg/kg, 40 pg/kg, 50 pg/kg, 60 pg/kg, 70 pg/kg, 80 pg/kg, 90 pg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg, 90 mg/kg, 95 mg/kg, 100 mg/kg, 200 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, or 1,000 mg/kg,. In particular embodiments, the T cell engaging therapy is administered orally, intravenously, intraperitoneally, transdermally, intrathecally, intramuscularly, intranasally, transmucosally, subcutaneously, or rectally.
2. CELL THERAPY
[03181 In some embodiments, the therapy, e.g. immunotherapy or cell therapy, is a cell-based therapy that is or comprises administration of cells, such as immune cells, for example T cell or NK cells, that target a molecule expressed on the surface of a lesion, such as a tumor or a cancer. In some aspects, the cell therapy is a tumor infiltrating lymphocytic (TIL) therapy, a natural kill (NK) cell therapy, a transgenic TCR therapy, or a recombinant-receptor expressing cell therapy, which optionally is a T cell therapy, which optionally is a chimeric antigen receptor(CAR)-expressing cell therapy. In some embodiments, the T cell therapy includes administering T cells engineered to express a chimeric antigen receptor (CAR). In some aspects, the T cell therapy is an adoptive T cell therapy comprising T cells that specifically recognize and/or target an antigen associated with the cancer, such as an antigen associated with a B cell malignancy, e.g. a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL). In some aspects, the T cell therapy is an adoptive T cell therapy comprising T cells that specifically recognize and/or target an antigen associated with the cancer, such as an antigen associated with a B cell malignancy, e.g. a chronic lymphocytic leukemia (CLL). In some aspects, the T cell therapy is an adoptive T cell therapy comprising T cells that specifically recognize and/or target an antigen associated with the cancer, such as an antigen associated with a B cell malignancy, e.g. a small lymphocytic lymphoma (SLL). In some aspects, the T cell therapy comprises T cells engineered with a chimeric antigen receptor (CAR) comprising an antigen binding domain that binds, such as specifically binds, to the antigen. In some cases, the antigen targeted by the T cell therapy is CD19.
[0319] In some embodiments, the immune cells express a T cell receptor (TCR) or other antigenbinding receptor. In some embodiments, the immune cells express a recombinant receptor, such as a transgenic TCR or a chimeric antigen receptor (CAR). In some embodiments, the cells are autologous to the subject. In some embodiments, the cells are allogeneic to the subject. Exemplary of such cell therapies, e.g. T cell therapies, for use in the provided methods are described below.
[0320] In some embodiments, the provided cells express and/or are engineered to express receptors, such as recombinant receptors, including those containing ligand-binding domains or binding fragments thereof, and T cell receptors (TCRs) and components thereof, and/or functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs). In some embodiments, the recombinant receptor contains an extracellular ligand-binding domain that specifically binds to an antigen. In some embodiments, the recombinant receptor is a CAR that contains an extracellular antigen-recognition domain that specifically binds to an antigen. In some embodiments, the ligand, such as an antigen, is a protein expressed on the surface of cells. In some embodiments, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which, like a TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule.
[0321] In some embodiments, the cells for use in or administered in connection with the provided methods contain or are engineered to contain an engineered receptor, e.g., an engineered antigen receptor, such as a chimeric antigen receptor (CAR), or a T cell receptor (TCR). Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients, in accord with the provided methods, and/or with the provided articles of manufacture or compositions.
[0322] Among the engineered cells, including engineered cells containing recombinant receptors, are described in Section II below. Exemplary recombinant receptors, including CARs and recombinant TCRs, as well as methods for engineering and introducing the receptors into cells, include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, W02013/123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Patent Nos.: 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the genetically engineered antigen receptors include a CAR as described in U.S. Patent No.: 7,446,190, and those described in International Patent Application Publication No.: WO/2014055668 Al.
[0323] Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods, compositions and articles of manufacture and kits. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al', US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338.
[0324] In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject. [0325] In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.
[0326] The cells of the T cell therapy can be administered in a composition formulated for administration, or alternatively, in more than one composition (e.g., two compositions) formulated for separate administration. The dose(s) of the cells may include a particular number or relative number of cells or of the engineered cells, and/or a defined ratio or compositions of two or more sub-types within the composition, such as CD4 vs.CD8 T cells.
[0327] The cells can be administered by any suitable means. The cells are administered in a dosing regimen to achieve a therapeutic effect, such as a reduction in tumor burden. Dosing and administration may depend in part on the schedule of administration of the BCL2 inhibitor, which can be administered subsequent to initiation of administration of the cell therapy, such as T cell therapy, e.g. CAR T cell therapy. Various dosing schedules of the cell therapy include but are not limited to single or multiple administrations over various time -points, bolus administration, and pulse infusion.
A. COMPOSITIONS AND FORMULATIONS
[0328] In some embodiments, the dose of cells of the cell therapy, such as a T cell therapy comprising cells engineered with a recombinant antigen receptor, e.g. CAR or TCR, is provided as a composition or formulation, such as a pharmaceutical composition or formulation. Such compositions can be used in accord with the provided methods and/or with the provided articles of manufacture or compositions, such as in the treatment of a B cell malignancy (e.g. CLL or SLL).
[0329] The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
[0330] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
[0331] In some embodiments, the cell therapy, such as engineered T cells e.g. CAR T cells), are formulated with a pharmaceutically acceptable carrier. In some aspects, the choice of carrier is determined in part by the particular cell or agent and/or by the method of administration. Accordingly, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Carriers are described, e.g., by Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes e.g. Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG).
[0332] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some aspects, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams & Wilkins; 21st ed. (May 1, 2005).
[0333] The formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells or agents, where the respective activities do not adversely affect one another. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Thus, in some embodiments, the pharmaceutical composition further includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
[0334] The pharmaceutical composition in some embodiments contains cells in amounts effective to treat the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic efficacy in some embodiments is monitored by periodic assessment of treated subjects. For repeated administrations over several days or longer, depending on the condition, the treatment is repeated until a desired suppression of disease symptoms occurs. However, other dosage regimens may be useful and can be determined. The desired dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition.
[0335| The cells may be administered using standard administration techniques, formulations, and/or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. With respect to cells, administration can be autologous or heterologous. For example, immunoresponsive cells or progenitors can be obtained from one subject, and administered to the same subject or a different, compatible subject. Peripheral blood derived immunoresponsive cells or their progeny (e.g., in vivo, ex vivo or in vitro derived) can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modified immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
[0336] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. In some embodiments, the agent or cell populations are administered parenterally. The term “parenteral,” as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the agent or cell populations are administered to a subject using peripheral systemic delivery by intravenous, intraperitoneal, or subcutaneous injection.
[0337] Compositions in some embodiments are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues. Liquid or viscous compositions can comprise carriers, which can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol) and suitable mixtures thereof.
[0338] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.
[0339] The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. B. DOSING
[0340] The cells can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon’s injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration of the cells. In some embodiments, it is administered by multiple bolus administrations of the cells, for example, over a period of no more than 3 days, or by continuous infusion administration of the cells. In some embodiments, administration of the cell dose or any additional therapies, e.g., the lymphodepleting therapy, intervention therapy and/or combination therapy, is carried out via outpatient delivery.
[0341] For the treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, previous therapy, the subject’s clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.
[0342] In some embodiments, a dose of cells is administered to subjects in accord with the provided methods, and/or with the provided articles of manufacture or compositions. In some embodiments, the size or timing of the doses is determined as a function of the particular disease or condition (e.g., cancer, e.g., B cell malignancy) in the subject. In some cases, the size or timing of the doses for a particular disease in view of the provided description may be empirically determined.
[0343] In some embodiments, the dose of cells comprises between at or about 2 x 105 of the cells/kg and at or about 2 x 106 of the cells/kg, such as between at or about 4 x 105 of the cells/kg and at or about 1 x 106 of the cells/kg or between at or about 6 x 105 of the cells/kg and at or about 8 x 105 of the cells/kg. In some embodiments, the dose of cells comprises no more than 2 x 105 of the cells (e.g. antigen-expressing, such as CAR-expressing cells) per kilogram body weight of the subject (cells/kg), such as no more than at or about 3 x 105 cells/kg, no more than at or about 4 x 105 cells/kg, no more than at or about 5 x 105 cells/kg, no more than at or about 6 x 105 cells/kg, no more than at or about 7 x 105 cells/kg, no more than at or about 8 x 105 cells/kg, no more than at or about 9 x 105 cells/kg, no more than at or about 1 x 106 cells/kg, or no more than at or about 2 x 106 cells/kg. In some embodiments, the dose of cells comprises at least or at least about or at or about 2 x 105 of the cells e.g. antigenexpressing, such as CAR-expressing cells) per kilogram body weight of the subject (cells/kg), such as at least or at least about or at or about 3 x 105 cells/kg, at least or at least about or at or about 4 x 105 cells/kg, at least or at least about or at or about 5 x 105 cells/kg, at least or at least about or at or about 6 x 105 cells/kg, at least or at least about or at or about 7 x 105 cells/kg, at least or at least about or at or about 8 x 105 cells/kg, at least or at least about or at or about 9 x 105 cells/kg, at least or at least about or at or about 1 x 106 cells/kg, or at least or at least about or at or about 2 x 106 cells/kg.
[0344] In certain embodiments, the cells, or individual populations of sub-types of cells, are administered to the subject at a range of at or about one million to at or about 100 billion cells and/or that amount of cells per kilogram of body weight, such as, e.g. , 1 million to at or about 50 billion cells (e.g. , at or about 5 million cells, at or about 25 million cells, at or about 500 million cells, at or about 1 billion cells, at or about 5 billion cells, at or about 20 billion cells, at or about 30 billion cells, at or about 40 billion cells, or a range defined by any two of the foregoing values), at or about 1 million to at or about 50 billion cells (e.g., at or about 5 million cells, at or about 25 million cells, at or about 500 million cells, at or about 1 billion cells, at or about 5 billion cells, at or about 20 billion cells, at or about 30 billion cells, at or about 40 billion cells, or a range defined by any two of the foregoing values), such as at or about 10 million to at or about 100 billion cells e.g., at or about 20 million cells, at or about 30 million cells, at or about 40 million cells, at or about 60 million cells, at or about 70 million cells, at or about 80 million cells, at or about 90 million cells, at or about 10 billion cells, at or about 25 billion cells, at or about 50 billion cells, at or about 75 billion cells, at or about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases at or about 100 million cells to at or about 50 billion cells (e.g., at or about 120 million cells, at or about 250 million cells, at or about 350 million cells, at or about 450 million cells, at or about 650 million cells, at or about 800 million cells, at or about 900 million cells, at or about 3 billion cells, at or about 30 billion cells, at or about 45 billion cells) or any value in between these ranges and/or per kilogram of body weight. Dosages may vary depending on attributes particular to the disease or disorder and/or patient and/or other treatments.
|0345] In some embodiments, the dose of cells comprises from at or about 1 x 105 to at or about 5 x 108 total CAR-expressing T cells, from at or about 1 x 105 to at or about 2.5 x 108 total CAR-expressing T cells, from at or about 1 x 105 to at or about 1 x 108 total CAR-expressing T cells, from at or about 1 x 105 to at or about 5 x 107 total CAR-expressing T cells, from at or about 1 x 105 to at or about 2.5 x 107 total CAR-expressing T cells, from at or about 1 x 105 to at or about 1 x 107 total CAR-expressing T cells, from at or about 1 x 105 to at or about 5 x 106 total CAR-expressing T cells, from at or about 1 x
105 to at or about 2.5 x 106 total CAR-expressing T cells, from at or about 1 x 105 to at or about 1 x 106 total CAR-expressing T cells, from at or about 1 x 106 to at or about 5 x 108 total CAR-expressing T cells, from at or about 1 x 106 to at or about 2.5 x 108 total CAR-expressing T cells, from at or about 1 x
106 to at or about 1 x 108 total CAR-expressing T cells, from at or about 1 x 106 to at or about 5 x 107 total CAR-expressing T cells, from at or about 1 x 106 to at or about 2.5 x 107 total CAR-expressing T cells, from at or about 1 x 106 to at or about 1 x 107 total CAR-expressing T cells, from at or about 1 x 106 to at or about 5 x 106 total CAR-expressing T cells, from at or about 1 x 106 to at or about 2.5 x 106 total CAR-expressing T cells, from at or about 2.5 x 106 to at or about 5 x 108 total CAR-expressing T cells, from at or about 2.5 x 106 to at or about 2.5 x 108 total CAR-expressing T cells, from at or about 2.5 x 106 to at or about 1 x 108 total CAR-expressing T cells, from at or about 2.5 x 106 to at or about 5 x 107 total CAR-expressing T cells, from at or about 2.5 x 106 to at or about 2.5 x 107 total CAR- expressing T cells, from at or about 2.5 x 106 to at or about 1 x 107 total CAR-expressing T cells, from at or about 2.5 x 106 to at or about 5 x 106 total CAR-expressing T cells, from at or about 5 x 106 to at or about 5 x 108 total CAR-expressing T cells, from at or about 5 x 106 to at or about 2.5 x 108 total CAR- expressing T cells, from at or about 5 x 106 to at or about 1 x 108 total CAR-expressing T cells, from at or about 5 x 106 to at or about 5 x 107 total CAR-expressing T cells, from at or about 5 x 106 to at or about 2.5 x 107 total CAR-expressing T cells, from at or about 5 x 106 to at or about 1 x 107 total CAR- expressing T cells, from at or about 1 x 107 to at or about 5 x 108 total CAR-expressing T cells, from at or about 1 x 107 to at or about 2.5 x 108 total CAR-expressing T cells, from at or about 1 x 107 to at or about 1 x 108 total CAR-expressing T cells, from at or about 1 x 107 to at or about 5 x 107 total CAR- expressing T cells, from at or about 1 x 107 to at or about 2.5 x 107 total CAR-expressing T cells, from at or about 2.5 x 107 to at or about 5 x 108 total CAR-expressing T cells, from at or about 2.5 x 107 to at or about 2.5 x 108 total CAR-expressing T cells, from at or about 2.5 x 107 to at or about 1 x 108 total CAR- expressing T cells, from at or about 2.5 x 107 to at or about 5 x 107 total CAR-expressing T cells, from at or about 5 x 107 to at or about 5 x 108 total CAR-expressing T cells, from at or about 5 x 107 to at or about 2.5 x 108 total CAR-expressing T cells, from at or about 5 x 107 to at or about 1 x 108 total CAR- expressing T cells, from at or about 1 x 108 to at or about 5 x 108 total CAR-expressing T cells, from at or about 1 x 108 to at or about 2.5 x 108 total CAR-expressing T cells, from at or about or 2.5 x 108 to at or about 5 x 108 total CAR-expressing T cells.
[0346] In some embodiments, the dose of cells comprises at least or at least about 1 x 105 CAR- expressing cells, at least or at least about 2.5 x 105 CAR-expressing cells, at least or at least about 5 x 105 CAR-expressing cells, at least or at least about 1 x 106 CAR-expressing cells, at least or at least about 2.5 x 106 CAR-expressing cells, at least or at least about 5 x 106 CAR-expressing cells, at least or at least about 1 x 107 CAR-expressing cells, at least or at least about 2.5 x 107 CAR-expressing cells, at least or at least about 5 x 107 CAR-expressing cells, at least or at least about 1 x 108 CAR-expressing cells, at least or at least about 2.5 x 108 CAR-expressing cells, or at least or at least about 5 x 108 CAR-expressing cells.
[0347] In some embodiments, the dose of cells is a flat dose of cells or fixed dose of cells such that the dose of cells is not tied to or based on the body surface area or weight of a subject.
[0348] In some embodiments, for example, where the subject is a human, the dose includes fewer than at or about 5 x 108 total recombinant receptor (e.g., CAR)-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs), e.g., in the range of at or about 1 x 106 to at or about 5 x 108 such cells, such as at or about 2 x 106, 5 x 106, 1 x 107, 5 x 107, 1 x 108 2 x 108, 3 x 108, 4 x 108 or 5 x 108 total such cells, or the range between any two of the foregoing values. In some embodiments, where the subject is a human, the dose includes between at or about 1 x 106 and at or 3 x 108 total recombinant receptor (e.g., CAR)-expressing cells, e.g., in the range of at or about 1 x 107 to at or about 2 x 108 such cells, such as at or about 1 x 107, 5 x 107, 1 x 108 or 1.5 x 108 total such cells, or the range between any two of the foregoing values. In some embodiments, the patient is administered multiple doses, and each of the doses or the total dose can be within any of the foregoing values. In some embodiments, the dose of cells comprises the administration of from at or about 1 x 105 to at or about 5 x 108 total recombinant receptor (e.g. CAR)-expressing T cells or total T cells, from at or about 1 x 105 to at or about 1 x 108 total recombinant receptor e.g. CAR)-expressing T cells or total T cells, from at or about 5 x 105 to at or about 1 x 107 total recombinant receptor (e.g. CAR)-expressing T cells or total T cells, or from at or about 1 x 106 to at or about 1 x 107 total recombinant receptor (e.g. CAR)-expressing T cells or total T cells, each inclusive. In some embodiments, the dose of cells comprises the administration of from at or about 2.5 x 107 total recombinant receptor (e.g. CAR)-expressing T cells. In some embodiments, the dose of cells comprises the administration of from at or about 1 x 108 total recombinant receptor (e.g. CAR)- expressing T cells.
[0349] In some embodiments, the T cells of the dose include CD4+ T cells, CD8+ T cells or CD4+ and CD8+ T cells. In some embodiments, the T cells of the dose include CD4+ T cells. In some embodiments, the T cells of the dose include CD8+ T cells. In some embodiments, the T cells of the dose include CD4+ and CD8+ T cells.
[0350| In some embodiments, for example, where the subject is human, the CD8+ T cells of the dose, including in a dose including CD4+ and CD8+ T cells, includes between at or about 1 x 106 and at or about 1 x 108 total recombinant receptor (e.g., CAR)-expressing CD8+cells, e.g., in the range of at or about 5 x 106 to at or about 1 x 108 such cells, such cells at or about 1 x 107, 2.5 x 107, 5 x 107, 7.5 x 107, 1 x 108, 1.5 x 108, or 5 x 108 total such cells, or the range between any two of the foregoing values. In some embodiments, the patient is administered multiple doses, and each of the doses or the total dose can be within any of the foregoing values. In some embodiments, the dose of cells comprises the administration of from at or about 1 x 107 to at or about 0.75 x 108 total recombinant receptor-expressing CD8+ T cells, from at or about 1 x 107 to at or about 2.5 x 107 total recombinant receptor-expressing CD8+ T cells, from at or about 1 x 107 to at or about 0.75 x 108 total recombinant receptor-expressing CD8+ T cells, each inclusive. In some embodiments, the dose of cells comprises the administration of at or about 1 x 107, 2.5 x 107, 5 x 107, 7.5 x 107, 1 x 108, 1.5 x 108, or 5 x 108 total recombinant receptorexpressing CD8+ T cells.
[0351] In some embodiments, for example, where the subject is human, the CD4+ T cells of the dose, including in a dose including CD4+ and CD8+ T cells, includes between at or about 1 x 106 and at or about 1 x 108 total recombinant receptor (e.g., CAR)-expressing CD4+cells, e.g., in the range of at or about 5 x 106 to 1 x 108 such cells, such at or about 1 x 107, 2.5 x 107, 5 x 107, 7.5 x 107, 1 x 108, 1.5 x 108, or 5 x 108 total such cells, or the range between any two of the foregoing values. In some embodiments, the patient is administered multiple doses, and each of the doses or the total dose can be within any of the foregoing values. In some embodiments, the dose of cells comprises the administration of from at or about 1 x 107 to at or about 0.75 x 108 total recombinant receptor-expressing CD4+ T cells, from at or about 1 x 107 to at or about 2.5 x 107 total recombinant receptor-expressing CD4+ T cells, from at or about 1 x 107 to at or about 0.75 x 108 total recombinant receptor-expressing CD4+ T cells, each inclusive. In some embodiments, the dose of cells comprises the administration of at or about 1 x 107, 2.5 x 107, 5 x 107 7.5 x 107, 1 x 108, 1.5 x 108, or 5 x 108total recombinant receptor-expressing CD4+ T cells.
[0352] In some embodiments, the dose of cells, e.g., recombinant receptor-expressing T cells, is administered to the subject as a single dose or is administered only one time within a period of two weeks, one month, three months, six months, 1 year or more.
[0353] In the context of adoptive cell therapy, administration of a given “dose” encompasses administration of the given amount or number of cells as a single composition and/or single uninterrupted administration, e.g., as a single injection or continuous infusion, and also encompasses administration of the given amount or number of cells as a split dose or as a plurality of compositions, provided in multiple individual compositions or infusions, over a specified period of time, such as over no more than 3 days. Thus, in some contexts, the dose is a single or continuous administration of the specified number of cells, given or initiated at a single point in time. In some contexts, however, the dose is administered in multiple injections or infusions over a period of no more than three days, such as once a day for three days or for two days or by multiple infusions over a single day period.
[0354] Thus, in some aspects, the cells of the dose are administered in a single pharmaceutical composition. In some embodiments, the cells of the dose are administered in a plurality of compositions, collectively containing the cells of the dose.
[0355] In some embodiments, the term “split dose” refers to a dose that is split so that it is administered over more than one day. This type of dosing is encompassed by the present methods and is considered to be a single dose.
[0356] Thus, the dose of cells may be administered as a split dose, e.g., a split dose administered over time. For example, in some embodiments, the dose may be administered to the subject over 2 days or over 3 days. Exemplary methods for split dosing include administering 25% of the dose on the first day and administering the remaining 75% of the dose on the second day. In other embodiments, 33% of the dose may be administered on the first day and the remaining 67% administered on the second day. In some aspects, 10% of the dose is administered on the first day, 30% of the dose is administered on the second day, and 60% of the dose is administered on the third day. In some embodiments, the split dose is not spread over more than 3 days. [0357] In some embodiments, cells of the dose may be administered by administration of a plurality of compositions or solutions, such as a first and a second, optionally more, each containing some cells of the dose. In some aspects, the plurality of compositions, each containing a different population and/or sub-types of cells, are administered separately or independently, optionally within a certain period of time. For example, the populations or sub-types of cells can include CD8+ and CD4+ T cells, respectively, and/or CD8+ and CD4+-enriched populations, respectively, e.g., CD4+ and/or CD8+ T cells each individually including cells genetically engineered to express the recombinant receptor. In some embodiments, the administration of the dose comprises administration of a first composition comprising a dose of CD8+ T cells or a dose of CD4+ T cells and administration of a second composition comprising the other of the dose of CD4+ T cells and the CD8+ T cells.
[0358] In some embodiments, the administration of the composition or dose, e.g., administration of the plurality of cell compositions, involves administration of the cell compositions separately. In some aspects, the separate administrations are carried out simultaneously, or sequentially, in any order. In some embodiments, the dose comprises a first composition and a second composition, and the first composition and second composition are administered from at or about 0 to at or about 12 hours apart, from at or about 0 to at or about 6 hours apart or from at or about 0 to at or about 2 hours apart. In some embodiments, the initiation of administration of the first composition and the initiation of administration of the second composition are carried out no more than at or about 2 hours, no more than at or about 1 hour, or no more than at or about 30 minutes apart, no more than at or about 15 minutes, no more than at or about 10 minutes or no more than at or about 5 minutes apart. In some embodiments, the initiation and/or completion of administration of the first composition and the completion and/or initiation of administration of the second composition are carried out no more than at or about 2 hours, no more than at or about 1 hour, or no more than at or about 30 minutes apart, no more than at or about 15 minutes, no more than at or about 10 minutes or no more than at or about 5 minutes apart.
[0359] In some embodiments, the first composition and the second composition are mixed prior to the administration into the subject. In some embodiments, the first composition and the second composition are mixed shortly e.g., within at or about 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1.5 hours, 1 hour, or 0.5 hour) before the administration, In some embodiments, the first composition and the second composition are mixed immediately before the administration.
[0360] In some composition, the first composition, e.g., first composition of the dose, comprises CD4+ T cells. In some composition, the first composition, e.g., first composition of the dose, comprises CD8+ T cells. In some embodiments, the first composition is administered prior to the second composition.
[0361] In some embodiments, the dose or composition of cells includes a defined or target ratio of CD4+ cells expressing a recombinant receptor to CD8+ cells expressing a recombinant receptor and/or of CD4+ cells to CD8+ cells, which ratio optionally is approximately 1:1 or is between approximately 1:3 and approximately 3:1, such as approximately 1:1. In some embodiments, the dose or composition of cells includes a defined or target ratio of CD4+ cells expressing a recombinant receptor to CD8+ cells expressing a recombinant receptor and/or of CD4+ cells to CD8+ cells, which ratio optionally is approximately 1:1. In some embodiments, the dose or composition of cells includes a defined or target ratio of CD4+ cells expressing a recombinant receptor to CD8+ cells expressing a recombinant receptor and/or of CD4+ cells to CD8+ cells, which ratio optionally is between approximately 1:3 and approximately 3:1. In some aspects, the administration of a composition or dose with the target or desired ratio of different cell populations (such as CD4+:CD8+ ratio or CAR+CD4+:CAR+CD8+ ratio, e.g., 1:1) involves the administration of a cell composition containing one of the populations and then administration of a separate cell composition comprising the other of the populations, where the administration is at or approximately at the target or desired ratio. In some aspects, administration of a dose or composition of cells at a defined ratio leads to improved expansion, persistence and/or antitumor activity of the T cell therapy.
[0362] In some embodiments, the subject receives multiple doses, e.g., two or more doses or multiple consecutive doses, of the cells. In some embodiments, two doses are administered to a subject. In some embodiments, the subject receives the consecutive dose, e.g., second dose, approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days after the first dose. In some embodiments, multiple consecutive doses are administered following the first dose, such that an additional dose or doses are administered following administration of the consecutive dose. In some aspects, the number of cells administered to the subject in the additional dose is the same as or similar to the first dose and/or consecutive dose. In some embodiments, the additional dose or doses are larger than prior doses.
[0363] In some aspects, the size of the first and/or consecutive dose is determined based on one or more criteria such as response of the subject to prior treatment, e.g. chemotherapy, disease burden in the subject, such as tumor load, bulk, size, or degree, extent, or type of metastasis, stage, and/or likelihood or incidence of the subject developing toxic outcomes, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or a host immune response against the cells and/or recombinant receptors being administered.
[0364] In some aspects, the time between the administration of the first dose and the administration of the consecutive dose is about 9 to about 35 days, about 14 to about 28 days, or 15 to 27 days. In some embodiments, the administration of the consecutive dose is at a time point more than about 14 days after and less than about 28 days after the administration of the first dose. In some aspects, the time between the first and consecutive dose is about 21 days. In some embodiments, an additional dose or doses, e.g. consecutive doses, are administered following administration of the consecutive dose. In some aspects, the additional consecutive dose or doses are administered at least about 14 and less than about 28 days following administration of a prior dose. In some embodiments, the additional dose is administered less than about 14 days following the prior dose, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days after the prior dose. In some embodiments, no dose is administered less than about 14 days following the prior dose and/or no dose is administered more than about 28 days after the prior dose.
[0365] In some embodiments, the dose of cells, e.g., recombinant receptor-expressing cells, comprises two doses e.g., a double dose), comprising a first dose of the T cells and a consecutive dose of the T cells, wherein one or both of the first dose and the second dose comprises administration of the split dose of T cells.
[0366] In some embodiments, the dose of cells is generally large enough to be effective in reducing disease burden.
[0367] In some embodiments, the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type(s) and/or a desired ratio of cell types. Thus, the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+ to CD8+ ratio. In some embodiments, the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types. In some embodiments, the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.
[0368] In some embodiments, the populations or sub-types of cells, such as CD8+ and CD4+ T cells, are administered at or within a tolerated difference of a desired dose of total cells, such as a desired dose of T cells. In some aspects, the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg. In some aspects, the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight. In some aspects, among the total cells, administered at the desired dose, the individual populations or sub-types are present at or near a desired output ratio (such as CD4+ to CD8+ ratio), e.g., within a certain tolerated difference or error of such a ratio.
[0369] In some embodiments, the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or sub-types of cells, such as a desired dose of CD4+ cells and/or a desired dose of CD8+ cells. In some aspects, the desired dose is a desired number of cells of the sub-type or population, or a desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg. In some aspects, the desired dose is at or above a minimum number of cells of the population or sub-type, or minimum number of cells of the population or sub-type per unit of body weight.
[0370] Thus, in some embodiments, the dosage is based on a desired fixed dose of total cells and a desired ratio, and/or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or sub-populations. Thus, in some embodiments, the dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4+ to CD8+ cells, and/or is based on a desired fixed or minimum dose of CD4+ and/or CD8+ cells. [0371] In some embodiments, the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4+ and CD8+ cells or sub-types. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios, for example, in some embodiments, the desired ratio (e.g., ratio of CD4+to CD8+ cells) is between at or about 5:1 and at or about 5:1 (or greater than about 1:5 and less than about 5:1), or between at or about 1:3 and at or about 3:1 (or greater than about 1:3 and less than about 3:1), such as between at or about 2:1 and at or about 1:5 (or greater than about 1:5 and less than about 2:1, such as at or about 5:1, 4.5:1, 4:1, 3.5:1, 3:1, 2.5:1, 2:1, 1.9:1, 1.8:1, 1.7:1, 1.6:1, 1.5:1, 1.4:1, 1.3:1, 1.2:1, 1.1:1, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9: 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In some aspects, the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges.
[0372] In particular embodiments, the numbers and/or concentrations of cells refer to the number of recombinant receptor (e.g., CAR)-expressing cells. In other embodiments, the numbers and/or concentrations of cells refer to the number or concentration of all cells, T cells, or peripheral blood mononuclear cells (PBMCs) administered.
[0373] In some aspects, the size of the dose is determined based on one or more criteria such as response of the subject to prior treatment, e.g. chemotherapy, disease burden in the subject, such as tumor load, bulk, size, or degree, extent, or type of metastasis, stage, and/or likelihood or incidence of the subject developing toxic outcomes, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or a host immune response against the cells and/or recombinant receptors being administered.
[0374] In some embodiments, the methods also include administering one or more additional doses of cells expressing a chimeric antigen receptor (CAR) and/or lymphodepleting therapy, and/or one or more steps of the methods are repeated. In some embodiments, the one or more additional dose is the same as the initial dose. In some embodiments, the one or more additional dose is different from the initial dose, e.g., higher, such as 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold or 10-fold or more higher than the initial dose, or lower, such as e.g., higher, such as 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold or 10-fold or more lower than the initial dose. In some embodiments, administration of one or more additional doses is determined based on response of the subject to the initial treatment or any prior treatment, disease burden in the subject, such as tumor load, bulk, size, or degree, extent, or type of metastasis, stage, and/or likelihood or incidence of the subject developing toxic outcomes, e.g., CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and/or a host immune response against the cells and/or recombinant receptors being administered.
[0375] Following administration of the cells, the biological activity of the engineered cell populations in some embodiments is measured, e.g., by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable known methods, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying expression and/or secretion of one or more cytokines, such as CD107a, IFNy, IL -2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.
C. BRIDGING THERAPY
[0376] In some embodiments, the BCL2 inhibitor, e.g. venetoclax, is also administered as a “bridging therapy” prior to administration of the immunotherapy or cell therapy, such as a T cell therapy (e.g. CAR T cells). In some aspects, the bridging therapy is administered to a subject after collection of autologous cells from a subject to be treated. In some aspects, the bridging therapy is administered to a subject prior to administration of a lymphodepleting chemotherapy, as described in Subsection D below. Thus, in some aspects, the bridging therapy is administered after collection of autologous cells from the subject and prior to administration of a lymphodepleting chemotherapy to the subject. In some aspects, the bridging therapy is ceased at least or at least about 1 day prior to administration of the lymphodepleting chemotherapy. In some aspects, the bridging therapy is ceased at or at about 1 day prior to administration of the lymphodepleting chemotherapy. In some aspects, this interval is known as a “washout” period, such that there is a washout period of at least about 1 day between cessation of the bridging therapy and initiation of administration of the lymphodepleting chemotherapy.
[0377] In some embodiments, the bridging therapy comprises administration of a BCL2 inhibitor in weekly escalating doses. In some embodiments, the inhibitor is administered to the subject in escalating doses until a maximum dose of 100 mg daily is reached. In some embodiments, the inhibitor is administered to the subject in escalating doses until a maximum dose of 200 mg daily is reached. In some embodiments, the inhibitor is administered to the subject in escalating doses until a maximum dose of 400 mg daily is reached.
[0378] In some embodiments, the bridging therapy comprises daily administration of between about 20 mg and 400 mg of the BCL2 inhibitor. In some embodiments, the bridging therapy comprises daily administration of a first dose of the BCL2 inhibitor for a first week, daily administration of a second dose of the BCL2 inhibitor for a second week, and daily administration of a third dose of the BCL2 inhibitor for a third week. In some embodiments, the second dose is an increased amount of BCL2 inhibitor compared to the first dose, and the third dose is an increased amount of BCL2 inhibitor compared to the second dose.
[0379] In some embodiments, a subject is administered 20 mg per day of a BCL2 inhibitor for a first week. In some embodiments, a subject is administered 50 mg per day of a BCL2 inhibitor for a second week. In some embodiments, a subject is administered 100 mg per day of a BCL2 inhibitor for a third week. Thus, in some embodiments, a subject is administered 20 mg per day of a BCL2 inhibitor for a first week, 50 mg per day of a BCL2 inhibitor for a second week, and 100 mg per day of a BCL2 inhibitor for a third week. In some embodiments, bridging therapy comprises administration of 20 mg per day of a BCL2 inhibitor for a first week, 50 mg per day of a BCL2 inhibitor for a second week, and 100 mg per day of a BCL2 inhibitor for a third week. In some embodiments, the bridging therapy ceases after the third week of treatment.
[0380] In some embodiments, the bridging therapy further comprises administration of an anti- CD20 antibody. In some embodiments, the bridging therapy further comprises administration of an anti- CD20 antibody if the subject has been previously treated with a BCL2 inhibitor (e.g. venetoclax). In some embodiments, if the subject has not been previously treated with a BCL2 inhibitor (e.g. venetoclax), the bridging therapy does not further comprise administration of the inhibitor.
[0381] In some embodiments, the bridging therapy further comprises administration of a BTK inhibitor ( e.g. ibrutinib.) In some embodiments, the bridging therapy further comprises administration of the BTK inhibitor (e.g. ibrutinib) if the subject was receiving treatment with ibrutinib prior to treatment with the combination therapy. Thus, in some cases, the subject is administered a BTK inhibitor (e.g. ibrutinib) until autologous cells are collected from the subject and again during the bridging therapy.
[0382] In some embodiments, the BCL2 inhibitor provided in the bridging therapy is venetoclax. In some embodiments, the BCL2 inhibitor provided in the bridging therapy is the same BCL2 inhibitor provided in the dosing regimen following administration of the cell therapy, e.g. CAR T cells, to the subject. Thus, in some cases, a subject receives BCL2 inhibitor bridging therapy for about three weeks prior to administration of CAR T cells, and a BCL2 inhibitor dosing regimen after administration of CAR T cells.
[0383] In some cases, the BCL2 inhibitor administered in the bridging therapy and the BCL2 inhibitor administered in the dosing regimen are the same BCL2 inhibitor. In some cases, the BCL2 inhibitor administered in the bridging therapy and the BCL2 inhibitor administered in the dosing regimen are different BCL2 inhibitors. In some embodiments, the BCL2 inhibitor is venetoclax or navitoclax. In some embodiments, the BCL2 inhibitor is venetoclax. In some embodiments, the BCL2 inhibitor administered in the bridging therapy is venetoclax. In some embodiments, the BCL2 inhibitor administered in the bridging therapy is navitoclax.
[0384] Thus, in some aspects, a subject undergoes collection of autologous cells (e.g. leukapheresis), bridging therapy with the BCL2 inhibitor (e.g. venetoclax) for about three weeks, a washout period of about 1 day, lymphodepletion, initiation of administration of CAR T cells, and initiation of administration of the dosing regmen of the BCL2 inhibitor (e.g., venetoclax) about 1 day after initiation of administration of the CAR T cells, in that order. D. LYMPHODEPLETING TREATMENT
[0385] In some aspects, the provided methods can further include administering one or more lymphodepleting therapies, such as prior to or simultaneous with initiation of administration of the immunotherapy or cell thearpy, such as a T cell therapy (e.g. CAR-expressing T cells). In some embodiments, the lymphodepleting therapy comprises administration of a phosphamide, such as cyclophosphamide. In some embodiments, the lymphodepleting therapy can include administration of fludarabine.
