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WO2025006561A2 - Méthodes de traitement utilisant des immunothérapies ciblant cd19 - Google Patents

Méthodes de traitement utilisant des immunothérapies ciblant cd19 Download PDF

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Publication number
WO2025006561A2
WO2025006561A2 PCT/US2024/035555 US2024035555W WO2025006561A2 WO 2025006561 A2 WO2025006561 A2 WO 2025006561A2 US 2024035555 W US2024035555 W US 2024035555W WO 2025006561 A2 WO2025006561 A2 WO 2025006561A2
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cells
dose
subject
administered
car
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WO2025006561A3 (fr
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David Richard SHOOK
Nishi KOTHARI
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Nkarta Inc
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Nkarta Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/35—Cytokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
    • 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
    • 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
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/10—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the structure of the chimeric antigen receptor [CAR]
    • A61K2239/22—Intracellular domain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/48—Blood cells, e.g. leukemia or lymphoma
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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)

Definitions

  • the present disclosure relates to cells engineered to express chimeric antigen receptors directed to CD19, and administration of such cells in accordance with certain dosing regimens.
  • BACKGROUND [0003] As further knowledge is gained about various cancers and what characteristics a cancerous cell has that can be used to specifically distinguish that cell from a healthy cell, therapeutics that leverage the distinct features of a cancerous cell, and dosing regimens for administration of the same, are under development. Immunotherapies that employ engineered immune cells are one approach to treating cancers.
  • all three doses are administered to the subject within between about 4 days and about 10 days.
  • the second dose is administered to the subject between 2-4 days after administration of the first dose to the subject;
  • the third dose is administered to the subject between 2-4 days after administration of the second dose to the subject; and each of the first, second and third doses comprises at least about 2 ⁇ 10 9 CAR-expressing NK cells.
  • a method of treating a subject having a cancer comprising administering to the subject a first dosing cycle comprising a first dose of natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells.
  • the second dose is administered to the subject between 2-4 days after administration of the first dose to the subject;
  • the third dose is administered to the subject between 2-4 days after administration of the second dose to the subject; and each of the first, second and third doses comprises at least about 1 ⁇ 10 9 CAR- expressing NK cells.
  • Also provided herein is a method of treating a subject having a cancer comprising administering to the subject a first dosing cycle comprising a first dose of natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: all three doses are administered to the subject within between about 4 days and about 10 days; and each of the first, second and third doses comprises at least about 1 ⁇ 10 9 CAR-expressing NK cells.
  • NK natural killer
  • CAR chimeric antigen receptor
  • a method of treating a subject having a cancer comprising administering to the subject a first dosing cycle comprising a first dose of natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: all three doses are administered to the subject within between about 4 days and about 10 days; the subject is administered a lymphodepleting therapy prior to administration of the first dosing cycle; and the lymphodepleting therapy comprises cyclophosphamide and does not comprise fludarabine.
  • NK natural killer
  • CAR chimeric antigen receptor
  • each dose is separated by between about 24 hours and about 72 hours. In some embodiments, each dose is separated by at least about 24 hours. In some embodiments, each dose is separated by about 24 hours. In some embodiments, each dose is separated by at least about 48 hours. In some embodiments, each dose is separated by about 48 hours. In some embodiments, each dose is separated by at least about 72 hours. In some embodiments, each dose is separated by about 72 hours. [0010] In some embodiments, the first dose is administered on about Day 0 of the dosing cycle.
  • the second dose is administered on about Day 2 of the dosing cycle. In some embodiments, the second dose is administered on about Day 3 of the dosing cycle. In some embodiments, the second dose is administered on about Day 4 of the dosing cycle. In some embodiments, the third dose is administered on about Day 4 of the dosing cycle. In some embodiments, the third dose is administered on about Day 5 of the dosing cycle. In some embodiments, the third dose is administered on about Day 6 of the dosing cycle. In some embodiments, the third dose is administered on about Day 7 of the dosing cycle. In some embodiments, the third dose is administered on about Day 8 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 2 of the dosing cycle
  • the third dose is administered on about Day 4 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 2 of the dosing cycle
  • the third dose is administered on about Day 5 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 2 of the dosing cycle
  • the third dose is administered on about Day 6 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 3 of the dosing cycle
  • the third dose is administered on about Day 5 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 3 of the dosing cycle
  • the third dose is administered on about Day 6 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 3 of the dosing cycle
  • the third dose is administered on about Day 7 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 4 of the dosing cycle
  • the third dose is administered on about Day 6 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 4 of the dosing cycle
  • the third dose is administered on about Day 7 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 4 of the dosing cycle
  • the third dose is administered on about Day 8 of the dosing cycle.
  • each of the first, second and third doses comprises about 1 ⁇ 10 9 CAR-expressing NK cells. In some embodiments, each of the first, second and third doses comprises about 1.5 ⁇ 10 9 CAR-expressing NK cells. In some embodiments, each of the first, second and third doses comprises about 2 ⁇ 10 9 CAR- expressing NK cells. In some embodiments, each of the first, second and third doses comprises about 2.5 ⁇ 10 9 CAR-expressing NK cells. [0013] In some embodiments, the subject is administered a lymphodepleting therapy prior to initiation of the dosing cycle. In some embodiments, the subject is administered a lymphodepleting therapy prior to administration of the first dosing cycle.
  • the first dosing cycle is followed by an additional dosing cycle.
  • the method comprises an additional dosing cycle to deepen or consolidate the response.
  • the method comprises an additional dosing cycle to deepen the response.
  • the method comprises an additional dosing cycle as consolidation treatment.
  • the method comprises an additional dosing cycle as consolidation treatment.
  • the method comprises an additional dosing cycle as retreatment. In some embodiments, if the subject exhibits a PR following a dosing cycle and subsequently exhibits disease progression, the method comprises an additional dosing cycle as retreatment. In some embodiments, if the subject exhibits a CR following a dosing cycle and subsequently exhibits disease progression, the method comprises an additional dosing cycle as retreatment. In some embodiments, the method comprises between one dosing cycle and five dosing cycles. In some embodiments, the subject is administered a lymphodepleting therapy prior to each dosing cycle.
  • each dosing cycle is between about 14 days and about 35 days. In some embodiments, each dosing cycle is about 28 days. In some embodiments, if the subject exhibits a partial response to the treatment, the dosing regimen comprises an additional dosing cycle. In some embodiments, if the subject exhibits a complete response to the treatment, the dosing regimen comprises an additional dosing cycle. In some embodiments, if the subject exhibits an initial clinical response to the treatment and subsequent disease progression, the dosing regimen comprises an additional dosing cycle. [0014] In some embodiments, the lymphodepleting therapy comprises cyclophosphamide, optionally wherein a dose of cyclophosphamide is between about 300 mg/m2 and about 1000 mg/m2.
  • the lymphodepleting therapy comprises cyclophosphamide. In some embodiments a dose of cyclophosphamide is between about 300 mg/m2 and about 1000 mg/m2. In some embodiments, the lymphodepleting therapy comprises a single dose of cyclophosphamide. In some embodiments, the lymphodepleting therapy consists of a single dose of cyclophosphamide. In some embodiments, the single dose of cyclophosphamide is administered to the subject about 3 days prior to administration of the dosing cycle. In some embodiments, the single dose of cyclophosphamide is about 1000 mg/m2.
  • the lymphodepleting therapy comprises three doses of cyclophosphamide. In some embodiments, each dose of cyclophosphamide is about 500 mg/m2. In some embodiments, a dose of cyclophosphamide is administered to the subject on each of about 5 days prior, 4 days prior, and 3 days prior to administration of the first dose of the dosing cycle. In some embodiments, the lymphodepleting therapy does not comprise fludarabine. In some embodiments, the lymphodepleting therapy comprises cyclophosphamide and does not comprise fludarabine. In some embodiments, the lymphodepleting therapy consists of cyclophosphamide.
  • the lymphodepleting therapy does not comprise fludarabine.
  • the lymphodepleting therapy comprises fludarabine.
  • a dose of fludarabine is between about 20 mg/m2and about 40 mg/m2.
  • the lymphodepleting therapy comprises three doses of fludarabine.
  • each dose of fludarabine is about 30 mg/m2.
  • a dose of fludarabine is administered to the subject on each of about 5 days prior, 4 days prior, and 3 days prior to administration of the first dose of the dosing cycle.
  • the method further comprises administration of a therapeutic agent that targets CD20.
  • the subject is administered a therapeutic agent that targets CD20.
  • the therapeutic agent is an anti- CD20 monoclonal antibody.
  • the anti-CD20 antibody is rituximab.
  • the therapeutic agent that targets CD20 is administered in an amount between about 150 mg/m2 and about 500 mg/m2.
  • the therapeutic agent that targets CD20 is administered in an amount of about 375 mg/m2.
  • the therapeutic agent is administered to the subject at least one time and the at least one time is at least 2 days prior to administration of the first dose of the dosing cycle.
  • the therapeutic agent is administered to the subject one time 3 days prior to administration of the first dose of the dosing cycle.
  • the first dose of genetically engineered NK cells is administered to the subject about 2 to 5 days after administration of the lymphodepleting therapy has concluded. In some embodiments, the first dose of the genetically engineered NK cells is administered to the subject about 2 days after administration of the lymphodepleting therapy has concluded. In some embodiments, the first dose of the genetically engineered NK cells is administered to the subject about 3 days after administration of the lymphodepleting therapy has concluded. In some embodiments, the first dose of the genetically engineered NK cells is administered to the subject about 4 days after administration of the lymphodepleting therapy has concluded.
  • the first dose of the genetically engineered NK cells is administered to the subject about 5 days after administration of the lymphodepleting therapy has concluded.
  • a method of treating a subject having a cancer comprising administering to the subject a first dosing cycle comprising a first dose of natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: the second dose is administered to the subject about 3 days after administration of the first dose to the subject, the third dose is administered to the subject about 4 days after administration of the second dose to the subject; each of the first, second and third doses comprises between about 1.0 ⁇ 109 CAR-expressing NK cells and about 2 ⁇ 109 CAR-expressing NK cells; the subject is administered a lymphodepleting therapy prior to administration of the first dosing cycle; and the lymphodepleting
  • a method of treating a subject having a cancer comprising administering to the subject a first dosing cycle comprising a first dose of natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: the second dose is administered to the subject about 3 days after administration of the first dose to the subject, the third dose is administered to the subject about 4 days after administration of the second dose to the subject; each of the first, second and third doses comprises about 1.5 ⁇ 109 CAR-expressing NK cells, the subject is administered a lymphodepleting therapy comprising (i) a dose of about 500 mg/m2 of cyclophosphamide on each of 5 days, 4 days, and 3 days prior to administration of the first dose of the dosing cycle and (ii) a dose of about 30 mg/m2 of fludarabine on each of
  • a method of treating a subject having a cancer comprising administering to the subject a first dosing cycle comprising a first dose of natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: the second dose is administered to the subject about 3 days after administration of the first dose to the subject, the third dose is administered to the subject about 4 days after administration of the second dose to the subject; each of the first, second and third doses comprises about 2 ⁇ 109 CAR-expressing NK cells or about 2.5 ⁇ 109 CAR- expressing NK cells, the subject is administered a lymphodepleting therapy comprising (i) a dose of about 500 mg/m2 of cyclophosphamide on each of 5 days, 4 days, and 3 days prior to administration of the first dose of the dosing cycle and (ii) a dose
  • a method of preparing a subject having a cancer for treatment with natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19 comprising administering a lymphodepleting therapy to the subject prior to administration of a first dosing cycle of the genetically engineered NK cells to the subject, wherein: the first dosing cycle comprises a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells; each of the first, second and third doses comprises at least about 1 ⁇ 10 9 CAR-expressing NK cells; and all three doses are administered to the subject within between about 4 days and about 10 days.
  • NK natural killer
  • CAR chimeric antigen receptor
  • the lymphodepleting therapy comprises cyclophosphamide and does not comprise fludarabine. In some embodiments, the lymphodepleting therapy consists of cyclophosphamide.
  • a method of preparing a subject having a cancer for treatment with a composition comprising natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds to CD19 comprising administering a lymphodepleting therapy to the subject prior to administration of the composition to the subject, wherein the lymphodepleting therapy consists of cyclophosphamide.
  • NK natural killer
  • CAR chimeric antigen receptor
  • the composition is administered in a dosing cycle comprising a first dose of the CAR-expressing NK cells, a second dose of the CAR-expressing NK cells, and a third dose of the CAR-expressing NK cells.
  • each dose is separated by between about 24 hours and about 72 hours. In some embodiments, each dose is separated by at least about 24 hours. In some embodiments, each dose is separated by about 24 hours. In some embodiments, each dose is separated by at least about 48 hours. In some embodiments, each dose is separated by about 48 hours. In some embodiments, each dose is separated by at least about 72 hours. In some embodiments, each dose is separated by about 72 hours.
  • the first dose is administered on about Day 0 of the dosing cycle.
  • the second dose is administered on about Day 2 of the dosing cycle.
  • the second dose is administered on about Day 3 of the dosing cycle.
  • the second dose is administered on about Day 4 of the dosing cycle.
  • the third dose is administered on about Day 4 of the dosing cycle.
  • the third dose is administered on about Day 5 of the dosing cycle.
  • the third dose is administered on about Day 6 of the dosing cycle.
  • the third dose is administered on about Day 7 of the dosing cycle.