[0386] In some aspects, preconditioning subjects with immunodepleting (e.g., lymphodepleting) therapies can improve the effects of adoptive cell therapy (ACT). Preconditioning with lymphodepleting agents, including combinations of cyclosporine and fludarabine, have been effective in improving the efficacy of transferred tumor infiltrating lymphocyte (TIL) cells in cell therapy, including to improve response and/or persistence of the transferred cells. See, e.g., Dudley et al., Science, 298, 850-54 (2002); Rosenberg et al., Clin Cancer Res, 17(13):4550-4557 (2011). Likewise, in the context of CAR+ T cells, several studies have incorporated lymphodepleting agents, most commonly cyclophosphamide, fludarabine, bendamustine, or combinations thereof, sometimes accompanied by low-dose irradiation. See Han et al. Journal of Hematology & Oncology, 6:47 (2013); Kochenderfer et al., Blood, 119: 2709-2720 (2012); Kalos et al., Sci Transl Med, 3(95):95ra73 (2011); Clinical Trial Study Record Nos.: NCT02315612; NCT01822652.
[0387] Such preconditioning can be carried out with the goal of reducing the risk of one or more of various outcomes that could dampen efficacy of the therapy. These include the phenomenon known as “cytokine sink,” by which T cells, B cells, NK cells compete with TILs for homeostatic and activating cytokines, such as IL -2, IL-7, and/or IL-15; suppression of TILs by regulatory T cells, NK cells, or other cells of the immune system; impact of negative regulators in the tumor microenvironment. Muranski et al., Nat Clin Pract Oncol. December; 3(12): 668-681 (2006).
[0388] Thus in some embodiments, the provided method further involves administering a lymphodepleting therapy to the subject. In some embodiments, the method involves administering the lymphodepleting therapy to the subject prior to the initiation of the administration of the dose of cells. In some embodiments, the lymphodepleting therapy contains a chemotherapeutic agent such as fludarabine and/or cyclophosphamide. In some embodiments, the administration of the cells and/or the lymphodepleting therapy is carried out via outpatient delivery.
[0389] In some embodiments, the methods include administering a preconditioning agent, such as a lymphodepleting or chemotherapeutic agent, such as cyclophosphamide, fludarabine, or combinations thereof, to a subject prior to the initiation of the administration of the dose of cells. For example, the subject may be administered a preconditioning agent at least 2 days prior, such as at least 3, 4, 5, 6, or 7 days prior, to the first or subsequent dose. In some embodiments, the subject is administered a preconditioning agent no more than 7 days prior, such as no more than 6, 5, 4, 3, or 2 days prior, to the initiation of administration of the dose of cells. In some embodiments, the subject is administered a preconditioning agent between 2 and 7, inclusive, such as at 2, 3, 4, 5, 6, or 7, days prior to the initiation of the administration of the dose of cells.
[0390] In some embodiments, the subject is preconditioned with cyclophosphamide at a dose between or between about 20 mg/kg and 100 mg/kg, such as between or between about 40 mg/kg and 80 mg/kg. In some aspects, the subject is preconditioned with or with about 60 mg/kg of cyclophosphamide. In some embodiments, the cyclophosphamide can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some embodiments, the cyclophosphamide is administered once daily for one or two days. In some embodiments, where the lymphodepleting agent comprises cyclophosphamide, the subject is administered cyclophosphamide at a dose between or between about 100 mg/m2 and 500 mg/m2, such as between or between about 200 mg/m2 and 400 mg/m2, or 250 mg/m2 and 350 mg/m2, inclusive. In some instances, the subject is administered about 300 mg/m2 of cyclophosphamide. In some embodiments, the cyclophosphamide can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some embodiments, cyclophosphamide is administered daily, such as for 1-5 days, for example, for 3 to 5 days. In some instances, the subject is administered about 300 mg/m2 of cyclophosphamide, daily for 3 days, prior to initiation of the cell therapy.
[0391] In some embodiments, where the lymphodepleting agent comprises fludarabine, the subject is administered fludarabine at a dose between or between about 1 mg/m2 and 100 mg/m2, such as between or between about 10 mg/m2 and 75 mg/m2, 15 mg/m2 and 50 mg/m2, 20 mg/m2 and 40 mg/m2” 24 mg/m2 and 35 mg/m2, 20 mg/m2 and 30 mg/m2, or 24 mg/m2 and 26 mg/m2. In some instances, the subject is administered 25 mg/m2 of fludarabine. In some instances, the subject is administered about 30 mg/m2 of fludarabine. In some embodiments, the fludarabine can be administered in a single dose or can be administered in a plurality of doses, such as given daily, every other day or every three days. In some embodiments, fludarabine is administered daily, such as for 1-5 days, for example, for 3 to 5 days. In some instances, the subject is administered about 30 mg/m2 of fludarabine, daily for 3 days, prior to initiation of the cell therapy.
[0392] In some embodiments, the lymphodepleting agent comprises a combination of agents, such as a combination of cyclophosphamide and fludarabine. Thus, the combination of agents may include cyclophosphamide at any dose or administration schedule, such as those described above, and fludarabine at any dose or administration schedule, such as those described above. For example, in some aspects, the subject is administered 60 mg/kg (~2 g/m2) of cyclophosphamide and 3 to 5 doses of 25 mg/m2 fludarabine prior to the dose of cells. In some embodiments, the subject is administered about 300 mg/m2 cyclophosphamide and about 30 mg/m2 fludarabine each daily for 3 days. In some embodiments, the preconditioning administration schedule ends between 2 and 7, inclusive, such as at 2, 3, 4, 5, 6, or 7, days prior to the initiation of the administration of the dose of cells.
[0393] In one exemplary dosage regimen, prior to receiving the first dose of CAR-expressing cells, subjects receive a lymphodepleting preconditioning chemotherapy of cyclophosphamide and fludarabine (CY/FLU), which is administered at least two days before the first dose of CAR-expressing cells and generally no more than 7 days before administration of cells. In some cases a subject is treated with BCL2 inhibitor (e.g. venetoclax) bridging therapy prior to receiving a lymphodepleting preconditioning chemotherapy of cyclophosphamide and fludarabine (CY/FLU), wherein the bridging therapy is ceased at least about one day before the subject receives the lymphodepleting therapy. In some cases, this interval is known as a “washout” period. Thus, in some cases, there is a washout period of one day between conclusion of the bridging therapy and initiation of administration of the lymphodepleting preconditioning chemotherapy. After preconditioning treatment, subjects are administered the dose of CAR-expressing T cells as described above.
[0394] In some embodiments, the administration of the preconditioning agent prior to infusion of the dose of cells improves an outcome of the treatment. For example, in some aspects, preconditioning improves the efficacy of treatment with the dose or increases the persistence of the recombinant receptorexpressing cells (e.g., CAR-expressing cells, such as CAR-expressing T cells) in the subject. In some embodiments, preconditioning treatment increases disease-free survival, such as the percent of subjects that are alive and exhibit no minimal residual or molecularly detectable disease after a given period of time following the dose of cells. In some embodiments, the time to median disease-free survival is increased.
[0395] Once the cells (e.g. CAR T cells) are administered to the subject (e.g., human), the biological activity of the engineered cell populations in some aspects is measured by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009) , and Herman et al. J. Immunological Methods, 285(1): 25- 40 (2004). In certain embodiments, the biological activity of the cells also can be measured by assaying expression and/or secretion of certain cytokines, such as CD 107a, IFNy, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load. In some aspects, toxic outcomes, persistence and/or expansion of the cells, and/or presence or absence of a host immune response, are assessed.
[0396] In some embodiments, the administration of the preconditioning agent prior to infusion of the dose of cells improves an outcome of the treatment such as by improving the efficacy of treatment with the dose or increases the persistence of the recombinant receptor-expressing cells (e.g., CAR- expressing cells, such as CAR-expressing T cells) in the subject. Therefore, in some embodiments, the dose of preconditioning agent given in the method which is a combination therapy with the BCL2 inhibitor and cell therapy is higher or lower than the dose given in the method without the inhibitor. In some embodiments, the dose of preconditioning agent given in the method which is a combination therapy with the BCL2 inhibitor and cell therapy is higher than the dose given in the method without the inhibitor. In some embodiments, the dose of preconditioning agent given in the method which is a combination therapy with the BCL2 inhibitor and cell therapy is lower than the dose given in the method without the inhibitor.
II. CELL THERAPY AND CELL ENGINEERING
[0397] In some embodiments, the cells contain or are engineered to contain an engineered receptor, e.g., an engineered antigen receptor, such as a chimeric antigen receptor (CAR), or a T cell receptor (TCR). Also provided are populations of such cells, compositions containing such cells and/or enriched for such cells, such as in which cells of a certain type such as T cells or CD8+ or CD4+ cells are enriched or selected. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, e.g., patients.
[0398] Thus, in some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, gene transfer is accomplished by first stimulating the cells, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications.
A. RECOMBINANT RECEPTORS
[0399] In some embodiments, the cell therapy, e.g. T cell therapy, for use in accord with the provided combination therapy methods includes administering engineered cells expressing recombinant receptors designed to recognize and/or specifically bind to molecules associated with the disease or condition, such as a cancer (e.g., a CLL or SLL), and result in a response, such as an immune response against such molecules upon binding to such molecules. The receptors may include chimeric receptors, e.g., chimeric antigen receptors (CARs), and other transgenic antigen receptors including transgenic T cell receptors (TCRs).
Z CHIMERIC ANTIGEN RECEPTORS
[0400] In some embodiments of the provided methods and uses, the engineered cells, such as T cells, express a chimeric receptor, such as a chimeric antigen receptor (CAR), that contains one or more domains that combine a ligand-binding domain (e.g. antibody or antibody fragment) that provides specificity for a desired antigen (e.g., tumor antigen) with intracellular signaling domains. In some embodiments, the intracellular signaling domain is an activating intracellular domain portion, such as a T cell activating domain, providing a primary activation signal. In some embodiments, the intracellular signaling domain contains or additionally contains a costimulatory signaling domain to facilitate effector functions. Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immunostimulatory signal, such as an IT AM-transduced signal, into the cell, thereby promoting an immune response targeted to the disease or condition. In some embodiments, chimeric receptors when genetically engineered into immune cells can modulate T cell activity, and, in some cases, can modulate T cell differentiation or homeostasis, thereby resulting in genetically engineered cells with improved longevity, survival and/or persistence in vivo, such as for use in adoptive cell therapy methods.
[0401] Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012/129514, WO2014031687, WO2013/166321, WO2013/071154, W02013/123061, U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Patent Nos.: 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and/or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the antigen receptors include a CAR as described in U.S. Patent No.: 7,446,190, and those described in International Patent Application Publication No.: WO/2014055668 Al. Examples of the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, U.S. Patent No.: 7,446,190, US Patent No.: 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8,339,645, US 7,446,179, US 2013/0149337, U.S. Patent No.: 7,446,190, and US Patent No.: 8,389,282.
[0402] In some embodiments, the engineered cells, such as T cells, express a recombinant receptor such as a chimeric antigen receptor (CAR) with specificity for a particular antigen (or marker or ligand), such as an antigen expressed on the surface of a particular cell type. In some embodiments, the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and/or is expressed on the engineered cells.
[0403] The chimeric receptors, such as CARs, generally include an extracellular antigen binding domain that is an antigen-binding portion or portions of an antibody molecule. In some embodiments, the antigen-binding domain is a portion of an antibody molecule, generally a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment. In some embodiments, the antigen-binding domain is a single domain antibody (sdAb), such as sdFv, nanobody, VHH and VNAR. In some embodiments, an antigen-binding fragment comprises antibody variable regions joined by a flexible linker.
[0404] The chimeric receptors, such as CARs, generally include an extracellular antigen binding domain, such as a portion of an antibody molecule, generally a variable heavy (VH) chain region and/or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment. In some embodiments, the CAR contains an antibody or an antigen-binding fragment e.g. scFv) that specifically recognizes an antigen, such as an intact antigen, expressed on the surface of a cell.
[0405] Among the antigen receptors are a CAR containing an extracellular ntigen binding domain, such as antibody or antigen-binding fragment, that exhibits TCR-like specificity directed against peptide- MHC complexes, which also may be referred to as a TCR-like CAR. In some embodiments, the extracellular antigen binding domain specific for an MHC-peptide complex of a TCR-like CAR is linked to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s). In some embodiments, such molecules can typically mimic or approximate a signal through a natural antigen receptor, such as a TCR, and, optionally, a signal through such a receptor in combination with a costimulatory receptor.
[0406] Reference to “Major histocompatibility complex” (MHC) refers to a protein, generally a glycoprotein, that contains a polymorphic peptide binding site or binding groove that can, in some cases, complex with peptide antigens of polypeptides, including peptide antigens processed by the cell machinery. In some cases, MHC molecules can be displayed or expressed on the cell surface, including as a complex with peptide, i.e. MHC-peptide complex, for presentation of an antigen in a conformation recognizable by an antigen receptor on T cells, such as a TCRs or TCR-like antibody. Generally, MHC class I molecules are heterodimers having a membrane spanning a chain, in some cases with three a domains, and a non-covalently associated [32 microglobulin. Generally, MHC class II molecules are composed of two transmembrane glycoproteins, a and [3, both of which typically span the membrane. An MHC molecule can include an effective portion of an MHC that contains an antigen binding site or sites for binding a peptide and the sequences necessary for recognition by the appropriate antigen receptor. In some embodiments, MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where a MHC-peptide complex is recognized by T cells, such as generally CD8+ T cells, but in some cases CD4+ T cells. In some embodiments, MHC class II molecules deliver peptides originating in the vesicular system to the cell surface, where they are typically recognized by CD4+ T cells. Generally, MHC molecules are encoded by a group of linked loci, which are collectively termed H-2 in the mouse and human leukocyte antigen (HLA) in humans. Hence, typically human MHC can also be referred to as human leukocyte antigen (HLA). [0407] The term “MHC -peptide complex” or “peptide -MHC complex” or variations thereof, refers to a complex or association of a peptide antigen and an MHC molecule, such as, generally, by non- covalent interactions of the peptide in the binding groove or cleft of the MHC molecule. In some embodiments, the MHC -peptide complex is present or displayed on the surface of cells. In some embodiments, the MHC -peptide complex can be specifically recognized by an antigen receptor, such as a TCR, TCR-like CAR or antigen-binding portions thereof.
[0408] In some embodiments, a peptide, such as a peptide antigen or epitope, of a polypeptide can associate with an MHC molecule, such as for recognition by an antigen receptor. Generally, the peptide is derived from or based on a fragment of a longer biological molecule, such as a polypeptide or protein. In some embodiments, the peptide typically is about 8 to about 24 amino acids in length. In some embodiments, a peptide has a length of from or from about 9 to 22 amino acids for recognition in the MHC Class II complex. In some embodiments, a peptide has a length of from or from about 8 to 13 amino acids for recognition in the MHC Class I complex. In some embodiments, upon recognition of the peptide in the context of an MHC molecule, such as MHC -peptide complex, the antigen receptor, such as TCR or TCR-like CAR, produces or triggers an activation signal to the T cell that induces a T cell response, such as T cell proliferation, cytokine production, a cytotoxic T cell response or other response.
[0409] In some embodiments, a TCR-like antibody or antigen-binding portion, are known or can be produced by known methods (see e.g. US Published Application Nos. US 2002/0150914; US 2003/0223994; US 2004/0191260; US 2006/0034850; US 2007/00992530; US20090226474; US20090304679; and International PCT Publication No. WO 03/068201).
[04101 In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to a MHC-peptide complex, can be produced by immunizing a host with an effective amount of an immunogen containing a specific MHC-peptide complex. In some cases, the peptide of the MHC-peptide complex is an epitope of antigen capable of binding to the MHC, such as a tumor antigen, for example a universal tumor antigen, or other antigen as described below. In some embodiments, an effective amount of the immunogen is then administered to a host for eliciting an immune response, wherein the immunogen retains a three-dimensional form thereof for a period of time sufficient to elicit an immune response against the three-dimensional presentation of the peptide in the binding groove of the MHC molecule. Serum collected from the host is then assayed to determine if desired antibodies that recognize a three-dimensional presentation of the peptide in the binding groove of the MHC molecule is being produced. In some embodiments, the produced antibodies can be assessed to confirm that the antibody can differentiate the MHC-peptide complex from the MHC molecule alone, the peptide of interest alone, and a complex of MHC and irrelevant peptide. The desired antibodies can then be isolated.
[0411] In some embodiments, an antibody or antigen-binding portion thereof that specifically binds to an MHC-peptide complex can be produced by employing antibody library display methods, such as phage antibody libraries. In some embodiments, phage display libraries of mutant Fab, scFv or other antibody forms can be generated, for example, in which members of the library are mutated at one or more residues of a CDR or CDRs. See e.g. US published application No. US20020150914, US2014/0294841; and Cohen CJ. et al. (2003) J Mol. Recogn. 16:324-332.
[04121 The term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen binding (Fab) fragments, F(ab’)2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rlgG) fragments, variable heavy chain (VH) regions capable of specifically binding the antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
[0413] In some embodiments, the antigen-binding proteins, antibodies and antigen binding fragments thereof specifically recognize an antigen of a full-length antibody. In some embodiments, the heavy and light chains of an antibody can be full-length or can be an antigen-binding portion (a Fab, F(ab’)2, Fv or a single chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is chosen from, e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE, particularly chosen from, e.g., IgGl, IgG2, IgG3, and IgG4, more particularly, IgGl e.g., human IgGl). In another embodiment, the antibody light chain constant region is chosen from, e.g., kappa or lambda, particularly kappa.
[0414] Among the provided antibodies are antibody fragments. An “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; variable heavy chain (VH) regions, singlechain antibody molecules such as scFvs and single-domain VH single antibodies; and multispecific antibodies formed from antibody fragments. In particular embodiments, the antibodies are single -chain antibody fragments comprising a variable heavy chain region and/or a variable light chain region, such as scFvs.
[0415] The terms “complementarity determining region,” and “CDR,” synonymous with “hypervariable region” or “HVR,” are known, in some cases, to refer to non-contiguous sequences of amino acids within antibody variable regions, which confer antigen specificity and/or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). “Framework regions” and “FR” are known, in some cases, to refer to the non-CDR portions of the variable regions of the heavy and light chains. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR- H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).
[0416] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 (“Chothia” numbering scheme); MacCallum et al. , J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732- 745.” (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(l):55-77 (“IMGT” numbering scheme); Honegger A and Pliickthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, 2001 Jun 8;309(3):657-70, (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm,” PNAS, 1989, 86(23):9268-9272, (“AbM” numbering scheme).
[0417] The boundaries of a given CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Numbering for both the Kabat and Chothia schemes is based upon the most common antibody region sequence lengths, with insertions accommodated by insertion letters, for example, “30a,” and deletions appearing in some antibodies. The two schemes place certain insertions and deletions (“indels”) at different positions, resulting in differential numbering. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between Kabat and Chothia definitions based on that used by Oxford Molecular’s AbM antibody modeling software.
[0418] Table 2, below, lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between CDRs, for example, with FR-L1 located before CDR-L1, FR-L2 located between CDR- L1 and CDR-L2, FR-L3 located between CDR-L2 and CDR-L3 and so forth. It is noted that because the shown Kabat numbering scheme places insertions at H35A and H35B, the end of the Chothia CDR-H1 loop when numbered using the shown Kabat numbering convention varies between H32 and H34, depending on the length of the loop.
Figure imgf000103_0001
Figure imgf000104_0001
1 - Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD
2 - Al-Lazikani et al., (1997) JMB 273,927-948
[0419] Thus, unless otherwise specified, a “CDR” or “complementary determining region,” or individual specified CDRs (e.g., CDR-H1, CDR-H2, CDR-H3), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) complementary determining region as defined by any of the aforementioned schemes, or other known schemes. For example, where it is stated that a particular CDR (e.g., a CDR-H3) contains the amino acid sequence of a corresponding CDR in a given VH or VL region amino acid sequence, it is understood that such a CDR has a sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the aforementioned schemes, or other known schemes. In some embodiments, specific CDR sequences are specified. Exemplary CDR sequences of provided antibodies are described using various numbering schemes, although it is understood that a provided antibody can include CDRs as described according to any of the other aforementioned numbering schemes or other numbering schemes known to a skilled artisan.
[0420] Likewise, unless otherwise specified, a FR or individual specified FR(s) e.g., FR-H1, FR- H2, FR-H3, FR-H4), of a given antibody or region thereof, such as a variable region thereof, should be understood to encompass a (or the specific) framework region as defined by any of the known schemes. In some instances, the scheme for identification of a particular CDR, FR, or FRs or CDRs is specified, such as the CDR as defined by the Kabat, Chothia, AbM or Contact method, or other known schemes. In other cases, the particular amino acid sequence of a CDR or FR is given.
[0421] The term “variable region” or “variable domain” refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).
[0422] Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody. In some embodiments, the CAR comprises an antibody heavy chain domain that specifically binds the antigen, such as a cancer marker or cell surface antigen of a cell or disease to be targeted, such as a tumor cell or a cancer cell, such as any of the target antigens described herein or known.
[0423] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and/or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some embodiments, the antibody fragments are scFvs.
[0424] A “humanized” antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A “humanized form” of a non-human antibody, refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.
[0425] In some embodiments, the recombinant receptor, such as a chimeric receptor (e.g. CAR), includes an extracellular antigen binding domain, such as an antibody or antigen-binding fragment (e.g. scFv), that binds, such as specifically binds, to an antigen (or a ligand). Among the antigens targeted by the chimeric receptors are those expressed in the context of a disease, condition, or cell type to be targeted via the adoptive cell therapy. Among the diseases and conditions are proliferative, neoplastic, and malignant diseases and disorders, including cancers and tumors, including hematologic cancers, cancers of the immune system, such as lymphomas, leukemias, and/or myelomas, such as B, T, and myeloid leukemias, lymphomas, and multiple myelomas.
[0426] In some embodiments, the antigen targeted by the receptor is or comprises selected from among av(36 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer/testis antigen IB (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7/8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gplOO), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2/neu (receptor tyrosine kinase erb- B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight-melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen Al (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL -22 receptor alpha (IL-22Ra), IL-13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, LI cell adhesion molecule (Ll-CAM), CE7 epitope of Ll-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-Al, MAGE- A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta-isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal tag, and/or biotinylated molecules, and/or molecules expressed by HIV, HCV, HBV or other pathogens. Antigens targeted by the receptors in some embodiments include antigens associated with a B cell malignancy, such as any of a number of known B cell marker. In some embodiments, the antigen targeted by the receptor is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30. In some embodiments, the antigen targeted by the receptor is CD19. In some embodiments, the disease or condition is a B cell malignancy, and the antigen is CD19. In some embodiments, the disease or condition is chronic lymphocytic leukemia (CLL), and the antigen is CD 19. In some embodiments, the disease or condition is small lymphocytic lymphoma (SLL), and the antigen is CD19.
[0427] Antigens targeted by the receptors in some embodiments include antigens associated with a B cell malignancy, such as any of a number of known B cell marker. In some embodiments, the antigen targeted by the receptor is CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30. In particular aspects, the antigen is CD19. In some embodiments, any of such antigens are antigens expressed on human B cells.
[0428] In some embodiments, the antibody or an antigen-binding fragment (e.g. scFv or VH domain) specifically recognizes an antigen, such as CD19. In some embodiments, the antibody or antigen-binding fragment is derived from, or is a variant of, antibodies or antigen-binding fragment that specifically binds to CD19. In some embodiments, the antigen is CD19. In some embodiments, the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD 19. In some embodiments, the antibody or antibody fragment that binds CD 19 is a mouse derived antibody such as FMC63 and SJ25C1. In some embodiments, the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US 2016/0152723.
[0429] In some embodiments the antigen-binding domain includes a VH and/or VL derived from FMC63, which, in some aspects, can be an scFv. FMC63 generally refers to a mouse monoclonal IgGl antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, N. R., et al. (1987). Leucocyte typing HI. 302). In some embodiments, the FMC63 antibody comprises CDR-H1 and CDR-H2 set forth in SEQ ID NO: 38 and 39, respectively, and CDR-H3 set forth in SEQ ID NO: 40 or 54 and CDR-L1 set forth in SEQ ID NO: 35 and CDR-L2 set forth in SEQ ID NO: 36 or 55 and CDR-L3 sequences set forth in SEQ ID NO: 37 or 56. In some embodiments, the FMC63 antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 41 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 42.
[0430] In some embodiments, the scFv comprises a variable light chain containing the CDR-L1 sequence of SEQ ID NO:35, a CDR-L2 sequence of SEQ ID NO:36, and a CDR-L3 sequence of SEQ ID NO:37 and/or a variable heavy chain containing a CDR-H1 sequence of SEQ ID NO:38, a CDR-H2 sequence of SEQ ID NO:39, and a CDR-H3 sequence of SEQ ID NO:40, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the scFv comprises a variable heavy chain region of FMC63 set forth in SEQ ID NO:41 and a variable light chain region of FMC63 set forth in SEQ ID NO:42, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the variable heavy and variable light chains are connected by a linker. In some embodiments, the linker is set forth in SEQ ID NO:59. In some embodiments, the scFv comprises, in order, a VH, a linker, and a VL- In some embodiments, the scFv comprises, in order, a VL, a linker, and a VH- In some embodiments, the scFv is encoded by a sequence of nucleotides set forth in SEQ ID NO:57 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:57. In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO:43 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:43.
[0431] In some embodiments the antigen-binding domain includes a VH and/or VL derived from SJ25C1, which, in some aspects, can be an scFv. SJ25C1 is a mouse monoclonal IgGl antibody raised against Nalm-1 and -16 cells expressing CD19 of human origin (Ling, N. R., et al. (1987). Leucocyte typing III. 302). In some embodiments, the SJ25C1 antibody comprises CDR-H1, CDR-H2 and CDR- H3 set forth in SEQ ID NOS: 47-49, respectively, and CDR-L1, CDR-L2 and CDR-L3 sequences set forth in SEQ ID NOS: 44-46, respectively. In some embodiments, the SJ25C1 antibody comprises the heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 50 and the light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 51. In some embodiments, the scFv comprises a variable light chain containing a CDR-L1 sequence of SEQ ID NO:44, a CDR-L2 sequence of SEQ ID NO: 45, and a CDR-L3 sequence of SEQ ID NO:46 and/or a variable heavy chain containing a CDR-H1 sequence of SEQ ID NO:47, a CDR-H2 sequence of SEQ ID NO:48, and a CDR-H3 sequence of SEQ ID NO:49, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the scFv comprises a variable heavy chain region of SJ25C1 set forth in SEQ ID NO:50 and a variable light chain region of SJ25C1 set forth in SEQ ID NO:51, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the variable heavy and variable light chains are connected by a linker. In some embodiments, the linker is set forth in SEQ ID NO:52. In some embodiments, the scFv comprises, in order, a VH, a linker, and a VL- In some embodiments, the scFv comprises, in order, a VL, a linker, and a VH- In some embodiments, the scFv comprises the sequence of amino acids set forth in SEQ ID NO: 53 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 53.
[0432] In some aspects, the recombinant receptor, e.g., a chimeric antigen receptor, includes an extracellular portion containing one or more ligand- (e.g., antigen-) binding domains, such as an antibody or fragment thereof, and one or more intracellular signaling region or domain (also interchangeably called a cytoplasmic signaling domain or region). In some embodiments, the antibody or fragment includes an scFv. In some aspects, the chimeric antigen receptor includes an extracellular portion containing an antibody or fragment and an intracellular signaling region. In some embodiments, the intracellular signaling region comprises an intracellular signaling domain. In some embodiments, the intracellular signaling domain is or comprises a primary signaling domain, a signaling domain that is capable of inducing a primary activation signal in a T cell, a signaling domain of a T cell receptor (TCR) component, and/or a signaling domain comprising an immunoreceptor tyrosine-based activation motif (IT AM). In some aspects, the recombinant receptor, e.g., CAR, further includes a spacer and/or a transmembrane domain or portion. In some aspects, the spacer and/or transmembrane domain can link the extracellular portion containing the ligand- (e.g., antigen-) binding domain and the intracellular signaling region(s) or domain(s)
[04331 In some embodiments, the recombinant receptor such as the CAR, further includes a spacer, which may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region, e.g., an IgG4 hinge region, and/or a CH1/CL and/or Fc region. In some embodiments, the recombinant receptor further comprises a spacer and/or a hinge region. In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgGl. In some aspects, the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain. The spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer. In some examples, the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and including any integer between the endpoints of any of the listed ranges. In some embodiments, a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary spacers include IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain. Exemplary spacers include, but are not limited to, those described in Hudecek et al. (2013) Clin. Cancer Res., 19:3153, Hudecek et al. (2015) Cancer Immunol Res. 3(2): 125-135 or international patent application publication number WO2014031687, U.S. Patent No. 8,822,647 or published app. No. US2014/0271635.
[0434] In some embodiments, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgGl, such as the hinge only spacer set forth in SEQ ID NO: 1, and encoded by the sequence set forth in SEQ ID NO: 2. In some embodiments, the spacer is an Ig hinge, e.g., and IgG4 hinge, linked to a CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ ID NO:4. In some embodiments, the spacer the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only, such as set forth in SEQ ID NO: 3. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers. In some embodiments, the constant region or portion is of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 5. In some embodiments, the spacer has a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 1, 3, 4 and 5. In some embodiments, the spacer has a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity SEQ ID NO: 1. In some embodiments, the spacer comprises the sequence set forth in SEQ ID NO: 1.
[0435] In some aspects, the spacer is a polypeptide spacer that (a) comprises or consists of all or a portion of an immunoglobulin hinge or a modified version thereof or comprises about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region, (b) comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof and/or comprises about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region, or (c) is at or about 12 amino acids in length and/or comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4, or a modified version thereof; or (d) consists or comprises the sequence of amino acids set forth in SEQ ID NOS: 1, 3-5, 27-34 or 58, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or (e) comprises or consists of the formula X1PPX2P, where Xi is glycine, cysteine or arginine and X2 is cysteine or threonine.
[0436] In some embodiments, the antigen receptor comprises an intracellular domain linked directly or indirectly to the extracellular domain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an IT AM. For example, in some aspects, the antigen recognition domain (e.g. extracellular domain) generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and/or signal via another cell surface receptor. In some embodiments, the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g. scFv) and intracellular signaling domain. Thus, in some embodiments, the antigen-binding component (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains.
[0437] In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
[0438] The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 (4-1BB), or CD154. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and/or transmembrane domain(s). In some aspects, the transmembrane domain contains a transmembrane portion of CD28 or a variant thereof. The extracellular domain and transmembrane can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein.
[0439] In some embodiments, the transmembrane domain of the receptor, e.g., the CAR is a transmembrane domain of human CD28 or variant thereof, e.g., a 27-amino acid transmembrane domain of a human CD28 (Accession No.: P10747.1), or is a transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 8 or a sequence of amino acids that exhibits at least or at least about85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8. In some embodiments, the transmembrane-domain containing portion of the recombinant receptor comprises the sequence of amino acids set forth in SEQ ID NO: 9 or a sequence of amino acids having at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
[0440] In some embodiments, the recombinant receptor, e.g., CAR, includes at least one intracellular signaling component or components, such as an intracellular signaling region or domain. T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components. Among the intracellular signaling region are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and/or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.
[0441] In some embodiments, upon ligation of the CAR, the cytoplasmic domain or intracellular signaling region of the CAR activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling region of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling regions, e.g., comprising intracellular domain or domains, include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capability. In some embodiments, the intracellular signaling regions, e.g., comprising intracellular domain or domains, include the cytoplasmic sequences of a region or domain that is involved in providing costimulatory signal.
[0442] In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine -based activation motifs or IT AMs. Examples of IT AM containing primary cytoplasmic signaling sequences include those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.
[0443] In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen-binding portion is linked to one or more cell signaling modules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and/or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor y, CD8alpha, CD8beta, CD4, CD25, or CD16. For example, in some aspects, the CAR or other chimeric receptor includes a chimeric molecule between CD3-zeta (CD3-Q or Fc receptor y and CD8alpha, CD8beta, CD4, CD25 or CD 16.
[0444] In some embodiments, the intracellular (or cytoplasmic) signaling region comprises a human CD3 chain, optionally a CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD3^ (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Patent No.: 7,446,190 or U.S. Patent No. 8,911,993. In some embodiments, the intracellular signaling region comprises the sequence of amino acids set forth in SEQ ID NO: 13, 14 or 15 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, 14 or 15. In some embodiments, the intracellular signaling region comprises the sequence of amino acids set forth in SEQ ID NO: 13 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13. In some embodiments, the intracellular signaling region comprises the sequence of amino acids set forth in SEQ ID NO: 13.
[0445] In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.
[0446] In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some embodiments, the CAR includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, 0X40 (CD134), CD27, DAP10, DAP12, ICOS and/or other costimulatory receptors. In some embodiments, the CAR includes a costimulatory region or domain of CD28 or 4-1BB, such as of human CD28 or human 4-1BB.
[0447] In some embodiments, the intracellular signaling region or domain comprises an intracellular costimulatory signaling domain of human CD28 or functional variant or portion thereof, such as a 41 amino acid domain thereof and/or such a domain with an LL to GG substitution at positions 186-187 of a native CD28 protein. In some embodiments, the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 10 or 11 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10 or 11. In some embodiments, the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 10 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10. In some embodiments, the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 11 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 11. In some embodiments, the intracellular region comprises an intracellular costimulatory signaling domain of 4- IBB or functional variant or portion thereof, such as a 42-amino acid cytoplasmic domain of a human 4-1BB (Accession No. Q07011.1) or functional variant or portion thereof, such as the sequence of amino acids set forth in SEQ ID NO: 12 or a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12. In some embodiments, the intracellular region comprises an intracellular costimulatory signaling domain comprising the sequence of amino acids set forth in SEQ ID NO: 12.
[0448] In some aspects, the same CAR includes both the primary (or activating) cytoplasmic signaling regions and costimulatory signaling components.
[0449] In some embodiments, the activating domain is included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014/055668). In some aspects, the cells include one or more stimulatory or activating CAR and/or a costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sei. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than the one associated with and/or specific for the disease or condition whereby an activating signal delivered through the disease-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.
[0450] In some embodiments, the two receptors induce, respectively, an activating and an inhibitory signal to the cell, such that ligation of one of the receptor to its antigen activates the cell or induces a response, but ligation of the second inhibitory receptor to its antigen induces a signal that suppresses or dampens that response. Examples are combinations of activating CARs and inhibitory CARs (iCARs). Such a strategy may be used, for example, to reduce the likelihood of off-target effects in the context in which the activating CAR binds an antigen expressed in a disease or condition but which is also expressed on normal cells, and the inhibitory receptor binds to a separate antigen which is expressed on the normal cells but not cells of the disease or condition.
[0451] In some aspects, the chimeric receptor is or includes an inhibitory CAR (e.g. iCAR) and includes intracellular components that dampen or suppress an immune response, such as an ITAM- and/or co stimulatory-promoted response in the cell. Exemplary of such intracellular signaling components are those found on immune checkpoint molecules, including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptors, EP2/4 Adenosine receptors including A2AR. In some aspects, the engineered cell includes an inhibitory CAR including a signaling domain of or derived from such an inhibitory molecule, such that it serves to dampen the response of the cell, for example, that induced by an activating and/or costimulatory CAR.
[0452| In some cases, CARs are referred to as first, second, and/or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3-chain induced signal upon antigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD 137; in some aspects, a third generation CAR in some aspects is one that includes multiple costimulatory domains of different costimulatory receptors.
[0453] In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.
[0454] In some embodiments, the antigen receptor further includes a marker and/or cells expressing the CAR or other antigen receptor further includes a surrogate marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor. In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor, such as truncated version of such a cell surface receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. For example, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence.
[0455| An exemplary polypeptide for a truncated EGFR (e.g. tEGFR) comprises the sequence of amino acids set forth in SEQ ID NO: 7 or 16 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7 or 16. An exemplary T2A linker sequence comprises the sequence of amino acids set forth in SEQ ID NO: 6 or 17 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 6 or 17.
[0456] In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof. In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self’ by the immune system of the host into which the cells will be adoptively transferred.
[0457] In some embodiments, the marker serves no therapeutic function and/or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and/or dampen responses of the cells upon adoptive transfer and encounter with ligand.
[0458| In some embodiments, the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some embodiments, the CAR contains an antibody, e.g., antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4-1BB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some such embodiments, the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a hinge -only spacer.
[0459] In some embodiments, the transmembrane domain of the recombinant receptor, e.g., the CAR, is or includes a transmembrane domain of human CD28 (e.g. Accession No. P01747.1) or variant thereof, such as a transmembrane domain that comprises the sequence of amino acids set forth in SEQ ID NO: 8 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8; in some embodiments, the transmembrane -domain containing portion of the recombinant receptor comprises the sequence of amino acids set forth in SEQ ID NO: 9 or a sequence of amino acids having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the transmembrane domain comprises the sequence of amino acids set forth in SEQ ID NO: 8 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8. In some embodiments, the transmembrane domain comprises the sequence of amino acids set forth in SEQ ID NO: 8. In some embodiments, the transmembrane domain comprises the sequence of amino acids set forth in SEQ ID NO: 9 or a sequence of amino acids having at least at or about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. In some embodiments, the transmembrane domain comprises the sequence of amino acids set forth in SEQ ID NO: 9.
[0460] In some embodiments, the intracellular signaling component(s) of the recombinant receptor, e.g. the CAR, contains an intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof, such as a domain with an LL to GG substitution at positions 186-187 of a native CD28 protein. For example, the intracellular signaling domain can comprise the sequence of amino acids set forth in SEQ ID NO: 10 or 11 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 10 or 11. In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of 4-1BB (e.g. (Accession No. Q07011.1) or functional variant or portion thereof, such as the sequence of amino acids set forth in SEQ ID NO: 12 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12. In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain comrpising the sequence of amino acids set forth in SEQ ID NO: 12 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12. In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain comrpising the sequence of amino acids set forth in SEQ ID NO: 12.
[0461] In some embodiments, the intracellular signaling domain of the recombinant receptor, e.g. the CAR, comprises a human CD3 zeta stimulatory signaling domain or functional variant thereof, such as an 112 AA cytoplasmic domain of isoform 3 of human CD3^ (Accession No.: P20963.2) or a CD3 zeta signaling domain as described in U.S. Patent No.: 7,446,190 or U.S. Patent No. 8,911,993. For example, in some embodiments, the intracellular signaling domain comprises the sequence of amino acids as set forth in SEQ ID NO: 13, 14 or 15 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, 14 or 15. In some embodiments, the intracellular signaling domain comprises the sequence of amino acids as set forth in SEQ ID NO: 13 or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13. In some embodiments, the intracellular signaling domain comprises the sequence of amino acids as set forth in SEQ ID NO: 13.
[0462| In some embodiments, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgGl, such as the hinge only spacer set forth in SEQ ID NO: 1, and encoded by the sequence set forth in SEQ ID NO: 2. In some embodiments, the spacer is an Ig hinge, e.g., and IgG4 hinge, linked to a CH2 and/or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains, such as set forth in SEQ ID NO: 4. In some embodiments, the spacer the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to a CH3 domain only, such as set forth in SEQ ID NO: 3. In some embodiments, the spacer is or comprises a glycine-serine rich sequence or other flexible linker such as known flexible linkers. In some embodiments, the constant region or portion is of IgD. In some embodiments, the spacer has the sequence set forth in SEQ ID NO: 5. In some embodiments, the spacer has a sequence of amino acids that exhibits at least or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOS: 1, 3, 4 and 5.
[0463] For example, in some embodiments, the CAR includes an antibody such as an antibody fragment, including scFvs, a spacer, such as a spacer containing a portion of an immunoglobulin molecule, such as a hinge region and/or one or more constant regions of a heavy chain molecule, such as an Ig-hinge containing spacer, a transmembrane domain containing all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR includes an antibody or fragment, such as scFv, a spacer such as any of the Ig-hinge containing spacers, a CD28-derived transmembrane domain, a 4-lBB-derived intracellular signaling domain, and a CD3 zeta-derived signaling domain.
[0464] In some embodiments, nucleic acid molecules encoding such CAR constructs further includes a sequence encoding a T2A ribosomal skip element and/or a tEGFR sequence, e.g., downstream of the sequence encoding the CAR. In some embodiments, the sequence encodes a T2A ribosomal skip element set forth in SEQ ID NO: 6 or 17, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 6 or 17. In some embodiments, T cells expressing an antigen receptor (e.g. CAR) can also be generated to express a truncated EGFR (EGFRt) as a non-immunogenic selection epitope (e.g. by introduction of a construct encoding the CAR and EGFRt separated by a T2A ribosome switch to express two proteins from the same construct), which then can be used as a marker to detect such cells (see e.g. U.S. Patent No. 8,802,374). In some embodiments, the sequence encodes an tEGFR sequence set forth in SEQ ID NO: 7 or 16, or a sequence of amino acids that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7 or 16. In some cases, the peptide, such as T2A, can cause the ribosome to skip (ribosome skipping) synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream (see, for example, de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004)). Many 2A elements are known. Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein, without limitation, 2A sequences from the foot-and-mouth disease virus (F2A, e.g., SEQ ID NO: 21), equine rhinitis A virus (E2A, e.g., SEQ ID NO: 20), Thosea asigna virus (T2A, e.g., SEQ ID NO: 6 or 17), and porcine teschovirus-1 (P2A, e.g., SEQ ID NO: 18 or 19) as described in U.S. Patent Publication No.
20070116690.
[0465] In some of any of the embodiments, the CAR comprises, in order, an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is or comprises a 4- IBB, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is or comprises a CD3zeta signaling domain and optionally further includes a spacer between the transmembrane domain and the scFv;
[0466] In some of any of the embodiments, the CAR includes, in order, an scFv specific for the antigen, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is or comprises a 4- IBB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is a CD3zeta signaling domain.
[0467] In some of any of the embodiments, the CAR comprises or consists of, in order, an scFv specific for the antigen, a spacer, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4-1BB signaling domain, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is or comprises a CD3zeta signaling domain.In some aspects, the spacer is a polypeptide spacer that (a) comprises or consists of all or a portion of an immunoglobulin hinge or a modified version thereof or comprises about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region, (b) comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4 hinge, or a modified version thereof and/or comprises about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region, or (c) is at or about 12 amino acids in length and/or comprises or consists of all or a portion of an immunoglobulin hinge, optionally an IgG4, or a modified version thereof; or (d) has or consists of the sequence of SEQ ID NO: 1, a sequence encoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, or (e) comprises or consists of the formula X1PPX2P, where Xi is glycine, cysteine or arginine and X2 is cysteine or threonine; and/or the costimulatory domain comprises SEQ ID NO: 12 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and/or the primary signaling domain comprises SEQ ID NO: 13 or 14 or 15 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and/orthe scFv comprises a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and/or a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37) and/or a CDRH1 sequence of DYGVS (SEQ ID NO: 38), a CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and/or a CDRH3 sequence of YAMDYWG (SEQ ID NO: 40) or wherein the scFv comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC63 and/or a CDRL1 sequence of FMC63, a CDRL2 sequence of FMC63, a CDRL3 sequence of FMC63, a CDRH1 sequence of FMC63, a CDRH2 sequence of FMC63, and a CDRH3 sequence of FMC63 or binds to the same epitope as or competes for binding with any of the foregoing, and optionally wherein the scFv comprises, in order, a VH, a linker, optionally comprising SEQ ID NO: 59, and a VL, and/or the scFv comprises a flexible linker and/or comprises the amino acid sequence set forth as SEQ ID NO: 59.
[0468] In some embodiments, the spacer comprises or consists of SEQ ID NO: 1, the costimulatory domain comprises SEQ ID NO: 12 or variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, the transmembrane domain is of CD28 or comprises SEQ ID NO: 9 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, the scFv contains the binding domain of or CDRs of or VH and VL of FMC63, the primary signaling domain contains SEQ ID NO: 13, 14, or 15, and/or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
[0469] In some embodiments, the spacer comprises or consists of SEQ ID NO: 30, the costimulatory domain comprises SEQ ID NO: 12 or variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, the transmembrane domain is of CD28 or comprises SEQ ID NO: 9 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, the scFv contains the binding domain of or CDRs of or VH and VL of FMC63, the primary signaling domain contains SEQ ID NO: 13, 14, or 15, and/or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
[0470] In some embodiments, the spacer comprises or consists of SEQ ID NO: 31, the costimulatory domain comprises SEQ ID NO: 12 or variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, the transmembrane domain is of CD28 or comprises SEQ ID NO: 9 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, the scFv contains the binding domain of or CDRs of or VH and VL of FMC63, the primary signaling domain contains SEQ ID NO: 13, 14, or 15, and/or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
[0471] In some embodiments, the spacer comprises or consists of SEQ ID NO: 33, the costimulatory domain comprises SEQ ID NO: 12 or variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, the transmembrane domain is of CD28 or comprises SEQ ID NO: 9 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, the scFv contains the binding domain of or CDRs of or VH and VL of FMC63, the primary signaling domain contains SEQ ID NO: 13, 14, or 15, and/or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
[0472] In some embodiments, the spacer comprises or consists of SEQ ID NO: 34, the costimulatory domain comprises SEQ ID NO: 12 or variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, the transmembrane domain is of CD28 or comprises SEQ ID NO: 9 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, the scFv contains the binding domain of or CDRs of or VH and VL of FMC63, the primary signaling domain contains SEQ ID NO: 13, 14, or 15, and/or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto.
[0473] The recombinant receptors, such as CARs, expressed by the cells administered to the subject generally recognize or specifically bind to a molecule that is expressed in, associated with, and/or specific for the disease or condition or cells thereof being treated. Upon specific binding to the molecule, e.g., antigen, the receptor generally delivers an immunostimulatory signal, such as an ITAM-transduced signal, into the cell, thereby promoting an immune response targeted to the disease or condition. For example, in some embodiments, the cells express a CAR that specifically binds to an antigen expressed by a cell or tissue of the disease or condition or associated with the disease or condition.
2. / CELL RECEPTORS
[0474] In some embodiments, engineered cells, such as T cells, used in connection with the provided methods, uses, articles of manufacture or compositions are cells that express a T cell receptor (TCR) or antigen-binding portion thereof that recognizes an peptide epitope or T cell epitope of a target polypeptide, such as an antigen of a tumor, viral or autoimmune protein.
[0475] In some embodiments, a “T cell receptor” or “TCR” is a molecule that contains a variable a and (3 chains (also known as TCRa and TCR , respectively) or a variable y and 5 chains (also known as TCRa and TCR , respectively), or antigen-binding portions thereof, and which is capable of specifically binding to a peptide bound to an MHC molecule. In some embodiments, the TCR is in the a form. Typically, TCRs that exist in aP and y5 forms are generally structurally similar, but T cells expressing them may have distinct anatomical locations or functions. A TCR can be found on the surface of a cell or in soluble form. Generally, a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.
[0476] Unless otherwise stated, the term “TCR” should be understood to encompass full TCRs as well as antigen-binding portions or antigen-binding fragments thereof. In some embodiments, the TCR is an intact or full-length TCR, including TCRs in the a[3 form or y5 form. In some embodiments, the TCR is an antigen-binding portion that is less than a full-length TCR but that binds to a specific peptide bound in an MHC molecule, such as binds to an MHC -peptide complex. In some cases, an antigen-binding portion or fragment of a TCR can contain only a portion of the structural domains of a full-length or intact TCR, but yet is able to bind the peptide epitope, such as MHC-peptide complex, to which the full TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as variable a chain and variable [3 chain of a TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex. Generally, the variable chains of a TCR contain complementarity determining regions involved in recognition of the peptide, MHC and/or MHC-peptide complex.
[0477] In some embodiments, the variable domains of the TCR contain hypervariable loops, or complementarity determining regions (CDRs), which generally are the primary contributors to antigen recognition and binding capabilities and specificity. In some embodiments, a CDR of a TCR or combination thereof forms all or substantially all of the antigen-binding site of a given TCR molecule. The various CDRs within a variable region of a TCR chain generally are separated by framework regions (FRs), which generally display less variability among TCR molecules as compared to the CDRs (see, e.g., Jores et al., Proc. Nat’l Acad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the main CDR responsible for antigen binding or specificity, or is the most important among the three CDRs on a given TCR variable region for antigen recognition, and/or for interaction with the processed peptide portion of the peptide -MHC complex. In some contexts, the CDR1 of the alpha chain can interact with the N- terminal part of certain antigenic peptides. In some contexts, CDR1 of the beta chain can interact with the C-terminal part of the peptide. In some contexts, CDR2 contributes most strongly to or is the primary CDR responsible for the interaction with or recognition of the MHC portion of the MHC-peptide complex. In some embodiments, the variable region of the [3-chain can contain a further hypervariable region (CDR4 or HVR4), which generally is involved in superantigen binding and not antigen recognition (Kotb (1995) Clinical Microbiology Reviews, 8:411-426).
[0478] In some embodiments, a TCR also can contain a constant domain, a transmembrane domain and/or a short cytoplasmic tail (see, e.g., Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997). In some aspects, each chain of the TCR can possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end. In some embodiments, a TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction.
[0479| In some embodiments, a TCR chain contains one or more constant domain. For example, the extracellular portion of a given TCR chain (e.g., a-chain or [3-chain) can contain two immunoglobulin- like domains, such as a variable domain (e.g., Va or V|3; typically amino acids 1 to 116 based on Kabat numbering Kabat et al., “Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.) and a constant domain (e.g., a-chain constant domain or Ca, typically positions 117 to 259 of the chain based on Kabat numbering or |3 chain constant domain or Cp, typically positions 117 to 295 of the chain based on Kabat) adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two membrane -proximal constant domains, and two membrane -distal variable domains, which variable domains each contain CDRs. The constant domain of the TCR may contain short connecting sequences in which a cysteine residue forms a disulfide bond, thereby linking the two chains of the TCR. In some embodiments, a TCR may have an additional cysteine residue in each of the a and [3 chains, such that the TCR contains two disulfide bonds in the constant domains.
[0480] In some embodiments, the TCR chains contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules like CD3 and subunits thereof. For example, a TCR containing constant domains with a transmembrane region may anchor the protein in the cell membrane and associate with invariant subunits of the CD3 signaling apparatus or complex. The intracellular tails of CD3 signaling subunits (e.g. CD3y, CD35, CD3s and CD3^ chains) contain one or more immunoreceptor tyrosine -based activation motif or IT AM that are involved in the signaling capacity of the TCR complex.
[0481] In some embodiments, the TCR may be a heterodimer of two chains a and [3 (or optionally y and 5) or it may be a single chain TCR construct. In some embodiments, the TCR is a heterodimer containing two separate chains (a and [3 chains or y and 5 chains) that are linked, such as by a disulfide bond or disulfide bonds.
[0482| In some embodiments, the TCR can be generated from a known TCR sequence(s), such as sequences of Va,[3 chains, for which a substantially full-length coding sequence is readily available. Methods for obtaining full-length TCR sequences, including V chain sequences, from cell sources are well known. In some embodiments, nucleic acids encoding the TCR can be obtained from a variety of sources, such as by polymerase chain reaction (PCR) amplification of TCR-encoding nucleic acids within or isolated from a given cell or cells, or synthesis of publicly available TCR DNA sequences. [0483] In some embodiments, the TCR is obtained from a biological source, such as from cells such as from a T cell (e.g. cytotoxic T cell), T-cell hybridomas or other publicly available source. In some embodiments, the T-cells can be obtained from in vivo isolated cells. In some embodiments, the TCR is a thymically selected TCR. In some embodiments, the TCR is a neoepitope -restricted TCR. In some embodiments, the T- cells can be a cultured T-cell hybridoma or clone. In some embodiments, the TCR or antigen-binding portion thereof or antigen-binding fragment thereof can be synthetically generated from knowledge of the sequence of the TCR.
[0484] In some embodiments, the TCR is generated from a TCR identified or selected from screening a library of candidate TCRs against a target polypeptide antigen, or target T cell epitope thereof. TCR libraries can be generated by amplification of the repertoire of Va and V(3 from T cells isolated from a subject, including cells present in PBMCs, spleen or other lymphoid organ. In some cases, T cells can be amplified from tumor-infiltrating lymphocytes (TILs). In some embodiments, TCR libraries can be generated from CD4+ or CD8+ cells. In some embodiments, the TCRs can be amplified from a T cell source of a normal of healthy subject, i.e. normal TCR libraries. In some embodiments, the TCRs can be amplified from a T cell source of a diseased subject, i.e. diseased TCR libraries. In some embodiments, degenerate primers are used to amplify the gene repertoire of Va and V(3, such as by RT- PCR in samples, such as T cells, obtained from humans. In some embodiments, scTv libraries can be assembled from naive Va and V(3 libraries in which the amplified products are cloned or assembled to be separated by a linker. Depending on the source of the subject and cells, the libraries can be HLA allelespecific. Alternatively, in some embodiments, TCR libraries can be generated by mutagenesis or diversification of a parent or scaffold TCR molecule. In some aspects, the TCRs are subjected to directed evolution, such as by mutagenesis, e.g., of the a or (3 chain. In some aspects, particular residues within CDRs of the TCR are altered. In some embodiments, selected TCRs can be modified by affinity maturation. In some embodiments, antigen-specific T cells may be selected, such as by screening to assess CTL activity against the peptide. In some aspects, TCRs, e.g. present on the antigen-specific T cells, may be selected, such as by binding activity, e.g., particular affinity or avidity for the antigen.
[0485] In some embodiments, the TCR or antigen-binding portion thereof is one that has been modified or engineered. In some embodiments, directed evolution methods are used to generate TCRs with altered properties, such as with higher affinity for a specific MHC -peptide complex. In some embodiments, directed evolution is achieved by display methods including, but not limited to, yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci U S A, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), or T cell display (Chervin et al. (2008) J Immunol Methods, 339, 175-84). In some embodiments, display approaches involve engineering, or modifying, a known, parent or reference TCR. For example, in some cases, a wild-type TCR can be used as a template for producing mutagenized TCRs in which in one or more residues of the CDRs are mutated, and mutants with an desired altered property, such as higher affinity for a desired target antigen, are selected.
[0486] In some embodiments, peptides of a target polypeptide for use in producing or generating a TCR of interest are known or can be readily identified. In some embodiments, peptides suitable for use in generating TCRs or antigen-binding portions can be determined based on the presence of an HLA- restricted motif in a target polypeptide of interest, such as a target polypeptide described below. In some embodiments, peptides are identified using available computer prediction models. In some embodiments, for predicting MHC class I binding sites, such models include, but are not limited to, ProPredl (Singh and Raghava (2001) Bioinformatics 17(12): 1236-1237, and SYFPEITHI (see Schuler et al. (2007) Immunoinformatics Methods in Molecular Biology, 409(1): 75-93 2007). In some embodiments, the MHC -restricted epitope is HLA-A0201, which is expressed in approximately 39-46% of all Caucasians and therefore, represents a suitable choice of MHC antigen for use preparing a TCR or other MHC-peptide binding molecule.
[0487] HLA-A0201 -binding motifs and the cleavage sites for proteasomes and immune - proteasomes using computer prediction models are known. For predicting MHC class I binding sites, such models include, but are not limited to, ProPredl (described in more detail in Singh and Raghava, ProPred: prediction of HLA-DR binding sites. BIOINFORMATICS 17(12): 1236-1237 2001), and SYFPEITHI (see Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction, in Immunoinformatics Methods in Molecular Biology, vol 409(1): 75-93 2007)
[0488] In some embodiments, the TCR or antigen binding portion thereof may be a recombinantly produced natural protein or mutated form thereof in which one or more property, such as binding characteristic, has been altered. In some embodiments, a TCR may be derived from one of various animal species, such as human, mouse, rat, or other mammal. A TCR may be cell-bound or in soluble form. In some embodiments, for purposes of the provided methods, the TCR is in cell-bound form expressed on the surface of a cell.
[0489] In some embodiments, the TCR is a full-length TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some embodiments, the TCR is a single -chain TCR (sc-TCR). In some embodiments, a dTCR or scTCR have the structures as described in WO 03/020763, WO 04/033685, WO2011/044186.
[0490] In some embodiments, the TCR contains a sequence corresponding to the transmembrane sequence. In some embodiments, the TCR does contain a sequence corresponding to cytoplasmic sequences. In some embodiments, the TCR is capable of forming a TCR complex with CD3. In some embodiments, any of the TCRs, including a dTCR or scTCR, can be linked to signaling domains that yield an active TCR on the surface of a T cell. In some embodiments, the TCR is expressed on the surface of cells. [0491] In some embodiments a dTCR contains a first polypeptide wherein a sequence corresponding to a TCR a chain variable region sequence is fused to the N terminus of a sequence corresponding to a TCR a chain constant region extracellular sequence, and a second polypeptide wherein a sequence corresponding to a TCR P chain variable region sequence is fused to the N terminus a sequence corresponding to a TCR chain constant region extracellular sequence, the first and second polypeptides being linked by a disulfide bond. In some embodiments, the bond can correspond to the native interchain disulfide bond present in native dimeric aP TCRs. In some embodiments, the interchain disulfide bonds are not present in a native TCR. For example, in some embodiments, one or more cysteines can be incorporated into the constant region extracellular sequences of dTCR polypeptide pair. In some cases, both a native and a non-native disulfide bond may be desirable. In some embodiments, the TCR contains a transmembrane sequence to anchor to the membrane.
[0492] In some embodiments, a dTCR contains a TCR a chain containing a variable a domain, a constant a domain and a first dimerization motif attached to the C-terminus of the constant a domain, and a TCR P chain comprising a variable P domain, a constant P domain and a first dimerization motif attached to the C-terminus of the constant P domain, wherein the first and second dimerization motifs easily interact to form a covalent bond between an amino acid in the first dimerization motif and an amino acid in the second dimerization motif linking the TCR a chain and TCR P chain together.
[0493] In some embodiments, the TCR is a scTCR. Typically, a scTCR can be generated using methods known, See e.g., Soo Hoo, W. F. et al. PNAS (USA) 89, 4759 (1992); Wiilfing, C. and Pliickthun, A., J. Mol. Biol. 242, 655 (1994); Kurucz, I. et al. PNAS (USA) 90 3830 (1993); International published PCT Nos. WO 96/13593, WO 96/18105, W099/60120, WO99/18129, WO 03/020763, WO2011/044186; and Schlueter, C. J. et al. J. Mol. Biol. 256, 859 (1996). In some embodiments, a scTCR contains an introduced non-native disulfide interchain bond to facilitate the association of the TCR chains (see e.g. International published PCT No. WO 03/020763). In some embodiments, a scTCR is a non-disulfide linked truncated TCR in which heterologous leucine zippers fused to the C-termini thereof facilitate chain association (see e.g. International published PCT No. W099/60120). In some embodiments, a scTCR contain a TCRa variable domain covalently linked to a TCRP variable domain via a peptide linker (see e.g., International published PCT No. WO99/18129).
[0494] In some embodiments, a scTCR contains a first segment constituted by an amino acid sequence corresponding to a TCR a chain variable region, a second segment constituted by an amino acid sequence corresponding to a TCR P chain variable region sequence fused to the N terminus of an amino acid sequence corresponding to a TCR P chain constant domain extracellular sequence, and a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
[0495] In some embodiments, a scTCR contains a first segment constituted by an a chain variable region sequence fused to the N terminus of an a chain extracellular constant domain sequence, and a second segment constituted by a P chain variable region sequence fused to the N terminus of a sequence P chain extracellular constant and transmembrane sequence, and, optionally, a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
[0496] In some embodiments, a scTCR contains a first segment constituted by a TCR chain variable region sequence fused to the N terminus of a P chain extracellular constant domain sequence, and a second segment constituted by an a chain variable region sequence fused to the N terminus of a sequence a chain extracellular constant and transmembrane sequence, and, optionally, a linker sequence linking the C terminus of the first segment to the N terminus of the second segment.
[0497] In some embodiments, the linker of a scTCRs that links the first and second TCR segments can be any linker capable of forming a single polypeptide strand, while retaining TCR binding specificity. In some embodiments, the linker sequence may, for example, have the formula -P-AA-P- wherein P is proline and AA represents an amino acid sequence wherein the amino acids are glycine and serine. In some embodiments, the first and second segments are paired so that the variable region sequences thereof are orientated for such binding. Hence, in some cases, the linker has a sufficient length to span the distance between the C terminus of the first segment and the N terminus of the second segment, or vice versa, but is not too long to block or reduces bonding of the scTCR to the target ligand. In some embodiments, the linker can contain from or from about 10 to 45 amino acids, such as 10 to 30 amino acids or 26 to 41 amino acids residues, for example 29, 30, 31 or 32 amino acids. In some embodiments, the linker has the formula -PGGG-(SGGGG)5-P- wherein P is proline, G is glycine and S is serine (SEQ ID NO:22). In some embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO:23)
[0498| In some embodiments, the scTCR contains a covalent disulfide bond linking a residue of the immunoglobulin region of the constant domain of the a chain to a residue of the immunoglobulin region of the constant domain of the P chain. In some embodiments, the interchain disulfide bond in a native TCR is not present. For example, in some embodiments, one or more cysteines can be incorporated into the constant region extracellular sequences of the first and second segments of the scTCR polypeptide. In some cases, both a native and a non-native disulfide bond may be desirable.
[0499] In some embodiments of a dTCR or scTCR containing introduced interchain disulfide bonds, the native disulfide bonds are not present. In some embodiments, the one or more of the native cysteines forming a native interchain disulfide bonds are substituted to another residue, such as to a serine or alanine. In some embodiments, an introduced disulfide bond can be formed by mutating non-cysteine residues on the first and second segments to cysteine. Exemplary non-native disulfide bonds of a TCR are described in published International PCT No. W02006/000830.
[0500] In some embodiments, the TCR or antigen-binding fragment thereof exhibits an affinity with an equilibrium binding constant for a target antigen of between or between about 10-5 and 10-12 M and all individual values and ranges therein. In some embodiments, the target antigen is an MHC-peptide complex or ligand. [0501] In some embodiments, nucleic acid or nucleic acids encoding a TCR, such as a and [3 chains, can be amplified by PCR, cloning or other suitable means and cloned into a suitable expression vector or vectors. The expression vector can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses.
[0502] In some embodiments, the vector can a vector of the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), or the pEX series (Clontech, Palo Alto, Calif.). In some cases, bacteriophage vectors, such as G I 0. XGT11, XZapII (Stratagene), XEMBL4, and XNM1149, also can be used. In some embodiments, plant expression vectors can be used and include pBIOl, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). In some embodiments, animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). In some embodiments, a viral vector is used, such as a retroviral vector.
[0503] In some embodiments, the recombinant expression vectors can be prepared using standard recombinant DNA techniques. In some embodiments, vectors can contain regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate and taking into consideration whether the vector is DNA- or RNA-based. In some embodiments, the vector can contain a nonnative promoter operably linked to the nucleotide sequence encoding the TCR or antigenbinding portion (or other MHC -peptide binding molecule). In some embodiments, the promoter can be a non-viral promoter or a viral promoter, such as a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV promoter, and a promoter found in the long-terminal repeat of the murine stem cell virus. Other known promoters also are contemplated.
[0504] In some embodiments, to generate a vector encoding a TCR, the a and [3 chains are PCR amplified from total cDNA isolated from a T cell clone expressing the TCR of interest and cloned into an expression vector. In some embodiments, the a and [3 chains are cloned into the same vector. In some embodiments, the a and [3 chains are cloned into different vectors. In some embodiments, the generated a and [3 chains are incorporated into a retroviral, e.g. lentiviral, vector.
B. METHODS OF ENGINEERING
[0505] In some embodiments, the provided methods involve administering to a subject having a disease or condition (e.g. a cancer such as CLL or SLL) cells expressing a recombinant antigen receptor. Various methods for the introduction of genetically engineered components, e.g., recombinant receptors, e.g., CARs or TCRs, are well known and may be used with the provided methods and compositions. Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation. [0506] Among the cells expressing the receptors and administered by the provided methods are engineered cells. The genetic engineering generally involves introduction of a nucleic acid encoding the recombinant or engineered component into a composition containing the cells, such as by retroviral transduction, transfection, or transformation.
[0507] In particular embodiments, the engineered cells are produced by a process that generates an output composition of enriched T cells from one or more input compositions and/or from a single biological sample. In certain embodiments, the output composition contains cells that express a recombinant receptor, e.g., a CAR, such as an anti-CD19 CAR. In particular embodiments, the cells of the output compositions are suitable for administration to a subject as a therapy, e.g., an autologous cell therapy. In some embodiments, the output composition is a composition of enriched CD4+ or CD8+ T cells. In some embodiments, the output composition is a composition of enriched CD4+ and CD8+ T cells.
[0508] In some embodiments, the process for generating or producing engineered cells is by a process that includes some or all of the steps of: collecting or obtaining a biological sample; isolating, selecting, or enriching input cells from the biological sample; cry opreserving and storing the input cells; thawing and/or incubating the input cells under stimulating conditions; engineering the stimulated cells to express or contain a recombinant polynucleotide, e.g., a polynucleotide encoding a recombinant receptor such as a CAR; cultivating the engineered cells, e.g. to a threshold amount, density, or expansion; formulating the cultivated cells in an output composition; and/or cryopreserving and storing the formulated output cells until the cells are released for infusion and/or are suitable to be administered to a subject. In certain embodiments, the process is performed with two or more input compositions of enriched T cells, such as a separate CD4+ composition and a separate CD8+ composition, that are separately processed and engineered from the same starting or initial biological sample and re -infused back into the subject at a defined ratio, e.g. 1:1 ratio of CD4+ to CD8+ T cells. In some embodiments, the enriched T cells are or include engineered T cells, e.g., T cells transduced to express a recombinant receptor.
[0509] In particular embodiments, an output composition of engineered cells expressing a recombinant receptor (e.g. anti-CD19 CAR) is produced from an initial and/or input composition of cells. In some embodiments, the input composition is a composition of enriched CD3+ T cells, enriched CD4+ T cells, and/or enriched CD8+ T cells (herein after also referred to as compositions of enriched T cells, compositions of enriched CD4+ T cells, and compositions of enriched CD8+ T cells, respectively). In some embodiments, a composition enriched in CD4+ T cells contains at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% CD4+ T cells. In particular embodiments, the composition of enriched CD4+ T cells contains about 100% CD4+ T cells. In certain embodiments, the composition of enriched CD4+T cells includes or contains less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and/or contains no CD8+ T cells, and/or is free or substantially free of CD8+ T cells. In some embodiments, the populations of enriched CD4+T cells consist essentially of CD4+ T cells. In some embodiments, a composition enriched in CD8+ T cells contains at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% CD8+ T cells, or contains or contains about 100% CD8+ T cells. In certain embodiments, the composition of enriched CD8+ T cells includes or contains less than 20%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and/or contains no CD4+ T cells, and/or is free or substantially free of CD4+ T cells. In some embodiments, the populations of enriched CD8+T cells consist essentially of CD8+ T cells.
10510] In some embodiments, a composition enriched in CD3+ T cells contains at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% CD3+ T cells. In particular embodiments, the composition of enriched CD3+ T cells contains about 100% CD3+ T cells. In certain embodiments, the composition of enriched CD3+T cells includes CD4+ and CD8+ T cells that are at a ratio of CD4+ T cells to CD8+ T cells of between approximately 1:3 and approximately 3:1, such as approximately 1:1. In some embodiments, the ratio is between approximately 1:3 and approximately 3:1. In some embodiments, the ratio is approximately 1:1.
[0511] In certain embodiments, the process for producing engineered cells further can include one or more of: activating and/or stimulating a cells, e.g., cells of an input composition; genetically engineering the activated and/or stimulated cells, e.g., to introduce a polynucleotide encoding a recombinant protein by transduction or transfection; and/or cultivating the engineered cells, e.g., under conditions that promote proliferation and/or expansion. In particular embodiments, the provided methods may be used in connection with harvesting, collecting, and/or formulating output compositions produced after the cells have been incubated, activated, stimulated, engineered, transduced, transfected, and/or cultivated.
[0512] In some embodiments, engineered cells, such as those that express an anti-CD19 CAR, used in accord with the provided methods are produced or generated by a process for selecting, isolating, activating, stimulating, expanding, cultivating, and/or formulating cells. In some embodiments, such methods include any as described.
[0513] In some embodiments, at least one separate composition of enriched CD4+ T cells and at least one separate composition of enriched CD8+ T cells are isolated, selected, enriched, or obtained from a single biological sample, e.g., a sample of PBMCs or other white blood cells from the same donor such as a patient or healthy individual. In some embodiments, a separate composition of enriched CD4+ T cells and a separate composition of enriched CD8+ T cells originated, e.g., are initially isolated, selected, and/or enriched, from the same biological sample, such as a single biological sample obtained, collected, and/or taken from a single subject. In some embodiments, a biological sample is first subjected to selection of CD4+ T cells, where both the negative and positive fractions are retained, and the negative fraction is further subjected to selection of CD8+ T cells. In other embodiments, a biological sample is first subjected to selection of CD8+ T cells, where both the negative and positive fractions are retained, and the negative fraction is further subjected to selection of CD4+ T cells. In some embodiments, methods of selection are carried out as described in International PCT publication No. WO2015/164675. In some aspects, a biological sample is first positively selected for CD8+ T cells to generate at least one composition of enriched CD8+ T cells, and the negative fraction is then positively selected for CD4+ T cells to generate at least one composition of enriched CD4+ T cells, such that the at least one composition of enriched CD8+ T cells and the at least one composition of enriched CD4+ T cells are separate compositions from the same biological sample, e.g., from the same donor patient or healthy individual. In some aspects, two or more separate compositions of enriched T cells, e.g., at least one being a composition of enriched CD4+ T cells and at least one being a separate composition of enriched CD8+ T cells from the same donor, are separately frozen, e.g., cryoprotected or cryopreserved in a cryopreservation media.
[0514] In some aspects, two or more separate compositions of enriched T cells, e.g., at least one being a composition of enriched CD4+ T cells and at least one being a separate composition of enriched CD8+ T cells from the same biological sample, are activated and/or stimulated by contacting with a stimulatory reagent (e.g., by incubation with CD3/CD28 conjugated magnetic beads for T cell activation). In some aspects, each of the activated/stimulated cell composition is engineered, transduced, and/or transfected, e.g., using a viral vector encoding a recombinant protein (e.g. CAR), to express the same recombinant protein in the CD4+ T cells and CD8+ T cells of each cell composition. In some aspects, the method comprises removing the stimulatory reagent, e.g., magnetic beads, from the cell composition. In some aspects, a cell composition containing engineered CD4+ T cells and a cell compostion containing engineered CD8+ T cells are separately cultivated, e.g., for separate expansion of the CD4+ T cell and CD8+ T cell populations therein. In certain embodiments, a cell composition from the cultivation is harvested and/or collected and/or formulated, e.g., by washing the cell composition in a formulation buffer. In certain embodiments, a formulated cell composition comprising CD4+ T cells and a formulated cell composition comprising CD8+ T cells is frozen, e.g., cryoprotected or cryopreserved in a cryopreservation media. In some aspects, engineered CD4+ T cells and CD8+ T cells in each formulation originate from the same donor or biological sample and express the same recombination protein (e.g., CAR, such as anti-CD19 CAR). In some aspects, a separate engineered CD4+ formulation and a separate engineered CD8+ formulation are administered at a defined ratio, e.g. 1:1, to a subject in need thereof such as the same donor.
[0515] In some aspects, two or more separate compositions of enriched T cells, e.g., at least one being a composition of enriched CD4+ T cells and at least one being a separate composition of enriched CD8+ T cells from the same biological sample, selected from a sample from a subject and then are combined at a defined ratio, e.g. 1:1. In some embodiments, the combined composition enriched in CD4+ and CD8+ T cells are activated and/or stimulated by contacting with a stimulatory reagent (e.g., by incubation with CD3/CD28 conjugated magnetic beads for T cell activation). In some aspects, the activated/stimulated cell composition is engineered, transduced, and/or transfected, e.g., using a viral vector encoding a recombinant protein (e.g. CAR), to express the recombinant protein in the CD4+ T cells and CD8+ T cells of the cell composition. In some aspects, the method comprises removing the stimulatory reagent, e.g., magnetic beads, from the cell composition. In some aspects, the cell composition containing engineered CD4+ T cells and engineered CD8+ T cells are cultivated, e.g., for expansion of the CD4+ T cell and CD8+ T cell populations therein. In certain embodiments, a cell composition from the cultivation is harvested and/or collected and/or formulated, e.g., by washing the cell composition in a formulation buffer. In certain embodiments, a formulated cell composition comprising recombinant receptor (e.g. CAR) engineered CD4+ T cells and CD8+ T cells is frozen, e.g., cryoprotected or cryopreserved in a cryopreservation media. In some aspects, engineered CD4+ T cells and CD8+ T cells in the formulation originate from the same donor or biological sample and express the same recombinant protein (e.g., CAR, such as anti-CD19 CAR).
[0516] In some aspects, a composition of enriched CD3+ T cells is selected from a sample from a subject. In some embodiments, the composition enriched in CD3+ T cells is activated and/or stimulated by contacting with a stimulatory reagent (e.g., by incubation with CD3/CD28 conjugated magnetic beads for T cell activation). In some aspects, the activated/stimulated cell composition is engineered, transduced, and/or transfected, e.g., using a viral vector encoding a recombinant protein (e.g. CAR), to express the recombinant protein in the T cells of the cell composition. In some aspects, the method comprises removing the stimulatory reagent, e.g., magnetic beads, from the cell composition. In some aspects, the cell composition containing engineered CD3+ T cells are cultivated, e.g., for expansion of the T cells populations therein. In certain embodiments, a cell composition from the cultivation is harvested and/or collected and/or formulated, e.g., by washing the cell composition in a formulation buffer. In certain embodiments, a formulated cell composition comprising recombinant receptor (e.g. CAR) engineered CD3+ T cells is frozen, e.g., cryoprotected or cryopreserved in a cryopreservation media. In some aspects, engineered CD3+ T cells in the formulation express a CAR, such as anti-CD19 CAR.