  • the third dose is administered on about Day 8 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 2 of the dosing cycle
  • the third dose is administered on about Day 4 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 2 of the dosing cycle
  • the third dose is administered on about Day 5 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 2 of the dosing cycle
  • the third dose is administered on about Day 6 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 3 of the dosing cycle
  • the third dose is administered on about Day 5 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 3 of the dosing cycle
  • the third dose is administered on about Day 6 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 3 of the dosing cycle
  • the third dose is administered on about Day 7 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 4 of the dosing cycle
  • the third dose is administered on about Day 6 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 4 of the dosing cycle
  • the third dose is administered on about Day 7 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 4 of the dosing cycle
  • the third dose is administered on about Day 8 of the dosing cycle.
  • the cancer is a CD19-expressing cancer.
  • the cancer is a blood cancer. In some embodiments, the cancer is a leukemia or a lymphoma. In some embodiments, the cancer is a B cell cancer. In some embodiments, the cancer is a Non-Hodgkin lymphoma (NHL). In some embodiments, wherein the cancer is a large B-cell lymphoma (LBCL), optionally an aggressive LBCL. In some embodiments, the cancer is diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), Waldenström macroglobulinemia (WM), or B-cell acute lymphoblastic leukemia (B-ALL).
  • NHL B-cell lymphoblastic leukemia
  • the cancer is diffuse large B-cell lymphoma (DLBCL). In some embodiments, the cancer is follicular lymphoma (FL). In some embodiments, the cancer is marginal zone lymphoma (MZL). In some embodiments, the cancer is mantle cell lymphoma (MCL). In some embodiments, the cancer is Waldenström macroglobulinemia (WM). In some embodiments, the cancer is or B-cell acute lymphoblastic leukemia (B-ALL). In some embodiments, the cancer is a chronic lymphocytic leukemia (CLL) or a small lymphocytic lymphoma (SLL). In some embodiments, the cancer is a chronic lymphocytic leukemia (CLL).
  • DLBCL diffuse large B-cell lymphoma
  • the cancer is follicular lymphoma (FL). In some embodiments, the cancer is marginal zone lymphoma (MZL). In some embodiments, the cancer is mantle cell lymphoma (MCL). In some embodiment
  • the cancer is a small lymphocytic lymphoma (SLL). In some embodiments, the cancer is a relapsed/refractory (R/R) cancer.
  • the subject has less than or equal to 5% peripheral blasts. In some embodiments the subject has no evidence of extramedullary disease. In some embodiments, the subject has received at least 1 but not more than 7 lines of previous therapy, optionally wherein the subject has received at least 1 but not more than 4 lines of previous therapy. In some embodiments, the subject has received at least one line of previous therapy. In some embodiments, the subject has received at least two lines of previous therapy. In some embodiments, the subject has received at least three lines of previous therapy.
  • a line of previous therapy comprises an anti-CD20 monoclonal antibody and a cytotoxic chemotherapy.
  • the cytotoxic therapy is anthracycline.
  • a line of previous therapy comprises chimeric antigen receptor-expressing T (CAR T) cells.
  • a line of previous therapy comprises autologous anti-CD19 CAR T cells.
  • a line of previous therapy does not comprise CAR T cells.
  • a line of previous therapy does not comprise autologous anti-CD19 CAR T cells.
  • a line of previous therapy comprises an inhibitor of Bruton’s tyrosine kinase (BTKi).
  • the BTKi is ibrutinib.
  • a line of previous therapy comprises an inhibitor of Bcl-2.
  • the Bcl-2 inhibitor is venetoclax.
  • the CAR comprises: (a) an antigen-binding moiety that targets CD19; (b) a transmembrane domain; and (c) an intracellular signaling domain comprising an OX40 domain and a CD3zeta domain.
  • the antigen-binding moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a CDR-1, a CDR-2, and a CDR-3 comprising the amino acid sequences set forth in SEQ ID NOS: 24, 25, and 26, respectively; and the VL comprises a CDR-1, a CDR-2, and a CDR-3 comprising the amino acid sequences set forth in SEQ ID NOS: 27, 28, and 29, respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antigen-binding moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises a CDR-1, a CDR-2, and a CDR-3 comprising the amino acid sequences set forth in SEQ ID NOS: 30, 31, and 32, respectively; and the VL comprises a CDR-1, a CDR-2, and a CDR-3 comprising the amino acid sequences set forth in SEQ ID NOS: 33, 34, and 29, respectively.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antigen-binding moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the amino acid sequence set forth in SEQ ID NO: 35 and/or the VL comprises the amino acid sequence set forth in SEQ ID NO:36.
  • the VH comprises the amino acid sequence set forth in SEQ ID NO: 35.
  • the VL comprises the amino acid sequence set forth in SEQ ID NO:36.
  • the VH comprises the amino acid sequence set forth in SEQ ID NO: 35
  • the VL comprises the amino acid sequence set forth in SEQ ID NO:36.
  • the antigen-binding moiety is an scFv comprising the amino acid sequence of SEQ ID NO:37.
  • the genetically engineered NK cells are also engineered to express membrane-bound interleukin 15 (mbIL15).
  • the mbIL15 has at least 95% sequence identity to SEQ ID NO: 23 or 40.
  • the mbIL15 has at least 95% sequence identity to SEQ ID NO: 23.
  • the mbIL15 has at least 95% sequence identity to SEQ ID NO: 40.
  • the CAR and the mbIL15 are bicistronically encoded by the same nucleic acid molecule.
  • the nucleic acid sequences encoding the CAR and the mbIL15 are separated by a nucleic acid sequence encoding a T2A peptide.
  • the T2A peptide comprises the amino acid sequence set forth in SEQ ID NO:20.
  • the NK cells genetically engineered to express a CAR are also genetically edited.
  • the NK cells are genetically edited to increase IL15 signaling.
  • the methods comprise genetically editing the NK cells to increase IL15 signaling.
  • the NK cells are genetically edited to reduce expression of the CISH gene.
  • the methods comprise genetically editing the NK cells to reduce expression of the CISH gene.
  • the NK cells are genetically edited to reduce expression of the Cis protein.
  • the methods comprise genetically editing the NK cells to reduce expression of the Cis protein.
  • the NK cells comprise a disruption in one or both alleles of the CISH gene.
  • the NK cells comprise a disruption in one allele of the CISH gene.
  • the NK cells comprise a disruption in both alleles of the CISH gene.
  • the dosing cycle does not result in cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS)/neurotoxicity, and/or graft versus host disease.
  • the engineered NK cells are allogeneic with respect to the subject.
  • one dose of each dosing cycle is administered to the subject on an outpatient basis. In some embodiments, at least one dose of each dosing cycle is administered to the subject on an outpatient basis. In some embodiments, each dose of each dosing cycle is administered to the subject on an outpatient basis. In some embodiments, a dosing cycle is administered to the subject on an outpatient basis.
  • the overall response rate is at least about 50%, at least about 60%, at least about 70%, or at least about 80%. In some embodiments, at least about 50%, at least about 60%, at least about 70%, or at least about 80% of subjects treated according to the method exhibit a complete response (CR).
  • NK cells natural killer cells in the manufacture of a medicament for treating a subject having a cancer
  • the NK cells are genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19; and the NK cells are for administration as a first dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells.
  • CAR chimeric antigen receptor
  • the second dose is for administration to the subject between 2-4 days after administration of the first dose to the subject;
  • the third dose is for administration to the subject between 2-4 days after administration of the second dose to the subject; and each of the first, second and third doses comprises at least about 2 ⁇ 10 9 CAR-expressing NK cells.
  • NK cells in the manufacture of a medicament for treating a subject having a cancer
  • the NK cells are genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19
  • the NK cells are for administration as a first dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein all three doses are administered to the subject within between about 4 days and about 10 days; and each of the first, second and third doses comprises at least about 1 ⁇ 10 9 CAR-expressing NK cells.
  • CAR chimeric antigen receptor
  • NK cells natural killer cells in the manufacture of a medicament for treating a subject having a cancer
  • the NK cells are genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19
  • the NK cells are for administration as a first dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein all three doses are administered to the subject within between about 4 days and about 10 days; and the subject is administered a lymphodepleting therapy prior to administration of the first dosing cycle, wherein the lymphodepleting therapy comprises cyclophosphamide and does not comprise fludarabine.
  • CAR chimeric antigen receptor
  • the second dose is for administration to the subject between 2-4 days after administration of the first dose to the subject.
  • the third dose is for administration to the subject between 2-4 days after administration of the second dose to the subject.
  • the second dose is for administration to the subject between 2-4 days after administration of the first dose to the subject and the third dose is for administration to the subject between 2-4 days after administration of the second dose to the subject.
  • lymphodepleting therapy for preparing a subject having a cancer for treatment with a composition comprising natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds to CD19, wherein the lymphodepleting therapy consists of cyclophosphamide.
  • NK natural killer
  • CAR chimeric antigen receptor
  • lymphodepleting therapy in the manufacture of a medication for preparing a subject having a cancer for treatment with natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19
  • the lymphodepleting therapy is for administration to the subject prior to administration of a first dosing cycle of the genetically engineered NK cells to the subject, wherein: the first dosing cycle comprises a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells; each of the first, second and third doses comprises at least about 1 ⁇ 10 9 CAR- expressing NK cells; and all three doses are for administration to the subject within between about 4 days and about 10 days.
  • NK natural killer
  • CAR chimeric antigen receptor
  • the composition is administered in a dosing cycle. In some embodiments, the composition is administered in a dosing cycle comprising a first dose of the CAR-expressing NK cells, a second dose of the CAR-expressing NK cells, and a third dose of the CAR-expressing NK cells.
  • NK cells natural killer cells in the manufacture of a medicament for treating a subject having a cancer
  • the NK cells are genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19; and the NK cells are for administration as a first dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells.
  • CAR chimeric antigen receptor
  • the second dose is for administration to the subject between 2-4 days after administration of the first dose to the subject; the third dose is for administration to the subject between 2-4 days after administration of the second dose to the subject; and each of the first, second and third doses comprises at least about 1 ⁇ 10 9 CAR-expressing NK cells.
  • each of the first, second and third doses comprises about 1 ⁇ 10 9 CAR-expressing NK cells.
  • each of the first, second and third doses comprises about 1.5 ⁇ 10 9 CAR-expressing NK cells.
  • each of the first, second and third doses comprises about 2 ⁇ 10 9 CAR- expressing NK cells.
  • each of the first, second and third doses comprises about 2.5 ⁇ 10 9 CAR-expressing NK cells.
  • the NK cells are for administration as a first dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: the second dose is for administration to the subject between 2- 4 days after administration of the first dose to the subject, the third dose is for administration to the subject between 2-4 days after administration of the second dose to the subject, and each of the first, second and third doses comprises between about 1.0 ⁇ 10 9 CAR-expressing NK cells and about 2 ⁇ 10 9 CAR-expressing NK cells; the subject is administered a lymphodepleting therapy prior to administration of the first dosing cycle; and the lymphodepleting therapy consists of a dose of about
  • the second dose is for administration between about 24 hours and about 72 hours after administration of the first dose
  • the third dose is for administration between about 24 hours and about 72 hours after administration of the second dose.
  • the second dose is for administration at least about 24 hours after administration of the first dose
  • the third dose is for administration at least about 24 hours after administration of the second dose.
  • the second is for administration about 24 hours after administration of the first dose
  • the third dose is for administration about 24 hours after administration of the second dose.
  • the second dose is for administration at least about 48 hours after administration of the first dose
  • the third dose is for administration at least about 48 hours after administration of the second dose.
  • the second is for administration about 48 hours after administration of the first dose
  • the third dose is for administration about 48 hours after administration of the second dose.
  • the second dose is for administration at least about 72 hours after administration of the first dose
  • the third dose is for administration at least about 72 hours after administration of the second dose.
  • the second is for administration about 72 hours after administration of the first dose
  • the third dose is for administration about 72 hours after administration of the second dose.
  • the first dose is for administration on about Day 0 of the dosing cycle.
  • the second dose is for administration on about Day 2 of the dosing cycle.
  • the second dose is for administration on about Day 3 of the dosing cycle.
  • the second dose is for administration on about Day 4 of the dosing cycle.
  • the third dose is for administration on about Day 4 of the dosing cycle. In some embodiments, the third dose is for administration on about Day 5 of the dosing cycle. In some embodiments, the third dose is for administration on about Day 6 of the dosing cycle. In some embodiments, the third dose is for administration on about Day 7 of the dosing cycle. In some embodiments, the third dose is for administration on about Day 8 of the dosing cycle.
  • the first dose is for administration on about Day 0 of the dosing cycle
  • the second dose is for administration on about Day 2 of the dosing cycle
  • the third dose is for administration on about Day 4 of the dosing cycle.
  • the first dose is for administration on about Day 0 of the dosing cycle
  • the second dose is for administration on about Day 2 of the dosing cycle
  • the third dose is for administration on about Day 5 of the dosing cycle.
  • the first dose is for administration on about Day 0 of the dosing cycle
  • the second dose is for administration on about Day 2 of the dosing cycle
  • the third dose is for administration on about Day 6 of the dosing cycle.
  • the first dose is for administration on about Day 0 of the dosing cycle
  • the second dose is for administration on about Day 3 of the dosing cycle
  • the third dose is for administration on about Day 5 of the dosing cycle.
  • the first dose is for administration on about Day 0 of the dosing cycle
  • the second dose is for administration on about Day 3 of the dosing cycle
  • the third dose is for administration on about Day 6 of the dosing cycle.