Z CELLS AND PREPARATION OF CELLS FOR GENETIC ENGINEERING
[0517] In some embodiments, cells, such as T cells, used in connection with the provided methods, uses, articles of manufacture or compositions are cells have been genetically engineered to express a recombinant receptor, e.g., a CAR or a TCR described herein. In some embodiments, the engineered cells are used in the context of cell therapy, e.g., adoptive cell therapy. In some embodiments, the engineered cells are immune cells. In some embodiments, the engineered cells are T cells, such as CD4+ and CD8+ T cells, CD4+ T cells, or CD8+ T cells. [0518] In some embodiments, the nucleic acids, such as nucleic acids encoding a recombinant receptor, are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.
[0519] The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen. In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the- shelf technologies, the cells are pluripotent and/or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the methods include isolating cells from the subject, preparing, processing, culturing, and/or engineering them, and re-introducing them into the same subject, before or after cry opreservation.
[0520] Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells.
[0521] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils.
[0522] In some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and/or an organism from which such cell is derived. In some embodiments, the nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.
[0523] In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for introduction of the nucleic acid encoding the transgenic receptor such as the CAR, may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered.
[0524] Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.
[0525] In some aspects, the sample from which the cells are derived or isolated is blood or a blood- derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. In some aspects, the sample from which the cells are derived or isolated is an apheresis product. In some aspects, the sample from which the cells are derived or isolated is a leukapheresis product. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
[0526] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig.
[0527] In some embodiments, isolation of the cells includes one or more preparation and/or nonaffinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.
[0528] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets.
[0529] In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and/or magnesium and/or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer’s instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer’s instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++/Mg++ free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.
[0530] In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.
[0531] In some embodiments, at least a portion of the selection step includes incubation of cells with a selection reagent. The incubation with a selection reagent or reagents, e.g., as part of selection methods which may be performed using one or more selection reagents for selection of one or more different cell types based on the expression or presence in or on the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method using a selection reagent or reagents for separation based on such markers may be used. In some embodiments, the selection reagent or reagents result in a separation that is affinity- or immunoaffinity-based separation. For example, the selection in some aspects includes incubation with a reagent or reagents for separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
[0532] In some aspects of such processes, a volume of cells is mixed with an amount of a desired affinity-based selection reagent. The immunoaffinity-based selection can be carried out using any system or method that results in a favorable energetic interaction between the cells being separated and the molecule specifically binding to the marker on the cell, e.g., the antibody or other binding partner on the solid surface, e.g., particle. In some embodiments, methods are carried out using particles such as beads, e.g. magnetic beads, that are coated with a selection agent (e.g. antibody) specific to the marker of the cells. The particles (e.g. beads) can be incubated or mixed with cells in a container, such as a tube or bag, while shaking or mixing, with a constant cell density-to-particle (e.g., bead) ratio to aid in promoting energetically favored interactions. In other cases, the methods include selection of cells in which all or a portion of the selection is carried out in the internal cavity of a centrifugal chamber, for example, under centrifugal rotation. In some embodiments, incubation of cells with selection reagents, such as immunoaffinity-based selection reagents, is performed in a centrifugal chamber. In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus described in International Patent Application, Publication Number W02009/072003, or US 20110003380 Al. In one example, the system is a system as described in International Publication Number W02016/073602.
[0533] In some embodiments, by conducting such selection steps or portions thereof (e.g., incubation with antibody-coated particles, e.g., magnetic beads) in the cavity of a centrifugal chamber, the user is able to control certain parameters, such as volume of various solutions, addition of solution during processing and timing thereof, which can provide advantages compared to other available methods. For example, the ability to decrease the liquid volume in the cavity during the incubation can increase the concentration of the particles (e.g. bead reagent) used in the selection, and thus the chemical potential of the solution, without affecting the total number of cells in the cavity. This in turn can enhance the pairwise interactions between the cells being processed and the particles used for selection. In some embodiments, carrying out the incubation step in the chamber, e.g., when associated with the systems, circuitry, and control as described herein, permits the user to effect agitation of the solution at desired time(s) during the incubation, which also can improve the interaction.
[0534] In some embodiments, at least a portion of the selection step is performed in a centrifugal chamber, which includes incubation of cells with a selection reagent. In some aspects of such processes, a volume of cells is mixed with an amount of a desired affinity-based selection reagent that is far less than is normally employed when performing similar selections in a tube or container for selection of the same number of cells and/or volume of cells according to manufacturer’s instructions. In some embodiments, an amount of selection reagent or reagents that is/are no more than 5%, no more than 10%, no more than 15%, no more than 20%, no more than 25%, no more than 50%, no more than 60%, no more than 70% or no more than 80% of the amount of the same selection reagent(s) employed for selection of cells in a tube or container-based incubation for the same number of cells and/or the same volume of cells according to manufacturer’s instructions is employed.
[0535] In some embodiments, for selection, e.g., immunoaffinity-based selection of the cells, the cells are incubated in the cavity of the chamber in a composition that also contains the selection buffer with a selection reagent, such as a molecule that specifically binds to a surface marker on a cell that it desired to enrich and/or deplete, but not on other cells in the composition, such as an antibody, which optionally is coupled to a scaffold such as a polymer or surface, e.g., bead, e.g., magnetic bead, such as magnetic beads coupled to monoclonal antibodies specific for CD3, CD4 and/or CD8. In some embodiments, as described, the selection reagent is added to cells in the cavity of the chamber in an amount that is substantially less than (e.g. is no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the amount) as compared to the amount of the selection reagent that is typically used or would be necessary to achieve about the same or similar efficiency of selection of the same number of cells or the same volume of cells when selection is performed in a tube with shaking or rotation. In some embodiments, the incubation is performed with the addition of a selection buffer to the cells and selection reagent to achieve a target volume with incubation of the reagent of, for example, 10 mL to 200 mL, such as at least or at least about 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL or 200 mL. In some embodiments, the selection buffer and selection reagent are pre -mixed before addition to the cells. In some embodiments, the selection buffer and selection reagent are separately added to the cells. In some embodiments, the selection incubation is carried out with periodic gentle mixing condition, which can aid in promoting energetically favored interactions and thereby permit the use of less overall selection reagent while achieving a high selection efficiency.
[0536] In some embodiments, the total duration of the incubation with the selection reagent is from or from about 5 minutes to 6 hours, such as 30 minutes to 3 hours, for example, at least or at least about 30 minutes, 60 minutes, 120 minutes or 180 minutes.
[0537] In some embodiments, the incubation generally is carried out under mixing conditions, such as in the presence of spinning, generally at relatively low force or speed, such as speed lower than that used to pellet the cells, such as from or from about 600 rpm to 1700 rpm (e.g. at or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm), such as at an RCF at the sample or wall of the chamber or other container of from or from about 80g to 100g (e.g. at or about or at least 80 g, 85 g, 90 g, 95 g, or 100 g). In some embodiments, the spin is carried out using repeated intervals of a spin at such low speed followed by a rest period, such as a spin and/or rest for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, such as a spin at approximately 1 or 2 seconds followed by a rest for approximately 5, 6, 7, or 8 seconds.
[0538] In some embodiments, such process is carried out within the entirely closed system to which the chamber is integral. In some embodiments, this process (and in some aspects also one or more additional step, such as a previous wash step washing a sample containing the cells, such as an apheresis sample) is carried out in an automated fashion, such that the cells, reagent, and other components are drawn into and pushed out of the chamber at appropriate times and centrifugation effected, so as to complete the wash and binding step in a single closed system using an automated program.
[0539] In some embodiments, after the incubation and/or mixing of the cells and selection reagent and/or reagents, the incubated cells are subjected to a separation to select for cells based on the presence or absence of the particular reagent or reagents. In some embodiments, the separation is performed in the same closed system in which the incubation of cells with the selection reagent was performed. In some embodiments, after incubation with the selection reagents, incubated cells, including cells in which the selection reagent has bound are transferred into a system for immunoaffinity-based separation of the cells. In some embodiments, the system for immunoaffinity-based separation is or contains a magnetic separation column.
[0540] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.
[0541] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.
[0542| The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
[0543] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.
[0544] For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques.
[0545] In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker +) at a relatively higher level (markerhlgh) on the positively or negatively selected cells, respectively.
[0546] In particular embodiments, a biological sample, e.g., a sample of PBMCs or other white blood cells, are subjected to selection of CD4+ T cells, where both the negative and positive fractions are retained. In certain embodiments, CD8+ T cells are selected from the negative fraction. In some embodiments, a biological sample is subjected to selection of CD8+ T cells, where both the negative and positive fractions are retained. In certain embodiments, CD4+ T cells are selected from the negative fraction.
[0547] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations.
[0548| In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve longterm survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood.1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TcM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.
[0549] In embodiments, memory T cells are present in both CD62L+ and CD62L subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies.
[0550] In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.
[0551] In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+ cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.
[0552] CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO , CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L and CD45RO .
[0553] In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinitymagnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).
[0554] In some aspects, the sample or composition of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynalbeads™ or MACS® beads). The magnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.
[0555] In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.
[0556] The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.
[0557] In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.
[0558] In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.
[0559] In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and/or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.
[0560] In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS®) (Miltenyi Biotec, Auburn, CA). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.
[0561] In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and/or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and/or contamination. In one example, the system is a system as described in International Patent Application, Publication Number W02009/072003, or US 20110003380 Al.
[0562] In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and/or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and/or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and/or adjust various aspects of the processing, isolation, engineering, and formulation steps.
[0563] In some aspects, the separation and/or other steps is carried out using CliniMACS® system (Miltenyi Biotec), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.
[0564] The CliniMACS® system in some aspects uses antibody -coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is then connected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag. [0565] In certain embodiments, separation and/or other steps are carried out using the CliniMACS Prodigy® system (Miltenyi Biotec). The CliniMACS Prodigy® system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy® system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy® system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immuno ther. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and Wang et al. (2012) J Immuno ther. 35(9):689-701.
[0566] In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. l(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.
[0567| In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and/or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS), including preparative scale (FACS) and/or microelectromechanical systems (MEMS) chips, e.g., in combination with a flowcytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.
[0568] In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and/or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are generally then frozen to -80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.
[0569] In some embodiments, the isolation and/or selection results in one or more input compositions of enriched T cells, e.g., CD3+ T cells, CD4+ T cells, and/or CD8+ T cells. In some embodiments, two or more separate input composition are isolated, selected, enriched, or obtained from a single biological sample. In some embodiments, separate input compositions are isolated, selected, enriched, and/or obtained from separate biological samples collected, taken, and/or obtained from the same subject.
[0570] In certain embodiments, the one or more input compositions is or includes a composition of enriched T cells that includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or at about 100% CD3+ T cells. In particular embodiment, the input composition of enriched T cells consists essentially of CD3+ T cells.
[0571] In certain embodiments, the one or more input compositions is or includes a composition of enriched CD4+ T cells that includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or at about 100% CD4+ T cells. In certain embodiments, the input composition of CD4+ T cells includes less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD8+ T cells, and/or contains no CD8+ T cells, and/or is free or substantially free of CD8+ T cells. In some embodiments, the composition of enriched T cells consists essentially of CD4+ T cells.
[0572] In certain embodiments, the one or more compositions is or includes a composition of CD8+ T cells that is or includes at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at or at about 100% CD8+ T cells. In certain embodiments, the composition of CD8+ T cells contains less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 1%, less than 0.1%, or less than 0.01% CD4+ T cells, and/or contains no CD4+ T cells, and/or is free of or substantially free of CD4+ T cells. In some embodiments, the composition of enriched T cells consists essentially of CD8+ T cells.
2. A CTIVATION AND STIMULATION
[0573] In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. The incubation and/or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.
[0574] The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.
[0575] In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of stimulating or activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR, e.g. anti-CD3. In some embodiments, the stimulating conditions include one or more agent, e.g. ligand, which is capable of stimulating a costimulatory receptor, e.g., anti-CD28. In some embodiments, such agents and/or ligands may be, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti-CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include IL-2, IL- 15 and/or IL-7. In some aspects, the IL -2 concentration is at least about 10 units/mL.
[0576] For example, the stimulating conditions can include incubation using anti-CD3/anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).
[0577| In some aspects, incubation is carried out in accordance with techniques such as those described in US Patent No. 6,040,177 to Riddell et al., Klebanoff et al.(2012) J Immunother. 35(9): 651— 660, Terakura et al. (2012) Blood.1:72-82, and/or Wang et al. (2012) J Immunother. 35(9):689-701.
[0578] In some embodiments, the T cells are expanded by adding to a culture-initiating composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gammairradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.
[0579] In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees Celsius, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.
[0580] In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and/or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigenspecific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.
[0581] In some embodiments, at least a portion of the incubation in the presence of one or more stimulating conditions or a stimulatory agents is carried out in the internal cavity of a centrifugal chamber, for example, under centrifugal rotation, such as described in International Publication Number W02016/073602. In some embodiments, at least a portion of the incubation performed in a centrifugal chamber includes mixing with a reagent or reagents to induce stimulation and/or activation. In some embodiments, cells, such as selected cells, are mixed with a stimulating condition or stimulatory agent in the centrifugal chamber. In some aspects of such processes, a volume of cells is mixed with an amount of one or more stimulating conditions or agents that is far less than is normally employed when performing similar stimulations in a cell culture plate or other system.
[0582] In some embodiments, the stimulating agent is added to cells in the cavity of the chamber in an amount that is substantially less than (e.g. is no more than 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80% of the amount) as compared to the amount of the stimulating agent that is typically used or would be necessary to achieve about the same or similar efficiency of selection of the same number of cells or the same volume of cells when selection is performed without mixing in a centrifugal chamber, e.g. in a tube or bag with periodic shaking or rotation. In some embodiments, the incubation is performed with the addition of an incubation buffer to the cells and stimulating agent to achieve a target volume with incubation of the reagent of, for example, 10 mL to 200 mL, such as at least or at least about or about or 10 mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, 100 mL, 150 mL or 200 mL. In some embodiments, the incubation buffer and stimulating agent are pre-mixed before addition to the cells. In some embodiments, the incubation buffer and stimulating agent are separately added to the cells. In some embodiments, the stimulating incubation is carried out with periodic gentle mixing condition, which can aid in promoting energetically favored interactions and thereby permit the use of less overall stimulating agent while achieving stimulating and activation of cells.
[0583] In some embodiments, the incubation generally is carried out under mixing conditions, such as in the presence of spinning, generally at relatively low force or speed, such as speed lower than that used to pellet the cells, such as from or from about 600 rpm to 1700 rpm (e.g. at or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm), such as at an RCF at the sample or wall of the chamber or other container of from or from about 80g to 100g (e.g. at or about or at least 80 g, 85 g, 90 g, 95 g, or 100 g). In some embodiments, the spin is carried out using repeated intervals of a spin at such low speed followed by a rest period, such as a spin and/or rest for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 seconds, such as a spin at approximately 1 or 2 seconds followed by a rest for approximately 5, 6, 7, or 8 seconds.
[0584] In some embodiments, the total duration of the incubation, e.g. with the stimulating agent, is between or between about 1 hour and 96 hours, 1 hour and 72 hours, 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours or 12 hours and 24 hours, such as at least or at least about 6 hours, 12 hours, 18 hours, 24 hours, 36 hours or 72 hours. In some embodiments, the further incubation is for a time between or about between 1 hour and 48 hours, 4 hours and 36 hours, 8 hours and 30 hours or 12 hours and 24 hours, inclusive.
[0585] In particular embodiments, the stimulating conditions include incubating, culturing, and/or cultivating a composition of enriched T cells with and/or in the presence of one or more cytokines. In particular embodiments, the one or more cytokines are recombinant cytokines. In some embodiments, the one or more cytokines are human recombinant cytokines. In certain embodiments, the one or more cytokines bind to and/or are capable of binding to receptors that are expressed by and/or are endogenous to T cells. In particular embodiments, the one or more cytokines is or includes a member of the 4-alpha- helix bundle family of cytokines. In some embodiments, members of the 4-alpha-helix bundle family of cytokines include, but are not limited to, interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin 12 (IL- 12), interleukin 15 (IL- 15), granulocyte colony-stimulating factor (G-CSF), and granulocyte -macrophage colony-stimulating factor (GM-CSF).
[0586] In some embodiments, the stimulation results in activation and/or proliferation of the cells, for example, prior to transduction.
.7. VECTORS AND METHODS FOR GENETIC ENGINEERING
[0587] In some embodiments, engineered cells, such as T cells, used in connection with the provided methods, uses, articles of manufacture or compositions are cells have been genetically engineered to express a recombinant receptor, e.g., a CAR or a TCR described herein. In some embodiments, the cells are engineered by introduction, delivery or transfer of nucleic acid sequences that encode the recombinant receptor and/or other molecules.
[0588] In some embodiments, methods for producing engineered cells includes the introduction of a polynucleotide encoding a recombinant receptor (e.g. anti-CD19 CAR) into a cell, e.g., such as a stimulated or activated cell. In particular embodiments, the recombinant proteins are recombinant receptors, such as any described. Introduction of the nucleic acid molecules encoding the recombinant protein, such as recombinant receptor, in the cell may be carried out using any of a number of known vectors. Such vectors include viral and non-viral systems, including lentiviral and gammaretroviral systems, as well as transposon-based systems such as PiggyBac or Sleeping Beauty-based gene transfer systems. Exemplary methods include those for transfer of nucleic acids encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation. In some embodiments, the engineering produces one or more engineered compositions of enriched T cells. [0589] In certain embodiments, the one or more compositions of stimulated T cells are or include two separate stimulated compositions of enriched T cells. In particular embodiments, two separate compositions of enriched T cells, e.g., two separate compositions of enriched T cells that have been selected, isolated, and/or enriched from the same biological sample, are separately engineered. In certain embodiments, the two separate compositions include a composition of enriched CD4+ T cells. In particular embodiments, the two separate compositions include a composition of enriched CD8+ T cells. In some embodiments, two separate compositions of enriched CD4+ T cells and enriched CD8+ T cells are genetically engineered separately.
[0590] In some embodiments, gene transfer is accomplished by first stimulating the cell, such as by combining it with a stimulus that induces a response such as proliferation, survival, and/or activation, e.g., as measured by expression of a cytokine or activation marker, followed by transduction of the activated cells, and expansion in culture to numbers sufficient for clinical applications. In certain embodiments, the gene transfer is accomplished by first incubating the cells under stimulating conditions, such as by any of the methods described.
[0591] In some embodiments, methods for genetic engineering are carried out by contacting one or more cells of a composition with a nucleic acid molecule encoding the recombinant protein, e.g. recombinant receptor. In some embodiments, the contacting can be effected with centrifugation, such as spinoculation (e.g. centrifugal inoculation). Such methods include any of those as described in International Publication Number WO2016/073602. Exemplary centrifugal chambers include those produced and sold by Biosafe SA, including those for use with the Sepax® and Sepax® 2 system, including an A-200/F and A-200 centrifugal chambers and various kits for use with such systems. Exemplary chambers, systems, and processing instrumentation and cabinets are described, for example, in US Patent No. 6,123,655, US Patent No. 6,733,433 and Published U.S. Patent Application, Publication No.: US 2008/0171951, and published international patent application, publication no. WO 00/38762, the contents of each of which are incorporated herein by reference in their entirety. Exemplary kits for use with such systems include, but are not limited to, single -use kits sold by BioSafe SA under product names CS-430.1, CS-490.1, CS-600.1 or CS-900.2.
[0592] In some embodiments, the contacting can be effected with centrifugation, such as spinoculation (e.g., centrifugal inoculation). In some embodiments, the composition containing cells, the vector, e.g., viral particles and reagent can be rotated, generally at relatively low force or speed, such as speed lower than that used to pellet the cells, such as from or from about 600 rpm to 1700 rpm (e.g., at or about or at least 600 rpm, 1000 rpm, or 1500 rpm or 1700 rpm). In some embodiments, the rotation is carried at a force, e.g., a relative centrifugal force, of from or from about 100 g to 3200 g (e.g., at or about or at least at or about 100 g, 200 g, 300 g, 400 g, 500 g, 1000 g, 1500 g, 2000 g, 2500 g, 3000 g or 3200 g), as measured for example at an internal or external wall of the chamber or cavity. The term “relative centrifugal force” or RCF is generally understood to be the effective force imparted on an object or substance (such as a cell, sample, or pellet and/or a point in the chamber or other container being rotated), relative to the earth’s gravitational force, at a particular point in space as compared to the axis of rotation. The value may be determined using well-known formulas, taking into account the gravitational force, rotation speed and the radius of rotation (distance from the axis of rotation and the object, substance, or particle at which RCF is being measured).
[0593] In some embodiments, the system is included with and/or placed into association with other instrumentation, including instrumentation to operate, automate, control and/or monitor aspects of the transduction step and one or more various other processing steps performed in the system, e.g. one or more processing steps that can be carried out with or in connection with the centrifugal chamber system as described herein or in International Publication Number W02016/073602. This instrumentation in some embodiments is contained within a cabinet. In some embodiments, the instrumentation includes a cabinet, which includes a housing containing control circuitry, a centrifuge, a cover, motors, pumps, sensors, displays, and a user interface. An exemplary device is described in US Patent No. 6,123,655, US Patent No. 6,733,433 and US 2008/0171951.
[0594] In some embodiments, the system comprises a series of containers, e.g., bags, tubing, stopcocks, clamps, connectors, and a centrifuge chamber. In some embodiments, the containers, such as bags, include one or more containers, such as bags, containing the cells to be transduced and the viral vector particles, in the same container or separate containers, such as the same bag or separate bags. In some embodiments, the system further includes one or more containers, such as bags, containing medium, such as diluent and/or wash solution, which is pulled into the chamber and/or other components to dilute, resuspend, and/or wash components and/or compositions during the methods. The containers can be connected at one or more positions in the system, such as at a position corresponding to an input line, diluent line, wash line, waste line and/or output line.
[0595] In some embodiments, the chamber is associated with a centrifuge, which is capable of effecting rotation of the chamber, such as around its axis of rotation. Rotation may occur before, during, and/or after the incubation in connection with transduction of the cells and/or in one or more of the other processing steps. Thus, in some embodiments, one or more of the various processing steps is carried out under rotation, e.g., at a particular force. The chamber is typically capable of vertical or generally vertical rotation, such that the chamber sits vertically during centrifugation and the side wall and axis are vertical or generally vertical, with the end wall(s) horizontal or generally horizontal.
[0596] In some embodiments, during at least a part of the genetic engineering, e.g. transduction, and/or subsequent to the genetic engineering the cells are transferred to a bioreactor bag assembly for culture of the genetically engineered cells, such as for cultivation or expansion of the cells.
[0597] In some embodiments, recombinant nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038/gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557.
[0598] In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV) or spleen focus forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the gene to be expressed replaces the retroviral gag, pol and/or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.
[0599] Methods of lentiviral transduction are known. Exemplary methods are described in, e.g., Wang et al. (2012) J. Immunother. 35(9): 689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102(2): 497- 505.
[0600| In some embodiments, the viral vector particles contain a genome derived from a retroviral genome based vector, such as derived from a lentiviral genome based vector. In some aspects of the provided viral vectors, the heterologous nucleic acid encoding a recombinant receptor, such as an antigen receptor, such as a CAR, is contained and/or located between the 5' LTR and 3' LTR sequences of the vector genome.
[0601] In some embodiments, the viral vector genome is a lentivirus genome, such as an HIV-1 genome or an SIV genome. For example, lentiviral vectors have been generated by multiply attenuating virulence genes, for example, the genes env, vif, vpu and nef can be deleted, making the vector safer for therapeutic purposes. Lentiviral vectors are known. See Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136). In some embodiments, these viral vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection, and for transfer of the nucleic acid into a host cell. Known lentiviruses can be readily obtained from depositories or collections such as the American Type Culture Collection (“ATCC”; 10801 University Blvd., Manassas, Va. 20110-2209), or isolated from known sources using commonly available techniques.
[0602] Non-limiting examples of lentiviral vectors include those derived from a lentivirus, such as Human Immunodeficiency Virus 1 (HIV-1), HIV-2, an Simian Immunodeficiency Virus (SIV), Human T-lymphotropic virus 1 (HTLV-1), HTLV-2 or equine infection anemia virus (E1AV). For example, lentiviral vectors have been generated by multiply attenuating the HIV virulence genes, for example, the genes env, vif, vpr, vpu and nef are deleted, making the vector safer for therapeutic purposes. Lentiviral vectors are known in the art, see Naldini et al., (1996 and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Pat. Nos. 6,013,516; and 5,994,136). In some embodiments, these viral vectors are plasmid-based or virus-based, and are configured to carry the essential sequences for incorporating foreign nucleic acid, for selection, and for transfer of the nucleic acid into a host cell. Known lentiviruses can be readily obtained from depositories or collections such as the American Type Culture Collection (“ATCC”; 10801 University Blvd., Manassas, Va. 20110-2209), or isolated from known sources using commonly available techniques.
[0603] In some embodiments, the viral genome vector can contain sequences of the 5' and 3' LTRs of a retrovirus, such as a lentivirus. In some aspects, the viral genome construct may contain sequences from the 5' and 3' LTRs of a lentivirus, and in particular can contain the R and U5 sequences from the 5' LTR of a lentivirus and an inactivated or self-inactivating 3' LTR from a lentivirus. The LTR sequences can be LTR sequences from any lentivirus from any species. For example, they may be LTR sequences from HIV, SIV, FIV or BIV. Typically, the LTR sequences are HIV LTR sequences.
[0604] In some embodiments, the nucleic acid of a viral vector, such as an HIV viral vector, lacks additional transcriptional units. The vector genome can contain an inactivated or self-inactivating 3' LTR (Zufferey et al. J Virol 72: 9873, 1998; Miyoshi et al., J Virol 72:8150, 1998). For example, deletion in the U3 region of the 3' LTR of the nucleic acid used to produce the viral vector RNA can be used to generate self-inactivating (SIN) vectors. This deletion can then be transferred to the 5' LTR of the proviral DNA during reverse transcription. A self-inactivating vector generally has a deletion of the enhancer and promoter sequences from the 3' long terminal repeat (LTR), which is copied over into the 5' LTR during vector integration. In some embodiments enough sequence can be eliminated, including the removal of a TATA box, to abolish the transcriptional activity of the LTR. This can prevent production of full-length vector RNA in transduced cells. In some aspects, the U3 element of the 3' LTR contains a deletion of its enhancer sequence, the TATA box, Spl, and NF-kappa B sites. As a result of the selfinactivating 3' LTR, the provirus that is generated following entry and reverse transcription contains an inactivated 5' LTR. This can improve safety by reducing the risk of mobilization of the vector genome and the influence of the LTR on nearby cellular promoters. The self-inactivating 3' LTR can be constructed by any method known in the art. In some embodiments, this does not affect vector titers or the in vitro or in vivo properties of the vector.
[0605] Optionally, the U3 sequence from the lentiviral 5' LTR can be replaced with a promoter sequence in the viral construct, such as a heterologous promoter sequence. This can increase the titer of virus recovered from the packaging cell line. An enhancer sequence can also be included. Any enhancer/promoter combination that increases expression of the viral RNA genome in the packaging cell line may be used. In one example, the CMV enhancer/promoter sequence is used (U.S. Pat. No.
5,385,839 and U.S. Pat. No. 5,168,062).
[0606| In certain embodiments, the risk of insertional mutagenesis can be minimized by constructing the retroviral vector genome, such as lentiviral vector genome, to be integration defective. A variety of approaches can be pursued to produce a non-integrating vector genome. In some embodiments, a mutation(s) can be engineered into the integrase enzyme component of the pol gene, such that it encodes a protein with an inactive integrase. In some embodiments, the vector genome itself can be modified to prevent integration by, for example, mutating or deleting one or both attachment sites, or making the 3' LTR-proximal polypurine tract (PPT) non-functional through deletion or modification. In some embodiments, non-genetic approaches are available; these include pharmacological agents that inhibit one or more functions of integrase. The approaches are not mutually exclusive; that is, more than one of them can be used at a time. For example, both the integrase and attachment sites can be nonfunctional, or the integrase and PPT site can be non-functional, or the attachment sites and PPT site can be non-functional, or all of them can be non-functional. Such methods and viral vector genomes are known and available (see Philpott and Thrasher, Human Gene Therapy 18:483, 2007; Engelman et al. J Virol 69:2729, 1995; Brown et al J Virol 73:9011 (1999); WO 2009/076524; McWilliams et al., J Virol 77:11150, 2003; Powell and Levin J Virol 70:5288, 1996).
[0607] In some embodiments, the vector contains sequences for propagation in a host cell, such as a prokaryotic host cell. In some embodiments, the nucleic acid of the viral vector contains one or more origins of replication for propagation in a prokaryotic cell, such as a bacterial cell. In some embodiments, vectors that include a prokaryotic origin of replication also may contain a gene whose expression confers a detectable or selectable marker such as drug resistance.
[0608] The viral vector genome is typically constructed in a plasmid form that can be transfected into a packaging or producer cell line. Any of a variety of known methods can be used to produce retroviral particles whose genome contains an RNA copy of the viral vector genome. In some embodiments, at least two components are involved in making a virus-based gene delivery system: first, packaging plasmids, encompassing the structural proteins as well as the enzymes necessary to generate a viral vector particle, and second, the viral vector itself, i.e., the genetic material to be transferred. Biosafety safeguards can be introduced in the design of one or both of these components.
[0609] In some embodiments, the packaging plasmid can contain all retroviral, such as HIV-1, proteins other than envelope proteins (Naldini et al., 1998). In other embodiments, viral vectors can lack additional viral genes, such as those that are associated with virulence, e.g., vpr, vif, vpu and nef, and/or Tat, a primary transactivator of HIV. In some embodiments, lentiviral vectors, such as HIV-based lentiviral vectors, comprise only three genes of the parental virus: gag, pol and rev, which reduces or eliminates the possibility of reconstitution of a wild-type virus through recombination. [0610] In some embodiments, the viral vector genome is introduced into a packaging cell line that contains all the components necessary to package viral genomic RNA, transcribed from the viral vector genome, into viral particles. Alternatively, the viral vector genome may comprise one or more genes encoding viral components in addition to the one or more sequences, e.g., recombinant nucleic acids, of interest. In some aspects, in order to prevent replication of the genome in the target cell, however, endogenous viral genes required for replication are removed and provided separately in the packaging cell line.
[0611] In some embodiments, a packaging cell line is transfected with one or more plasmid vectors containing the components necessary to generate the particles. In some embodiments, a packaging cell line is transfected with a plasmid containing the viral vector genome, including the LTRs, the cis-acting packaging sequence and the sequence of interest, i.e. a nucleic acid encoding an antigen receptor, such as a CAR; and one or more helper plasmids encoding the virus enzymatic and/or structural components, such as Gag, pol and/or rev. In some embodiments, multiple vectors are utilized to separate the various genetic components that generate the retroviral vector particles. In some such embodiments, providing separate vectors to the packaging cell reduces the chance of recombination events that might otherwise generate replication competent viruses. In some embodiments, a single plasmid vector having all of the retroviral components can be used.
[0612] In some embodiments, the retroviral vector particle, such as lentiviral vector particle, is pseudotyped to increase the transduction efficiency of host cells. For example, a retroviral vector particle, such as a lentiviral vector particle, in some embodiments is pseudotyped with a VSV-G glycoprotein, which provides a broad cell host range extending the cell types that can be transduced. In some embodiments, a packaging cell line is transfected with a plasmid or polynucleotide encoding a nonnative envelope glycoprotein, such as to include xenotropic, polytropic or amphotropic envelopes, such as Sindbis virus envelope, GALV or VSV-G.
[0613] In some embodiments, the packaging cell line provides the components, including viral regulatory and structural proteins, that are required in trans for the packaging of the viral genomic RNA into lentiviral vector particles. In some embodiments, the packaging cell line may be any cell line that is capable of expressing lentiviral proteins and producing functional lentiviral vector particles. In some aspects, suitable packaging cell lines include 293 (ATCC CCL X), 293T, HeLA (ATCC CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL-10) and Cf2Th (ATCC CRL 1430) cells.
[0614] In some embodiments, the packaging cell line stably expresses the viral protein(s). For example, in some aspects, a packaging cell line containing the gag, pol, rev and/or other structural genes but without the LTR and packaging components can be constructed. In some embodiments, a packaging cell line can be transiently transfected with nucleic acid molecules encoding one or more viral proteins along with the viral vector genome containing a nucleic acid molecule encoding a heterologous protein, and/or a nucleic acid encoding an envelope glycoprotein.
[0615] In some embodiments, the viral vectors and the packaging and/or helper plasmids are introduced via transfection or infection into the packaging cell line. The packaging cell line produces viral vector particles that contain the viral vector genome. Methods for transfection or infection are well known. Non-limiting examples include calcium phosphate, DEAE-dextran and lipofection methods, electroporation and microinjection.
[0616] When a recombinant plasmid and the retroviral LTR and packaging sequences are introduced into a special cell line (e.g., by calcium phosphate precipitation for example), the packaging sequences may permit the RNA transcript of the recombinant plasmid to be packaged into viral particles, which then may be secreted into the culture media. The media containing the recombinant retroviruses in some embodiments is then collected, optionally concentrated, and used for gene transfer. For example, in some aspects, after cotransfection of the packaging plasmids and the transfer vector to the packaging cell line, the viral vector particles are recovered from the culture media and titered by standard methods used by those of skill in the art.
[0617] In some embodiments, a retroviral vector, such as a lentiviral vector, can be produced in a packaging cell line, such as an exemplary HEK 293T cell line, by introduction of plasmids to allow generation of lentiviral particles. In some embodiments, a packaging cell is transfected and/or contains a polynucleotide encoding gag and pol, and a polynucleotide encoding a recombinant receptor, such as an antigen receptor, for example, a CAR. In some embodiments, the packaging cell line is optionally and/or additionally transfected with and/or contains a polynucleotide encoding a rev protein. In some embodiments, the packaging cell line is optionally and/or additionally transfected with and/or contains a polynucleotide encoding a non-native envelope glycoprotein, such as VSV-G. In some such embodiments, approximately two days after transfection of cells, e.g., HEK 293T cells, the cell supernatant contains recombinant lentiviral vectors, which can be recovered and titered.
[0618] Recovered and/or produced retroviral vector particles can be used to transduce target cells using the methods as described. Once in the target cells, the viral RNA is reverse-transcribed, imported into the nucleus and stably integrated into the host genome. One or two days after the integration of the viral RNA, the expression of the recombinant protein, e.g., antigen receptor, such as CAR, can be detected.
[0619] In some embodiments, the provided methods involve methods of transducing cells by contacting, e.g., incubating, a cell composition comprising a plurality of cells with a viral particle. In some embodiments, the cells to be transfected or transduced are or comprise primary cells obtained from a subject, such as cells enriched and/or selected from a subject.
[0620] In some embodiments, the concentration of cells to be transduced of the composition is from or from about 1.0 x 105 cells/mL to 1.0 x 108 cells/mL, such as at least or at least about or about 1.0 x 105 cells/mL, 5 x 105 cells/mL, 1 x 106 cells/mL, 5 x 106 cells/mL, 1 x 107 cells/mL, 5 x 107 cells/mL or 1 x 108 cells/mL.
[0621] In some embodiments, the viral particles are provided at a certain ratio of copies of the viral vector particles or infectious units (IU) thereof, per total number of cells to be transduced (lU/cell). For example, in some embodiments, the viral particles are present during the contacting at or about or at least at or about 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or 60 IU of the viral vector particles per one of the cells.
[0622] In some embodiments, the titer of viral vector particles is between or between about 1 x 106 lU/mL and 1 x 108 lU/mL, such as between or between about 5 x 106 lU/mL and 5 x 107 lU/mL, such as at least 6 x 106 lU/mL, 7 x 106 lU/mL, 8 x 106 lU/mL, 9 x 106 lU/mL, 1 x 107 lU/mL, 2 x 107 lU/mL, 3 x 107 lU/mL, 4 x 107 lU/mL, or 5 xlO7 lU/mL.
[0623] In some embodiments, transduction can be achieved at a multiplicity of infection (MOI) of less than 100, such as generally less than 60, 50, 40, 30, 20, 10, 5 or less.
[0624] In some embodiments, the method involves contacting or incubating, the cells with the viral particles. In some embodiments, the contacting is for 30 minutes to 72 hours, such as 30 minute to 48 hours, 30 minutes to 24 hours or 1 hour to 24 hours, such as at least or at least about 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours or more.
[0625] In some embodiments, contacting is performed in solution. In some embodiments, the cells and viral particles are contacted in a volume of from or from about 0.5 mL to 500 mL, such as from or from about 0.5 mL to 200 mL, 0.5 mL to 100 mL, 0.5 mL to 50 mL, 0.5 mL to 10 mL, 0.5 mL to 5 mL, 5 mL to 500 mL, 5 mL to 200 mL, 5 mL to 100 mL, 5 mL to 50 mL, 5 mL to 10 mL, 10 mL to 500 mL, 10 mL to 200 mL, 10 mL to 100 mL, 10 mL to 50 mL, 50 mL to 500 mL, 50 mL to 200 mL, 50 mL to 100 mL, 100 mL to 500 mL, 100 mL to 200 mL or 200 mL to 500 mL.
[0626] In certain embodiments, the input cells are treated, incubated, or contacted with particles that comprise binding molecules that bind to or recognize the recombinant receptor that is encoded by the viral DNA.
[0627] In some embodiments, the incubation of the cells with the viral vector particles results in or produces an output composition comprising cells transduced with the viral vector particles.
[0628] In some embodiments, recombinant nucleic acids are transferred into T cells via electroporation (see, e.g., Chicaybam et al, (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al. (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al. (2010) Hum Gene Ther 21(4): 427-437; Sharma et al. (2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115- 126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. N.Y.), protoplast fusion, cationic liposome-mediated transfection; tungsten particle -facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).
[0629] Other approaches and vectors for transfer of the nucleic acids encoding the recombinant products are those described, e.g., in international patent application, Publication No.: WO2014055668, and U.S. Patent No. 7,446,190.
[0630] In some embodiments, the cells, e.g., T cells, may be transfected either during or after expansion e.g. with a T cell receptor (TCR) or a chimeric antigen receptor (CAR). This transfection for the introduction of the gene of the desired receptor can be carried out with any suitable retroviral vector, for example. The genetically modified cell population can then be liberated from the initial stimulus (the anti-CD3/anti-CD28 stimulus, for example) and subsequently be stimulated with a second type of stimulus e.g. via a de novo introduced receptor). This second type of stimulus may include an antigenic stimulus in form of a peptide/MHC molecule, the cognate (cross-linking) ligand of the genetically introduced receptor (e.g. natural ligand of a CAR) or any ligand (such as an antibody) that directly binds within the framework of the new receptor (e.g. by recognizing constant regions within the receptor). See, for example, Cheadle et al, “Chimeric antigen receptors for T-cell based therapy” Methods Mol Biol. 2012; 907:645-66 or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65: 333-347 (2014).
[0631] In some cases, a vector may be used that does not require that the cells, e.g., T cells, are activated. In some such instances, the cells may be selected and/or transduced prior to activation. Thus, the cells may be engineered prior to, or subsequent to culturing of the cells, and in some cases at the same time as or during at least a portion of the culturing.
[0632] Among additional nucleic acids, e.g., genes for introduction are those to improve the efficacy of therapy, such as by promoting viability and/or function of transferred cells; genes to provide a genetic marker for selection and/or evaluation of the cells, such as to assess in vivo survival or localization; genes to improve safety, for example, by making the cell susceptible to negative selection in vivo as described by Lupton S. D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319- 338 (1992); see also the publications of PCT/US91/08442 and PCT/US94/05601 by Lupton et al. describing the use of bifunctional selectable fusion genes derived from fusing a dominant positive selectable marker with a negative selectable marker. See, e.g., Riddell et al., US Patent No. 6,040,177, at columns 14-17.
CULTLVATLON EXPANSLON AND FORMULA TLON OF ENGLNEERED CELLS
[0633] In some embodiments, the methods for generating the engineered cells, e.g., for cell therapy in accord with any of provided methods, uses, articles of manufacture or compositions, include one or more steps for cultivating cells, e.g., cultivating cells under conditions that promote proliferation and/or expansion. In some embodiments, cells are cultivated under conditions that promote proliferation and/or expansion subsequent to a step of genetically engineering, e.g., introducing a recombinant polypeptide to the cells by transduction or transfection. In particular embodiments, the cells are cultivated after the cells have been incubated under stimulating conditions and transduced or transfected with a recombinant polynucleotide, e.g., a polynucleotide encoding a recombinant receptor. Thus, in some embodiments, a composition of CAR-positive T cells that has been engineered by transduction or transfection with a recombinant polynucleotide encoding the CAR, is cultivated under conditions that promote proliferation and/or expansion.
[0634] In certain embodiments, the one or more compositions of engineered T cells are or include two separate compositions of enriched T cells, such as two separate compositions of enriched T cells that have been engineered with a polynucleotide encoding a recombinant receptor, e.g. a CAR. In particular embodiments, two separate compositions of enriched T cells, e.g., two separate compositions of enriched T cells selected, isolated, and/or enriched from the same biological sample, are separately cultivated under stimulating conditions , such as subsequent to a step of genetically engineering, e.g., introducing a recombinant polypeptide to the cells by transduction or transfection. In certain embodiments, the two separate compositions include a composition of enriched CD4+ T cells, such as a composition of enriched CD4+ T cells that have been engineered with a polynucleotide encoding a recombinant receptor, e.g. a CAR. In particular embodiments, the two separate compositions include a composition of enriched CD8+ T cells, such as a composition of enriched CD4+ T cells that have been engineered with a polynucleotide encoding a recombinant receptor, e.g. a CAR. In some embodiments, two separate compositions of enriched CD4+ T cells and enriched CD8+ T cells, such as a composition of enriched CD4+ T cells and a composition of enriched CD8+ T cells that have each been separately engineered with a polynucleotide encoding a recombinant receptor, e.g. a CAR, are separately cultivated, e.g., under conditions that promote proliferation and/or expansion.
[0635] In some embodiments, cultivation is carried out under conditions that promote proliferation and/or expansion. In some embodiments, such conditions may be designed to induce proliferation, expansion, activation, and/or survival of cells in the population. In particular embodiments, the stimulating conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to promote growth, division, and/or expansion of the cells.
[0636] In particular embodiments, the cells are cultivated in the presence of one or more cytokines. In particular embodiments, the one or more cytokines are recombinant cytokines. In some embodiments, the one or more cytokines are human recombinant cytokines. In certain embodiments, the one or more cytokines bind to and/or are capable of binding to receptors that are expressed by and/or are endogenous to T cells. In particular embodiments, the one or more cytokines, e.g. a recombinant cytokine, is or includes a member of the 4-alpha-helix bundle family of cytokines. In some embodiments, members of the 4-alpha-helix bundle family of cytokines include, but are not limited to, interleukin-2 (IL -2), interleukin-4 (IL-4), interleukin-7 (IL-7), interleukin-9 (IL-9), interleukin 12 (IL-12), interleukin 15 (IL- 15), granulocyte colony-stimulating factor (G-CSF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). In some embodiments, the one or more recombinant cytokine includes IL -2, IL-7 and/or IL-15. In some embodiments, the cells, e.g., engineered cells, are cultivated in the presence of a cytokine, e.g., a recombinant human cytokine, at a concentration of between 1 lU/mL and 2,000 lU/mL, between 10 lU/mL and 100 lU/mL, between 50 lU/mL and 200 lU/mL, between 100 lU/mL and 500 lU/mL, between 100 lU/mL and 1,000 lU/mL, between 500 lU/mL and 2,000 lU/mL, or between 100 lU/mL and 1 ,500 lU/mL.
[0637] In some embodiments, the cultivation is performed under conditions that generally include a temperature suitable for the growth of primary immune cells, such as human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees Celsius, and generally at or about 37 degrees Celsius. In some embodiments, the composition of enriched T cells is incubated at a temperature of 25 to 38°C, such as 30 to 37°C, for example at or about 37 °C ± 2 °C. In some embodiments, the incubation is carried out for a time period until the culture, e.g. cultivation or expansion, results in a desired or threshold density, number or dose of cells. In some embodiments, the incubation is greater than or greater than about or is for about or 24 hours, 48 hours, 72 hours, 96 hours, 5 days, 6 days, 7 days, 8 days, 9 days or more.
[0638] In particular embodiments, the cultivation is performed in a closed system. In certain embodiments, the cultivation is performed in a closed system under sterile conditions. In particular embodiments, the cultivation is performed in the same closed system as one or more steps of the provided systems. In some embodiments the composition of enriched T cells is removed from a closed system and placed in and/or connected to a bioreactor for the cultivation. Examples of suitable bioreactors for the cultivation include, but are not limited to, GE Xuri W25, GE Xuri W5, Sartorius BioSTAT® RM 20 | 50, Finesse SmartRocker Bioreactor Systems, and Pall XRS Bioreactor Systems. In some embodiments, the bioreactor is used to perfuse and/or mix the cells during at least a portion of the cultivation step.
[0639] In some embodiments, the mixing is or includes rocking and/or motioning. In some cases, the bioreactor can be subject to motioning or rocking, which, in some aspects, can increase oxygen transfer. Motioning the bioreactor may include, but is not limited to rotating along a horizontal axis, rotating along a vertical axis, a rocking motion along a tilted or inclined horizontal axis of the bioreactor or any combination thereof. In some embodiments, at least a portion of the incubation is carried out with rocking. The rocking speed and rocking angle may be adjusted to achieve a desired agitation. In some embodiments the rock angle is 20°, 19°, 18°, 17°, 16°, 15°, 14°, 13°, 12°, 11°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2° or 1°. In certain embodiments, the rock angle is between 6-16°. In other embodiments, the rock angle is between 7-16°. In other embodiments, the rock angle is between 8-12°. In some embodiments, the rock rate is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 rpm. In some embodiments, the rock rate is between 4 and 12 rpm, such as between 4 and 6 rpm, inclusive.
[0640] In some embodiments, the bioreactor maintains the temperature at or near 37°C and CO2 levels at or near 5% with a steady air flow at, at about, or at least 0.01 L/min, 0.05 L/min, 0.1 L/min, 0.2 L/min, 0.3 L/min, 0.4 L/min, 0.5 L/min, 1.0 L/min, 1.5 L/min, or 2.0 L/min or greater than 2.0 L/min. In certain embodiments, at least a portion of the cultivation is performed with perfusion, such as with a rate of 290 ml/day, 580 ml/day, and/or 1160 ml/day, e.g., depending on the timing in relation to the start of the cultivation and/or density of the cultivated cells. In some embodiments, at least a portion of the cell culture expansion is performed with a rocking motion, such as at an angle of between 5° and 10°, such as 6°, at a constant rocking speed, such as a speed of between 5 and 15 RPM, such as 6 RPM or 10 RPM.
[0641] In some embodiments, the methods for manufacturing, generating or producing a cell therapy and/or engineered cells, in accord with the provided methods, uses or articles of manufacture, may include formulation of cells, such as formulation of genetically engineered cells resulting from the processing steps prior to or after the incubating, engineering, and cultivating, and/or one or more other processing steps as described. In some embodiments, one or more of the processing steps, including formulation of cells, can be carried out in a closed system. In some cases, the cells are processed in one or more steps (e.g. carried out in the centrifugal chamber and/or closed system) for manufacturing, generating or producing a cell therapy and/or engineered cells may include formulation of cells, such as formulation of genetically engineered cells resulting from the transduction processing steps prior to or after the culturing, e.g. cultivation and expansion, and/or one or more other processing steps as described. In some embodiments, the genetically engineered cells are formulated as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof.
[0642] In some embodiments, the dose of cells comprising cells engineered with a recombinant antigen receptor, e.g. CAR or TCR, is provided as a composition or formulation, such as a pharmaceutical composition or formulation. Such compositions can be used in accord with the provided methods, such as in the treatment of diseases, conditions, and disorders, or in detection, diagnostic, and prognostic methods, and uses and articles of manufacture. In some cases, the cells can be formulated in an amount for dosage administration, such as for a single unit dosage administration or multiple dosage administration.
[0643] In some embodiments, the cells can be formulated into a container, such as a bag or vial. In some embodiments, the vial may be an infusion vial. In some embodiments, the vial is formulated with a single unit dose of the engineered cells, such as including the number of cells for administration in a given dose or fraction thereof. [0644] In some embodiments, the cells are formulated in a pharmaceutically acceptable buffer, which may, in some aspects, include a pharmaceutically acceptable carrier or excipient. In some embodiments, the processing includes exchange of a medium into a medium or formulation buffer that is pharmaceutically acceptable or desired for administration to a subject. In some embodiments, the processing steps can involve washing the transduced and/or expanded cells to replace the cells in a pharmaceutically acceptable buffer that can include one or more optional pharmaceutically acceptable carriers or excipients. Exemplary of such pharmaceutical forms, including pharmaceutically acceptable carriers or excipients, can be any described below in conjunction with forms acceptable for administering the cells and compositions to a subject. The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount.
[0645] In some embodiments, the formulation buffer contains a cryopreservative. In some embodiments, the cell are formulated with a cyropreservative solution that contains 1.0% to 30% DMSO solution, such as a 5% to 20% DMSO solution or a 5% to 10% DMSO solution. In some embodiments, the cryopreservation solution is or contains, for example, PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. In some embodiments, the cryopreservative solution is or contains, for example, at least or about 7.5% DMSO. In some embodiments, the processing steps can involve washing the transduced and/or expanded cells to replace the cells in a cryopreservative solution. In some embodiments, the cells are frozen, e.g., cryoprotected or cryopreserved, in media and/or solution with a final concentration of or of about 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, or 5.0% DMSO, or between 1% and 15%, between 6% and 12%, between 5% and 10%, or between 6% and 8% DMSO. In particular embodiments, the cells are frozen, e.g., cryoprotected or cryopreserved, in media and/or solution with a final concentration of or of about 5.0%, 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.25%, 1.0%, 0.75%, 0.5%, or 0.25% HSA, or between 0.1% and 5%, between 0.25% and 4%, between 0.5% and 2%, or between 1% and 2% HSA.
[0646] In some embodiments, the formulation is carried out using one or more processing step including washing, diluting or concentrating the cells, such as the cultured or expanded cells. In some embodiments, the processing can include dilution or concentration of the cells to a desired concentration or number, such as unit dose form compositions including the number of cells for administration in a given dose or fraction thereof. In some embodiments, the processing steps can include a volumereduction to thereby increase the concentration of cells as desired. In some embodiments, the processing steps can include a volume-addition to thereby decrease the concentration of cells as desired. In some embodiments, the processing includes adding a volume of a formulation buffer to transduced and/or expanded cells. In some embodiments, the volume of formulation buffer is from or from about 10 mL to 1000 mL, such as at least or at least about or about or 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, 500 mL, 600 mL, 700 mL, 800 mL, 900 mL or 1000 mL.
[0647] In some embodiments, such processing steps for formulating a cell composition is carried out in a closed system. Exemplary of such processing steps can be performed using a centrifugal chamber in conjunction with one or more systems or kits associated with a cell processing system, such as a centrifugal chamber produced and sold by Biosafe SA, including those for use with the Sepax® or Sepax 2® cell processing systems. An exemplary system and process is described in International Publication Number WO2016/073602. In some embodiments, the method includes effecting expression from the internal cavity of the centrifugal chamber a formulated composition, which is the resulting composition of cells formulated in a formulation buffer, such as pharmaceutically acceptable buffer, in any of the above embodiments as described. In some embodiments, the expression of the formulated composition is to a container, such as the vials of the biomedical material vessels described herein, that is operably linked as part of a closed system with the centrifugal chamber. In some embodiments, the biomedical material vessels are configured for integration and or operable connection and/or is integrated or operably connected, to a closed system or device that carries out one or more processing steps. In some embodiments, the biomedical material vessel is connected to a system at an output line or output position. In some cases, the closed system is connected to the vial of the biomedical material vessel at the inlet tube. Exemplary close systems for use with the biomedical material vessels described herein include the Sepax® and Sepax® 2 system.
[0648] In some embodiments, the closed system, such as associated with a centrifugal chamber or cell processing system, includes a multi-port output kit containing a multi-way tubing manifold associated at each end of a tubing line with a port to which one or a plurality of containers can be connected for expression of the formulated composition. In some aspects, a desired number or plurality of vials, can be sterilely connected to one or more, generally two or more, such as at least 3, 4, 5, 6, 7, 8 or more of the ports of the multi-port output. For example, in some embodiments, one or more containers, e.g., biomedical material vessels, can be attached to the ports, or to fewer than all of the ports. Thus, in some embodiments, the system can effect expression of the output composition into a plurality of vials of the biomedical material vessels.
[0649] In some aspects, cells can be expressed to the one or more of the plurality of output containers, e.g., vials, in an amount for dosage administration, such as for a single unit dosage administration or multiple dosage administration. For example, in some embodiments, the vials, may each contain the number of cells for administration in a given dose or fraction thereof. Thus, each vial, in some aspects, may contain a single unit dose for administration or may contain a fraction of a desired dose such that more than one of the plurality of vials, such as two of the vials, or 3 of the vials, together constitute a dose for administration. In some embodiments, 4 vials together constitute a dose for administration. [0650] Thus, the containers, e.g. bags or vials, generally contain the cells to be administered, e.g., one or more unit doses thereof. The unit dose may be an amount or number of the cells to be administered to the subject or twice the number (or more) of the cells to be administered. It may be the lowest dose or lowest possible dose of the cells that would be administered to the subject. In some aspects, the provided articles of manufacture includes one or more of the plurality of output containers.
[0651] In some embodiments, each of the containers, e.g. bags or vials, individually comprises a unit dose of the cells. Thus in some embodiments, each of the containers comprises the same or approximately or substantially the same number of cells. In some embodiments, each unit dose contains at or about or at least or at least about 1 x 106, 2 x 106, 5 x 106, 1 x 107, 5 x 107, or 1 x 108 engineered cells, total cells, T cells, or PBMCs. In some embodiments, each unit dose contains at or about or at least or at least about 1 x 106, 2 x 106, 5 x 106, 1 x 107, 5 x 107, or 1 x 108 CAR+ T cells that are CD3+, such as CD4+ or CD8+, or a viable subset thereof. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is between at or about 10 mL and at or about 100 mL, such as at or about or at least or at least about 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL or 100 mL. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is between at or about 1 mL and at or about 10 mL, such as between at or about 1 mL and at or about 5 mL. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is between at or about 4 mL and at or about 5 mL. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is or is about 4.4 mL. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is or is about 4.5 mL. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is or is about 4.6 mL. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is or is about 4.7 mL. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is or is about 4.8 mL. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is or is about 4.9 mL. In some embodiments, the volume of the formulated cell composition in each container, e.g. bag or vial, is or is about 5.0 mL.
[0652] In some embodiments, the formulated cell composition has a concentration of greater than at or about 0.5 x 106 recombinant receptor-expressing (e.g. CAR+)/CD3+ cells or such viable cells per mL, greater than at or about 1.0 x 106 recombinant receptor-expressing (e.g. CAR+)/CD3+ cells or such viable cells per mL, greater than at or about 1.5 x 106 recombinant receptor-expressing (e.g. CAR+)/CD3+ cells or such viable cells per mL, greater than at or about 2.0 x 106 recombinant receptor-expressing (e.g. CAR+)/CD3+ cells or such viable cells per mL. greater than at or about 2.5 x 106 recombinant receptorexpressing (e.g. CAR+)/CD3+ cells or such viable cells per mL, greater than at or about 2.6 x 106 recombinant receptor-expressing (e.g. CAR+)/CD3+ cells or such viable cells per mL, greater than at or about 2.7 x 106 recombinant receptor-expressing (e.g. CAR+)/CD3+ cells or such viable cells per mL, greater than at or about 2.8 x 106 recombinant receptor-expressing (e.g. CAR+)/CD3+ cells or such viable cells per mL, greater than at or about 2.9 x 106 recombinant receptor-expressing (e.g. CAR+)/CD3+ cells or such viable cells per mL greater than at or about 3.0 x 106 recombinant receptor-expressing (e.g. CAR+)/CD3+ cells or such viable cells per mL, greater than at or about 3.5 x 106 recombinant receptorexpressing (e.g. CAR+)/CD3+ cells or such viable cells per mL, greater than at or about 4.0 x 106 recombinant receptor-expressing (e.g. CAR+)/CD3+ cells or such viable cells per mL, greater than at or about 4.5 x 106 recombinant receptor-expressing (e.g. CAR+)/CD3+ cells or such viable cells per mL or greater than at or about 5 x 106 recombinant receptor-expressing (e.g. CAR+)/CD3+ cells or such viable cells per mL. In some embodiments, the CD3+ cells are CD4+ T cells. In some embodiments, the CD3+ cells are CD8+ T cells. In some embodiments, the CD3+ T dels are CD4+ and CD8+ T cells.
[0653] In some embodiments, the cells in the container, e.g. bag or vials, can be cryopreserved. In some embodiments, the container, e.g. vials, can be stored in liquid nitrogen until further use.
[0654] In some embodiments, such cells produced by the method, or a composition comprising such cells, are administered to a subject for beating a disease or condition, for example, in accord with the methods, uses and articles of manufacture described herein.
III. EXEMPLARY TREATMENT OUTCOMES AND METHODS FOR ASSESSING THE SAME
[0655] In some embodiments of the methods, combinations, uses, kits and articles of manufacture provided herein, the provided combination therapy results in one or more treatment outcomes, such as a feature associated with any one or more of the parameters associated with the therapy or treatment, as described below. In some embodiments, the method includes assessment of the cytotoxicity of the T cells toward cancer cells, e.g., T cells administered for the T cell based therapy. In some embodiments, the method includes assessment of the exposure, persistence and proliferation of the T cells, e.g., T cells administered for the T cell based therapy. In some embodiments, the exposure, or prolonged expansion and/or persistence of the cells, and/or changes in cell phenotypes or functional activity of the cells, e.g., cells administered for immunotherapy, e.g. T cell therapy, in the methods provided herein, can be measured by assessing the characteristics of the T cells in vitro or ex vivo. In some embodiments, such assays can be used to determine or confirm the function of the T cells, e.g. T cell therapy, before, during, or after administering the combination therapy provided herein.
[0656] In some embodiments, the combination therapy can further include one or more screening steps to identify subjects for treatment with the combination therapy and/or continuing the combination therapy, and/or a step for assessment of heatment outcomes and/or monitoring treatment outcomes. In some embodiments, the step for assessment of heatment outcomes can include steps to evaluate and/or to monitor heatment and/or to identify subjects for administration of further or remaining steps of the therapy and/or for repeat therapy. In some embodiments, the screening step and/or assessment of treatment outcomes can be used to determine the dose, frequency, duration, timing and/or order of the combination therapy provided herein.
[0657] In some embodiments, any of the screening steps and/or assessment of treatment of outcomes described herein can be used prior to, during, during the course of, or subsequent to administration of one or more steps of the provided combination therapy, e.g., administration of the immunotherapy or cell therapy e.g. CAR-expressing T cells), and/or a BCL2 inhibitor, e.g. venetoclax. In some embodiments, assessment is made prior to, during, during the course of, or after performing any of the methods provided herein. In some embodiments, the assessment is made prior to performing the methods provided herein. In some embodiments, assessment is made after performing one or more steps of the methods provided herein. In some embodiments, the assessment is performed prior to administration of one or more steps of the provided combination therapy, for example, to screen and identify patients suitable and/or susceptible to receive the combination therapy. In some embodiments, the assessment is performed during, during the course of, or subsequent to administration of one or more steps of the provided combination therapy, for example, to assess the intermediate or final treatment outcome, e.g., to determine the efficacy of the treatment and/or to determine whether to continue or repeat the treatments and/or to determine whether to administer the remaining steps of the combination therapy.
[0658] In some embodiments, treatment of outcomes includes improved immune function, e.g., immune function of the T cells administered for cell based therapy and/or of the endogenous T cells in the body. In some embodiments, exemplary treatment outcomes include, but are not limited to, enhanced T cell proliferation, enhanced T cell functional activity, changes in immune cell phenotypic marker expression, such as such features being associated with the engineered T cells, e.g. CAR-T cells, administered to the subject. In some embodiments, exemplary treatment outcomes include decreased disease burden, e.g., tumor burden, improved clinical outcomes and/or enhanced efficacy of therapy.
[0659] In some embodiments, the screening step and/or assessment of treatment of outcomes includes assessing the survival and/or function of the T cells administered for cell based therapy. In some embodiments, the screening step and/or assessment of treatment of outcomes includes assessing the levels of cytokines or growth factors. In some embodiments, the screening step and/or assessment of treatment of outcomes includes assessing disease burden and/or improvements, e.g., assessing tumor burden and/or clinical outcomes. In some embodiments, either of the screening step and/or assessment of treatment of outcomes can include any of the assessment methods and/or assays described herein and/or known in the art, and can be performed one or more times, e.g., prior to, during, during the course of, or subsequently to administration of one or more steps of the combination therapy. Exemplary sets of parameters associated with a treatment outcome, which can be assessed in some embodiments of the methods provided herein, include peripheral blood immune cell population profile and/or tumor burden. [0660] In some embodiments, the methods affect efficacy of the cell therapy in the subject. In some embodiments, the cytotoxicity of recombinant receptor-expressing, e.g., CAR-expressing, cells in the subject following administration of the dose of cells in the method with a BCL2 inhibitor, e.g., venetoclax, is greater as compared to that achieved via a method without the administration of the inhibitor. In some embodiments, the cytotoxicity of recombinant receptor-expressing, e.g., CAR- expressing, cells in the subject following administration of the dose of cells in the method with a subtherapeutically effective amount of a BCL2 inhibitor, e.g., venetoclax, is greater as compared to that achieved via a method without the administration of the inhibitor. In some embodiments, cytotoxicity in the subject of the administered T cell therapy, e.g., CAR-expressing T cells is assessed as compared to a method in which the T cell therapy is administered to the subject in the absence of a BCL2 inhibitor, e.g., venetoclax. In some embodiments, the methods result in the administered T cells exhibiting increased or prolonged cytotoxicity in the subject as compared to a method in which the T cell therapy is administered to the subject in the absence of the inhibitor.
[0661] In some embodiments, the administration of a BCL2 inhibitor, e.g., venetoclax, decreases disease burden, e.g., tumor burden, in the subject as compared to a method in which the dose of cells expressing the recombinant receptor is administered to the subject in the absence of the inihibitor. In some embodiments, the administration of a subtherapeutically effective amount of a BCL2 inhibitor, e.g., venetoclax, decreases disease burden, e.g., tumor burden, in the subject as compared to a method in which the dose of cells expressing the recombinant receptor is administered to the subject in the absence the inhibitor. In some embodiments, the administration of a BCL2 inhibitor, e.g., venetoclax, decreases blast marrow in the subject as compared to a method in which the dose of cells expressing the recombinant receptor is administered to the subject in the absence of the inhibitor. In some embodiments, the administration of a BCL2 inhibitor, e.g., venetoclax, results in improved clinical outcomes, e.g., objective response rate (ORR), progression-free survival (PFS) and/or overall survival (OS), compared to a method in which the dose of cells expressing the recombinant receptor is administered to the subject in the absence of the inhibitor.
[0662] In some embodiments, the subject can be screened prior to the administration of one or more steps of the combination therapy. For example, the subject can be screened for characteristics of the disease and/or disease burden, e.g., tumor burden, prior to administration of the combination therapy, to determine suitability, responsiveness and/or susceptibility to administering the combination therapy. For example, the subject can be screened for characteristics of the disease, e.g., overexpression or aberrant expression of a prosurvival or proapoptotic BCL2 family protein, prior to administration of the combination therapy, to determine suitability, responsiveness and/or susceptibility to administering the combination therapy. In some embodiments, the screening step and/or assessment of treatment outcomes can be used to determine the dose, frequency, duration, timing and/or order of the combination therapy provided herein. [0663] In some embodiments, the subject can be screened after administration of one of the steps of the combination therapy, to determine and identify subjects to receive the remaining steps of the combination therapy and/or to monitor efficacy of the therapy. In some embodiments, the number, level or amount of administered T cells and/or proliferation and/or activity of the administered T cells is assessed after administration of the engineered T cells and/or after administration of a BCL2 inhibitor, e.g., venetoclax.
[0664] In some embodiments, a change and/or an alteration, e.g., an increase, an elevation, a decrease or a reduction, in levels, values or measurements of a parameter or outcome compared to the levels, values or measurements of the same parameter or outcome in a different time point of assessment, a different condition, a reference point and/or a different subject is determined or assessed. In some embodiments, the levels, values or measurements of two or more parameters are determined, and relative levels are compared. In some embodiments, the determined levels, values or measurements of parameters are compared to the levels, values or measurements from a control sample or an untreated sample. In some embodiments, the determined levels, values or measurements of parameters are compared to the levels from a sample from the same subject but at a different time point. The values obtained in the quantification of individual parameter can be combined for the purpose of disease assessment, e.g., by forming an arithmetical or logical operation on the levels, values or measurements of parameters by using multi-parametric analysis. In some embodiments, a ratio of two or more specific parameters can be calculated.
[0665] Assessment and determination of parameters associated with T cell health, function, activity, and/or outcomes, such as response, efficacy and/or toxicity outcomes, can be assessed at various time points. In some aspects, the assessment can be performed multiple times, e.g., prior to, during, and/or after manufacturing of the cells, prior to, during, and/or after the initiation of administrationof the cell therapy, and/or prior to, during, and/or after the initiation of administration of the BCL2 inhibitor, e.g., venetoclax.
[0666] In some embodiments, functional attributes of the administered cells and/or cell compositions include monitoring pharmacokinetic (PK) and pharmacodynamics parameters, expansion and persistence of the cells, cell functional assays (e.g., any described herein, such as cytotoxicity assay, cytokine secretion assay and in vivo assays), high-dimensional T cell signaling assessment, and assessment of exhaustion phenotypes and/or signatures of the T cells. In some aspects, other attributes that can be assessed or monitored include monitoring and assessment of minimal residual disease (MRD). In some aspects, other attributes that can be assessed or monitored include pharmacodynamic parameters of the BCL2 inhibitor, e.g., venetoclax. A. RESPONSE, EFFICACY, AND SURVIVAL
[0667] In some embodiments, parameters associated with therapy or a treatment outcome, which include parameters that can be assessed for the screening steps and/or assessment of treatment of outcomes and/or monitoring treatment outcomes, includes tumor or disease burden. The administration of the therapy that is an immunotherapy or cell therapy, such as a T cell therapy (e.g. CAR-expressing T cells) and/or a BCL2 inhibitor, e.g., venetoclax, can reduce or prevent the expansion or burden of the disease or condition in the subject. For example, where the disease or condition is a tumor, the methods generally reduce tumor size, bulk, metastasis, percentage of blasts in the bone marrow or molecularly detectable B cell malignancy and/or improve prognosis or survival or other symptom associated with tumor burden. In some embodiments, the disaease or condition is a cancer, such as a CLL or SLL.
[0668] In some aspects, the administration in accord with the provided methods, and/or with the provided articles of manufacture or compositions, generally reduces or prevents the expansion or burden of the disease or condition in the subject. For example, where the disease or condition is a tumor, the methods generally reduce tumor size, bulk, metastasis, percentage of blasts in the bone marrow or molecularly detectable B cell malignancy and/or improve prognosis or survival or other symptom associated with tumor burden.
[0669] In some embodiments, the provided methods result in a decreased tumor burden in treated subjects compared to alternative methods in which the therapy, such as a T cell therapy (e.g. CAR- expressing T cells) is given without administration of a BCL2 inhibitor, e.g., venetoclax. In some embodiments, the provided methods result in a decreased tumor burden in subjects treated with a subtherapeutically effective amount of a BCL2 inhibitor, e.g. venetoclax, compared to alternative methods in which the immunotherapy, such as a T cell therapy e.g. CAR-expressing T cells) is given without administration of the inhibitor. It is not necessary that the tumor burden actually be reduced in all subjects receiving the combination therapy, but that tumor burden is reduced on average in subjects treated, such as based on clinical data, in which a majority of subjects treated with such a combination therapy exhibit a reduced tumor burden, such as at least 50%, 60%, 70%, 80%, 90%, 95% or more of subjects treated with the combination therapy, exhibit a reduced tumor burden.
[0670] In some embodiments, the provided methods result in increased cytotoxic activity of the immunotherapy or cell therapy as compared to alternative methods in which the therapy, such as a T cell therapy (e.g. CAR-expressing T cells) is given without administration of a BCL2 inhibitor, e.g., venetoclax. In some embodiments, the provided methods result in increased cytotoxicity in subjects treated with a subtherapeutically effective amount of a BCL2 inhibitor, e.g. venetoclax, compared to alternative methods in which the immunotherapy, such as a T cell therapy (e.g. CAR-expressing T cells) is given without administration of the inhibitor. In some cases, the provided methods result in an increased cytotoxicity of the immunotherapy or cell therapy, optionally via perforin- and/or granzyme- mediate apoptosis, of one or more cancer cells, compared to alternative methods in which the immunotherapy, such as a T cell therapy (e.g. CAR-expressing T cells) is given without administration of a BCL2 inhibitor, e.g., venetoclax. It is not necessary that the cytotoxicity actually be increased in all subjects receiving the combination therapy, but that cytotoxicity is increased on average in subjects treated, such as based on clinical data, in which a majority of subjects treated with such a combination therapy exhibit a reduced tumor burden, such as at least 50%, 60%, 70%, 80%, 90%, 95% or more of subjects treated with the combination therapy, exhibit a reduced tumor burden.
[0671] Disease burden can encompass a total number of cells of the disease in the subject or in an organ, tissue, or bodily fluid of the subject, such as the organ or tissue of the tumor or another location, e.g., which would indicate metastasis. For example, tumor cells may be detected and/or quantified in the blood, lymph or bone marrow in the context of certain hematological malignancies. In some aspects, tumor cells may be detected and/or quantified in the peripheral blood (PB). In some aspects, tumor cells may be detected and/or quantified in the lymph nodes. In some aspects, tumor cells may be detected and/or quantified in the bone marrow. Disease burden can include, in some embodiments, the mass of a tumor, the number or extent of metastases and/or the percentage of blast cells present in the bone marrow.
[0672] In some embodiments, the subject has a myeloma, a lymphoma or a leukemia. The extent of disease burden can be determined by assessment of residual leukemia in blood or bone marrow. In some embodiments, the subject has a non-Hodgkin lymphoma (NHL), an acute lymphoblastic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a small lymphocytic lymphoma (SLL) a diffuse large B-cell lymphoma (DLBCL) or a myeloma, e.g., a multiple myeloma (MM). In some embodiments, the subject has a leukemia or lymphoma. In some embodiments, the subject has a leukemia. In some cases, the leukemia is CLL. In some embodiments, the subject has a lymphoma. In some cases, the subject has a NHL, including DBCBL. In some cases, the lymphoma is SLL.
[0673] In some aspects, response rates in subjects, such as subjects with NHL, are based on the Lugano criteria. (Cheson et al., (2014) JCO., 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323- 338; Cheson, B.D. (2015) Chin. Clin. Oncol. 4(1):5). In some aspects, response assessment utilizes any of clinical, hematologic, and/or molecular methods. In some aspects, response assessed using the Lugano criteria involves the use of positron emission tomography (PET)-computed tomography (CT) and/or CT as appropriate. PET-CT evaluations may further comprise the use of fluorodeoxyglucose (FDG) for FDG-avid lymphomas. In some aspects, where PET-CT will be used to assess response in FDG-avid histologies, a 5-point scale may be used. In some respects, the 5-point scale comprises the following criteria: 1, no uptake above background; 2, uptake < mediastinum; 3, uptake > mediastinum but < liver; 4, uptake moderately > liver; 5, uptake markedly higher than liver and/or new lesions; X, new areas of uptake unlikely to be related to lymphoma. [0674] In some aspects, a complete response as described using the Lugano criteria involves a complete metabolic response and a complete radiologic response at various measureable sites. In some aspects, these sites include lymph nodes and extralymphatic sites, wherein a CR is described as a score of 1, 2, or 3 with or without a residual mass on the 5-point scale, when PET -CT is used. In some aspects, in Waldeyer’s ring or extranodal sites with high physiologic uptake or with activation within spleen or marrow (e.g., with chemotherapy or myeloid colony-stimulating factors), uptake may be greater than normal mediastinum and/or liver. In this circumstance, complete metabolic response may be inferred if uptake at sites of initial involvement is no greater than surrounding normal tissue even if the tissue has high physiologic uptake. In some aspects, response is assessed in the lymph nodes using CT, wherein a CR is described as no extralymphatic sites of disease and target nodes/nodal masses must regress to < 1.5 cm in longest transverse diameter of a lesion (LDi). Further sites of assessment include the bone marrow wherein PET-CT-based assessment should indicate a lack of evidence of FDG-avid disease in marrow and a CT-based assessment should indicate a normal morphology, which if indeterminate should be IHC negative. Further sites may include assessment of organ enlargement, which should regress to normal. In some aspects, nonmeasured lesions and new lesions are assessed, which in the case of CR should be absent (Cheson et al., (2014) JCO., 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323-338; Cheson, B.D. (2015) Chin. Clin. Oncol. 4(1):5).