  • the first dose is for administration on about Day 0 of the dosing cycle
  • the second dose is for administration on about Day 3 of the dosing cycle
  • the third dose is for administration on about Day 7 of the dosing cycle.
  • the first dose is for administration on about Day 0 of the dosing cycle
  • the second dose is for administration on about Day 4 of the dosing cycle
  • the third dose is for administration on about Day 6 of the dosing cycle.
  • the first dose is for administration on about Day 0 of the dosing cycle
  • the second dose is for administration on about Day 4 of the dosing cycle
  • the third dose is for administration on about Day 7 of the dosing cycle.
  • the first dose is for administration on about Day 0 of the dosing cycle
  • the second dose is for administration on about Day 4 of the dosing cycle
  • the third dose is for administration on about Day 8 of the dosing cycle.
  • NK cells natural killer cells in the manufacture of a medicament for treating a subject having a cancer
  • the NK cells are genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19
  • the NK cells are for administration as a first dosing cycle comprising a first dose of the generally engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: the second dose is for administration to the subject about 3 days after administration of the first dose to the subject, the third dose is for administration to the subject about 4 days after administration of the second dose to the subject; each of the first, second and third doses comprises about 2 ⁇ 10 9 CAR-expressing NK cells or about 2.5 ⁇ 10 9 CAR-expressing NK cells; the subject was administered a lymphodepleting therapy comprising (i) a dose of about 500 mg/m2 of cyclophosphamide on each of 5 days, 4 days
  • CAR chimeric antigen receptor
  • NK cells natural killer cells in the manufacture of a medicament for treating a subject having a cancer
  • the NK cells are genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19
  • the NK cells are for administration as a first dosing cycle comprising a first dose of the generally engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: the second dose is for administration to the subject about 3 days after administration of the first dose to the subject, the third dose is for administration to the subject about 4 days after administration of the second dose to the subject; each of the first, second and third doses comprises at least about 1 ⁇ 10 9 CAR-expressing NK cells; the subject was administered a lymphodepleting therapy comprising (i) a dose of about 500 mg/m2 of cyclophosphamide on each of 5 days, 4 days, and 3 days prior to administration of the first dose of
  • CAR chimeric antigen receptor
  • each of the first, second and third doses comprises about 1 ⁇ 109 CAR-expressing NK cells. In some embodiments, each of the first, second and third doses comprises about 1.5 ⁇ 109 CAR-expressing NK cells. In some embodiments, each of the first, second and third doses comprises about 2 ⁇ 109 CAR- expressing NK cells. In some embodiments, each of the first, second and third doses comprises about 2.5 ⁇ 109 CAR-expressing NK cells. [0051] In some embodiments, the CAR-expressing NK cells also express a membrane-bound interleukin-15 (mbIL15).
  • mbIL15 membrane-bound interleukin-15
  • Figure 1 depicts non-limiting schematics of CD19-directed chimeric antigen receptors (CARs).
  • Figures 2A-2B depict non-limiting schematics of a dosing cycle for treating a CD19-related disease (e.g., cancer) with CD19 CAR-expressing NK cells.
  • a CD19-related disease e.g., cancer
  • Figure 3 shows the concentration of CD19 CAR-expressing NK cells in two subjects with CD19+ B cell malignancies who were administered a lymphodepleting therapy of cyclophosphamide and fludarabine (cy/flu) prior to a first dosing cycle and a lymphodepleting therapy of cyclophosphamide only (cy) prior to a second dosing cycle.
  • DETAILED DESCRIPTION Some embodiments of the methods and compositions provided herein relate to engineered immune cells (e.g., natural killer cells) and use of the same for immunotherapy (e.g., cancer immunotherapy).
  • the immune cells are engineered to express a chimeric antigen receptor (CAR) that targets CD19.
  • CAR chimeric antigen receptor
  • the methods are for treatment of a cancer, such as a hematologic malignancy.
  • a cancer such as a hematologic malignancy.
  • genetically engineered immune cells for use in accord with the provided methods includes administering engineered NK cells expressing a recombinant receptor (e.g. CAR) designed to recognize and/or specifically bind to an antigen associated with a disease such as cancer.
  • a recombinant receptor e.g. CAR
  • the antigen that is bound or recognized by the CAR is CD19.
  • binding to the antigen results in a response, such as an immune response against such antigen.
  • binding to the antigen results in the reduction or depletion of cells expressing the antigen (e.g., B cells expressing CD19, or a subset thereof).
  • the genetically engineered cells contain or are engineered to contain the CAR.
  • the CAR generally includes an extracellular antigen-binding domain specific to the antigen (e.g., CD19), which is linked to one or more intracellular signaling components, in some aspects via linkers and/or transmembrane domain(s).
  • the genetically engineered cells are provided as pharmaceutical compositions and formulations suitable for administration to a subjects, such as for cell therapy. Also provided are therapeutic methods for administering the cells and compositions to subjects, uses of the compositions for treatment of subjects, and uses of the compositions in the manufacture of medicaments for treating subjects. A.
  • CARs that specifically bind to CD19, such as receptors comprising an anti-CD19 antibody, e.g., antibody fragment.
  • immune cells e.g., NK cells
  • the CARs generally include an extracellular antigen-binding domain that includes an anti-CD19 antibody.
  • Such CARs include antibodies (including antigen-binding fragments thereof) that specifically bind to CD19 proteins, such as human CD19 protein (e.g., SEQ ID NO:39).
  • the antibodies include those that are multi-domain antibodies, such as those containing VH and VL domains.
  • the antibodies include a variable heavy chain and a variable light chain, such as scFvs.
  • the provided anti-CD19 antibodies are human and humanized antibodies.
  • 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 (rIgG) 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 antibody (e.g., sdAb, sdFv, nanobody) fragments.
  • Fab fragment antigen binding
  • rIgG Fab′ fragments
  • 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.
  • CDR complementarity determining region
  • HVR hypervariable region
  • 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).
  • FR-H1, FR-H2, FR-H3, and FR-H4 four FRs in each full-length heavy chain variable region
  • FR-L1, FR-L2, FR-L3, and FR-L4 four FRs in each full-length light chain variable region.
  • the boundaries of a given CDR or FR may vary depending on the scheme used for identification.
  • the Kabat scheme is based 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.
  • Table 1 lists non-limiting position boundaries of CDR-L1, CDR- L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, 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 between CDR-L1 and CDR-L2, and so forth.
  • a “CDR” or “complementary determining region,” or individual specified CDRs (e.g., “CDR-H1, CDR-H2), 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.
  • 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 amino acid sequence
  • 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.
  • FR or individual specified FR(s) e.g., FR-H1, FR-H2
  • FR-H1, FR-H2 FR-H2
  • the scheme for identification of a particular CDR, FR, or FRs or CDRs is specified, such as the CDR as defined by the IMGT, Kabat, Chothia, or Contact method.
  • 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. A single VH or VL domain may be sufficient to confer antigen- binding specificity.
  • FRs conserved framework regions
  • a single VH or VL domain may be sufficient to confer antigen- binding specificity.
  • 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, single-chain 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 a variable light chain region, such as scFvs.
  • 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.
  • 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 or humanized single-domain antibody.
  • 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
  • a “human antibody” is an antibody with an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or non-human source that utilizes human antibody repertoires or other human antibody-encoding sequences, including human antibody libraries.
  • the term excludes humanized forms of non-human antibodies comprising non- human antigen-binding regions, such as those in which all or substantially all CDRs are non- human.
  • the term includes antigen-binding fragments of human antibodies.
  • monoclonal antibodies including monoclonal antibody fragments.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from or within a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical, except for possible variants containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations which typically include different antibodies directed against different epitopes
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen.
  • 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 antibodies and antibody chains and other peptides, e.g., linkers and CD19-binding 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, phosphorylation, and the like.
  • the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity.
  • the antigen-binding domain may be or comprise any antibody (e.g., anti-CD19 antibody) as described herein.
  • the extracellular antigen-binding domain comprises a heavy chain variable region (VH) having a CDR-1, a CDR-2, and a CDR-3 comprising the amino acid sequences set forth in SEQ ID NOS: 24, 25, and 26, respectively.
  • VH heavy chain variable region
  • the extracellular antigen-binding domain comprises a light chain variable region (VL) having a CDR-1, a CDR-2, and a CDR-3 comprising the amino acid sequences set forth in SEQ ID NOS: 27, 28, and 29, respectively.
  • VL light chain variable region
  • the extracellular antigen-binding domain comprises a VH having a CDR-1, a CDR-2, and a CDR- 3 comprising the amino acid sequences set forth in SEQ ID NOS: 24, 25, and 26, respectively; and a VL having a CDR-1, a CDR-2, and a CDR-3 comprising the amino acid sequences set forth in SEQ ID NOS: 27, 28, and 29, respectively.
  • the extracellular antigen-binding domain comprises a heavy chain variable region (VH) having a CDR-1, a CDR-2, and a CDR-3 comprising the amino acid sequences set forth in SEQ ID NOS: 30, 31, 32, respectively.
  • the extracellular antigen-binding domain comprises a light chain variable region (VL) having a CDR-1, a CDR-2, and a CDR-3 comprising the amino acid sequences set forth in SEQ ID NOS: 33, 34, and 29, respectively.
  • the extracellular antigen-binding domain comprises a VH having a CDR-1, a CDR-2, and a CDR-3 comprising the amino acid sequences set forth in SEQ ID NOS: 30, 31, and 32, respectively; and a VL having a CDR-1, a CDR-2, and a CDR-3 comprising the amino acid sequences set forth in SEQ ID NOS: 33, 34, and 29, respectively.
  • the VH comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:35.
  • the VH comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:35. In some embodiments, the VH comprises an amino acid sequence having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:35. In some embodiments, the VH comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:35. In some embodiments, the VH comprises an amino acid sequence having at least about 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:35. In some embodiments, the VH comprises the amino acid sequence set forth in SEQ ID NO:35.
  • the VL comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:36. In some embodiments, the VL comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:36. In some embodiments, the VL comprises an amino acid sequence having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:36. In some embodiments, the VL comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:36.
  • the VL comprises an amino acid sequence having at least about 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:36. In some embodiments, the VL comprises the amino acid sequence set forth in SEQ ID NO:36. In some embodiments, the VH comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:35; and the VL comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:36.
  • the VH comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:35; and the VL comprises an amino acid sequence having at least about 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:36. In some embodiments, the VH comprises an amino acid sequence having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:35; and the VL comprises an amino acid sequence having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:36.
  • the VH comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:35; and the VL comprises an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:36.
  • the VH comprises an amino acid sequence having at least about 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:35; and the VL comprises an amino acid sequence having at least about 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:36.
  • the VH comprises the amino acid sequence set forth in SEQ ID NO:35, and the VL comprises the amino acid sequence set forth in SEQ ID NO:36.
  • the antigen-binding domain is an scFv comprising a VH and a VL joined by a linker (e.g., a linker comprising any of SEQ ID NOS:1- 3).
  • the linker comprises the amino acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:3.
  • the extracellular antigen-binding domain is an scFv comprising the linker set forth in SEQ ID NO:1.
  • the antigen- binding domain is an scFv comprising an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:37. In some embodiments, the antigen- binding domain is an scFv comprising an amino acid sequence having at least about 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:37. In some embodiments, the antigen-binding domain is an scFv comprising an amino acid sequence having at least about 85% sequence identity to the amino acid sequence set forth in SEQ ID NO:37.
  • the antigen-binding domain is an scFv comprising an amino acid sequence having at least about 90% sequence identity to the amino acid sequence set forth in SEQ ID NO:37. In some embodiments, the antigen-binding domain is an scFv comprising an amino acid sequence having at least about 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:37. IIn some embodiments, the antigen-binding domain is an scFv comprising the amino acid sequence set forth in SEQ ID NO:37. In some embodiments, the extracellular antigen-binding domain is an scFv comprising the linker set forth in SEQ ID NO:2.
  • the extracellular antigen-binding domain is an scFv comprising the linker set forth in SEQ ID NO:3.
  • Additional CD19-binding domains are known and described in the art, including any of those as described in PCT Application Nos. PCT/US2015/024671, PCT/US2018/029107, PCT/US2020/020824, PCT/US2020/033559, PCT/IB2021/060213, and PCT/CN2021/106892, each of which is incorporated herein in its entirety by reference.
  • recombinant receptors e.g., CARs
  • the extracellular antigen-binding domain generally is linked to an intracellular signaling domain comprising 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.
  • the extracellular antigen-binding domain of a CAR is linked to an intracellular signaling domain by a transmembrane domain.
  • the CD19-binding molecule e.g., antibody
  • a CAR comprises an extracellular antigen-binding domain that binds to CD19, a transmembrane domain, and an intracellular signaling domain comprising a co-stimulatory signal region and a primary signaling domain (e.g., CD3zeta).
  • the transmembrane domain is fused to the extracellular domain.
  • 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 (e.g., comprising at least the transmembrane region(s) of) CD3, CD4, CD5, CD8, CD9, CD 16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.
  • the transmembrane domain in some embodiments is synthetic.
  • the transmembrane domain comprises at least a portion of CD8, a transmembrane glycoprotein normally expressed on both T cells and NK cells.
  • the transmembrane domain comprises CD8alpha (CD8a).
  • the transmembrane domain comprises a CD8 (e.g., CD8a) hinge and a CD8 (e.g., CD8a) transmembrane region.