[0675] In some aspects, a partial response (PR) as described using the Lugano criteria involves a partial metabolic and/or radiological response at various measureable sites. In some aspects, these sites include lymph nodes and extralymphatic sites, wherein a PR is described as a score of 4 or 5 with reduced uptake compared with baseline and residual mass(es) of any size, when PET-CT is used. At interim, such findings can indicate responding disease. At the end of treatment, such findings can indicate residual disease. In some aspects, response is assessed in the lymph nodes using CT, wherein a PR is described as >50% decrease in SPD of up to 6 target measureable nodes and extranodal sites. If a lesion is too small to measure on CT, 5 mm x 5 mm is assigned as the default value; if the lesion is no longer visible, the value is 0 mm x 0 mm; for a node >5 mm x 5 mm, but smaller than normal, actual measurements are used for calculation. Further sites of assessment include the bone marrow wherein PET-CT-based assessment should indicate residual uptake higher than uptake in normal marrow but reduced compared with baseline (diffuse uptake compatible with reactive changes from chemotherapy allowed). In some aspects, if there are persistent focal changes in the marrow in the context of a nodal response, consideration should be given to further evaluation with MRI or biopsy, or an interval scan. In some aspects, further sites may include assessment of organ enlargement, where the spleen must have regressed by >50% in length beyond normal. In some aspects, nonmeasured lesions and new lesions are assessed, which in the case of PR should be absent/normal, regressed, but no increase. No response/stable disease (SD) or progressive disease (PD) can also be measured using PET-CT and/or CT based assessments. (Cheson et al., (2014) JCO., 32(27):3059-3067; Johnson et al., (2015) Radiology 2:323-338; Cheson, B.D. (2015) Chin. Clin. Oncol., 4(1):5).
[0676] In some respects, progression-free survival (PFS) is described as the length of time during and after the treatment of a disease, such as a B cell malignancy, that a subject lives with the disease but it does not get worse. In some aspects, objective response (OR) is described as a measurable response. In some aspects, objective response rate (ORR) is described as the proportion of patients who achieved CR or PR. In some aspects, overall survival (OS) is described as the length of time from either the date of diagnosis or the start of treatment for a disease, such as a B cell malignancy, that subjects diagnosed with the disease are still alive. In some aspects, event-free survival (EFS) is described as the length of time after treatment for a B cell malignancy ends that the subject remains free of certain complications or events that the treatment was intended to prevent or delay. These events may include the return of the B cell malignancy or the onset of certain symptoms, such as bone pain from B cell malignancy that has spread to the bone, or death.
[0677] In some embodiments, the measure of duration of response (DOR) includes the time from documentation of tumor response to disease progression. In some embodiments, the parameter for assessing response can include durable response, e.g., response that persists after a period of time from initiation of therapy. In some embodiments, durable response is indicated by the response rate at approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18 or 24 months after initiation of therapy. In some embodiments, the response is durable for greater than 3 months or greater than 6 months.
[0678] In some aspects, the RECIST criteria is used to determine objective tumor response. (Eisenhauer et al., European Journal of Cancer 45 (2009) 228-247.) In some aspects, the RECIST criteria is used to determine objective tumor response for target lesions. In some respects, a complete response as determined using RECIST criteria is described as the disappearance of all target lesions and any pathological lymph nodes (whether target or non-target) must have reduction in short axis to <10 mm. In other aspects, a partial response as determined using RECIST criteria is described as at least a 30% decrease in the sum of diameters of target lesions, taking as reference the baseline sum diameters. In other aspects, progressive disease (PD) is described as at least a 20% increase in the sum of diameters of target lesions, taking as reference the smallest sum on study (this includes the baseline sum if that is the smallest on study). In addition to the relative increase of 20%, the sum must also demonstrate an absolute increase of at least 5 mm (in some aspects the appearance of one or more new lesions is also considered progression). In other aspects, stable disease (SD) is described as neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, taking as reference the smallest sum diameters while on study.
[0679] In some aspects, exemplary parameters to assess the extent of disease burden include such parameters as number of clonal plasma cells (e.g., >10% on bone marrow biopsy or in any quantity in a biopsy from other tissues; plasmacytoma), presence of monoclonal protein (paraprotein) in either serum or urine, evidence of end-organ damage felt related to the plasma cell disorder (e.g., hypercalcemia (corrected calcium >2.75 mmol/1); renal insufficiency attributable to myeloma; anemia (hemoglobin <10 g/dl); and/or bone lesions (lytic lesions or osteoporosis with compression fractures)).
[0680| In some aspects, such as in the case of DLBCL, exemplary parameters to assess the extent of disease burden include such parameters as cellular morphology (e.g., centroblastic, immunobias tic, and anaplastic cells), gene expression, miRNA expression and protein expression (e.g., expression of BCL2, BCL6, MUM1, LM02, MYC, and p21).
[0681] In some aspects, response rates in subjects, such as subjects with CLL, are based on the International Workshop on Chronic Lymphocytic Leukemia (IWCLL) response criteria (Hallek, et al., Blood 2008, Jun 15; 111(12): 5446-5456). In some aspects, these criteria are described as follows: complete remission (CR), which in some aspects requires the absence of peripheral blood clonal lymphocytes by immunophenotyping, absence of lymphadenopathy, absence of hepatomegaly or splenomegaly, absence of constitutional symptoms and satisfactory blood counts; complete remission with incomplete marrow recovery (CRi), which in some aspects is described as CR above, but without normal blood counts; partial remission (PR), which in some aspects is described as > 50% fall in lymphocyte count, > 50% reduction in lymphadenopathy or > 50% reduction in liver or spleen, together with improvement in peripheral blood counts; progressive disease (PD), which in some aspects is described as > 50% rise in lymphocyte count to > 5 xlO9/L, > 50% increase in lymphadenopathy, > 50% increase in liver or spleen size, Richter’s transformation, or new cytopenias due to CLL; and stable disease, which in some aspects is described as not meeting criteria for CR, CRi, PR or PD.
[0682| In some embodiments, the subjects exhibits a CR or OR if, within 1 month of the administration of the dose of cells, lymph nodes in the subject are less than at or about 20 mm in size, less than at or about 10 mm in size or less than at or about 10 mm in size.
[0683] In some embodiments, an index clone of the CLL is not detected in the bone marrow of the subject (or in the bone marrow of greater than 50%, 60%, 70%, 80%, 90% or more of the subjects treated according to the methods. In some embodiments, an index clone of the CLL is assessed by IgH deep sequencing. In some embodiments, the index clone is not detected at a time that is at or about or at least at or about 1, 2, 3, 4, 5, 6, 12, 18 or 24 months following the administration of the cells.
[0684] In some embodiments, a subject exhibits morphologic disease if there are greater than or equal to 5% blasts in the bone marrow, for example, as detected by light microscopy, such as greater than or equal to 10% blasts in the bone marrow, greater than or equal to 20% blasts in the bone marrow, greater than or equal to 30% blasts in the bone marrow, greater than or equal to 40% blasts in the bone marrow or greater than or equal to 50% blasts in the bone marrow. In some embodiments, a subject exhibits morphologic disease if there are greater than or equal to 5% blasts in the bone marrow. In some embodiments, a subject exhibits complete or clinical remission if there are less than 5% blasts in the bone marrow. [0685] In some embodiments, a subject may exhibit complete remission, but a small proportion of morphologically undetectable (by light microscopy techniques) residual leukemic cells are present. A subject is said to exhibit minimum residual disease (MRD) if the subject exhibits less than 5% blasts in the bone marrow and exhibits molecularly detectable B cell malignancy. In some embodiments, molecularly detectable B cell malignancy can be assessed using any of a variety of molecular techniques that permit sensitive detection of a small number of cells. In some aspects, such techniques include PCR assays, which can determine unique Ig/T-cell receptor gene rearrangements or fusion transcripts produced by chromosome translocations. In some embodiments, flow cytometry can be used to identify B cell malignancy cell based on leukemia-specific immunophenotypes. In some embodiments, molecular detection of B cell malignancy can detect as few as 1 leukemia cell in 100,000 normal cells. In some embodiments, a subject exhibits MRD that is molecularly detectable if at least or greater than 1 leukemia cell in 100,000 cells is detected, such as by PCR or flow cytometry. In some embodiments, the disease burden of a subject is molecularly undetectable or MRD , such that, in some cases, no leukemia cells are able to be detected in the subject using PCR or flow cytometry techniques.
[0686] In the case of leukemia, the extent of disease burden can be determined by assessment of residual leukemia in blood or bone marrow. In some embodiments, a subject exhibits morphologic disease if there are greater than or equal to 5% blasts in the bone marrow, for example, as detected by light microscopy. In some embodiments, a subject exhibits complete or clinical remission if there are less than 5% blasts in the bone marrow.
[0687] In some embodiments, the methods and/or administration of an immunotherapy or cell therapy, such as a T cell therapy (e.g. CAR-expressing T cells) and/or a BCL2 inhibitor, e.g., venetoclax, decrease(s) disease burden as compared with disease burden at a time immediately prior to the administration of the immunotherapy or cell therapy, e.g., T cell therapy and/or the BCL2 inhibitor, e.g., venetoclax.
[0688] In some aspects, administration of the immunotherapy or cell therapy, e.g. T cell therapy and/or a BCL2 inhibitor, e.g., venetoclax, may prevent an increase in disease burden, and this may be evidenced by no change in disease burden.
[0689] In some embodiments, the method reduces the burden of the disease or condition, e.g., number of tumor cells, size of tumor, duration of patient survival or event-free survival, to a greater degree and/or for a greater period of time as compared to the reduction that would be observed with a comparable method using an alternative therapy, such as one in which the subject receives immunotherapy or cell therapy, e.g. T cell therapy alone, in the absence of administration of a BCL2 inhibitor, e.g., venetoclax. In some embodiments, disease burden is reduced to a greater extent or for a greater duration following the combination therapy of administration of the immunotherapy or cell therapy, e.g., T cell therapy, and the BCL2 inhibitor, e.g., venetoclax, compared to the reduction that would be effected by administering each of the agent alone, e.g., administering a BCL2 inhibitor, e.g., venetoclax, to a subject having not received the immunotherapy or cell therapy, e.g. T cell therapy; or administering the immunotherapy or cell therapy, e.g. T cell therapy, to a subject having not received a BCL2 inhibitor, e.g., venetoclax.
[0690| In some embodiments, the burden of a disease or condition in the subject is detected, assessed, or measured. Disease burden may be detected in some aspects by detecting the total number of disease or disease-associated cells, e.g., tumor cells, in the subject, or in an organ, tissue, or bodily fluid of the subject, such as blood or serum. In some embodiments, disease burden, e.g. tumor burden, is assessed by measuring the number or extent of metastases. In some aspects, survival of the subject, survival within a certain time period, extent of survival, presence or duration of event-free or symptom- free survival, or relapse -free survival, is assessed. In some embodiments, any symptom of the disease or condition is assessed. In some embodiments, the measure of disease or condition burden is specified. In some embodiments, exemplary parameters for determination include particular clinical outcomes indicative of amelioration or improvement in the disease or condition, e.g., tumor. Such parameters include: duration of disease control, including complete response (CR), partial response (PR) or stable disease (SD) (see, e.g., Response Evaluation Criteria In Solid Tumors (RECIST) guidelines), objective response rate (ORR), progression-free survival (PFS) and overall survival (OS). Specific thresholds for the parameters can be set to determine the efficacy of the method of combination therapy provided herein.
[0691] In some aspects, disease burden is measured or detected prior to administration of the immunotherapy or cell therapy, e.g. T cell therapy, following the administration of the immunotherapy or cell therapy, e.g. T cell therapy but prior to administration of a BCL2 inhibitor, e.g., venetoclax, and/or following the administration of both the immunotherapy or cell therapy, e.g. T cell therapy and the BCL2 family inhibitor, e.g., venetoclax. In the context of multiple administration of one or more steps of the combination therapy, disease burden in some embodiments may be measured prior to, or following administration of any of the steps, doses and/or cycles of administration, or at a time between administration of any of the steps, doses and/or cycles of administration.
[0692] In some embodiments, the burden is decreased by or by at least at or about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 percent by the provided methods compared to immediately prior to the administration of a BCL2 inhibitor, e.g., venetoclax, and/or the immunotherapy or cell therpay, e.g. T cell therapy. In some embodiments, disease burden, tumor size, tumor volume, tumor mass, and/or tumor load or bulk is reduced following administration of the immunotherapy or cell therpay, e.g. T cell therapy and the BCL2 inhibitor, e.g., venetoclax, by at least at or about 10, 20, 30, 40, 50, 60, 70, 80, 90% or more compared to that immediately prior to the administration.
[0693] In some embodiments, reduction of disease burden by the method comprises an induction in morphologic complete remission, for example, as assessed at 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more than 6 months, after administration of, e.g., initiation of, the combination therapy.
[0694] In some aspects, an assay for minimal residual disease, for example, as measured by multiparametric flow cytometry, is negative, or the level of minimal residual disease is less than about 0.3%, less than about 0.2%, less than about 0.1%, or less than about 0.05%.
[0695] In some embodiments, the event-free survival rate or overall survival rate of the subject is improved by the methods, as compared with other methods. For example, in some embodiments, event- free survival rate or probability for subjects treated by the methods at 6 months following the method of combination therapy provided herein, is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some aspects, overall survival rate is greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some embodiments, the subject treated with the methods exhibits event-free survival, relapse-free survival, or survival to at least 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. In some embodiments, the time to progression is improved, such as a time to progression of greater than at or about 6 months, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.
[0696] In some embodiments, following treatment by the method, the probability of relapse is reduced as compared to other methods. For example, in some embodiments, the probability of relapse at 6 months following the method of combination therapy, is less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, or less than about 10%.
[0697] In some cases, the pharmacokinetics of administered cells, e.g., adoptively transferred cells are determined to assess the availability, e.g., bioavailability of the administered cells. Methods for determining the pharmacokinetics of adoptively transferred cells may include drawing peripheral blood from subjects that have been administered engineered cells, and determining the number or ratio of the engineered cells in the peripheral blood. Approaches for selecting and/or isolating cells may include use of chimeric antigen receptor (CAR)-specific antibodies (e.g., Brentjens et al., Sci. Transl. Med. 2013 Mar; 5(177): 177ra38) Protein L (Zheng et al., J. Transl. Med. 2012 Feb; 10:29), epitope tags, such as Strep-Tag sequences, introduced directly into specific sites in the CAR, whereby binding reagents for Strep-Tag are used to directly assess the CAR (Liu et al. (2016) Nature Biotechnology, 34:430; international patent application Pub. No. WO2015095895) and monoclonal antibodies that specifically bind to a CAR polypeptide see international patent application Pub. No. WO2014190273). Extrinsic marker genes may in some cases be utilized in connection with engineered cell therapies to permit detection or selection of cells and, in some cases, also to promote cell suicide. A truncated epidermal growth factor receptor (EGFRt) in some cases can be co-expressed with a transgene of interest (e.g., a CAR) in transduced cells (see e.g. U.S. Patent No. 8,802,374). EGFRt may contain an epitope recognized by the antibody cetuximab (Erbitux®) or other therapeutic anti-EGFR antibody or binding molecule, which can be used to identify or select cells that have been engineered with the EGFRt construct and another recombinant receptor, such as a chimeric antigen receptor (CAR), and/or to eliminate or separate cells expressing the receptor. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. 2016 April; 34(4): 430-434).
[0698] In some embodiments, the number of CAR+ T cells in a biological sample obtained from the patient, e.g., blood, can be determined at a period of time after administration of the cell therapy, e.g., to determine the pharmacokinetics of the cells. In some embodiments, number of CAR+ T cells, optionally CAR+ CD8+ T cells and/or CAR+ CD4+ T cells, detectable in the blood of the subject, or in a majority of subjects so treated by the method, is greater than 1 cells per pL, greater than 5 cells per pL or greater than per 10 cells per pL.
B. SAFETY
[0699] In some embodiments, toxicity, adverse events and/or side effects of treatment can be monitored and used to assess the administration of the BCL2 inhibitor (e.g. venetoclax) and/or to adjust the dose and/or frequency of the BCL2 inhibitor; and/or to adjust dose and/or frequency of administration of the recombinant receptor, e.g., CAR, cells, and or compositions. For example, adverse events and laboratory abnormalities can be monitored and used to adjust dose and/or frequency of administration. Adverse events include infusion reactions, cytokine release syndrome (CRS), neurotoxicity (NT), macrophage activation syndrome (MAS)/hemophagocytic lympho-histiocytosis (HLH) and tumor lysis syndrome (TLS). Any of such events can establish dose-limiting toxicities and warrant decrease in dose and/or a termination of treatment. Other side effects or adverse events which can be used as a guideline for establishing dose and/or frequency of administration include non-hematologic adverse events, which include but are not limited to fatigue, fever or febrile neutropenia, increase in transaminases for a set duration (e.g., less than or equal to 2 weeks or less than or equal to 7 days), headache, bone pain, hypotension, hypoxia, chills, diarrhea, nausea/vomiting, neurotoxicity (e.g., confusion, aphasia, seizures, convulsions, lethargy, and/or altered mental status), disseminated intravascular coagulation, other asymptomatic non-hematological clinical laboratory abnormalities, such as electrolyte abnormalities. Other side effects or adverse events which can be used as a guideline for establishing dose and/or frequency of administration include hematologic adverse events, which include but are not limited to neutropenia, leukopenia, thrombocytopenia, animal, and/or B-cell aplasia and hypogammaglobinemia.
[0700] In some embodiments, a toxic outcome in a subject to administration of a therapeutic agent (e.g. CAR T-cells) can be assessed or monitored. In some embodiments, the toxic outcome is or is associated with the presence of a toxic event, such as cytokine release syndrome (CRS), severe CRS (sCRS), macrophage activation syndrome (MAS), tumor lysis syndrome, fever of at least at or about 38 degrees Celsius for three or more days and a plasma level of C-reactive protein (CRP) of at least at or about 20 mg/dL, neurotoxicity (NT) and/or severe neurotoxicity (sNT). In some embodiments, the toxic outcome is a sign, or symptom, particular signs, and symptoms and/or quantities or degrees thereof which presence or absence may specify a particular extent, severity or level of toxicity in a subject. It is within the level of a skilled artisan to specify or determine a particular sign, symptom and/or quantities or degrees thereof that are related to an undesired toxic outcome of a therapeutic agent (e.g. CAR- T cells).
[0701] In some aspects, the toxic outcome is or is associated with or indicative of cytokine release syndrome (CRS) or severe CRS (sCRS). CRS, e.g., sCRS, can occur in some cases following adoptive T cell therapy and administration to subjects of other biological products. See Davila et al., Sci Transl Med 6, 224ra25 (2014); Brentjens et al., Sci. Transl. Med. 5, 177ra38 (2013); Grupp et al., N. Engl. J. Med. 368, 1509-1518 (2013); and Kochenderfer et al., Blood 119, 2709-2720 (2012); Xu et al., Cancer Letters 343 (2014) 172-78.
[0702] Typically, CRS is caused by an exaggerated systemic immune response mediated by, for example, T cells, B cells, NK cells, monocytes, and/or macrophages. Such cells may release a large amount of inflammatory mediators such as cytokines and chemokines. Cytokines may trigger an acute inflammatory response and/or induce endothelial organ damage, which may result in microvascular leakage, heart failure, or death. Severe, life-threatening CRS can lead to pulmonary infiltration and lung injury, renal failure, or disseminated intravascular coagulation. Other severe, life-threatening toxicities can include cardiac toxicity, respiratory distress, neurologic toxicity and/or hepatic failure. In some aspects, fever, especially high fever (> 38.5°C or > 101.3°F), is associated with CRS. In some cases, features or symptoms of CRS mimic infection. In some embodiments, infection is also considered in subjects presenting with CRS symptoms, and monitoring by cultures and empiric antibiotic therapy can be administered. Other symptoms associated with CRS can include cardiac dysfunction, adult respiratory distress syndrome, renal and/or hepatic failure, coagulopathies, disseminated intravascular coagulation, and capillary leak syndrome.
[0703] In the context of administering CAR-expressing cells, CRS typically occurs 6-20 days after infusion of cells that express a CAR. See Xu et al., Cancer Letters 343 (2014) 172-78. In some cases, CRS occurs less than 6 days or more than 20 days after CAR T cell infusion. The incidence and timing of CRS may be related to baseline cytokine levels or tumor burden at the time of infusion. Commonly, CRS involves elevated serum levels of interferon (IFN)-y, tumor necrosis factor (TNF)-a, and/or interleukin (IL)-2. Other cytokines that may be rapidly induced in CRS are IL-1[3, IL-6, IL-8, and IL-10. CRS has been described to be more severe in subjects with multiple myeloma with higher disease burden and is associated with increased serum cytokines including IL-6, IFN-y, and other cytokines together with elevation of inflammatory markers, C reactive protein (CRP) and ferritin (Cohen et al., J Clin Invest. 2019:1-12.; Brudno et al., Blood 2016;127(26):3321 3330; Lee et al., Blood 2015; 126(8): 104; Davila et al., Sci Transl Med 2014;6(224):224ra225).
[0704] Exemplary signs or symptoms associated with CRS include fever, rigors, chills, hypotension, dyspnea, acute respiratory distress syndrome (ARDS), encephalopathy, aspartate transaminase (AST)/alanine transaminase (ALT) elevation, renal failure, cardiac disorders, hypoxia, neurologic disturbances, and death. Neurological complications include delirium, seizure -like activity, confusion, word-finding difficulty, aphasia, and/or becoming obtunded. Other CRS-related signs or outcomes include fatigue, nausea, headache, seizure, tachycardia, myalgias, rash, acute vascular leak syndrome, liver function impairment, and renal failure. In some aspects, CRS is associated with an increase in one or more factors such as serum-ferritin, d-dimer, aminotransferases, lactate dehydrogenase and triglycerides, or with hypofibrinogenemia or hepatosplenomegaly. Other exemplary signs or symptoms associated with CRS include hemodynamic instability, febrile neutropenia, increase in serum C-reactive protein (CRP), changes in coagulation parameters (for example, international normalized ratio (INR), prothrombin time (PTI) and/or fibrinogen), changes in cardiac and other organ function, and/or absolute neutrophil count (ANC).
[0705] In some embodiments, signs or symptoms associated with CRS include one or more of: persistent fever, e.g., fever of a specified temperature, e.g., greater than at or about 38 degrees Celsius, for two or more, e.g., three or more, e.g., four or more days or for at least three consecutive days; fever greater than at or about 38 degrees Celsius; elevation of cytokines (e.g. IFNy or IL-6); and/or at least one clinical sign of toxicity, such as hypotension (e.g., as measured by at least one intravenous vasoactive pressor); hypoxia (e.g., plasma oxygen (PO2) levels of less than at or about 90%); and/or one or more neurologic disorders (including mental status changes, obtundation, and seizures). In some embodiments, neurotoxicity (NT) can be observed concurrently with CRS.
[0706] Exemplary CRS-related outcomes include increased or high serum levels of one or more factors, including cytokines and chemokines and other factors associated with CRS. Exemplary outcomes further include increases in synthesis or secretion of one or more of such factors. Such synthesis or secretion can be by the T cell or a cell that interacts with the T cell, such as an innate immune cell or B cell.
[0707] CRS criteria that appear to correlate with the onset of CRS to predict which patients are more likely to be at risk for developing sCRS have been developed (see Davila et al. Sci Transl Med 2014;6(224):224ra225). Factors include fevers, hypoxia, hypotension, neurologic changes, elevated serum levels of inflammatory cytokines whose treatment-induced elevation can correlate well with both pretreatment tumor burden and sCRS symptoms. Other guidelines on the diagnosis and management of CRS are known (see e.g., Lee et al, Blood. 2014;124(2):188-95). In some embodiments, the criteria reflective of CRS grade are those detailed in Table 3 below.
Figure imgf000177_0001
[0708] In some embodiments, a criteria reflective of CRS grade are those detailed in Table 4 below.
Table 4. Exemplary Grading Criteria for CRS
Figure imgf000177_0002
[0709] In some embodiments, high-dose vasopressor therapy include those described in Table 5 below.
Figure imgf000178_0001
[0710] In some embodiments, the toxic outcome is severe CRS. In some embodiments, the toxic outcome is the absence of severe CRS (e.g. moderate or mild CRS). In some embodiments, severe CRS includes CRS with a grade of 3 or greater, such as set forth in Table 3 and Table 4. In some embodiments, severe CRS includes CRS with a grade of 2 or higher, such as grades 2, 3, 4 or 5 CRS.
[0711] In some aspects, the toxic outcome is or is associated with neurotoxicity. In some embodiments, signs or symptoms associated with a clinical risk of neurotoxicity include confusion, delirium, aphasia, expressive aphasia, obtundation, myoclonus, lethargy, altered mental status, convulsions, seizure -like activity, seizures (optionally as confirmed by electroencephalogram (EEG)), elevated levels of beta amyloid (A[3), elevated levels of glutamate, and elevated levels of oxygen radicals. In some embodiments, neurotoxicity is graded based on severity (e.g., using a Grade 1-5 scale (see, e.g., Guido Cavaletti & Paola Marmiroli Nature Reviews Neurology 6, 657-666 (December 2010); National Cancer Institute — Common Toxicity Criteria version 4.03 (NCI-CTCAE v4.03). In some embodiments, a subject is deemed to develop “severe neurotoxicity” in response to or secondary to administration of a cell therapy or dose of cells thereof, if, following administration, the subject displays symptoms that limit self-care (e.g. bathing, dressing and undressing, feeding, using the toilet, taking medications) from among: 1) symptoms of peripheral motor neuropathy, including inflammation or degeneration of the peripheral motor nerves; 2) symptoms of peripheral sensory neuropathy, including inflammation or degeneration of the peripheral sensory nerves, dysesthesia, such as distortion of sensory perception, resulting in an abnormal and unpleasant sensation, neuralgia, such as intense painful sensation along a nerve or a group of nerves, and/or paresthesia, such as functional disturbances of sensory neurons resulting in abnormal cutaneous sensations of tingling, numbness, pressure, cold and warmth in the absence of stimulus. In some embodiments, severe neurotoxicity includes neurotoxicity with a grade of 3 or greater, such as set forth in Table 6. In some embodiments, severe neurotoxicity includes neurotoxicity with a grade of 2 or higher, such as grades 2, 3, 4 or 5 neurotoxicity.
Figure imgf000179_0001
[0712] In some aspects, neurotoxicity is may be associated with CRS or may be independent from or separate from CRS. In some aspects, neurotoxicity can be associated with early onset of CRS and rapid elevation of inflammatory cytokines both within the serum and central nervous system (CNS), possibly resulting in the disruption of the blood-brain barrier (BBB) (Gus et al., Cancer Discov 2017;7(12):1404 1419). In some aspects, increases in peak serum IL-6, IFN-y, and MIP-la can be associated with neurotoxicity. In some cases, neurotoxicity is also associated with an increase in peak endogenous IL-IRa, an endogenous inhibitor of the pro-inflammatory effects of IL-1 alpha (IL- la) and IL-1 beta (IL-1[3), which in some cases are involved in neurotoxicity. In some aspects, levels of cytokines usually associated with a systemic inflammation (e.g., IL-6, IL-10, and interferon-gamma (IFNy)) are observed to be higher in cases of severe neurotoxicity.
[0713] In some embodiments, the toxic outcome is a dose-limiting toxicity. In some embodiments, the toxic outcome is the absence of a dose-limiting toxicity. In some embodiments, a dose-limiting toxicity (DLT) is defined as any grade 3 or higher toxicity as assessed by any known or published guidelines for assessing the particular toxicity, such as any described above and including the National Cancer Institute (NCI) Common Terminology Criteria for Adverse Events (CTCAE) version 4.0.
[07141 In some embodiments, treatment according to the provided methods can result in a lower rate and/or lower degree of toxicity, toxic outcome or symptom, toxicity-promoting profile, factor, or property, such as a symptom or outcome associated with or indicative of cytokine release syndrome (CRS) or neurotoxicity, such as severe CRS or severe neurotoxicity, for example, compared to administration of other therapies. In some embodiments, treatment according to the provided methods can result in both a higher response rate, e.g., higher rate of OR, CR, sCR, VGPR or PR, and/or a more durable response, together with a lower rate and/or lower degree of toxicity, toxic outcome or symptom, toxicity-promoting profile, factor, or property, such as a symptom or outcome associated with or indicative of cytokine release syndrome (CRS) or neurotoxicity, such as severe CRS or severe neurotoxicity, for example, compared to administration of other therapies. In some embodiments, treatment according to the provided methods can result in both a higher response rate and a lower rate or degree of toxicity. In some aspects, such results can also be accompanied by higher expansion or prolonged persistence of the administered cells, compared to administration of other therapies
IV. ARTICLES OF MANUFACTURE AND KITS
[0715] Also provided provided are articles of manufacture containing a BCL2 inhibitor, e.g., venetoclax, and components for the immunotherapy or cell therapy, e.g., a T cell therapy, e.g. CAR T cells, and/or compositions thereof. The articles of manufacture may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container in some embodiments holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition. In some embodiments, the container has a sterile access port. Exemplary containers include an intravenous solution bags, vials, including those with stoppers pierceable by a needle for injection, or bottles or vials for orally administered agents. The label or package insert may indicate that the composition is used for treating a disease or condition.
[0716] The article of manufacture may include (a) a first container with a composition contained therein, wherein the composition includes the engineered cells used for the cell therapy, e.g. an engineered T cell therapy; and (b) a second container with a composition contained therein, wherein the composition includes a BCL2 inhibitor, e.g., venetoclax.
[0717] The article of manufacture may include (a) a first container with a composition contained therein, wherein the composition includes the engineered cells used for the cell therapy, e.g. an engineered T cell therapy; (b) a second container with a composition contained therein, wherein the composition includes the engineered cells used for the cell therapy, e.g. an engineered T cell therapy; and (c) a second container with a composition contained therein, wherein the composition includes a BCL2 inhibitor, e.g., venetoclax.
[0718] In some embodiments, the first container comprises a first composition and a second composition, wherein the first composition comprises a first population of the engineered cells used for the immunotherapy, e.g., CD4+ T cell therapy, and the second composition comprises a second population of the engineered cells, wherein the second population may be engineered separately from the first population, e.g., CD8+ T cell therapy. In some embodiments, the first and second cell compositions contain a defined ratio of the engineered cells, e.g., CD4+ and CD8+ cells (e.g., 1:1 ratio of CD4+:CD8+ CAR+ T cells).
[0719] The article of manufacture may further include a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further include another or the same container comprising a pharmaceutically- acceptable buffer. It may further include other materials such as other buffers, diluents, filters, needles, and/or syringes.
V. DEFINITIONS
[0720] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
[0721] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided receptors and other polypeptides, e.g., linkers or peptides, may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site- directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
[0722| As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human. In some embodiments, the subject, e.g., patient, to whom an inhibitor of BCL2 protein, e.g., venetoclax, engineered cells, and/or compositions are administered, is a mammal, typically a primate, such as a human. In some embodiments, the primate is a monkey or an ape. The subject can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, such as a rodent.
[0723] As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.
[0724] As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and/or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated. In some embodiments, sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.
[0725] “Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided cells and compositions are used to delay development of a disease or to slow the progression of a disease.
[0726] As used herein, to “suppress” a function or activity is to reduce the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another condition. For example, cells that suppress tumor growth reduce the rate of growth of the tumor compared to the rate of growth of the tumor in the absence of the cells.
[0727] An “effective amount” of an agent, e.g., engineered cells, an inhibitor of BCL2 protein, and/or or a pharmaceutical formulation or composition thereof, in the context of administration, refers to an amount effective, at dosages/amounts and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic result.
[0728] A “therapeutically effective amount” of an agent, e.g., engineered cells, an inhibitor of BCL2 protein, and/or a pharmaceutical formulation or composition thereof, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and/or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered. In some embodiments, the provided methods involve administering the cells and/or compositions at effective amounts, e.g., therapeutically effective amounts. In some embodiments, the provided methods involve administering a BCL2 inhibitor, e.g., venetoclax, engineered cells e.g. cell therapy), and/or compositions at effective amounts, e.g., therapeutically effective amounts. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In the context of lower tumor burden, the prophylactically effective amount in some aspects will be higher than the therapeutically effective amount.
[0729] A “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
[0730] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.
[0731] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or more.” It is understood that aspects and variations described herein include “consisting” and/or “consisting essentially of’ aspects and variations.
[0732] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.
[0733| As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, nonaqueous or any combination thereof.
[0734] As used herein, “enriching” when referring to one or more particular cell type or cell population, refers to increasing the number or percentage of the cell type or population, e.g., compared to the total number of cells in or volume of the composition, or relative to other cell types, such as by positive selection based on markers expressed by the population or cell, or by negative selection based on a marker not present on the cell population or cell to be depleted. The term does not require complete removal of other cells, cell type, or populations from the composition and does not require that the cells so enriched be present at or even near 100% in the enriched composition.
[0735] As used herein, a statement that a cell or population of cells is “positive” for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is detectable by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions and/or at a level substantially similar to that for cell known to be positive for the marker, and/or at a level substantially higher than that for a cell known to be negative for the marker.
[0736| As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control or fluorescence minus one (FMO) gating control under otherwise identical conditions, and/or at a level substantially lower than that for cell known to be positive for the marker, and/or at a level substantially similar as compared to that for a cell known to be negative for the marker.
[0737] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “expression vectors.”
[0738] As used herein, a composition refers to any mixture of two or more products, substances, or a BCL2 inhibitor, e.g., venetoclax, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.
[0739| As used herein, a “subject” is a mammal, such as a human or other animal, and typically is human.
VI. Exemplary Embodiments
[0740] Among the provided embodiments are:
1. A method of treating cancer, the method comprising:
(1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and
(2) administering to the subject a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy; and
(ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
2. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the BCL2 inhibitor is administered in a dosing regimen comprising:
(i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of a cell therapy, wherein the cell therapy comprises a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) and was administered to the subject on Day 1; and
(ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
3. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy; and
(ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
4. The method of any of embodiments 1-3, wherein the daily administration of the first dose of the BCL2 inhibitor begins about 1 day after or within about 1 day after initiation of administration of the cell therapy. 5. The method of any of embodiments 1-4, wherein the daily administration of the first dose of the BCL2 inhibitor begins about 1 day after initiation of administration of the cell therapy.
6. The method of any of embodiments 1-5, wherein the daily administration of the first dose of the BCL2 inhibitor begins within about 1 day after initiation of administration of the cell therapy.
7. The method of any of embodiments 1-6, wherein the daily administration of the first dose of the BCL2 inhibitor begins at or after a time when activation-induced cell death (AICD) of the cells of the cell therapy has peaked following initiation of administration of the cell therapy.
8. The method of any of embodiments 1-7, wherein the at least one subsequent dose comprises a second dose, and the dosing regimen comprises:
(i) daily administration of a first dose of the BCL2 inhibitor for a first predetermined period; and
(ii) daily administration of a second dose of the BCL2 inhibitor for a second predetermined period beginning after the first predetermined period.
9. The method of any of embodiments 1-8, wherein the at least one subsequent dose comprises a second dose and a third dose, and the dosing regimen comprises:
(i) daily administration of a first dose of the BCL2 inhibitor for a first predetermined period;
(ii) daily administration of a second dose of the BCL2 inhibitor for a second predetermined period beginning after the first predetermined period; and
(iii) daily administration of a third dose of the BCL2 inhibitor for a third predetermined period beginning after the second predetermined period.
10. The method of any of embodiments 1-9, wherein the first predetermined period is from about Day 2 to about Day 30
11. The method of any of embodiments 1-10, wherein the second predetermined period is from about Day 31 to about Day 90. .
12. The method of any of embodiments 1-9, wherein the first predetermined period is from about Day 2 to about Day 7.
13. The method of any of embodiments 1-9 and 12, wherein the second predetermined period is from about Day 8 to about Day 30.
14. The method of any of embodiments 1-10, wherein the second predetermined period is from about Day 31 to about Day 37.
15. The method of any of embodiments 9, 12 and 13, wherein the third predetermined period is from about Day 31 to about Day 90.
16. The method of any of embodiments 9, 10 and 14, wherein the third predetermined period is from about Day 38 to about Day 90.
17. The method of any of embodiments 1-16, wherein the first dose is at least about 20 mg of the BCL2 inhibitor. 18. The method of any of embodiments 1-17, wherein no dose of the dosing regimen is greater than about 400 mg of the BCL2 inhibitor.
19. The method of any of embodiments 1-18, wherein the first dose is between about 20 mg and about 100 mg of the BCL2 inhibitor.
20. The method of any of embodiments 1-19, wherein the first dose is about 20 mg, about 50 mg, or about 100 mg of the BCL2 inhibitor.
21. The method of any of embodiments 8-20, wherein the second dose is between about 50 mg and about 200 mg of the BCL2 inhibitor.
22. The method of any of embodiments 8-21, wherein the second dose is about 50 mg, about 100 mg, or about 200 mg of the BCL2 inhibitor.
23. The method of any of embodiments 9-22, wherein the third dose is about 100 mg and 400 mg of the BCL2 inhibitor.
24. The method of any of embodiments 9-23, wherein the third dose is about 100 mg, about 200 mg, or about 400 mg of the BCL2 inhibitor.
25. A method of treating cancer, the method comprising:
(1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and
(2) administering to the subject a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 20 mg of the BCL2 inhibitor from about Day 2 to about Day 7;
(ii) daily administration of about 50 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and
(iii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
26. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the subject has been administered a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1, and wherein the BCL2 inhibitor is administered in a dosing regimen comprising:
(i) daily administration of about 20 mg of the BCL2 inhibitor from about Day 2 to about Day 7 ;
(ii) daily administration of about 50 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and
(iii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day
90. 27. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 20 mg of the BCL2 inhibitor from about Day 2 to about Day 7 ;
(ii) daily administration of about 50 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and
(iii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
28. A method of treating cancer, the method comprising:
(1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and
(2) administering to the subject a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and
(ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
29. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the subject has been administered a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1, and wherein the BCL2 inhibitor is administered in a dosing regimen comprising:
(i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and
(ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
30. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and (ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day
90.
31. A method of treating cancer, the method comprising:
(1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and
(2) administering to the subject a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 7;
(ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and
(iii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
32. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the subject has been administered a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, and the BCL2 inhibitor is administered in a dosing regimen comprising:
(i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 7 ;
(ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and
(iii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
33. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 7 ;
(ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and
(iii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
34. A method of treating cancer, the method comprising: (1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and
(2) administering to the subject a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and
(ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
35. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the subject has been administered a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1, and wherein the BCL2 inhibitor is administered in a dosing regimen comprising:
(i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and
(ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
36. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and
(ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
37. A method of treating cancer, the method comprising:
(1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and
(2) administering to the subject a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30; (ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 37 ; and
(iii) daily administration of about 400 mg of the BCL2 inhibitor from about Day 38 to about Day 90.
38. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the subject has been administered a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1, and wherein the BCL2 inhibitor is administering in a dosing regimen comprising:
(i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30;
(ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 37; and
(iii) daily administration of about 400 mg of the BCL2 inhibitor from about Day 38 to about Day 90.
39. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is be administered a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30;
(ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 37; and
(iii) daily administration of about 400 mg of the BCL2 inhibitor from about Day 38 to about Day 90.
40. The method of any of embodiments 1-39, wherein the BCL2 inhibitor is administered for no more than about 24 months.
41. The method of any of embodiments 1-40, wherein the BCL2 inhibitor is administered for no more than about 12 months.
42. The method of any of embodiments 1-41, wherein if the subject exhibits minimum residual disease (MRD) in peripheral blood (PB) at Day 90, the BCL2 inhibitor is administered for up to 12 months or for up to 24 months.
43. The method of any of embodiments 1-42, wherein if the subject does not exhibit a complete response (CR) at Day 90, the BCL2 inhibitor is administered for up to 12 months or for up to 24 months. 44. The method of any of embodiments 1-39, wherein daily administration of the BCL2 inhibitor is discontinued after or about after Day 90.
45. The method of any of embodiments 1-39, wherein the BCL2 inhibitor is administered daily until at or about Day 90.
46. The method of any of embodiments 1-45, wherein the subject has a chronic lymphocytic leukemia (CLL).
47. The method of embodiment 46, wherein the CLL is a relapsed or refractory (r/r) CLL.
48. The method of any of embodiments 1-45, wherein the subject has a small lymphocytic lymphoma (SLL).
49. The method of embodiment 48, wherein the SLL is a relapsed or refractory (r/r) SLL.
50. The method of any of embodiments 1-49, further comprising, prior to initiation of administration of the cell therapy, administering a lymphodepleting therapy to the subject.
51. The method of any of embodiments 1-50, wherein the subject has been preconditioned with a lymphodepleting therapy prior to initiation of administration of the cell therapy.
52. The method of embodiment 50 or embodiment 51, wherein the lymphodepleting therapy comprises the administration of fludarabine and/or cyclophosphamide.
53. The method of any of embodiments 50-52, wherein the lymphodepleting therapy comprises administration of cyclophosphamide at about 200-400 mg/m2, optionally at or about 300 mg/m2, inclusive, and/or fludarabine at about 20-40 mg/m2, optionally 30 mg/m2, daily for 2-4 days, optionally for 3 days.
54. The method of any of embodiments 50-53, wherein the lymphodepleting therapy comprises administration of cyclophosphamide at or about 300 mg/m2 and fludarabine at about 30 mg/m2 daily for 3 days, optionally wherein the cell therapy is administered at least at or about 2-7 days after the lymphodepleting therapy or at least at or about 2-7 days after the initiation of the lymphodepleting therapy.
55. The method of any of embodiments 1-54, further comprising administering to the subject a bridging therapy comprising a BCL2 inhibitor.
56. The method of any of embodiments 1-55, wherein the subject is administered a bridging therapy comprising a BCL2 inhibitor.
57. The method of embodiment 55 or embodiment 56, wherein the bridging therapy is administered prior to initiation of administration of a lymphodepleting therapy.
58. The method of any of embodiments 55-57, wherein the bridging therapy is administered at a time between collecting of autologous cells from the subject and prior to administering a lymphodepleting therapy to the subject, optionally wherein the collecting is by apheresis or leukapheresis. 59. The method of any of embodiments 55-58, wherein the bridging therapy comprises daily administration of a first dose of the BCL2 inhibitor for a first week, daily administration of a second dose of the BCL2 inhibitor for a second week, and daily administration of a third dose of the BCL2 inhibitor for a third week.
60. The method of any of embodiments 55-59, wherein the bridging therapy comprises daily administration of between about 20 mg and about 400 mg of the BCL2 inhibitor.
61. The method of embodiment 59 or embodiment 60, wherein the second dose of the bridging therapy is an increased amount of BCL2 inhibitor compared to the first dose of the bridging therapy, and the third dose of the bridging therapy is an increased amount of BCL2 inhibitor compared to the second dose of the bridging therapy.
62. The method of any of embodiments 59-61, wherein the first dose of the bridging therapy is about 20 mg of the BCL2 inhibitor, the second dose of the bridging therapy is about 50 mg of the BCL2 inhibitor, and the third dose of the bridging therapy is about 100 mg of the BCL2 inhibitor.
63. The method of any of embodiments 55-62, wherein the bridging therapy is ceased about or at least about 1 day prior to initiation of administration of a lymphodepleting therapy.
64. The method of any of embodiments 1-63, wherein the BCL2 inhibitor is venetoclax or navitoclax.
65. The method of any of embodiments 1-64, wherein the BCL2 inhibitor is venetoclax.
66. The method of any of embodiments 55-65, wherein the BCL2 inhibitor administered in the bridging therapy and the BCL2 inhibitor administered in the dosing regimen are the same BCL2 inhibitor, optionally wherein the BCL2 inhibitor administered in the bridging therapy and the dosing regimen is venetoclax.
67. The method of any of embodiments 55-65, wherein the BCL2 inhibitor administered in the bridging therapy and the BCL2 inhibitor administered in the dosing regimen are different BCL2 inhibitors.
68. The method of any of embodiments 55-67, wherein the bridging therapy further comprises administration of an anti-CD20 antibody to the subject.
69. The method of any of embodiments 55-68, wherein if the subject has been previously treated with a BCL2 inhibitor, the bridging therapy further comprises administration of an anti-CD20 antibody to the subject.
70. The method of any of embodiments 55-69, wherein the bridging therapy further comprises administration of a BTK inhibitor to the subject.
71. The method of any of embodiments 1-70, wherein the subject has been previously treated with a Bruton’s tyrosine kinase (BTK) inhibitor.
72. The method of any of embodiments 1-71, wherein the subject is ineligible for treatment with a Bruton’s tyrosine kinase (BTK) inhibitor. 73. The method of any of embodiments 70-72, wherein the BTK inhibitor is or comprises ibrutinib.
74. The method of any of embodiments 1-73, wherein, prior to the administration of the cell therapy, the subject has been treated with one or more prior therapies for the CLL or the SLL, other than another dose of cells expressing a CAR or a lymphodepleting therapy, optionally at least two prior therapies.
75. The method of any of embodiments 1-74, wherein, prior to the administration of the cell therapy, the subject has relapsed following remission after treatment with, or become refractory to, failed and/or was intolerant to treatment with two or more prior therapies.
76. The method of embodiment 74 or embodiment 75, wherein the one or more prior therapies are selected from a kinase inhibitor, optionally an inhibitor of Bruton’s tyrosine kinase (BTK), optionally ibrutinib; venetoclax; a combination therapy comprising fludarabine and rituximab; radiation therapy; and hematopoietic stem cell transplantation (HSCT).
77. The method of any of embodiments 74-76, wherein the one or more prior therapies comprises an inhibitor of Bruton’s tyrosine kinase (BTK), optionally ibrutinib.
78. The method of any of embodiments 74-77, wherein the one or more prior therapies comprises ibrutinib and a BCL2 inhibitor, optionally venetoclax.
79. The method of any of embodiments 1-78, wherein the subject has not been previously treated with venetoclax.
80. The method of any of embodiments 1-78, wherein the subject has been previously treated with venetoclax.
81. The method of any of embodiments 1-79, wherein the subject is not intolerant to venetoclax at the time of initiation of administration of the cell therapy and/or at the time of initiation of administration of the BCL2 inhibitor.
82. The method of embodiment 80 or embodiment 81, wherein more than six months have passed since the last dose of the previous treatment with venetoclax if the subject’s best overall response (BOR) during or within 6 months of discontinuing the previous treatment with venetoclax was stable disease (SD) or progressive disease (PD).
83. The method of any of embodiments 1-82, wherein prior to administration of the cell therapy and/or prior to administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes and/or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen.
84. The method of any of embodiments 1-83, wherein prior to administration of the cell therapy and/or prior to administration of the BCL2 inhibitor, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 104. 85. The method of any of embodiments 1-84, wherein the subject has Eastern Cooperative
Oncology Group (ECOG) performance status of 0 or 1.
86. The method of any of embodiments 1-85, wherein the dose of engineered cells comprises a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR, optionally wherein the ratio is between approximately 1:3 and approximately 3:1.
87. The method of any of embodiments 1-86, wherein the dose of engineered cells comprises a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR that is or is approximately 1:1.
88. The method of any of embodiments 1-87, wherein the dose of engineered cells comprises between about 2.5 x 107 total CAR-expressing cells and about 1.0 x 108 total CAR-expressing cells.
89. The method of any of embodiments 1-88, wherein the dose of engineered cells comprises at or about 2.5 x 107 total CAR-expressing cells.
90. The method of any of embodiments 1-88, wherein the dose of engineered cells comprises at or about 5 x 107 total cells or total CAR-expressing cells.
91. The method of any of embodiments 1-88, wherein the dose of engineered cells comprises at or about 1 x 108 total cells or total CAR-expressing cells.
92. The method of any of embodiments 1-91, wherein administration of the cell therapy comprises administering a plurality of separate compositions, wherein the plurality of separate compositions comprises a first composition comprising one of the CD4+ T cells and the CD8+ T cells and a second composition comprising the other of the CD4+ T cells and the CD8+ T cells.
93. The method of embodiment 92, wherein the first composition comprises the CD8+ T cells and the second composition comprises the CD4+ T cells.
94. The method of embodiment 92 or embodiment 93, wherein: the first composition and the second composition are administered 0 to 12 hours apart, 0 to 6 hours apart, or 0 to 2 hours apart, or wherein the administration of the first composition and the administration of the second composition are carried out on the same day, between about 0 and about 12 hours apart, between about 0 and about 6 hours apart, or between about 0 and 2 hours apart; and/or the initiation of administration of the first composition and the initiation of administration of the second composition are carried out between about 1 minute and about 1 hour apart, or between about 5 minutes and about 30 minutes apart.
95. The method of any of embodiments 92-94, wherein the first composition and the second composition are administered no more than 2 hours, no more than 1 hour, no more than 30 minutes, no more than 15 minutes, no more than 10 minutes, or no more than 5 minutes apart.
96. The method of any of embodiments 92-95, wherein the first composition is administered prior to the second composition. 97. The method of any of embodiments 92-96, wherein the CAR comprised by the CD4+ T cells and/or the CAR comprised by the CD8+ T cells comprises a CAR that is the same and/or wherein the CD4+ T cells and/or the CD8+ T cells are genetically engineered to express a CAR that is the same.
98. The method of any of embodiments 1-97, wherein: the CAR comprises an extracellular antigen-binding domain specific for CD 19, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4- 1BB, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is a CD3zeta; and/or the CAR comprises, in order, an extracellular antigen-binding domain specific for CD19, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule.
99. The method of embodiment 98, wherein the antigen-binding domain is an scFv.
100. The method of embodiment 99, wherein: the scFv comprises a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and/or a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37) and/or a CDRH1 sequence of DYGVS (SEQ ID NO: 38), a CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and/or a CDRH3 sequence of YAMDYWG (SEQ ID NO: 40); the scFv comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC63 and/or a CDRL1 sequence of FMC63, a CDRL2 sequence of FMC63, a CDRL3 sequence of FMC63, a CDRH1 sequence of FMC63, a CDRH2 sequence of FMC63, and a CDRH3 sequence of FMC63 or binds to the same epitope as or competes for binding with any of the foregoing; the scFv comprises a VH set forth in SEQ ID NO: 41 and a VL set forth in SEQ ID NO: 42, optionally wherein the VH and VL are separated by a flexible linker, optionally wherein the flexible linker is or comprises the sequence set forth in SEQ ID NO: 59; and/or the scFv is or comprises the sequence set forth in SEQ ID NO: 43.
101. The method of any of embodiments 98-100, wherein the costimulatory signaling domain is a signaling domain of CD28 or 4- IBB.
102. The method of any of embodiments 98-101, wherein the costimulatory signaling domain is a signaling domain of 4- IBB.
103. The method of any of embodiments 98-102, wherein the costimulatory signaling domain comprises SEQ ID NO: 12 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.
104. The method of any of embodiments 98-103, wherein the primary signaling domain is a CD3zeta signaling domain. 105. The method of any of embodiments 98-104, wherein the primary signaling domain comprises SEQ ID NO: 13,14, or 15, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, 14 or 15.
106. The method of any of embodiments 98-105, wherein the primary signaling domain comprises SEQ ID NO: 13 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13.
107. The method of any of embodiments 99-106, wherein the CAR further comprises a spacer between the transmembrane domain and the scFv.
108. The method of embodiment 107, wherein the spacer is a polypeptide spacer that comprises or consists of all or a portion of an immunoglobulin hinge or a modified version thereof, optionally an IgG4 hinge, or a modified version thereof.
109. The method of embodiment 107 or embodiment 108, wherein the spacer is about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region.
110. The method of any of embodiments 107-109, wherein the spacer is at or about 12 amino acids in length.
111. The method of any of embodiments 107-110, wherein the spacer: comprises or consists of the sequence of SEQ ID NO: 1, a sequence encoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and/or comprises or consists of the formula X1PPX2P, where Xi is glycine, cysteine or arginine and X2 is cysteine or threonine.
112. The method of any of embodiments 99-111, wherein the scFv comprises, in order, a VH, a linker, optionally comprising SEQ ID NO: 59, and a VL-
113. The method of any of embodiments 107-112, wherein: the antigen binding domain comprises an scFv that comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC63; the spacer is a polypeptide spacer that comprises the sequence of SEQ ID NO: 1; the costimulatory domain comprises SEQ ID NO: 12; and the primary signaling domain comprises SEQ ID NO: 13, 14 or 15.
114. The method of any of embodiments 107-113, wherein: the antigen binding domain comprises an scFv that comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC63; the spacer is a polypeptide spacer that comprises the sequence of SEQ ID NO: 1; the costimulatory domain comprises SEQ ID NO: 12; and the primary signaling domain comprises SEQ ID NO: 13.
115. The method of any of embodiments 1-114, wherein the engineered cells are primary T cells obtained from a subject.
116. The method of any of embodiments 1-115, wherein the engineered cells are autologous to the subject.
117. The method of any of embodiments 1-116, wherein the subject is a human subject.
118. A cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) and a BCL2 inhibitor for use in a method of treating a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL), where the method comprises:
(1) administering to the subject the cell therapy on Day 1; and
(2) administering to the subject the BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy; and
(ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
119. A BCL2 inhibitor for use in a method of treating a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) that has been treated with a cell therapy comprising a dose of engineered T cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19), wherein the method comprises administering the BCL2 inhibitor to the subject in dosing regimen comprising:
(i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy, wherein the cell therapy was administered to the subject on Day 1; and
(ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30. 120. A cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) for use in a method of treating a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL), wherein the method comprises administering the cell therapy to the subject on Day 1, and the subject is to be administered a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy; and
(ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
121. Use of a BCL2 inhibitor in the manufacture of a medicament for treatment of a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) that has been treated with a cell therapy comprising a dose of engineered T cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19), wherein the medicament is to be administered to the subject in dosing regimen comprising:
(i) daily administration of a first dose of the medicament for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy, wherein the cell therapy was administered to the subject on Day 1; and
(ii) daily administration of at least one subsequent dose of the medicament, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the medicament, wherein: each of the at least one subsequent dose is an increased amount of the medicament compared to the preceding dose; and the medicament is administered at no more than or no more than about 100 mg per day through Day 30.
122. Use of a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) in the manufacture of a medicament for the treatment of a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) that is to be administered a BCL2 inhibitor in a dosing regimen comprising: (i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the medicament, wherein the medicament is administered to the subject on Day 1; and
(ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
123. An article of manufacture comprising (i) a composition for a cell therapy, or one of a plurality of compositions for a cell therapy, comprising T cells expressing a CAR that binds CD 19 and (ii) an inhibitor of BCL2 protein, optionally venetoclax, and instructions for administering the cell therapy and the BCL2 inhibitor to the subject, wherein the instructions specify administering the cell therapy and the BCL2 inhibitor according to the methods of any of claims 1-117 or the uses of any of embodiments 118-122.
VII. Examples
[0741] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.
Example 1 : Assessment of CAR T-Mediated Killing of Tumor Cells
[0742] T cell compositions containing anti-CD19 CAR-expressing T cells were generated from leukapheresis samples from two human adult donors by a process including immunoaffinity-based selection of T cells (including CD4+ and CD8+ cells) from the samples, resulting in two compositions, enriched for CD8+ and CD4+ cells, respectively. Cells of the enriched CD4+ and CD8+ compositions were separately activated with anti-CD3/anti-CD28 beads and subjected to lentiviral transduction with a vector encoding an anti-CD19 CAR. The anti-CD19 CAR contained an anti-CD19 scFv derived from a murine antibody (variable region derived from FMC63), an immunoglobulin-derived spacer, a transmembrane domain derived from CD28, a costimulatory region derived from 4-1BB, and a CD3-zeta intracellular signaling domain. The expression construct in the viral vector further contained sequences encoding a truncated receptor, which served as a surrogate marker for CAR expression, which was separated from the CAR sequence by a T2A ribosome skip sequence. Transduced populations then were separately incubated in the presence of stimulating reagents for cell expansion. Expanded CD8+ and CD4+ cells were formulated and cryopreserved separately and stored. [0743] The cyropreserved CD4+ and CD8+ anti-CD19 CAR-expressing cells from each donor were thawed, and combined at approximately a 1:1 CAR+ CD4+:CD8+ ratio prior to use.
[0744] Anti-CD19 CAR T cell compositions were cultured with CD19-expressing target cells, including K562 cells transduced to express CD19 (K562.CD19) and the CD19-expressing lymphoma cell lines DOHH2, WSU-FSCLL, and RL. As a control, anti-CD19 CAR T cell compositions were cocultured with K562 cells not expressing CD19 (K562 parental). Co-cultures with T cells not expressing the CAR (mock) were also used as controls. The CAR+ T cell compositions and cell lines were cocultured for 24 hours at effector cell to target cell ratios (E:T) between 0:1 to 10:1 (0:1, 0.3:1, 1: 1, 3:1, and 10:1). The target cells were labeled with CellTrace Violet (CTV). Following culture, target cells were harvested and stained with LIVE/DEAD Fixable Near-IR Dead Cell Stain and analyzed by flow cytometry. Viable target cells were identified as CTV positive, LIVE/DEAD negative. As shown in FIG. 1A, the percentage of viable target cells decreased with increasing E:T ratios in co-cultures of anti-CD19 CAR T cells with CD19-expressing DOHH2, WSU-FSCLL, and K562.CD19 target cell lines. By contrast, the RL cells were more resistant to cytolytic killing by anti-CD19 CAR T cells generated from both donors.
[0745] Separately, RL spheroids were generated by culturing CD19-expressing RL cells engineered with a lentiviral reagent to express NucLight Red in culture plates for 72 hours. An anti-CD19 CAR- expressing T cell composition was generated from a human adult donor, substantially as described above. The anti-CD19 CAR T cells were co-cultured with the RL spheroids for up to 196 hours at E:T ratios of 0.25:1, 0.5:1, and 1:1. Spheroid size was measured over time by the red object total area (pm2/image). The size of spheroids co-cultured with anti-CD19 CAR T cells over time is shown in FIG. IB.
[0746] The cytolytic activity of anti-CD19 CAR T cells against two additional CD19-expressing lymphoma target cells lines, SUDHL6 and WSU-DLCL2, was assessed and compared to cytolytic activity against K562.CD19 target cells. Target cells were engineered with a lentiviral reagent to express NucLight Red (NLR) to permit tracking of target cells by microscopy. Anti-CD19 CAR-expressing T cell compositions were generated from two human adult donors, substantially as described above. Target cells were co-cultured with the donor-derived CAR T cell compositions for up to 120 hours at an E:T ratio of 2.5:1. Red target cell counts were tracked over time by live cell imaging and counts were normalized to the number of red target cells at baseline to determine the fold-change in target cells over time. As shown in FIG. 2, anti-CD19 CAR T cells exhibited substantial cytolytic activity against K562.CD19 and SUDHL6 lymphoma target cells. However, the fold-change in cell count of WSU- DLCL2 lymphoma target cells increased over the course of the assay, indicating that these cells were less susceptible to killing by anti-CD19 CAR T cells. These results indicate certain CD19-expressing cells are resistant to CD19-directed CAR-mediated cell killing and supports a hypothesis that some CD 19- expressing tumors may be less sensitive to CD19-directed CAR-mediated cell killing. Example 2 : Effect of a BH3 Family Member (BCL-2) Inhibitor on Anti-CD19 CAR T Cells
[0747] Anti-CD19 CAR-expressing T cell compositions were generated from three human adult donors, substantially as described in Example 1. Anti-CD19 CAR T cells were stimulated for 96 hours with an anti-idiotypic antibody in the presence of an exemplary BCL2 inhibitor venetoclax solubilized in dimethylsulfoxide (DMSO). Control anti-CD19 CAR T cells were stimulated for 96 hours with the anti- idiopathic antibody in the presence of an equal amount of DMSO. Specifically, anti-CD19 CAR- expressing cells were added to the wells of a 96-well plate that had been pre-coated with an anti-idiotypic antibody specific to the scFv of the anti-CD19 CAR-expressing T cells described in Example 1 at a concentration of 30 p.g/ml. The cells were cultured in the presence of 5% human serum at 37°C and 5% CO2. After 96 hours, cells were lysed and ATP from viable cells was detected via a luciferin reporter assay and reported as relative luminescence units. Venetoclax-treated CAR T cell counts were assessed and normalized to DMSO control CAR T cell counts.
[0748] As shown in FIG. 3, the percentage of viable anti-CD 19 CAR-expressing T cells decreased with increasing doses of venetoclax. Among the three donors, the average IC50 of venetoclax was 7.9 pM, and the average ICw of venetoclax was 0.88 pM. In some cases, clinical observations have revealed that a daily 400 milligram dose of venetoclax yields a maximum serum concentration (CmaX) of 2.4 ± 1.3 pM. These results are consistent with an observation that potentiation of anti-CD 19 CAR-T cell activity can be seen at lower concentrations than those that may have a detrimental effect on CAR T-cell viability, under certain conditions.
Example 3 : Effect of a BH3 Family Member (BCL-2) Inhibitor on CD19-Directed Tumor Cell Resistance
[0749] Anti-CD 19 CAR-expressing T cell compositions were generated as described in Example 1 and were cultured with a CD19-expressing lymphoma target cell lines, either SUDHL6, WSU-DLCL2, or Granta-519 at an E:T ratio of 2.5:1. The co-cultures were carried out in the presence of 20 nM or 200 nM of the exemplary BCL2 inhibitor, venetoclax. As controls, target tumor cell lines were incubated only in the presence of the inhibitor or the anti-CD 19 CAR T cells. Target cells were labeled with NucLight Red (NLR) to permit tracking of target cells by microscopy and target cell killing was monitored by the loss of fluorescent signal over time, substantially as described in Example 1.
[0750] As shown in FIG. 4A, anti-CD19 CAR T cells exhibited cytolytic activity against SUDHL6 cells but WSU-DLCL2 and Granta-519 cell lines were more resistant to CD19-directed CAR T cell killing. The presence of venetoclax alone at either 20 nM or 200 nM did not substantially impact tumor cell killing in this assay for the resistant tumor cell lines, although killing of the sensitive tumor cell line SUDHL6 was achieved in cultures incubated with 200 nM venetoclax. CD19-directed CAR T cell killing of tumor resistant cell lines was enhanced in co-cultures that were incubated with 20 or 200 nM venetoclax. These results suggest that even low-dose venetoclax, e.g. 20 nM, can sensitize tumor cells to CD19-directed CAR T cell-mediated cytotoxicity that are otherwise resistant.
[0751] In a further experiment, anti-CD19 CAR-expressing T cell compositions, generated as described above, were cultured with the Granta-519 CD19-expressing lymphoma target cell line. Granta- 519 cells were engineered with a lenti viral reagent to express NucLight Red (NLR) to permit tracking of target cells by microscopy. As described above, the Granta-519 cell line was determined to be relatively resistant to CD19-directed CAR T cell-mediated cytotoxicity, such that co-culture of the CAR T cells and the target cells at an E:T ratio of 1:1 represented a suboptimal dose of CAR T cells. The anti-CD19 CAR T cells were provided in the co-cultures at the suboptimal dose, and the co-cultures were carried out in the presence of 0, 0.01, 0.1, or 1 pM of the exemplary BCL2 inhibitor venetoclax. For comparison, target tumor cell lines were incubated only in the presence of 0, 0.01, 0.1, or 1 pM of the inhibitor. Red target cell counts were enumerated by live cell imaging and tracked over time.
[0752] As shown in FIG. 4B, the lymphoma target cells were relatively resistant to CD19-directed CAR T cell killing at the suboptimal dose of CAR T cells, as indicated by a modest decrease of target cell number following co-culture with the anti-CD19 CAR T cells in the absence of venetoclax. The presence of venetoclax alone at 0.01 pM did not substantially impact target cell number in this assay, whereas the presence of venetoclax alone at 0.1 pM and at 1.0 pM had a modest effect on target cell number. However, co-cultures containing target lymphoma cells and anti-CD19 CAR T cells in the presence of even 0.01 pM of venetoclax demonstrated a decrease in the number of target cells, compared to target cells treated with only 0.01 pM of the inhibitor, consistent with a potentially synergistic effect of the combination. Co-cultures containing target lymphoma cells and anti-CD19 CAR T cells in the presence of 0.1 pM and 1.0 pM venetoclax demonstrated an even greater CD19-directed CAR T cell killing.
[0753] These results are consistent with an observation that low doses of venetoclax may sensitize cells that are otherwise relatively resistant to a suboptimal dose of anti-CD19 CAR T cells.
Example 4 : Effect of a BH3 family member (BCL-2) Inhibitor on RL Cell Resistance to CD19- Targeting CAR T Cells
[0754] Anti-CD19 CAR-expressing T cell compositions, generated as described in Example 1, were cultured with the CD19-expressing lymphoma target cell line RL. Target cells were engineered with a lentiviral reagent to express NucLight Red (NLR) to permit tracking of the cells by microscopy. The RL cell line was determined to be relatively resistant to CD19-directed CAR T cell-mediated cytotoxicity, such that co-culture of the CAR T cells and the target cells at an E:T ratio of 1:1 represented a suboptimal dose of CAR T cells. The anti-CD19 CAR T cells were co-cultured with target RL cells at an E:T ratio of 1:1, and the co-cultures were carried out in the presence of 0.1 pM of the exemplary BCL2 inhibitor venetoclax. For comparison, target cells were incubated only in the presence of the CAR T cells or 0.1 pM of the inhibitor.
[0755] As shown in FIG. 5A, the RL cells were relatively resistant to CD19-directed CAR T cells alone, but were sensitized to the CD19-directed CAR T cell killing in the presence of 0.1 pM venetoclax.
[0756] The resistance of the RL cell line to CD19-directed CAR T cells was further assessed in a 3D tumor model. An anti-CD19 CAR-expressing T cell composition was generated from a human adult donor, substantially as described in Example 1. RL tumor cells engineered to express NLR were allowed to form spheroids for 72 hours, then co-cultured for 9 days with the anti-CD19 CAR T cells at an E:T ratio of 1:1. A subtherapeutic concentration of the exemplary BCL2 inhibitor venetoclax (0.1 pM) was added at the beginning of co-culture, and tumor volume was measured over time (FIG. 5B). While initial tumor volume was not reduced in the presence of the anti-CD19 CAR T cells alone, the addition of 0.1 pM venetoclax resulted in decreased tumor volume.
[0757] In a related experiment, spheroids were generated from CD19-expressing RL cells as described, and co-cultured for 9 days with anti-CD19 CAR T cells and increasing concentrations of venetoclax (0.01 pM, 0.1 pM, or 1.0 pM), or with only corresponding concentrations of venetoclax. As shown in FIG. 5C, at day 9 of the co-culture, larger concentrations of venetoclax resulted in larger decreases in tumor size. However, the size of the RL spheroids cultured with both anti-CD19 CAR T cells and venetoclax decreased substantially more compared to that of RL spheroids cultured with just venetoclax.
[0758] In both RL spheroid experiments, the combination of venetoclax and anti-CD19 CAR T cells was observed to induce apoptosis of tumor cells (data not shown), despite such effects not being observed in the presence of the CAR T cells alone. These results indicated that venetoclax may sensitize tumor cells to anti-CD19 CAR T cell-mediated cell death, such that the combination exhibits more potent antitumor activity than venetoclax or the anti-CD19 CAR T cells alone.
Example 5 : Effect of a BCL-2 Inhibitor on Chronically Stimulated Anti-CD19 CAR T Cells Against CD19-Directed Tumor Cell Resistance
[0759] Anti-CD19 CAR-expressing T cells, generated from healthy donors as described above, were chronically stimulated under conditions to exhaust T cell activity by incubation for 8 days with beads coated in anti-idiotypic (anti-ID) antibody directed against the CAR. After the 8 days of chronic stimulation, the anti-CD19 CAR-expressing T cells were harvested and subsequently co-cultured with RL cells engineered to express NLR for 8 days at an E:T ratio of 1:1. As a control, RL cells were cultured in the absence of anti-CD19 CAR T cells. Subtherapeutic concentrations of the exemplary BCL2 inhibitor venetoclax (0.01 pM or 0.1 pM) were added to the cultures, and the number of cells was assessed over time (FIG. 6A). [0760] In another experiment, spheroids were generated from CD19-expressing RL cells as previously described, and co-cultured for 8 days with anti-CD19 CAR T cells at an E:T ratio of 1:1 and increasing concentrations of venetoclax (0.01 pM, 0.1 pM, or 1.0 pM), or with only corresponding concentrations of venetoclax. As before, the anti-CD19 CAR T cells were chronically stimulated with anti-ID antibody-coated beads prior to co-culture with the RL spheroids. As shown in FIG. 6B, decreases in tumor size were only observed with 1 pM venetoclax when target cells were cultured in the absence of CD 19 -targeting CAR T cells. By contrast, greater decreases in tumor size were seen with increasing concentrations of venetoclax when target cells were cultured in the presence of CD19-targeting CAR T cells. Overall, the size of the RL spheroids cultured with both anti-CD19 CAR T cells and venetoclax decreased more, and with lower concentrations of venetoclax, compared to that of RL spheroids cultured with just venetoclax.
[0761] These data indicate that treatment with a BCL2 inhibitor enhanced the potency of chronically stimulated anti-CD19 CAR T cells. Without wishing to be bound by theory, the results indicate that addition of a BCL2 inhibitor may restore or reverse an exhausted or chronically stimulated state in CAR T cells, such as may be found in patients with terminally differentiated CAR T cells and/or high disease burden.
Example 6 : Effects of Chronic BCL2 Inhibition on Anti-CD19 CAR T Cells Co-Cultured with Target Cells
[0762] Anti-CD19 CAR-expressing T cell compositions were generated from three healthy human donors, substantially as described in Example 1. Expression of BCL2 by the CAR T cells was confirmed by flow cytometry and compared to a fluorescence minor one (FMO) control, as shown in FIG. 7A (mean fluorescence intensity; MFI).
[0763] Anti-CD19 CAR-expressing T cell compositions from healthy donors were incubated with anti-ID antibody-coated beads at 37°C in the presence of increasing concentrations of venetoclax (0.01 pM, 0.1 pM, 1 pM, or 10 pM) for 48 hours. As a control, CAR T cells were incubated under the same conditions, but without venetoclax. Caspase 3 expression, viability, and expansion kinetics of the CAR T cells were subsequently assessed. As shown in FIG. 7B (each dot represents an individual donor), at 48 hours post-stimulation, increasing concentrations of venetoclax resulted in larger percentages of CAR+ T cells expressing caspase 3, indicative of cell death.
[0764] Following culture with the anti-ID beads, and optionally venetoclax, similar expansion profiles of CAR+ T cells were observed, regardless of venetoclax treatment or dose (FIGS. 7C and 7D). Further, the cytolytic activity of anti-CD19 CAR T cells was not affected when co-cultured with JeKo-1 target cells for 100 hours in the presence of 1.0 pM venetoclax. As shown in FIG. 8, the number of JeKo-1 target cells was not reduced by venetoclax treatment alone, but was substantially decreased when co-cultured with CD19-targeting CAR T cells in the presence or absence of venetoclax. Without wishing to be bound by theory, the data indicate that venetoclax may affect the cell viability of anti-CD19 CAR T cells, but that the viability, expansion kinetics, and cytolytic activity of viable CAR T cells are unaffected.
[0765] Venetoclax can bind human plasma proteins. Thus, to determine whether serum concentration may modulate the effects of venetoclax on CAR T cells, anti-CD19 CAR T cells were cultured in the presence of 5%, 10%, or 20% serum and treated with increasing concentrations of venetoclax. The IC50 of venetoclax against anti-CD19 CAR T cells was found to increase with increasing concentrations of serum (FIG. 9), indicating that the effects of venetoclax on CAR T cell viability may depend on serum concentration.
Example 7 : Effects of a BCL-2 Inhibitor on Anti-CD19 CAR T Cells in a Murine Model of Mantle Cell Lymphoma
[0766] NOD scid gamma (NSG) mice were injected intravenously with 2 x 106 JeKo-1 mantle cell lymphoma cells (Day -7) and treated daily with 12.5, 25, 50, or 100 mg/kg of the exemplary BCL2 inhibitor venetoclax for 21 days (Day 0 through Day 21). Tumor burden (FIG. 10A) and body weight (FIG. 10B) were assessed over the course of treatment.
[0767] Consistent with the results in Example 7, the JeKo-1 lymphoma cell line was observed to be resistant to treatment with venetoclax alone, regardless of dose level (FIG. 10A showing tumor burden, as assessed by bioluminescence imaging; BLI).
[0768] To determine the effects of venetoclax on anti-CD19 CAR T cells in a venetoclax-resistant murine model of MCL, NSG mice were injected intravenously with 2 x 106 JeKo-1 cells on Day -7, and then treated with intravenous infusions of 1 x 106 anti-CD19 CAR T cells, generated substantially as described in previous Example, on Day -1 and Day 0. Venetoclax was administered daily for 21 days (Day 0 to Day 21) at 6.25, 25, or 100 mg/kg. As a control, a subset of mice were only treated with venetoclax. Tumor burden (FIG. 11A), survival (FIGS. 11B and 11C), and CAR T cell number (FIG. 12) were assessed.
[0769] Tumor burden increased similarly in untreated mice and mice treated with venetoclax alone. However, treatment with CD19-targeting CAR T cells, alone or in combination with venetoclax, resulted in reduced tumor volume at all measured time points, as compared to untreated and venetoclax-only treated mice (FIG. 11A). Similar effects on survival were observed, as shown in FIGS. 11B and 11C.
[0770] The number of CD4+ and CD8+ CAR T cells was assessed in mice treated with CD19- targeting CAR T cells, alone or in combination with venetoclax, on days 7, 13, and 19. Dose- wise decreases in both CD4+ and CD8+ CAR T cell subsets were observed with increasing concentrations of venetoclax (FIG. 12).
[0771] Together, these data indicate that the cytolytic ability of the CD19-targeting CAR T may not be compromised by the presence of the exemplary BCL2 inhibitor venetoclax, despite losses in CAR T cell viability observed with higher doses of the inhibitor.