  • the transmembrane domain comprises a hinge, e.g. a CD8a hinge.
  • the sequence encoding the CD8a hinge is truncated or modified.
  • the CD8a hinge is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:5.
  • the CD8a hinge comprises the nucleic acid sequence of SEQ ID NO:5. In several embodiments, the CD8a hinge is truncated or modified. In some embodiments, the CD8a hinge has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence of SEQ ID NO:6. In several embodiments, the hinge of CD8a comprises the amino acid sequence of SEQ ID NO:6. [0085] In several embodiments, the transmembrane domain comprises a CD8a transmembrane region. In several embodiments, the CD8a transmembrane region is truncated or modified.
  • the CD8a transmembrane region is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the sequence of SEQ ID NO:7.
  • the CD8a transmembrane region is encoded by a nucleic acid sequence of SEQ ID NO:7.
  • the CD8a transmembrane region is truncated or modified.
  • the CD8a transmembrane region has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the sequence of SEQ ID NO:8.
  • the CD8a transmembrane region comprises the amino acid sequence of SEQ ID NO:8.
  • the CD8 transmembrane domain is truncated or modified and is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the sequence of SEQ ID NO:9.
  • the CD8 transmembrane domain is encoded by the nucleic acid sequence of SEQ ID NO:9.
  • the CD28 transmembrane domain has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence of SEQ ID NO:11. In several embodiments, the CD28 transmembrane domain comprises the amino acid sequence of SEQ ID NO:11. [0088]
  • the receptor e.g., the CAR, generally includes an intracellular signaling domain comprising intracellular signaling components. 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.
  • the antigen-binding portion is linked to one or more cell signaling modules.
  • the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g., NK cell engineered to express the CAR.
  • the CAR induces a function of an immune cell (e.g., NK cell) such as cytolytic activity and/or secretion of cytokines or other factors.
  • a truncated portion of an intracellular signaling domain 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 domain includes 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 receptors to initiate signal transduction following antigen receptor engagement.
  • TCR T cell receptor
  • 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 receptor.
  • 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 receptor includes one or both of such signaling components.
  • the receptor 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 ITAMs.
  • ITAM containing primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d.
  • cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3zeta.
  • immune cells engineered according to several embodiments disclosed herein may comprise at least one subunit of the CD3 T cell receptor complex (or a fragment thereof).
  • the signaling domain comprises the CD3 zeta subunit.
  • the CD3zeta can be truncated or modified.
  • the CD3zeta is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:17.
  • the CD3zeta is encoded by the nucleic acid sequence of SEQ ID NO:17.
  • the intracellular signaling domain comprises an intracellular signaling region of 4-1BB. In some embodiments, the intracellular signaling domain comprises an intracellular signaling region of OX40. In some embodiments, the intracellular signaling domain comprises an intracellular signaling region of DAP10. In some embodiments, the intracellular signaling domain comprises an intracellular signaling region of ICOS. In some embodiments, the intracellular signaling domain does not include DAP10 and/or DAP12. In some embodiments, the intracellular signaling domain does not include DAP10. In some embodiments, the intracellular signaling domain does not include DAP12. In some aspects, the same receptor includes both a CD3zeta and a costimulatory signaling region.
  • the intracellular signaling domain of the recombinant receptor comprises a CD3zeta intracellular domain and a costimulatory signaling region.
  • the intracellular signaling domain comprises an intracellular signaling region of OX40.
  • the OX40 intracellular signaling region is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:13.
  • the OX40 intracellular signaling region is encoded by the nucleic acid sequence of SEQ ID NO:13.
  • the OX40 intracellular signaling region comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence of SEQ ID NO:14. In several embodiments, the OX40 intracellular signaling region comprises the amino acid sequence of SEQ ID NO:14. In several embodiments, OX40 is used as the sole intracellular signaling component in the construct, however, in several embodiments, OX40 can be used with one or more other components. For example, combinations of OX40 and CD3zeta are used in some embodiments. In some embodiments, the intracellular signaling domain comprises an OX40 costimulatory signaling region linked to CD3zeta.
  • the CAR comprises an extracellular antigen- binding domain comprising the sequence set forth in SEQ ID NO:37, a CD8alpha transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:8, an OX40 intracellular signaling region comprising the amino acid sequence set forth in SEQ ID NO:14, and a CD3zeta domain comprising the amino acid sequence set forth in SEQ ID NO:18.
  • the CAR comprises an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the amino acid sequence set forth in SEQ ID NO:38.
  • the CAR comprises the amino acid sequence set forth in SEQ ID NO:38.
  • the intracellular signaling domain comprises an intracellular signaling region of 4-1BB.
  • the 4-1BB intracellular signaling region is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO:15.
  • the 4-1BB intracellular signaling region is encoded by the nucleic acid sequence of SEQ ID NO:15.
  • the 4- 1BB intracellular signaling region comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence of SEQ ID NO:16.
  • the 4-1BB intracellular signaling region comprises the amino acid sequence of SEQ ID NO:16.
  • 4-1BB is used as the sole intracellular signaling component in the construct, however, in several embodiments, 4-1BB can be used with one or more other components. For example, combinations of 4-1BB and CD3zeta are used in some embodiments.
  • the intracellular signaling domain comprises a 4-1BB costimulatory signaling region linked to CD3zeta.
  • the intracellular signaling domain comprises an intracellular signaling region of CD28.
  • the CD28 intracellular signaling region comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence of SEQ ID NO:12.
  • the CD28 intracellular signaling region comprises the amino acid sequence of SEQ ID NO:12.
  • CD28 is used as the sole intracellular signaling component in the construct, however, in several embodiments, CD28 can be used with one or more other components.
  • the intracellular signaling domain comprises a CD28 costimulatory signaling region linked to CD3zeta.
  • CD28, OX40, 4-1BB and/or CD3zeta are used in some embodiments.
  • Additional CD19-directed CARs are known and described in the art, including any of those as described in Kalos et al., Sci Transl Med 3:95ra73 (2011); Porter et al., NEJM 365:725-733 (2011); Grupp et al., NEJM 368: 1509-1518 (2013); and PCT Application Nos.
  • the nucleic acid encoding the chimeric receptor, or a portion thereof is codon-optimized.
  • the polynucleotides are optimized, or contain certain features designed for optimization, such as for codon usage, to reduce RNA heterogeneity and/or to modify, e.g., increase or render more consistent among cell product lots, expression, such as surface expression, of the encoded receptor.
  • polynucleotides, encoding chimeric receptors are modified as compared to a reference polynucleotide, such as to remove cryptic or hidden splice sites, to reduce RNA heterogeneity.
  • polynucleotides, encoding chimeric receptors are codon optimized, such as for expression in a mammalian, e.g., human, cell such as in a human T cell.
  • the modified polynucleotides result in improved, e.g., increased or more uniform or more consistent level of, expression, e.g., surface expression, when expressed in a cell.
  • the genetic engineering involves introduction of a nucleic acid encoding the genetically engineered component or other component for introduction into the cell, such as a component encoding a gene-disrupting protein or nucleic acid.
  • 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. i.
  • binding molecules e.g., anti-CD19 binding molecules
  • recombinant receptors e.g., CARs
  • NK cells the genetically engineered immune cells
  • one or more binding molecules, including recombinant receptors can be genetically engineered into cells or a plurality of cells.
  • the genetic engineering generally involves introduction of a nucleic acid encoding the recombinant or engineered component into the cell, such as by retroviral transduction, transfection, or transformation.
  • polynucleotides encoding the antibodies and chimeric antigen receptors and/or portions, e.g., chains, thereof.
  • the provided polynucleotides are those encoding the anti-CD19 chimeric antigen receptors (e.g., antigen-binding fragment) described herein.
  • polynucleotides encoding one or more antibodies and/or portions thereof e.g., those encoding one or more of the anti-CD19 antibodies (e.g., antigen- binding fragment) described herein and/or other antibodies and/or portions thereof, e.g., antibodies and/or portions thereof that binds other target antigens.
  • the polynucleotides may include those encompassing natural and/or non-naturally occurring nucleotides and bases, e.g., including those with backbone modifications.
  • the terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide” may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA.
  • Nucleic acid sequence refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide. Also provided are polynucleotides that have been optimized for codon usage.
  • vectors containing the polynucleotides such as any of the polynucleotides described herein, and cells containing the vectors, e.g., for producing the antibodies or antigen-binding fragments thereof.
  • the vector is a viral vector.
  • the vector is a retroviral vector.
  • the vector is a lentiviral vector.
  • the nucleic acid may encode an amino acid sequence comprising the VL region and/or an amino acid sequence comprising the VH region of the antibody (e.g., the light and/or heavy chains of the antibody).
  • the nucleic acid may encode one or more amino acid sequence comprising the VL region and/or an amino acid sequence comprising the VH region of the antibody (e.g., the light and/or heavy chains of the antibody).
  • one or more vectors e.g., expression vectors
  • a host cell comprising such polynucleotides is provided.
  • a host cell comprises (e.g., has been transformed with) (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL region of the antibody and an amino acid sequence comprising the VH region of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL region of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH region of the antibody.
  • a host cell comprises (e.g., has been transformed with) one or more vectors comprising one or more nucleic acid that encodes one or more an amino acid sequence comprising one or more antibodies and/or portions thereof, e.g., antigen-binding fragments thereof.
  • one or more such host cells are provided.
  • a composition containing one or more such host cells are provided.
  • the one or more host cells can express different antibodies, or the same antibody.
  • each of the host cells can express more than one antibody.
  • a nucleic acid sequence encoding a chimeric receptor antibody may be isolated and inserted into one or more vectors for further cloning and/or expression in a host cell.
  • Such nucleic acid sequences may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).
  • a method of making the anti- CD19 chimeric antigen receptor is provided, wherein the method comprises culturing a host cell comprising a nucleic acid sequence encoding the antibody, as provided above, under conditions suitable for expression of the receptor.
  • immune cells such as human immune cells are used to express the provided polypeptides encoding chimeric antigen receptors.
  • the immune cells are NK cells including primary NK cells.
  • gene transfer is accomplished by transduction of the immune cells (e.g., activated immune cells), and expansion in culture to numbers sufficient for clinical applications.
  • the cells further are engineered to promote expression of cytokines or other factors.
  • Various methods for the introduction of genetically engineered components, e.g., antigen receptors, e.g., CARs are well known and may be used with the provided methods and compositions.
  • Non-limiting examples of methods include those for transfer of polynucleotides encoding the receptors, including via viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.
  • recombinant polynucleotides are transferred into immune cells (e.g., NK cells) using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV).
  • infectious virus particles such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV).
  • recombinant polynucleotides are transferred into immune cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors.
  • 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), spleen focus forming virus (SFFV), or human immunodeficiency virus type 1 (HIV-1).
  • LTR long terminal repeat sequence
  • 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.
  • 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.
  • the polynucleotide containing nucleic acid sequences encoding the CD19-binding receptor e.g., chimeric antigen receptor (CAR)
  • the signal sequence may encode a signal peptide derived from a native polypeptide.
  • the signal sequence may encode a heterologous or non-native signal peptide.
  • a non-limiting example of a signal peptide comprises a CD8 alpha (CD8a) signal peptide set forth in SEQ ID NO:4.
  • the vector or construct can contain promoter and/or enhancer or regulatory elements to regulate expression of the encoded recombinant receptor.
  • the promoter and/or enhancer or regulatory elements can be condition-dependent promoters, enhancers, and/or regulatory elements. In some examples these elements drive expression of the transgene.
  • the vector or construct can contain a single promoter that drives the expression of one or more nucleic acid molecules.
  • nucleic acid molecules e.g., transcripts
  • transcription units can be engineered as a bicistronic unit containing an IRES (internal ribosome entry site), which allows coexpression of gene products (e.g. encoding a chimeric receptor and membrane-bound interleukin-15) by a message from a single promoter.
  • IRES internal ribosome entry site
  • a single promoter may direct expression of an RNA that contains, in a single open reading frame (ORF), two or three genes (e.g.
  • a chimeric receptor and membrane-bound interleukin-15 separated from one another by sequences encoding a self-cleavage peptide (e.g., 2A cleavage sequences) or a protease recognition site.
  • the ORF thus encodes a single polypeptide, which, either during (in the case of T2A) or after translation, is cleaved into the individual proteins.
  • 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.
  • Many 2A elements are known.
  • 2A peptides that can be used in the methods and polynucleotides disclosed herein, without limitation, 2A peptides from the foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A, e.g. SEQ ID NO:20, encoded by SEQ ID NO:19), and porcine teschovirus-1 (P2A).
  • the one or more different or separate promoters drive the expression of a nucleic acid molecule encoding a binding molecule, e.g., recombinant receptor and a nucleic acid encoding membrane-bound interleukin-15.
  • any of the immune cells as provided herein are engineered to express interleukin 15 (IL15).
  • any of the immune cells as provided herein are engineered to express a membrane-bound interleukin 15 (mbIL15).
  • mbIL15 expression on the immune cell e.g., NK cell
  • the IL15 is expressed from a separate cassette on the construct comprising any one of the CARs disclosed herein.
  • the IL15 is expressed from the same cassette as any one of the CARs disclosed herein.
  • the chimeric receptor and IL15 are separated by a nucleic acid sequence encoding a cleavage site, for example, a proteolytic cleavage site or a T2A, P2A, E2A, or F2A self-cleaving peptide cleavage site.