Example 8 : Effects of Concurrent or Delayed Administration of a BCL-2 Inhibitor in Combination with Anti-CD19 CAR T Cells
A. Administration of a BCL-2 Inhibitor In Vitro
[0772] RL target cells were co-cultured with CD19-targeting CAR T cells, generated substantially as described in Example 1 , for 6 days in the absence or presence of the exemplary BCL2 inhibitor venetoclax (0.01 pM, 0.1 pM, or 1.0 pM). Venetoclax was added concurrently with co-culture or 24, 48, or 72 hours after the start of co-culture. As a control, RL target cells were cultured in the presence of venetoclax, but without CAR T cells.
[0773] After 6 days of co-culture, the number of CD3+ CAR T cells and target cells was assessed. As shown in FIG. 13A, the number of CAR T cells was reduced when 0.1 pM or 1.0 pM of venetoclax was added concurrently with co-culture or 24 hours later. By contrast, a reduction in CD3+ CAR T cells was not observed when venetoclax was added 48 or 72 hours following co-culture, regardless of the concentration of venetoclax. For all time points assessed, 0.01 pM of venetoclax was not observed to reduce CD3+ CAR T cell number. As shown in FIG. 13B, similar decreases in target cell number were observed for all conditions, except for target cells cultured only in the presence of venetoclax.
[0774] These results indicate that delaying treatment with a BCL2 inhibitor may reduce effects of the inhibitor on CAR T cell viability, while preserving CAR T cell cytolytic activity.
B. Administration of a BCL-2 Inhibitor in an Anti-CD19 CAR T Cell-Resistant Murine
Model of Lymphoma
[0775] To determine the effects of concurrent or delayed administration of venetoclax with anti- CD19 CAR T cells in an anti-CD19 CAR T cell-resistant murine model of MCL, NSG mice were injected intravenously with 0.5 x 106 Granta-519 cells on Day -14, and then treated on Day 0 with intravenous infusions of 2 x 106 anti-CD19 CAR T cells, generated substantially as described in previous Examples. Venetoclax was administered daily at 25 or 100 mg/kg, either for a total of 26 days beginning on Day -1 prior to CAR T cell administration, or for a total of 21 days beginning on Day 5 following CAR T cell administration. As controls, subsets of mice were treated with only venetoclax or only the CD- 19 targeting CAR T cells.
[0776] Survival of all groups was assessed (FIGS. 14A and 14B). As compared to mice treated with only CD19-targeting CAR T cells, a significant increase in percent survival was found in both groups treated with CD19-targeting CAR T cells and venetoclax, regardless of venetoclax dose and timing of administration. These data are consistent with an observation that both concurrent and delayed administration of venetoclax are effective to overcome resistance to anti-CD19 CAR T cells. Example 9 : Gene Expression Data from Subjects with Diffuse Large B-Cell Lymphoma
[0777] Therapeutic CAR T cell compositions containing autologous T cells expressing a chimeric antigen-receptor (CAR) specific for CD19 were administered to subjects with B cell malignancies, and expression of genes in pre -treatment tumor biopsies that correlated to response in subjects administered the CAR T cell compositions was determined for a subset of subjects.
[0778] Specifically, autologous anti-CD19 directed therapeutic T cell compositions were generated from and used to treat adult human subjects with relapsed or refractory (R/R) aggressive non-Hodgkin’s lymphoma (NHL), including diffuse large B-cell lymphoma (DLBCL), de novo or transformed from indolent lymphoma (NOS), high-grade B-cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements with DLBCL histology (double/triple hit), DLBCL transformed from chronic lymphocytic leukemia (CLL) or marginal zone lymphomas (MZL), primary mediastinal large b-cell lymphoma (PMBCL), and follicular lymphoma grade 3b (FL3B) after failure of 2 lines of therapy. Among the subjects treated were those having Eastern Cooperative Oncology Group (ECOG) scores of between 0 and 2 (median follow-up 3.2 months). No subjects were excluded based on prior allogeneic stem cell transplantation (SCT), secondary central nervous system (CNS) involvement or an ECOG score of 2, and there was no minimum absolute lymphocyte count (ALC) for apheresis required.
[0779] The therapeutic T cell compositions administered had been generated by a process including immunoaffinity-based (e.g., immunomagnetic selection) enrichment of CD4+ and CD8+ cells from leukapheresis samples from the individual subjects to be treated. Isolated CD4+ and CD8+ T cells were separately activated and independently transduced with a viral vector (e.g., lentiviral vector) encoding an anti-CD19 CAR, followed by separate expansion and cryopreservation of the engineered cell populations. The CAR contained an anti-CD19 scFv derived from a murine antibody (variable region derived from FMC63, VL-linker-Vn orientation), an immunoglobulin-derived spacer, a transmembrane domain derived from CD28, a costimulatory region derived from 4-1BB, and a CD3-zeta intracellular signaling domain. The viral vector further contained sequences encoding a truncated receptor, which served as a surrogate marker for CAR expression and was separated from the CAR sequence by a T2A ribosome skip sequence.
[0780] The cryopreserved cell compositions were thawed prior to intravenous administration. The therapeutic T cell dose was administered as a defined cell composition by administering a formulated CD4+ CAR+ cell population and a formulated CD8+ CAR+ population administered at a target ratio of approximately 1:1. Subjects were administered a single or double dose of CAR-expressing T cells (each single dose via separate infusions of CD4+ CAR-expressing T cells and CD8+ CAR-expressing T cells, respectively) as follows: a single dose of dose level 1 (DL-1) containing 5 x 107 total CAR-expressing T cells, a double dose of DL1 in which each dose was administered approximately fourteen (14) days apart (administered on day 1 and day 14), or a single dose of dose level 2 (DL-2) containing 1 x 108 total CAR-expressing T cells. The target dose level and the numbers of T cell subsets for the administered compositions are set forth in Table El.
Figure imgf000209_0001
[07811 Beginning at prior to CAR+ T cell infusion, subjects received a lymphodepleting chemotherapy with fludarabine (flu, 30 mg/m2) and cyclophosphamide (Cy, 300mg/m2) for three (3) days. The subjects received CAR-expressing T cells 2-7 days after lymphodepletion.
[0782] After administration of the CAR T cell composition, subjects were monitored for clinical response, including at 3 months after administration, and response to the CAR T cell composition was determined by assessing whether the subject had progressive disease (PD), partial response (PR), or complete response (CR).
[0783] Tumor biopsies from a subset of patients (n=36) were collected prior to administration of the CAR T cells and analyzed by RNA sequencing (RNA-seq) for gene expression on the complementary DNA (cDNA) samples prepared from the RNA isolated from the tumor biopsies Principal component analysis (PCA) was performed for the RNA-seq data sets, generated from DESeq2-normalized counts. Expression of genes by RNA-Seq from the pretreatment tumor biopsies were correlated, post facto, to response following administration of the autologous therapeutic CAR-T cell composition.
[0784] Results in FIG. 15 are shown for 36 tumor biopsy samples that were analyzed among the subset of subjects in an ongoing clinical trial. A hypothetical threshold was set assuming, at 3 months following administration of the CD19-directed CAR T cell composition, 30% (11/36) of subjects will not respond to CAR-T cell treatment and 70% (25/36) of subjects will be able to respond; actual response results are shown. The results show a separation of gene expression between subjects who did not respond to treatment (subjects exhibited progressive disease (PD); designated resistant tumors) and subjects that were more responsive to treatment (designated sensitive tumors). Among samples from subjects with resistant tumors, increased expression of genes associated with dysregulated cell cycle and cell proliferation pathways was observed. For example, higher expression of genes related to Myc targets, p53 signaling and EZH2 upregulated genes was observed. These results are consistent with a finding that upregulation of survival machinery, such as anti-apoptotic factors, in tumor cells may lead to tumor resistance and poor response to tumor-targeted immunotherapies. Example 10 : Treatment of Chronic Lymphocytic Leukemia and Small Lymphocytic
Lymphoma with CD19-Targeting CAR T Cells and Venetoclax
[0785] Therapeutic CAR T cell compositions containing autologous T cells expressing a chimeric antigen-receptor (CAR) specific for CD 19 and venetoclax were administered as a combination therapy to subjects with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) as a combination therapy.
[0786] Specifically, autologous anti-CD19 directed therapeutic T cell compositions were generated by a process including immunoaffinity-based (e.g., immunomagnetic selection) enrichment of CD4+ and CD8+ cells from leukapheresis samples from adult (i.e. > 18 years of age) human subjects with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL) who had failed one or more prior lines of therapy and used to treat the subjects. All subjects had been previously treated with and failed an inhibitor of Bruton’s tyrosine kinase (BTK, e.g. ibrutinib) or were ineligible to receive a BTK inhibitor (e.g. ibrutinib) due to requirement for full dose anticoagulation or history of arrhythmia. Subjects either had not been previously treated with venetoclax (venetoclax naive) or had been previously treated with venetoclax (venetoclax exposed). Among venetoclax exposed subjects, the subjects were not venetoclax- intolerant and more than six months had passed since the subjects received the previous dose of venetoclax if the best overall response (BOR) was stable disease (SD), or progressive disease (PD) either (1) during the previous treatment with venetoclax or (2) within six months of discontinuing the previous treatment with venetoclax. Eligible subjects exhibited measurable disease (lymph nodes >1.5 cm in greater transverse diameter; GTD) or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen, and evidence of disease (e.g. CLL) in the blood by local testing, such as by flow cytometry (e.g. minimum residual disease (MRD) in PB of greater than or equal to 104). Subjects were screened at baseline (e.g. prior to administration of a lymphodepleting chemotherapy) for expression of BCL2 family proteins, and subjects who were previously treated with venetoclax were additionally screened for BCL2 mutations. Subjects were not excluded on the basis of BCL2 mutation status.
[0787] T cell compositions enriched for CD4+ and/or CD8+ cells from leukapheresis samples from individual subjects to be treated were administered to the subjects. The isolated compositions enriched for CD4+ and/or CD8+ T cells were separately activated with anti-CD3/anti-CD28 antibody reagents and independently transduced with a viral vector (e.g., lentiviral vector) encoding an anti-CD19 CAR, followed by separate expansion and cryopreservation of each of the engineered CD4+ and CD8+ cell populations. The CAR contained an anti-CD19 scFv derived from a murine antibody (variable region derived from FMC63, VL-linker-Vn orientation), an immunoglobulin-derived spacer linking the antigenbinding domain to a transmembrane domain, a transmembrane domain derived from human CD28), a costimulatory region derived from human 4-1BB, and a human CD3-zeta intracellular signaling domain. The viral vector further contained sequences encoding a truncated receptor, which is separated from the CAR sequence by a T2A ribosome skip sequence and serves as a surrogate marker for CAR expression.
[0788] The cryopreserved cell compositions were separately thawed prior to intravenous administration. Subjects were administered a dose of 1 x 108 total CAR-expressing T cells (e.g. via separate infusions of CD4+ CAR-expressing T cells and CD8+ CAR-expressing T cells provided at approximately a 1:1 ratio of CD4+ to CD8+ cells.
[0789] Prior to CAR+ T cell infusion, leukapheresis samples were obtained from subjects. Subjects were then administered venetoclax bridging therapy for three weeks prior to lymphodepleting therapy. As bridging therapy, subjects were administered 20 milligrams (mg) of venetoclax daily for the first week, 50 mg of venetoclax daily for the second week, and 100 mg of venetoclax daily for the third week. Optionally, if a subject was previously treated with venetoclax, the subject was additionally administered an anti-CD20 antibody during the bridging therapy. Subjects were able to receive treatment with ibrutinib up until the time of leukapheresis and/or during bridging therapy.
[0790] Following venetoclax washout (e.g. 1 day), subjects received a lymphodepleting chemotherapy with fludarabine (Flu, 30 mg/m2) and cyclophosphamide (Cy, 300 mg/m2) for 3 days. The subjects received the dose of CAR-expressing T cells (e.g. 1 x 108 total CAR-expressing T cells) 2-7 days after lymphodepletion, on Day 1. Venetoclax administration was initiated on Day 2 and continued through Day 90.
[0791] Subjects were assigned to one of three dosing cohorts (see FIG. 16; DC1: Dosing cohort 1; DC2: Dosing cohort 2; DC3: Dosing cohort 3). Dosing cohort 1 received a dosing regimen of (1) 50 mg venetoclax daily from Day 2 through Day 30; and (2) 100 mg venetoclax daily from Day 31 through Day 90. Dosing cohort 2 receives a dosing regimen of (1) 100 mg venetoclax daily from Day 2 through Day 30; and (2) 200 mg venetoclax daily from Day 31 through Day 90. Dosing cohort 3 receives a dosing regimen of (1) 100 mg venetoclax daily from Day 2 through Day 30; (2) 200 mg venetoclax daily from Day 31 through Day 37; and (3) 400 mg venetoclax daily from Day 38 through Day 90. Optionally, if subjects in dosing cohort 1 experienced a dose limiting toxicity, the dosing regimen was modified (i.e. de-escalated) to (1) 20 mg venetoclax daily from Day 2 through Day 7; (2) 50 mg venetoclax daily from Day 8 through Day 30; and (3) 100 mg venetoclax daily from Day 31 through Day 90. Similarly, if subjects in dosing cohort 2 experienced a dose limiting toxicity, the dosing regimen wasoptionally modified (i.e. de-escalated) to (1) 50 mg venetoclax daily from Day 2 through Day 7; (2) 100 mg venetoclax daily from Day 8 through Day 30; and (3) 200 mg venetoclax daily from Day 31 through Day 90
[0792] Subjects were monitored for safety, pharmacokinetics and pharmacodynamics, and clinical response. Determining clinical response to the treatment included assessing whether the subject exhibits progressive disease (PD), partial response (PR), complete response (CR), and/or peripheral blood minimum residual disease-negative (PB MRDneg) status. [0793] In some cases, subjects who had minimum residual disease (MRD) (> 104 in PB) or who did not exhibit complete response (CR) by the end of the treatment, e.g. by Day 90, optionally continued treatment with venetoclax per standard of care, such as for a total treatment duration of 12 or 24 months.
[0794] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.
Sequences
Figure imgf000213_0001
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Figure imgf000215_0001

Claims

Claims WHAT IS CLAIMED:
1. A method of treating cancer, the method comprising:
(1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and
(2) administering to the subject a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy; and
(ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
2. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the BCL2 inhibitor is administered in a dosing regimen comprising:
(i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of a cell therapy, wherein the cell therapy comprises a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) and was administered to the subject on Day 1; and
(ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
3. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy; and
(ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
4. The method of any of claims 1-3, wherein the daily administration of the first dose of the BCL2 inhibitor begins about 1 day after or within about 1 day after initiation of administration of the cell therapy.
5. The method of any of claims 1-4, wherein the daily administration of the first dose of the BCL2 inhibitor begins about 1 day after initiation of administration of the cell therapy.
6. The method of any of claims 1-5, wherein the daily administration of the first dose of the BCL2 inhibitor begins within about 1 day after initiation of administration of the cell therapy.
7. The method of any of claims 1-6, wherein the daily administration of the first dose of the BCL2 inhibitor begins at or after a time when activation-induced cell death (AICD) of the cells of the cell therapy has peaked following initiation of administration of the cell therapy.
8. The method of any of claims 1-7, wherein the at least one subsequent dose comprises a second dose, and the dosing regimen comprises:
(i) daily administration of a first dose of the BCL2 inhibitor for a first predetermined period; and
(ii) daily administration of a second dose of the BCL2 inhibitor for a second predetermined period beginning after the first predetermined period.
9. The method of any of claims 1-8, wherein the at least one subsequent dose comprises a second dose and a third dose, and the dosing regimen comprises:
(i) daily administration of a first dose of the BCL2 inhibitor for a first predetermined period; (ii) daily administration of a second dose of the BCL2 inhibitor for a second predetermined period beginning after the first predetermined period; and
(iii) daily administration of a third dose of the BCL2 inhibitor for a third predetermined period beginning after the second predetermined period.
10. The method of any of claims 1-9, wherein the first predetermined period is from about Day 2 to about Day 30.
11. The method of any of claims 1-10, wherein the second predetermined period is from about Day 31 to about Day 90.
12. The method of any of claims 1-9, wherein the first predetermined period is from about Day 2 to about Day 7.
13. The method of any of claims 1-9 and 12, wherein the second predetermined period is from about Day 8 to about Day 30.
14. The method of any of claims 1-10, wherein the second predetermined period is from about Day 31 to about Day 37.
15. The method of any of claims 9, 12 and 13, wherein the third predetermined period is from about Day 31 to about Day 90.
16. The method of any of claims 9, 10 and 14, wherein the third predetermined period is from about Day 38 to about Day 90.
17. The method of any of claims 1-16, wherein the first dose is at least about 20 mg of the BCL2 inhibitor.
18. The method of any of claims 1-17, wherein no dose of the dosing regimen is greater than about 400 mg of the BCL2 inhibitor.
19. The method of any of claims 1-18, wherein the first dose is between about 20 mg and about 100 mg of the BCL2 inhibitor.
20. The method of any of claims 1-19, wherein the first dose is about 20 mg, about 50 mg, or about 100 mg of the BCL2 inhibitor.
21. The method of any of claims 8-20, wherein the second dose is between about 50 mg and about 200 mg of the BCL2 inhibitor.
22. The method of any of claims 8-21, wherein the second dose is about 50 mg, about 100 mg, or about 200 mg of the BCL2 inhibitor.
23. The method of any of claims 9-22, wherein the third dose is about 100 mg and 400 mg of the BCL2 inhibitor.
24. The method of any of claims 9-23, wherein the third dose is about 100 mg, about 200 mg, or about 400 mg of the BCL2 inhibitor.
25. A method of treating cancer, the method comprising:
(1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and
(2) administering to the subject a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 20 mg of the BCL2 inhibitor from about Day 2 to about Day 7;
(ii) daily administration of about 50 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and
(iii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
26. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the subject has been administered a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1, and wherein the BCL2 inhibitor is administered in a dosing regimen comprising:
(i) daily administration of about 20 mg of the BCL2 inhibitor from about Day 2 to about Day 7 ;
(ii) daily administration of about 50 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and
217 (iii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day
90.
27. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 20 mg of the BCL2 inhibitor from about Day 2 to about Day 7 ;
(ii) daily administration of about 50 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and
(iii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
28. A method of treating cancer, the method comprising:
(1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and
(2) administering to the subject a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and
(ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
29. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the subject has been administered a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1, and wherein the BCL2 inhibitor is administered in a dosing regimen comprising:
(i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and
(ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day
90.
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30. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and
(ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
31. A method of treating cancer, the method comprising:
(1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and
(2) administering to the subject a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 7;
(ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and
(iii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
32. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the subject has been administered a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, and the BCL2 inhibitor is administered in a dosing regimen comprising:
(i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 7 ;
(ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and
(iii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
33. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising
219 a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 50 mg of the BCL2 inhibitor from about Day 2 to about Day 7 ;
(ii) daily administration of about 100 mg of the BCL2 inhibitor from about Day 8 to about Day 30; and
(iii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
34. A method of treating cancer, the method comprising:
(1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and
(2) administering to the subject a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and
(ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
35. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the subject has been administered a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1, and wherein the BCL2 inhibitor is administered in a dosing regimen comprising:
(i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30; and
(ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
36. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is to be administered a BCL2 inhibitor in a dosing regimen comprising:
220 (i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day
30; and
(ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 90.
37. A method of treating cancer, the method comprising:
(1) administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1; and
(2) administering to the subject a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30;
(ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 37 ; and
(iii) daily administration of about 400 mg of the BCL2 inhibitor from about Day 38 to about Day 90.
38. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a BCL2 inhibitor, wherein the subject has been administered a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) on Day 1, and wherein the BCL2 inhibitor is administering in a dosing regimen comprising:
(i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day 30;
(ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 37; and
(iii) daily administration of about 400 mg of the BCL2 inhibitor from about Day 38 to about Day 90.
39. A method of treating cancer, the method comprising administering to a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) on Day 1, wherein the subject is be administered a BCL2 inhibitor in a dosing regimen comprising:
221 (i) daily administration of about 100 mg of the BCL2 inhibitor from about Day 2 to about Day
30;
(ii) daily administration of about 200 mg of the BCL2 inhibitor from about Day 31 to about Day 37; and
(iii) daily administration of about 400 mg of the BCL2 inhibitor from about Day 38 to about Day 90.
40. The method of any of claims 1-39, wherein the BCL2 inhibitor is administered for no more than about 24 months.
41. The method of any of claims 1-40, wherein the BCL2 inhibitor is administered for no more than about 12 months.
42. The method of any of claims 1-41, wherein, if the subject exhibits minimum residual disease (MRD) in peripheral blood (PB) at Day 90, the BCL2 inhibitor is administered for up to 12 months or for up to 24 months.
43. The method of any of claims 1-42, wherein if the subject does not exhibit a complete response (CR) at Day 90, the BCL2 inhibitor is administered for up to 12 months or for up to 24 months.
44. The method of any of claims 1-39, wherein daily administration of the BCL2 inhibitor is discontinued after or about after Day 90.
45. The method of any of claims 1-39, wherein the BCL2 inhibitor is administered daily until at or about Day 90.
46. The method of any of claims 1-45, wherein the subject has a chronic lymphocytic leukemia (CLL).
47. The method of claim 46, wherein the CLL is a relapsed or refractory (r/r) CLL.
48. The method of any of claims 1-45, wherein the subject has a small lymphocytic lymphoma (SLL).
49. The method of claim 48, wherein the SLL is a relapsed or refractory (r/r) SLL.
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50. The method of any of claims 1-49, further comprising, prior to initiation of administration of the cell therapy, administering a lymphodepleting therapy to the subject.
51. The method of any of claims 1-50, wherein the subject has been preconditioned with a lymphodepleting therapy prior to initiation of administration of the cell therapy.
52. The method of claim 50 or claim 51, wherein the lymphodepleting therapy comprises the administration of fludarabine and/or cyclophosphamide.
53. The method of any of claims 50-52, wherein the lymphodepleting therapy comprises administration of cyclophosphamide at about 200-400 mg/m2, optionally at or about 300 mg/m2, inclusive, and/or fludarabine at about 20-40 mg/m2, optionally 30 mg/m2, daily for 2-4 days, optionally for 3 days.
54. The method of any of claims 50-53, wherein the lymphodepleting therapy comprises administration of cyclophosphamide at or about 300 mg/m2 and fludarabine at about 30 mg/m2 daily for 3 days, optionally wherein the cell therapy is administered at least at or about 2-7 days after the lymphodepleting therapy or at least at or about 2-7 days after the initiation of the lymphodepleting therapy.
55. The method of any of claims 1-54, further comprising administering to the subject a bridging therapy comprising a BCL2 inhibitor.
56. The method of any of claims 1-55, wherein the subject is administered a bridging therapy comprising a BCL2 inhibitor.
57. The method of claim 55 or claim 56, wherein the bridging therapy is administered prior to initiation of administration of a lymphodepleting therapy.
58. The method of any of claims 55-57, wherein the bridging therapy is administered at a time between collecting of autologous cells from the subject and prior to administering a lymphodepleting therapy to the subject, optionally wherein the collecting is by apheresis or leukapheresis.
59. The method of any of claims 55-58, wherein the bridging therapy comprises daily administration of a first dose of the BCL2 inhibitor for a first week, daily administration of a second dose
223 of the BCL2 inhibitor for a second week, and daily administration of a third dose of the BCL2 inhibitor for a third week.
60. The method of any of claims 55-59, wherein the bridging therapy comprises daily administration of between about 20 mg and about 400 mg of the BCL2 inhibitor.
61. The method of claim 59 or claim 60, wherein the second dose of the bridging therapy is an increased amount of BCL2 inhibitor compared to the first dose of the bridging therapy, and the third dose of the bridging therapy is an increased amount of BCL2 inhibitor compared to the second dose of the bridging therapy.
62. The method of any of claims 59-61, wherein the first dose of the bridging therapy is 20 mg of the BCL2 inhibitor, the second dose of the bridging therapy is 50 mg of the BCL2 inhibitor, and the third dose of the bridging therapy is 100 mg of the BCL2 inhibitor.
63. The method of any of claims 55-62, wherein the bridging therapy is ceased about or at least about 1 day prior to initiation of administration of a lymphodepleting therapy.
64. The method of any of claims 1-63, wherein the BCL2 inhibitor is venetoclax or navitoclax.
65. The method of any of claims 1-64, wherein the BCL2 inhibitor is venetoclax.
66. The method of any of claims 55-65, wherein the BCL2 inhibitor administered in the bridging therapy and the BCL2 inhibitor administered in the dosing regimen are the same BCL2 inhibitor, optionally wherein the BCL2 inhibitor administered in the bridging therapy and the dosing regimen is venetoclax.
67. The method of any of claims 55-65, wherein the BCL2 inhibitor administered in the bridging therapy and the BCL2 inhibitor administered in the dosing regimen are different BCL2 inhibitors.
68. The method of any of claims 55-67, wherein the bridging therapy further comprises administration of an anti-CD20 antibody to the subject.
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69. The method of any of claims 55-68, wherein if the subject has been previously treated with a BCL2 inhibitor, the bridging therapy further comprises administration of an anti-CD20 antibody to the subject.
70. The method of any of claims 55-69, wherein the bridging therapy further comprises administration of a BTK inhibitor to the subject.
71. The method of any of claims 1-70, wherein the subject has been previously treated with a Bruton’ s tyrosine kinase (BTK) inhibitor.
72. The method of any of claims 1-71, wherein the subject is ineligible for treatment with a Bruton’ s tyrosine kinase (BTK) inhibitor.
73. The method of any of claims 70-72, wherein the BTK inhibitor is or comprises ibrutinib.
74. The method of any of claims 1-73, wherein, prior to the administration of the cell therapy, the subject has been treated with one or more prior therapies for the CLL or the SLL, other than another dose of cells expressing a CAR or a lymphodepleting therapy, optionally at least two prior therapies.
75. The method of any of claims 1-74, wherein, prior to the administration of the cell therapy, the subject has relapsed following remission after treatment with, or become refractory to, failed and/or was intolerant to treatment with two or more prior therapies.
76. The method of claim 74 or claim 75, wherein the one or more prior therapies are selected from a kinase inhibitor, optionally an inhibitor of Bruton’s tyrosine kinase (BTK), optionally ibrutinib; venetoclax; a combination therapy comprising fludarabine and rituximab; radiation therapy; and hematopoietic stem cell transplantation (HSCT).
77. The method of any of claims 74-76, wherein the one or more prior therapies comprises an inhibitor of Bruton’s tyrosine kinase (BTK), optionally ibrutinib.
78. The method of any of claims 74-77, wherein the one or more prior therapies comprises ibrutinib and a BCL2 inhibitor, optionally venetoclax.
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79. The method of any of claims 1-78, wherein the subject has not been previously treated with venetoclax.
80. The method of any of claims 1-78, wherein the subject has been previously treated with venetoclax.
81. The method of any of claims 1-79, wherein the subject is not intolerant to venetoclax at the time of initiation of administration of the cell therapy and/or at the time of initiation of administration of the BCL2 inhibitor.
82. The method of claim 80 or claim 81, wherein more than six months have passed since the last dose of the previous treatment with venetoclax if the subject’s best overall response (BOR) during or within 6 months of discontinuing the previous treatment with venetoclax was stable disease (SD) or progressive disease (PD).
83. The method of any of claims 1-82, wherein prior to administration of the cell therapy and/or prior to administration of the BCL2 inhibitor, the subject has measurable disease in the lymph nodes and/or evaluable or measurable disease in peripheral blood (PB), bone marrow (BM), liver, or spleen.
84. The method of any of claims 1-83, wherein prior to administration of the cell therapy and/or prior to administration of the BCL2 inhibitor, the subject has minimum residual disease (MRD) in peripheral blood (PB) of greater than or equal to 104.
85. The method of any of claims 1-84, wherein the subject has Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1.
86. The method of any of claims 1-85, wherein the dose of engineered cells comprises a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR, optionally wherein the ratio is between approximately 1:3 and approximately 3:1.
87. The method of any of claims 1-86, wherein the dose of engineered cells comprises a defined ratio of CD4+ cells expressing the CAR to CD8+ cells expressing the CAR that is or is approximately 1:1.
226
88. The method of any of claims 1-87, wherein the dose of engineered cells comprises between about 2.5 x 107 total CAR-expressing cells and about 1.0 x 108 total CAR-expressing cells.
89. The method of any of claims 1-88, wherein the dose of engineered cells comprises at or about 2.5 x 107 total CAR-expressing cells.
90. The method of any of claims 1-88, wherein the dose of engineered cells comprises at or about 5 x 107 total cells or total CAR-expressing cells.
91. The method of any of claims 1-88, wherein the dose of engineered cells comprises at or about 1 x 108 total cells or total CAR-expressing cells.
92. The method of any of claims 1-91, wherein administration of the cell therapy comprises administering a plurality of separate compositions, wherein the plurality of separate compositions comprises a first composition comprising one of the CD4+ T cells and the CD8+ T cells and a second composition comprising the other of the CD4+ T cells and the CD8+ T cells.
93. The method of claim 92, wherein the first composition comprises the CD8+ T cells and the second composition comprises the CD4+ T cells.
94. The method of claim 92 or claim 93, wherein: the first composition and the second composition are administered 0 to 12 hours apart, 0 to 6 hours apart, or 0 to 2 hours apart, or wherein the administration of the first composition and the administration of the second composition are carried out on the same day, between about 0 and about 12 hours apart, between about 0 and about 6 hours apart, or between about 0 and 2 hours apart; and/or the initiation of administration of the first composition and the initiation of administration of the second composition are carried out between about 1 minute and about 1 hour apart, or between about 5 minutes and about 30 minutes apart.
95. The method of any of claims 92-94, wherein the first composition and the second composition are administered no more than 2 hours, no more than 1 hour, no more than 30 minutes, no more than 15 minutes, no more than 10 minutes, or no more than 5 minutes apart.
96. The method of any of claims 92-95, wherein the first composition is administered prior to the second composition.
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97. The method of any of claims 92-96, wherein the CAR comprised by the CD4+ T cells and/or the CAR comprised by the CD8+ T cells comprises a CAR that is the same and/or wherein the CD4+ T cells and/or the CD8+ T cells are genetically engineered to express a CAR that is the same.
98. The method of any of claims 1-97, wherein: the CAR comprises an extracellular antigen-binding domain specific for CD 19, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, which optionally is a 4- 1BB, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule, which optionally is a CD3zeta; and/or the CAR comprises, in order, an extracellular antigen-binding domain specific for CD19, a transmembrane domain, a cytoplasmic signaling domain derived from a costimulatory molecule, and a cytoplasmic signaling domain derived from a primary signaling ITAM-containing molecule.
99. The method of claim 98, wherein the antigen-binding domain is an scFv.
100. The method of claim 99, wherein: the scFv comprises a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 35), a CDRL2 sequence of SRLHSGV (SEQ ID NO: 36), and/or a CDRL3 sequence of GNTLPYTFG (SEQ ID NO: 37) and/or a CDRH1 sequence of DYGVS (SEQ ID NO: 38), a CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO: 39), and/or a CDRH3 sequence of YAMDYWG (SEQ ID NO: 40); the scFv comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC63 and/or a CDRL1 sequence of FMC63, a CDRL2 sequence of FMC63, a CDRL3 sequence of FMC63, a CDRH1 sequence of FMC63, a CDRH2 sequence of FMC63, and a CDRH3 sequence of FMC63 or binds to the same epitope as or competes for binding with any of the foregoing; the scFv comprises a VH set forth in SEQ ID NO: 41 and a VL set forth in SEQ ID NO: 42, optionally wherein the VH and VL are separated by a flexible linker, optionally wherein the flexible linker is or comprises the sequence set forth in SEQ ID NO: 59; and/or the scFv is or comprises the sequence set forth in SEQ ID NO: 43.
101. The method of any of claims 98-100, wherein the costimulatory signaling domain is a signaling domain of CD28 or 4- IBB.
102. The method of any of claims 98-101, wherein the costimulatory signaling domain is a signaling domain of 4- IBB.
228
103. The method of any of claims 98-102, wherein the costimulatory signaling domain comprises SEQ ID NO: 12 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12.
104. The method of any of claims 98-103, wherein the primary signaling domain is a CD3zeta signaling domain.
105. The method of any of claims 98-104, wherein the primary signaling domain comprises SEQ ID NO: 13, 14, or 15, or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13, 14, or 15.
106. The method of any of claims 98-105, wherein the primary signaling domain comprises SEQ ID NO: 13 or a variant thereof having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 13.
107. The method of any of claims 99-106, wherein the CAR further comprises a spacer between the transmembrane domain and the scFv.
108. The method of claim 107, wherein the spacer is a polypeptide spacer that comprises or consists of all or a portion of an immunoglobulin hinge or a modified version thereof, optionally an IgG4 hinge, or a modified version thereof.
109. The method of claim 107 or claim 108, wherein the spacer is about 15 amino acids or less, and does not comprise a CD28 extracellular region or a CD8 extracellular region.
110. The method of any of claims 107-109, wherein the spacer is at or about 12 amino acids in length.
111. The method of any of claims 107-110, wherein the spacer: comprises or consists of the sequence of SEQ ID NO: 1, a sequence encoded by SEQ ID NO: 2, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, or a variant of any of the foregoing having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto; and/or comprises or consists of the formula X1PPX2P, where Xi is glycine, cysteine or arginine and X2 is cysteine or threonine.
229
112. The method of any of claims 99-111, wherein the scFv comprises, in order, a VH, a linker, optionally comprising SEQ ID NO: 59, and a VL-
113. The method of any of claims 107-112, wherein: the antigen binding domain comprises an scFv that comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC63; the spacer is a polypeptide spacer that comprises the sequence of SEQ ID NO: 1; the costimulatory domain comprises SEQ ID NO: 12; and the primary signaling domain comprises SEQ ID NO: 13, 14 or 15.
114. The method of any of claims 107-113, wherein: the antigen binding domain comprises an scFv that comprises a variable heavy chain region of FMC63 and a variable light chain region of FMC63; the spacer is a polypeptide spacer that comprises the sequence of SEQ ID NO: 1; the costimulatory domain comprises SEQ ID NO: 12; and the primary signaling domain comprises SEQ ID NO: 13.
115. The method of any of claims 1-114, wherein the engineered cells are primary T cells obtained from a subject.
116. The method of any of claims 1-115, wherein the engineered cells are autologous to the subject.
117. The method of any of claims 1-116, wherein the subject is a human subject.
118. A cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19) and a BCL2 inhibitor for use in a method of treating a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL), where the method comprises:
(1) administering to the subject the cell therapy on Day 1; and
(2) administering to the subject the BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy; and
(ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein:
230 each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
119. A BCL2 inhibitor for use in a method of treating a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) that has been treated with a cell therapy comprising a dose of engineered T cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19), wherein the method comprises administering the BCL2 inhibitor to the subject in dosing regimen comprising:
(i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy, wherein the cell therapy was administered to the subject on Day 1; and
(ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
120. A cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) for use in a method of treating a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL), wherein the method comprises administering the cell therapy to the subject on Day 1, and the subject is to be administered a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy; and
(ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
231
121. Use of a BCL2 inhibitor in the manufacture of a medicament for treatment of a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) that has been treated with a cell therapy comprising a dose of engineered T cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD19), wherein the medicament is to be administered to the subject in dosing regimen comprising:
(i) daily administration of a first dose of the medicament for a predetermined period beginning within about 2 days after initiation of administration of the cell therapy, wherein the cell therapy was administered to the subject on Day 1; and
(ii) daily administration of at least one subsequent dose of the medicament, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the medicament, wherein: each of the at least one subsequent dose is an increased amount of the medicament compared to the preceding dose; and the medicament is administered at no more than or no more than about 100 mg per day through Day 30.
122. Use of a cell therapy comprising a dose of engineered cells comprising T cells expressing a chimeric antigen receptor (CAR) that binds cluster of differentiation 19 (CD 19) in the manufacture of a medicament for the treatment of a subject having a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL) that is to be administered a BCL2 inhibitor in a dosing regimen comprising:
(i) daily administration of a first dose of the BCL2 inhibitor for a predetermined period beginning within about 2 days after initiation of administration of the medicament, wherein the medicament is administered to the subject on Day 1; and
(ii) daily administration of at least one subsequent dose of the BCL2 inhibitor, wherein each subsequent dose is individually administered for a predetermined period beginning after a predetermined period of a preceding dose of the BCL2 inhibitor, wherein: each of the at least one subsequent dose is an increased amount of the BCL2 inhibitor compared to the preceding dose; and the BCL2 inhibitor is administered at no more than or no more than about 100 mg per day through Day 30.
123. An article of manufacture comprising (i) a composition for a cell therapy, or one of a plurality of compositions for a cell therapy, comprising T cells expressing a CAR that binds CD 19 and (ii) an inhibitor of BCL2 protein, optionally venetoclax, and instructions for administering the cell therapy and the BCL2 inhibitor to the subject, wherein the instructions specify administering the cell
232 therapy and the BCL2 inhibitor according to the methods of any of claims 1-117 or the use of any of claim 118-122.
233
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