  • the chimeric receptor and IL15 are separated by a T2A peptide (e.g., SEQ ID NO:20, encoded by SEQ ID NO:19).
  • the IL15 is a membrane-bound IL15 (mbIL15).
  • the mbIL15 comprises a native IL15 sequence, such as a human native IL15 sequence (e.g., SEQ ID NO:22, encoded by SEQ ID NO:21), and at least one transmembrane domain (e.g., CD8a).
  • IL15 is encoded by the nucleic acid sequence of SEQ ID NO: 21.
  • IL15 can be truncated or modified, such that it is encoded by a nucleic acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 21.
  • the IL15 comprises the amino acid sequence of SEQ ID NO: 22.
  • the IL15 is truncated or modified, such that it has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 22.
  • any of the CARs as described herein are encoded by the same nucleic acid sequence as a mbIL15.
  • a nucleic acid sequence encoding the CAR and a nucleic acid sequence encoding the mbIL15 are separated by a 2A (e.g., T2A, P2A, E2A, or F2A)-encoding sequence.
  • a nucleic acid sequence encoding the CAR and a nucleic acid sequence encoding the mbIL15 are separated by a T2A-encoding sequence (e.g., SEQ ID NO:19).
  • any of the engineered cells as described herein express a CD19-targeting recombinant receptor (e.g., CAR) and a mbIL15.
  • the mbIL15 is membrane-bound by virtue of the fusion of IL15 to a transmembrane domain.
  • mbIL15 comprises a transmembrane domain.
  • the transmembrane domain comprises a CD8a transmembrane domain.
  • the transmembrane domain comprises a hinge and/or a transmembrane region.
  • the transmembrane domain comprises a hinge and a transmembrane region.
  • the hinge is a CD8a hinge sequence (e.g., SEQ ID NO:6).
  • the transmembrane region is a CD8a transmembrane region (e.g., SEQ ID NO:8).
  • the mbIL15 comprises a native IL15 sequence, such as a human native IL15 sequence, and at least one transmembrane domain (e.g., CD8a transmembrane domain).
  • the CD8a transmembrane domain comprises the sequence of SEQ ID NO:10.
  • the mbIL15 is truncated or modified such that it comprises an amino acid sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% sequency identity to the amino acid sequence of SEQ ID NO:23.
  • the mbIL15 comprises the amino acid sequence of SEQ ID NO:23.
  • Membrane- bound IL15 sequences are described in PCT publications WO 2018/183385 and WO 2020/056045, each of which is hereby expressly incorporated by reference in its entirety.
  • ii. Cell Types [00116] Some embodiments of the methods and compositions provided herein relate to a cell such as an immune cell.
  • an immune cell such as an NK cell or a T cell, may be engineered to express a CD19-targeting CAR.
  • targeted therapy is a cancer treatment that employs certain drugs that target specific genes or proteins found in cancer cells or cells supporting cancer growth, (like blood vessel cells) to reduce or arrest cancer cell growth.
  • genetic engineering has enabled approaches to be developed that harness certain aspects of the immune system to fight cancers.
  • Various types of immune cells can be used, such as T cells, Natural Killer (NK cells), or combinations thereof, as described in more detail below.
  • polypeptides comprising a CAR, as well as polynucleotides and vectors encoding the same.
  • the CAR comprises a target binding moiety (e.g., an extracellular antigen- binding domain) operably coupled to a cytotoxic signaling complex.
  • a target binding moiety e.g., an extracellular antigen- binding domain
  • some embodiments include a CAR comprising an extracellular antigen-binding domain that is directed against a tumor marker, for example, CD19, to facilitate targeting of an immune cell to a CD19-expressing cancer.
  • engineered immune cells e.g., NK cells
  • compositions comprising engineered immune cells (e.g., NK cells) expressing such CARs.
  • engineered immune cells e.g., NK cells
  • Methods of treating cancer and other uses of such cells for cancer immunotherapy are also provided for herein.
  • cells of the immune system are engineered to have enhanced cytotoxic effects against target cells, such as tumor cells.
  • a cell of the immune system may be engineered to include a tumor-directed chimeric antigen receptor (CAR) as described herein.
  • CAR tumor-directed chimeric antigen receptor
  • white blood cells or leukocytes are used, since their native function is to defend the body against growth of abnormal cells and infectious disease.
  • White blood cells include granulocytes and agranulocytes (presence or absence of granules in the cytoplasm, respectively).
  • Granulocytes include basophils, eosinophils, neutrophils, and mast cells.
  • Agranulocytes include lymphocytes and monocytes.
  • Cells such as those listed above or those that follow or are otherwise described herein may be engineered to express a CAR, for example by providing to the cell a nucleic acid encoding the CAR.
  • the immune cells are also engineered to express interleukin 15, such as a membrane-bound interleukin 15 (mbIL15).
  • interleukin 15 mbIL15
  • the cells are engineered to express a CAR and IL15 (e.g., mbIL15).
  • CAR and IL15 e.g., mbIL15.
  • the immune cells comprise monocytes. Monocytes are a subtype of leukocyte. Monocytes can differentiate into macrophages and myeloid lineage dendritic cells. Monocytes are associated with the adaptive immune system and serve the main functions of phagocytosis, antigen presentation, and cytokine production.
  • a monocyte is positive for cell surface expression of a marker selected from among the group consisting of CCR2, CCR5, CD11c, CD14, CD16, CD62L, CD68+, CX3CR1, HLA-DR, or any combination thereof.
  • a monocyte is positive for cell surface expression of CD14.
  • a monocyte is positive for cell surface expression of CCR2.
  • a monocyte is positive for cell surface expression of CCR5.
  • a monocyte is positive for cell surface expression of CD62L.
  • monocytes are used in connection with one or more additional engineered cells as disclosed herein. Some embodiments of the methods and compositions described herein relate to a monocyte that expresses a CAR that binds to CD19, or a nucleic acid encoding the CAR. [00123] In some embodiments, the monocytes are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, the monocytes engineered to express a CAR are engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbIL15) domain.
  • mbIL15 membrane-bound interleukin 15
  • the monocytes are engineered to bicistronically express the CAR and mbIL15.
  • the monocytes are allogeneic cells.
  • the monocytes are obtained from a donor who does not have a cancer.
  • the monocytes are autologous cells.
  • the immune cells comprise lymphocytes. Lymphocytes, the other primary sub-type of leukocyte include T cells (cell-mediated, cytotoxic adaptive immunity), natural killer cells (cell-mediated, cytotoxic innate immunity), and B cells (humoral, antibody-driven adaptive immunity).
  • the immune cells comprise T cells.
  • the immune cells comprise NK cells.
  • the immune cells comprise T cells and NK cells.
  • the immune cells comprise B cells.
  • lymphocytes are used in connection with one or more additional engineered cells as disclosed herein. Some embodiments of the methods and compositions described herein relate to a lymphocyte that expresses a CAR that binds to CD19, or a nucleic acid encoding the CAR.
  • the lymphocytes are engineered to express a membrane-bound interleukin 15 (mbIL15) domain.
  • the lymphocytes engineered to express a CAR are engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbIL15) domain.
  • lymphocytes are engineered to bicistronically express the CAR and mbIL15.
  • the lymphocytes are allogeneic cells.
  • the lymphocytes are obtained from a donor who does not have a cancer.
  • the lymphocytes are autologous cells. c.
  • the immune cells comprise T cells.
  • T cells are distinguishable from other lymphocytes sub-types (e.g., B cells or NK cells) based on the presence of a T-cell receptor on the cell surface.
  • T cells can be divided into various different subtypes, including effector T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cell, mucosal associated invariant T cells and gamma delta T cells.
  • a specific subtype of T cell is engineered.
  • a T cell is positive for cell surface expression of a marker selected from among the group consisting of CD3, CD4, and/or CD8.
  • a T cell is positive for cell surface expression of CD3. In some embodiments, a T cell is positive or cell surface expression of CD4. In some embodiments, a T cell is positive or cell surface expression of CD8.
  • CD3+ T cells are engineered.
  • CD4+ T cells are engineered.
  • CD8+ T cells are engineered.
  • regulatory T cells are engineered.
  • gamma delta T cells are engineered.
  • a mixed pool of T cell subtypes is engineered. For example, in some embodiments, CD4+ and CD8+ T cells are engineered.
  • cytotoxic receptor complexes there is no specific selection of a type of T cells to be engineered to express the cytotoxic receptor complexes disclosed herein.
  • specific techniques such as use of cytokine stimulation are used to enhance expansion/collection of T cells with a specific marker profile.
  • activation of certain human T cells e.g. CD4+ T cells, CD8+ T cells is achieved through use of CD3 and/or CD28 as stimulatory molecules.
  • a method of treating or preventing a cancer comprising administering T cells expressing a cytotoxic receptor complex as described herein.
  • the engineered T cells are autologous cells, while in some embodiments, the T cells are allogeneic cells. In some embodiments, the T cells are allogeneic cells. In some embodiments, the T cells are obtained from a donor who does not have a cancer. [00135] Several embodiments of the methods and compositions disclosed herein relate to T cells engineered to express a CAR that binds to CD19. In some embodiments, the T cells are engineered to express a membrane-bound interleukin 15 (mbIL15) domain. In some embodiments, the T cells engineered to express a CAR are engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbIL15) domain.
  • mbIL15 membrane-bound interleukin 15
  • the T cells are engineered to bicistronically express the CAR and mbIL15.
  • the immune cells comprise T cells and NK cells (either from the same donor or from different donors).
  • the T cells are autologous cells.
  • NK Natural Killer
  • the immune cells comprise natural killer (NK) cells.
  • a method of treating or preventing a cancer comprising administering natural killer (NK) cells expressing a CD19-targeting CAR as described herein.
  • the engineered NK cells are autologous cells, while in some embodiments, the NK cells are allogeneic cells.
  • the NK cells are autologous cells. In some embodiments, the NK cells are allogeneic. In some embodiments, the NK cells are derived from a donor who does not have a cancer. [00139] In several embodiments, NK cells are preferred because the natural cytotoxic potential of NK cells is relatively high. In several embodiments, it is unexpectedly beneficial that the engineered cells disclosed herein can further upregulate the cytotoxic activity of NK cells, leading to an even more effective activity against target cells (e.g., tumor or other diseased cells).
  • target cells e.g., tumor or other diseased cells
  • a NK cell is positive for cell surface expression of a marker selected from among the group consisting of CCR7, CD16, CD56, CD57, CD11, CX3CR1, a Killer Ig-like receptor (KIR), NKp30, NKp44, NKp46, or any combination thereof.
  • a NK cell is positive for cell surface expression of CD16.
  • a NK cell is positive for cell surface expression of CD56.
  • a NK cell is positive for cell surface expression of a Killer Ig-like receptor.
  • the NK cells are engineered to a membrane-bound interleukin 15 (mbIL15) domain.
  • the NK cells engineered to express a CAR are engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbIL15) domain.
  • the NK cells are engineered to bicistronically express the CAR and mbIL15.
  • the NK cells are derived from cell line NK-92.
  • NK-92 cells are derived from NK cells, but lack major inhibitory receptors displayed by normal NK cells, while retaining the majority of activating receptors.
  • NK-92 cells described herein related to NK-92 cell engineered to silence certain additional inhibitory receptors for example, SMAD3, allowing for upregulation of interferon- ⁇ (IFN ⁇ ), granzyme B, and/or perforin production. Additional information relating to the NK-92 cell line is disclosed in WO 1998/49268 and U.S. Patent Application Publication No. 2002-0068044 and incorporated in their entireties herein by reference.
  • the NK cells are used in combination with T cells.
  • the immune cells comprise T cells and NK cells (either from the same donor or from different donors).
  • primary NK cells are used in combination with primary T cells.
  • HSCs Hematopoietic Stem Cells
  • the immune cells comprise hematopoietic stem cells (HSCs).
  • HSCs are used in the methods disclosed herein.
  • the cells are engineered to express a CAR that binds to CD19.
  • a HSC is positive for cell surface expression of a marker selected from among the group consisting of CD34, CD59, and CD90.
  • a HSC is positive for cell surface expression of CD34.
  • a HSC is positive for cell surface expression of CD59.
  • a HSC is positive for cell surface expression of CD90.
  • allogeneic HSCs are used, while in some embodiments, autologous HSCs are used.
  • HSCs are used in combination with one or more additional engineered cell type disclosed herein.
  • Some embodiments of the methods and compositions described herein relate to a stem cell, such as a HSC engineered to express a CAR that binds to CD19, or a nucleic acid encoding the CAR.
  • the HSCs are engineered to express a membrane-bound interleukin 15 (mbIL15) domain.
  • the HSCs engineered to express a CAR are engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbIL15) domain.
  • HSCs are engineered to bicistronically express the CAR and mbIL15.
  • the HSCs are allogeneic cells.
  • the HSCs are obtained from a donor who does not have a cancer.
  • the HSCs are autologous cells. e.
  • immune cells are derived (differentiated) from pluripotent stem cells (PSCs).
  • PSCs pluripotent stem cells
  • immune cells e.g., NK cells
  • iPSCs induced pluripotent stem cells
  • NK cells are derived from iPSCs.
  • induced pluripotent stem cells are used in a method disclosed herein.
  • iPSCs are used, in several embodiments, to leverage their ability to differentiate and derive into non-pluripotent cells, including, but not limited to, CD34 cells, hemogenic endothelium cells, HSCs (hematopoietic stem and progenitor cells), hematopoietic multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, NKT cells, NK cells, and B cells comprising one or several genetic modifications at selected sites through differentiating iPSCs or less differentiated cells comprising the same genetic modifications at the same selected sites.
  • the iPSCs are used to generate iPSC-derived NK cells.
  • iPSCs engineered to express a CAR are engineered to also express (e.g., bicistronically express) a membrane-bound interleukin 15 (mbIL15).
  • mbIL15 membrane-bound interleukin 15
  • the engineered iPSCs are differentiated into NK, T, or other immune cells, such as for use in a composition or method provided herein.
  • the engineered iPSCs are differentiated into NK cells.
  • the iPSCs are allogeneic cells.
  • the iPSCs are obtained from a donor who does not have a cancer.
  • the iPSCs are autologous cells.
  • preparation of the engineered cells includes one or more culture and/or preparation steps.
  • the cells for introduction of the recombinant receptor e.g., CAR
  • CAR may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject.
  • the sample is an apheresis (e.g., leukapheresis) sample.
  • the subject from which the cells are isolated is one not having the cancer or in need of a cell therapy or not to which a cell therapy will be administered.
  • the cells are isolated from a subject that is different than the subject in need of a cell therapy or to which a cell therapy will be administered.
  • the cells are allogeneic to the subject to whom they are administered.
  • the subject from which the cells are isolated is one having the cancer or in need of a cell therapy or to which a cell therapy will be administered.
  • the cells are isolated from the subject to which a cell therapy will be administered.
  • the cells are autologous to the subject to whom they are administered.
  • 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 (e.g., a leukapheresis) product.
  • 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
  • Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.
  • the cells in some embodiments are primary cells, e.g., primary human cells.
  • the cells are immune cells, e.g. primary NK cells.
  • isolation of the cells includes one or more preparation and/or non affinity-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 (e.g., leukapheresis).
  • the samples contain lymphocytes, including NK cells, T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contain cells other than red blood cells and platelets.
  • 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.
  • 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.
  • NK cells or specific subpopulations thereof such as cells positive or expressing high levels of one or more surface markers, e.g., CD56+, CCR7+, CD16+, CD57+, CD11+, CX3CR1+, a Killer Ig-like receptor (KIR) +, NKp30+, NKp44+, or NKp46+ NK cells, are isolated by positive or negative selection techniques.
  • CD56+ NK cells can be positively selected using anti-CD56 conjugated magnetic beads.
  • the cells e.g., NK cells
  • the cells are expanded in culture prior to genetic engineering. In some embodiments, the cells are expanded in culture following genetic engineering. In some embodiments, the cells are expanded in culture prior to and following genetic engineering. Methods for expanding cells are known in the art and include any of those described in US Patent Nos. 7,435,596 and 8,026,097; and Patent Application Nos. PCT/SG2018/050138; PCT/US2020/044033; PCT/US2021/071330; and PCT/US2022/074164. [00170] In some embodiments, expanding the cells in culture comprises co- culturing the cells with feeder cells. In some embodiments, the feeder cells express IL15 (e.g., membrane-bound IL15) and 4-1BBL.
  • IL15 e.g., membrane-bound IL15
  • the feeder cells express membrane-bound interleukin 15 (mbIL15) and 4-1BBL. In some embodiments, the feeder cells do not express MHCI molecules. In some embodiments, the feeder cells do not express MHCII molecules. In some embodiments, the feeder cells are immune cells. In some embodiments, the feeder cells are K562 cells. Engineered feeder cells are disclosed in, for example, International Patent Application PCT/SG2018/050138. [00171] In some embodiments, expanding the cells in culture comprising culturing the cells in the presence of IL2, IL12, and/or IL18. In some embodiments, the cells are cultured in the presence of IL2. In some embodiments, the cells are cultured in the presence of IL12.
  • the cells are cultured in the presence of IL18. In some embodiments, the cells are cultured in the presence of IL12 and IL18. In some embodiments, the cells are cultured in the presence of IL2, IL12, and IL18.
  • the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, engineering, and/or expansion. In some embodiments, the cells are suspended in a freezing solution. In some embodiments, a composition provided herein is cryopreserved (e.g., prior to infusion into a subject). Any of a variety of known freezing solutions and parameters in some aspects may be used. D.
  • the immune cells are genetically edited to increase or decrease expression of a target protein.
  • the immune cells are genetically edited to increase expression of a target protein.
  • the immune cells are genetically edited to decrease expression of a target protein.
  • the methods comprise genetically editing the immune cells, such as to increase or decrease expression of a target protein.
  • the methods comprise genetically editing the immune cells to increase expression of a target protein.
  • the methods comprise genetically editing the immune cells to decrease expression of a target protein.
  • a target protein can be reduced by disrupting a gene (a target gene) encoding the target protein or a portion thereof.
  • the immune cells can be genetically edited at any point prior to, during, and/or after the genetic engineering. In some embodiments, the immune cells are genetically edited prior to the genetic engineering. In some embodiments, the immune cells are genetically edited contemporaneously with the genetic engineering. In some embodiments, the immune cells are genetically edited after the genetic engineering. [00175] As discussed below, in several embodiments, genetic editing is employed to reduce or eliminate expression of a target protein, for example by disrupting a gene encoding the protein.
  • genetic editing can reduce transcription of a target gene by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amount between those listed).
  • genetic editing reduces transcription of a target gene by at least about 30%.
  • genetic editing reduces transcription of a target gene by at least about 40%.
  • genetic editing reduces transcription of a target gene by at least about 50%.
  • genetic editing reduces transcription of a target gene by at least about 60%.
  • genetic editing reduces transcription of a target gene by at least about 70%.
  • genetic editing reduces transcription of a target gene by at least about 80%. In several embodiments, genetic editing reduces transcription of a target gene by at least about 90%. In several embodiments, genetic editing reduces transcription of a target gene by at least about 95%. In several embodiments, genetic editing reduces transcription of a target gene by at least about 99%. In several embodiments, the gene is completely knocked out, such that transcription of the target gene is eliminated (undetectable). [00176] In several embodiments, genetic editing can reduce expression of a target protein by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amount between those listed).
  • genetic editing reduces expression of a target protein by at least about 30%. In several embodiments, genetic editing reduces expression of a target protein by at least about 40%. In several embodiments, genetic editing reduces expression of a target protein by at least about 50%. In several embodiments, genetic editing reduces expression of a target protein by at least about 60%. In several embodiments, genetic editing reduces expression of a target protein by at least about 70%. In several embodiments, genetic editing reduces expression of a target protein by at least about 80%. In several embodiments, genetic editing reduces expression of a target protein by at least about 90%. In several embodiments, genetic editing reduces expression of a target protein by at least about 95%. In several embodiments, genetic editing reduces expression of a target protein by at least about 99%.
  • the gene is completely knocked out, such that expression of the target protein is eliminated (undetectable).
  • genetic editing is used to “knock in” or otherwise increase transcription of a target gene.
  • transcription of a target gene is increased by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amount between those listed).
  • transcription of a target gene is increased by at least about 30%.
  • transcription of a target gene is increased by at least about 40%.
  • transcription of a target gene is increased by at least about 50%.
  • transcription of a target gene is increased by at least about 60%. In several embodiments, transcription of a target gene is increased by at least about 70%. In several embodiments, transcription of a target gene is increased by at least about 80%. In several embodiments, transcription of a target gene is increased by at least about 90%. In several embodiments, transcription of a target gene is increased by at least about 100%. [00178] In several embodiments, genetic editing is used to “knock in” or otherwise enhance expression of a target protein.
  • expression of a target protein can be enhanced by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amount between those listed).
  • expression of a target protein is increased by at least about 30%.
  • expression of a target protein is increased by at least about 40%.
  • expression of a target protein is increased by at least about 50%.
  • expression of a target protein is increased by at least about 60%.
  • expression of a target protein is increased by at least about 70%.
  • expression of a target protein is increased by at least about 80%.
  • expression of a target protein is increased by at least about 90%. In several embodiments, expression of a target protein is increased by at least about 100%.
  • Genetic editing can be used to reduce, eliminate (e.g., knockout), or increase expression of a target gene. For example, the transcription of the target gene and/or the translation of a protein encoded by the target gene (e.g., a target protein) can be reduced, eliminated (e.g., knocked out), or increased.
  • the target gene can be implicated in the immune functionality of the cell or be a part of a signaling pathway for which an increase or decrease in function is desired.
  • NK cells can increase activity and/or persistence of those immune cells.
  • Methods of Genetic Editing (whether knock out or knock in) of a target gene is accomplished through targeted introduction of DNA breakage, and a subsequent DNA repair mechanism.
  • double strand breaks of DNA are repaired by non-homologous end joining (NHEJ), wherein enzymes are used to directly join the DNA ends to one another to repair the break. NHEJ is an error-prone process.
  • NHEJ non-homologous end joining
  • HDR homology directed repair
  • HDR pathway can occur by way of the canonical HDR pathway or the alternative HDR pathway.
  • HDR or “homology-directed repair” as used herein encompasses both canonical HDR and alternative HDR.
  • Canonical HDR or “canonical homology-directed repair” or “cHDR,” are used interchangeably, and refers to the process of repairing DNA damage using a homologous nucleic acid (e.g., an endogenous homologous sequence, such as a sister chromatid; or an exogenous nucleic acid, such as a donor template).
  • Canonical HDR typically acts when there has been a significant resection at the DSB, forming at least one single-stranded portion of DNA.
  • canonical HDR typically involves a series of steps such as recognition of the break, stabilization of the break, resection, stabilization of single-stranded DNA, formation of a DNA crossover intermediate, resolution of the crossover intermediate, and ligation.
  • the canonical HDR process requires RAD51 and BRCA2, and the homologous nucleic acid, e.g., repair template, is typically double-stranded.
  • a double- stranded polynucleotide e.g., a double-stranded repair template
  • a double-stranded repair template which comprises a sequence that is homologous to the targeting sequence, and which will either be directly integrated into the targeting sequence or will be used as a template to insert the sequence, or a portion the sequence, of the repair template into the target gene.
  • repair can progress by different pathways, e.g., by the double Holliday junction model (also referred to as the double strand break repair, or DSBR, pathway), or by the synthesis-dependent strand annealing (SDSA) pathway.
  • the double Holliday junction model also referred to as the double strand break repair, or DSBR, pathway
  • SDSA synthesis-dependent strand annealing
  • strand invasion occurs by the two single stranded overhangs of the targeting sequence to the homologous sequences in the double-stranded polynucleotide, e.g., double stranded donor template, which results in the formation of an intermediate with two Holliday junctions.
  • the junctions migrate as new DNA is synthesized from the ends of the invading strand to fill the gap resulting from the resection.
  • the end of the newly synthesized DNA is ligated to the resected end, and the junctions are resolved, resulting in the insertion at the targeting sequence, or a portion of the targeting sequence that includes the gene variant.
  • Crossover with the polynucleotide may occur upon resolution of the junctions.
  • the polynucleotide e.g., donor template
  • new DNA is synthesized from the end of the invading strand to fill the gap resulting from resection.
  • the newly synthesized DNA then anneals to the remaining single stranded overhang, new DNA is synthesized to fill in the gap, and the strands are ligated to produce the modified DNA duplex.
  • Alternative HDR or “alternative homology-directed repair,” or “alternative HDR,” are used interchangeably, and refers, in some embodiments, to the process of repairing DNA damage using a homologous nucleic acid (e.g., an endogenous homologous sequence, such as a sister chromatid; or an exogenous nucleic acid, such as a repair template).
  • a homologous nucleic acid e.g., an endogenous homologous sequence, such as a sister chromatid; or an exogenous nucleic acid, such as a repair template.
  • Alternative HDR is distinct from canonical HDR in that the process utilizes different pathways from canonical HDR, and can be inhibited by the canonical HDR mediators, RAD51 and BRCA2.
  • alternative HDR is also distinguished by the involvement of a single- stranded or nicked homologous nucleic acid template, e.g., repair template
  • canonical HDR generally involves a double-stranded homologous template.
  • a single strand template polynucleotide e.g., repair template
  • a nick, single strand break, or DSB at the cleavage site, for altering a desired target site, e.g., a gene variant in a target gene is mediated by a nuclease molecule, and resection at the break occurs to reveal single stranded overhangs.
  • HDR is carried out by introducing, into a cell, one or more agent(s) capable of inducing a DSB, and a repair template, e.g., a single-stranded oligonucleotide.
  • the introducing can be carried out by any suitable delivery.
  • the conditions under which HDR is allowed to occur can be any conditions suitable for carrying out HDR in a cell.
  • gene editing is accomplished by one or more of a variety of engineered nucleases.
  • restriction enzymes are used, particularly when double strand breaks are desired at multiple regions.
  • a bioengineered nuclease is used.
  • ZFN Zinc Finger Nuclease
  • TALEN transcription-activator like effector nuclease
  • CRISPR/Cas9 clustered regularly interspaced short palindromic repeats
  • Meganucleases are characterized by their capacity to recognize and cut large DNA sequences (from 14 to 40 base pairs).
  • a meganuclease from the LAGLIDADG family is used, and is subjected to mutagenesis and screening to generate a meganuclease variant that recognizes a unique sequence(s), such as a specific site in a target gene, or any other target gene disclosed herein.
  • two or more meganucleases, or functions fragments thereof are fused to create a hybrid enzyme that recognizes a desired target sequence within the target gene.
  • ZFNs and TALEN function based on a non-specific DNA cutting catalytic domain which is linked to specific DNA sequence recognizing peptides such as zinc fingers or transcription activator-like effectors (TALEs).
  • the ZFNs and TALENs thus allow sequence-independent cleavage of DNA, with a high degree of sequence-specificity in target recognition.
  • Zinc finger motifs naturally function in transcription factors to recognize specific DNA sequences for transcription.
  • the C- terminal part of each finger is responsible for the specific recognition of the DNA sequence.
  • sequences recognized by ZFNs are relatively short, (e.g., ⁇ 3 base pairs)
  • combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10 or more zinc fingers whose recognition sites have been characterized are used, thereby allowing targeting of specific sequences.
  • TALENs Transcription activator-like effector nucleases
  • TALENs are specific DNA-binding proteins that feature an array of 33 or 34-amino acid repeats. Like ZFNs, TALENs are a fusion of a DNA cutting domain of a nuclease to TALE domains, which allow for sequence-independent introduction of double stranded DNA breaks with highly precise target site recognition.
  • TALENs can create double strand breaks at the target site that can be repaired by error-prone non-homologous end-joining (NHEJ), resulting in gene disruptions through the introduction of small insertions or deletions.
  • NHEJ error-prone non-homologous end-joining
  • TALENs are used in several embodiments, at least in part due to their higher specificity in DNA binding, reduced off-target effects, and ease in construction of the DNA-binding domain.
  • CRISPRs Clustered Regularly Interspaced Short Palindromic Repeats
  • Cas CRISPR associated proteins
  • CRISPR is used to disrupt a target gene.
  • a Class 1 or Class 2 Cas is used.
  • a Class 1 Cas is used, and the Cas type is selected from the following types: I, IA, IB, IC, ID, IE, IF, IU, III, IIIA, IIIB, IIIC, IIID, IV IVA, IVB, and combinations thereof.
  • the Cas is selected from the group consisting of Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1, Cse2, Csy1, Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Cas10, Csx11, Csx10, Csf1, and combinations thereof.
  • the Cas is Cas3.
  • a Class 2 Cas is used, and the Cas type is selected from the following types: II, IIA, IIB, IIC, V, VI, and combinations thereof.
  • the Cas is selected from the group consisting of Cas9, Csn2, Cas4, Cas12a (previously known as Cpf1), C2c1, C2c3, Cas13a (previously known as C2c2), Cas13b, Cas13c, CasX, CasY and combinations thereof.
  • the Cas is Cas9.
  • class 2 CasX is used, wherein CasX can form a complex with a guide nucleic acid and wherein the complex can bind to a target DNA, and wherein the target DNA comprises a non-target strand and a target strand.
  • class 2 CasY is used, wherein CasY is capable of binding and modifying a target nucleic acid and/or a polypeptide associated with target nucleic acid.
  • Target Genes [00191]
  • the immune cells are genetically edited at a target gene. In several embodiments, editing of a target gene advantageously imparts to the edited cells enhanced expansion, cytotoxicity and/or persistence.
  • immune cells are genetically edited to increase IL15 levels and/or signaling.
  • genetically editing the cells to increase IL15 levels and/or signaling may obviate the need to provide a lymphodepleting therapy containing fludarabine to subjects prior to administration of genetically engineered cells.
  • the increased IL15 bioavailability afforded by fludarabine is not necessary in immune cells genetically edited to increase IL15 levels and/or signaling.
  • the immune cells are genetically edited at a target gene to increase IL15 levels, such as by reducing or eliminating expression of the cytokine-inducible SH2-containing protein (Cis) (e.g., by disrupting the CISH gene encoding Cis).
  • the immune cells e.g., NK cells
  • IL15 is a positive regulator of NK cells, which as disclosed herein, can enhance one or more of NK cell homing, NK cell migration, NK cell expansion/proliferation, NK cell cytotoxicity, and/or NK cell persistence.
  • CISH actively silences TCR signaling to maintain tumor tolerance, and CISH has been shown to be a downstream negative regulator of IL-15 receptor signaling (Palmer et al., J. Exp. Med. (2015) 212(12):2095-2113).
  • CISH plays a role in checkpoint maturation and proliferation (Delconte et al., Nature Immunol (2016) 17:816-24).
  • CISH genetic editing increases the persistence, proliferation, and/or cytotoxicity, or otherwise enhances the efficacy, of immune cells (e.g., NK cells) as disclosed herein.
  • CISH genetic editing activates or inhibits a wide variety of pathways.
  • the CIS protein is a negative regulator of IL15 signaling by way of, for example, inhibiting JAK-STAT signaling pathways. These pathways would typically lead to transcription of IL15-responsive genes (including CISH).
  • disruption of CISH disinhibits JAK-STAT (e.g., JAK1-STAT5) signaling and there is enhanced transcription of IL15-responsive genes.
  • disruption of CISH yields enhanced signaling through mammalian target of rapamycin (mTOR), with corresponding increases in expression of genes related to cell metabolism and respiration.
  • disruption of CISH yields IL15 induced increased expression of IL-2R ⁇ (CD25), but not IL-15R ⁇ or IL-2/15R ⁇ , enhanced NK cell membrane binding of IL15 and/or IL2, increased phosphorylation of STAT-3 and/or STAT-5, and elevated expression of the antiapoptotic proteins, such as Bcl-2.
  • CISH disruption results in IL15- induced upregulation of selected genes related to mitochondrial functions (e.g., electron transport chain and cellular respiration) and cell cycle.
  • CISH disruption alters the state (e.g., activates or inactivates) signaling via or through one or more of CXCL-10, IL2, TNF, IFNg, IL13, IL4, Jnk, PRF1, STAT5, PRKCQ, IL2 receptor Beta, SOCS2, MYD88, STAT3, STAT1, TBX21, LCK, JAK3, IL& receptor, ABL1, IL9, STAT5A, STAT5B, Tcf7, PRDM1, and/or EOMES.
  • CISH editing endows an NK cell with enhanced ability to home to a target site.
  • CIS expression is knocked down or knocked out through genetic editing of the CISH gene, for example, by use of CRISPR-Cas editing.
  • the immune cells e.g., NK cells
  • Small interfering RNA, antisense RNA, TALENs or zinc fingers are used in other embodiments.
  • Information on CISH editing can be found, for example, in International Patent Application Nos. PCT/US2023/060850 and PCT/US2020/035752, which are each incorporated in their entirety by reference herein.
  • genetic editing reduces transcription of CISH by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amount between those listed). In several embodiments, genetic editing reduces transcription of CISH by at least about 30%. In several embodiments, genetic editing reduces transcription of CISH by at least about 40%. In several embodiments, genetic editing reduces transcription of CISH by at least about 50%. In several embodiments, genetic editing reduces transcription of CISH by at least about 60%. In several embodiments, genetic editing reduces transcription of CISH by at least about 70%. In several embodiments, genetic editing reduces transcription of CISH by at least about 80%.
  • genetic editing reduces expression of Cis by about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or more (including any amount between those listed). In several embodiments, genetic editing reduces expression of Cis by at least about 30%. In several embodiments, genetic editing reduces expression of Cis by at least about 40%. In several embodiments, genetic editing reduces expression of Cis by at least about 50%. In several embodiments, genetic editing reduces expression of Cis by at least about 60%. In several embodiments, genetic editing reduces expression of Cis by at least about 70%. In several embodiments, genetic editing reduces expression of Cis by at least about 80%.
  • genetic editing reduces expression of Cis by at least about 90%. In several embodiments, genetic editing reduces expression of Cis by at least about 95%. In several embodiments, genetic editing reduces expression of Cis by at least about 99%.
  • immune cells e.g., NK cells
  • TGFBR2 transforming growth factor-beta receptor 2 protein
  • Cbl-b Casitas B- lineage lymphoma-b protein
  • the immune cells are genetically edited to reduce expression of TGFbR2.
  • the immune cells are genetically edited to reduce expression of Cbl-b.
  • compositions comprising genetically engineered NK cells expressing a CD19-directed CAR, a plurality of genetically engineered NK cells expressing a CD19-directed CAR, and/or additional agents for combination treatment or therapy.
  • the pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier.
  • the composition includes at least one additional therapeutic agent.
  • a composition comprising immune cells e.g., NK cells
  • a composition comprising immune cells comprises a cryopreservative.
  • a composition comprising immune cells e.g., NK cells
  • composition 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 choice of carrier is determined in part by the particular cell, binding molecule, and/or antibody, and/or by the method of administration. Accordingly, there are a variety of suitable formulations.
  • a buffer is included in the composition.
  • Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts.
  • a mixture of two or more buffers is used.
  • the buffering agent or mixtures thereof are typically present in an amount of from about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known.
  • Formulations of the antibodies described herein can include lyophilized formulations and aqueous solutions.
  • Suitable pharmaceutically acceptable acid addition salts include those derived from mineral acids, such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric, and sulphuric acids, and organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic acids, for example, p-toluenesulphonic acid.
  • the pharmaceutical composition in some embodiments contains the engineered cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects.
  • the liquid composition can comprise a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof.
  • a carrier such as an admixture with a suitable carrier, diluent, or excipient (e.g., sterile water, saline, glucose, dextrose, and the like).
  • a suitable carrier e.g., sterile water, saline, glucose, dextrose, and the like.
  • Formulations for in vivo administration are typically sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.
  • a lymphodepleting therapy is administered concurrently with the one or more additional therapeutic agents. In some embodiments, the lymphodepleting therapy is administered after the one or more additional therapeutic agents.
  • the additional therapeutic agent includes chemotherapy, radiation therapy, surgery, transplantation, adoptive cell therapy, antibodies, cytotoxic agents, chemotherapeutic agents, cytokines, growth inhibitory agents, anti-hormonal agents, kinase inhibitors, anti-angiogenic agents, cardioprotectants, immunostimulatory agents, immunosuppressive agents, immune checkpoint inhibitors, antibiotics, angiogenesis inhibitors, metabolic modulators or other therapeutic agents or any combination thereof.
  • the additional therapeutic agent includes surgery, chemotherapy, radiation therapy, transplantation, administration of cells expressing a recombinant receptor, e.g., CAR, kinase inhibitor, immune checkpoint inhibitor, mTOR pathway inhibitor, immunosuppressive agents, immunomodulators, antibodies, immunoablative agents, antibodies and/or antigen binding fragments thereof, antibody conjugates, other antibody therapies, cytotoxins, steroids, cytokines, peptide vaccines, hormone therapy, antimetabolites, metabolic modulators, alkylating agents, anthracyclines, vinca alkaloids, proteasome inhibitors, protein kinase inhibitors, and/or other types of immunotherapy.
  • a recombinant receptor e.g., CAR, kinase inhibitor, immune checkpoint inhibitor, mTOR pathway inhibitor, immunosuppressive agents, immunomodulators, antibodies, immunoablative agents, antibodies and/or antigen binding fragments thereof, antibody conjugates, other antibody therapies, cytotoxins, steroids, cytokines, peptide vaccines, hormone
  • the additional therapeutic agent or treatment is bone marrow transplantation, T cell ablative therapy using chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, and/or antibody therapy.
  • the therapeutic agent is an NK cell engager (e.g., a molecule that binds both an antigen expressed by cells of the cancer and an antigen expressed by NK cells).
  • the NK cell engager binds to an activating receptor on an NK cell and an antigen expressed by cells of the cancer.
  • the activating receptor on the NK cell is selected from the group consisting of CD16, NKp30, NKp46, NKG2D, and any combination thereof.
  • the additional agent includes an immune checkpoint inhibitor.
  • Immune checkpoint inhibitors include any agent that blocks or inhibits in a statistically significant manner, the inhibitory pathways of the immune system. Such inhibitors may include small molecule inhibitors or may include antibodies, or antigen binding fragments thereof, that bind to and block or inhibit immune checkpoint receptors, ligands and/or receptor-ligand interaction. In some embodiments, modulation, enhancement and/or stimulation of particular receptors can overcome immune checkpoint pathway components.
  • Illustrative immune checkpoint molecules that may be targeted for blocking, inhibition, modulation, enhancement and/or stimulation include, but are not limited to, PD-1 (CD279), PD-L1 (CD274, B7-H1), CTLA-4, LAG-3 (CD223), TIM-3, 4-1BB (CD137), 4-1BBL (CD137L), GITR (TNFRSF18, AITR), CD40, OX40 (CD134, TNFRSF4), B7-H3, B7-H4, B7H3, B7H4, VISTA, KIR, 2B4, CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), TIGIT, and LAIR1.
  • PD-1 CD279
  • PD-L1 CD274, B7-H1
  • CTLA-4 LAG-3
  • TIM-3 4-1BB
  • 4-1BBL CD137L
  • GITR TNFRSF18, AITR
  • CD40 OX40
  • the additional agent is a CD20 inhibitor, e.g., an anti-CD20 antibody (e.g., an anti-CD20 mono- or bi-specific antibody) or a fragment thereof, antibody-drug conjugate (ADC), or engineered toxin body (ETB).
  • the CD20 inhibitor is an anti-CD20 antibody.
  • the CD20 inhibitor is an anti- CD20 antibody or an antigen-binding fragment thereof.
  • Exemplary anti-CD20 antibodies include rituximab (RITUXAN®) and obinutuzumab (GAZYVA®).
  • the additional agent is or includes rituximab.
  • the additional agent is or includes obinutuzumab.
  • the antibody targets CD20.
  • the anti-CD20 antibody is rituximab.
  • biosimilar rituximab-abbs, rituximab-arrx, and/or rituximab-pvvr are used.
  • ocrelizumab, ofatumumab, obinutuzumab, ibritumomab, ibritumomab or combinations thereof are used.
  • the dose of the anti-CD20 antibody ranges from between about 150 mg/m2 and about 500 mg/m2, including about 150 mg/m2, about 200 mg/m2, about 250 mg/m2, about 300 mg/m2, about 350 mg/m2, about 375 mg/m2, about 400 mg/m2, about 425 mg/m2, about 450 mg/m2, or about 500 mg/m2 (or any dose between those listed).
  • the dose of the anti-CD20 antibody is about 375 mg/m2.
  • the anti-CD20 antibody is rituximab and the dose is about 375 mg/m2.
  • the dose of the anti-CD20 antibody is about 500 mg/m2.
  • the anti-CD20 antibody is rituximab and the dose is about 500 mg/m2.
  • the anti-CD20 antibody will be administered 1, 2, 3, or more times.
  • the anti-CD20 antibody will be administered 1, 2, 3, 4 or more days prior to administration of cells at the initial time point in a dosing cycle.
  • a single dose of 375 mg/m2 rituximab is administered during the dosing cycle.
  • a single dose of 375 mg/m2 rituximab is administered during the first dosing cycle, and a single dose of 500 mg/m2 rituximab is administered during each subsequent dosing cycle.
  • the single dose of rituximab is administered prior to administration of the engineered immune cells, (e.g., about 3 days prior to administration). In some embodiments, the single dose of rituximab is administered about 3 days prior to administration of the engineered immune cells. In several embodiments, the anti-CD20 antibody will be administered 3 days prior to administration of cells.
  • the CD20 inhibitor is an ADC. In some embodiments, the CD20 inhibitor is an ETB. In some embodiments, the CD20 inhibitor is a small molecule.
  • the additional agent is a CD22 inhibitor, e.g., an anti-CD22 antibody (e.g., an anti-CD22 mono- or bi-specific antibody) or a fragment thereof, antibody-drug conjugate (ADC), or engineered toxin body (ETB).
  • the CD22 inhibitor is an anti-CD22 antibody or an antigen-binding fragment thereof.
  • the CD22 inhibitor is an ADC.
  • the CD22 inhibitor is an ETB.
  • the CD22 inhibitor is a small molecule.
  • the additional agent is an EGFR inhibitor, e.g., an anti-EGFR antibody (e.g., an anti-EGFR mono- or bi-specific antibody) or a fragment thereof, antibody-drug conjugate (ADC), or engineered toxin body (ETB).
  • the EGFR inhibitor is an anti-EGFR antibody.
  • the EGFR inhibitor is an anti-EGFR antibody or an antigen-binding fragment thereof.
  • Exemplary anti- EGFR antibodies include cetuximab, panitumumab (VECTIBIX®), nimotuzumab, and necitumumab (PORTRAZZATM).
  • the anti-EGFR antibody comprises cetuximab. In several embodiments, the anti-EGFR antibody is cetuximab. In several embodiments, the anti-EGFR antibody comprises panitumumab. In several embodiments, the anti-EGFR antibody is panitumumab. In several embodiments, the anti-EGFR antibody comprises nimotuzumab. In several embodiments, the anti-EGFR antibody is nimotuzumab. In several embodiments, the anti-EGFR antibody comprises necitumumab. In several embodiments, the anti-EGFR antibody is necitumumab. In several embodiments, the anti-EGFR antibody will be administered 1, 2, 3, or more times.
  • the anti- EGFR antibody will be administered 1, 2, 3, 4 or more days prior to administration of cells at the initial time point in a dosing cycle. In some embodiments, a single dose of an anti-EGFR antibody is administered during the dosing cycle. In some embodiments, the anti-EGFR antibody is administered prior to, concurrent with, and/or subsequent to administration of the engineered immune cells. In some embodiments, the anti-EGFR antibody is administered prior to administration of the engineered immune cells, (e.g., about 3 days prior to administration). In some embodiments, the anti-EGFR antibody is administered concurrent with administration of the engineered immune cells. In some embodiments, the anti-EGFR antibody is administered subsequent to administration of the engineered immune cells.
  • the additional agent is or includes cetuximab.
  • the EGFR inhibitor is an ADC.
  • the EGFR inhibitor is an ETB.
  • the EGFR inhibitor is a small molecule.
  • the dose of the additional agent can be any therapeutically effective amount, e.g., any dose amount described herein, and the appropriate dosage of the additional agent may depend on the type of disease to be treated, the type, dose and/or frequency of the recombinant receptor, cell and/or composition administered, the severity and course of the disease, whether the recombinant receptor, cell and/or composition is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the recombinant receptor, cell and/or composition, and the discretion of the attending physician.
  • the recombinant receptor, cell and/or composition and/or the additional agent and/or therapy can be administered to the patient at one time, repeated or administered over a series of treatments. II.
  • 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, test tubes, IV solution bags, etc.
  • the containers may be formed from a variety of materials such as glass or plastic.
  • the container has a sterile access port.
  • Non-limiting examples of containers include intravenous solution bags and vials, including those with stoppers pierceable by a needle for injection.
  • the article of manufacture or kit may further include a package insert indicating that the composition can be used to treat a particular condition such as a condition described herein (e.g., cancer).
  • the article of manufacture or kit 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.
  • the label or package insert may indicate that the composition is used for treating a disease (e.g., cancer) in an individual.
  • the label or a package insert, which is on or associated with the container may indicate directions for reconstitution and/or use of the formulation.
  • the label or package insert may further indicate that the formulation is useful or intended for subcutaneous, intravenous, or other modes of administration for treating or preventing a disease (e.g., cancer) in an individual.
  • a disease e.g., cancer
  • 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 disease (e.g.,cancer).
  • the article of manufacture or kit may include (a) a first container with a composition contained therein (i.e., first medicament), wherein the composition includes the engineered NK cells; and (b) a second container with a composition contained therein (i.e., second medicament), wherein the composition includes a further agent, such as a cytotoxic or otherwise therapeutic agent, and which article or kit further comprises instructions on the label or package insert for treating the subject with the second medicament, in an effective amount.
  • a further agent such as a cytotoxic or otherwise therapeutic agent
  • the method includes treating or preventing cancer. In some embodiments, the method includes administering a therapeutically effective amount of immune cells expressing a tumor-directed chimeric antigen receptor as described herein. Examples of types of cancer that may be treated as such are described herein. [00223] Disclosed herein are methods of treating cancer in a subject. In some embodiments, the methods comprise administering to the subject any one of the CD19 binding domains disclosed herein, any one of the CD19-directed CARs disclosed herein, or any one of the CAR-expressing cells disclosed herein, or any combination thereof.
  • treatment of a subject with a genetically engineered cell(s) described herein achieves one, two, three, four, or more of the following effects, including, for example: (i) reduction or amelioration the severity of disease or symptom associated therewith; (ii) reduction in the duration of a symptom associated with a disease; (iii) protection against the progression of a disease or symptom associated therewith; (iv) regression of a disease or symptom associated therewith; (v) protection against the development or onset of a symptom associated with a disease; (vi) protection against the recurrence of a symptom associated with a disease; (vii) reduction in the hospitalization of a subject; (viii) reduction in the hospitalization length; (ix) an increase in the survival of a subject with a disease; (x) a reduction in the number of symptoms associated with a disease; and (xi) an enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s)
  • each of these comparisons are versus, for example, a different therapy for a disease, which includes a cell-based immunotherapy for a disease using cells that do not express the constructs disclosed herein.
  • the engineered NK and/or T cells disclosed herein further enhance one or more of the above.
  • Such unexpected effects are due, at least in part, to providing an increased number of genetically engineered NK cells during a time period in which a subject’s immune response has not yet fully recovered following lymphodepleting therapy, thereby allowing increased engraftment of adoptively transferred immune cells (e.g., NK cells).
  • a time period may include, for example, within about 7 days of administration of the genetically engineered NK cells and/or within about 14 days of administration of a first dose of lymphodepleting therapy.
  • the efficacy of genetically engineered NK cells may be improved.
  • NK cells are graft vs. host disease and toxicity associated with NK cells. This is in contrast with, for example, cytokine release syndrome (CRS) and neurotoxicity frequently observed with adoptive transfer of engineered T cells, such as CAR T cells.
  • CRS cytokine release syndrome
  • CAR T cells such as CAR T cells.
  • the relatively limited persistence of CAR NK cells in vivo combined with the methods of treatment described herein, may allow for modification of the standard lymphodepletion regimens used with CAR T cell therapies, such that the risk of potential toxicities can be mitigated.
  • lymphodepleting therapy has been an integral part of CAR T cell clinical trials.
  • use of high-dose lymphodepletion regimens prior to adoptive transfer of NK cell therapies has yielded in vivo NK cell expansion and persistence, whereas low-dose lymphodepletion regimens did not (Kilgour et al., Front. Immunol. (2023) 14:1166038).
  • a lymphodepletion regimen of fludarabine and cyclophosphamide has historically been associated with the ability to detect adoptively transferred immune cells.
  • the benefit of eliminating lymphocytes via a fludarabine-containing lymphodepleting therapy may be realized not just through reduced rejection of adoptively transferred immune cells, but also through improved availability of cytokines such as interleukin 15 (IL15) (Gauthier et al., Blood (2020) 136(Supp.1):37–38).
  • IL15 interleukin 15
  • CD19 CAR-expressing NK cells as provided herein are expected to have much of their activity shortly after administration to a subject, such that the primary benefit of lymphodepletion for NK cells may be from cyclophosphamide, which has activity against lymphocytes earlier than fludarabine.
  • cyclophosphamide which has activity against lymphocytes earlier than fludarabine.
  • the nadir of white blood cell counts has been reported to be approximately 13 days after fludarabine treatment (FLUDARA® USPI 2010) compared to approximately 9 days after cyclophosphamide treatment (Buckner et al., Cancer (1972) 29(2):357-65).
  • the provision of all doses within the peak activity window of cyclophosphamide may be particularly advantageous.
  • the inventors therefore contemplate the combination of a cyclophosphamide-only lymphodepleting therapy in combination with a dosing cycle in which all doses of CD19 CAR-expressing NK cells are provided within about 7-10 days of administration of the cyclophosphamide-only lymphodepleting therapy.
  • a single dose of cyclophosphamide can be provided about 3 days prior to administration of the first dose of CD19 CAR NK cells (Day -3), and the doses of CD19 CAR NK cells can be provided on Days 0, 3, and 7.
  • a single dose of cyclophosphamide can be provided about 3 days prior to administration of the first dose of CD19 CAR NK cells (Day -3), and the doses of CD19 CAR NK cells can be provided on Days 0, 2, and 4 or on Days 0, 2, and 5.
  • a dosing cycle comprising administration of a single dose of cyclophosphamide on Day -3 and administration of the CD19 CAR NK cells on Days 0, 2, and 4 could be particularly convenient in an outpatient setting, where cyclophosphamide could be provided e.g., on Friday, and doses of CD19 CAR NK cells could be provided e.g., on the following Monday, Wednesday, and Friday.
  • each of the doses of CD19 CAR-expressing NK cells within about 7-10 days of the administration of cyclophosphamide may allow for improved peak concentration and/or persistence of the NK cells as compared to a dosing regimen in which one or more doses of the dosing cycle are provided later in time.
  • increased cytokine e.g., IL15
  • mbIL15 membrane-bound interleukin 15
  • fludarabine may increase not only short-term toxicity (Hay et al., Blood (2017) 130(21):2295-2306) but also the potential for secondary malignancies, removing fludarabine from lymphodepleting therapy may improve the risk-benefit profile.
  • use of the CD19 CAR-expressing NK cells as provided herein to treat a cancer may not require a fludarabine-containing lymphodepletion regimen, as is commonly used for hematologic malignancies. Rather, a lymphodepletion regimen of only cyclophosphamide may be sufficient to achieve efficacy and reduce potential toxicities associated with LD.
  • NK cells can be determined for a given subject based on their body mass, disease type and state, and desired aggressiveness of treatment, but range, depending on the embodiments, from about 105 cells per kg to about 1012 cells per kg (e.g., 105-107, 107-1010, 1010-1012 and overlapping ranges therein).
  • a range of immune cells such as NK cells is administered, for example between about 1 x 108 cells/kg to about 1 x 107 cells/kg.
  • a range of immune cells such as NK cells is administered, for example between about 1 x 109 CAR- expressing immune cells to about 3 x 109 CAR-expressing immune cells.
  • a dosing cycle comprises administration of two doses of NK cells.
  • a dosing cycle consists of two doses of NK cells.
  • a dosing cycle comprises administration of three doses of NK cells.
  • a dosing cycle consists of three doses of NK cells.
  • a dosing cycle comprises administration of four doses of NK cells.
  • a dosing cycle consists of four doses of NK cells.
  • a dosing cycle comprises administration of five doses of NK cells.
  • a dosing cycle consists of five doses of NK cells. Such multi- dose cycles can be repeated one or more times, as needed to treat a cancer or disease progression. [00233] In some embodiments, all doses of the dosing cycle are administered to the subject within about 10 days, within about 9 days, within about 8 days, within about 7 days, within about 6 days, within about 5 days, or within about 4 days. In some embodiments, all doses of the dosing cycle are administered to the subject within about 10 days. In some embodiments, all doses of the dosing cycle are administered to the subject within about 9 days. In some embodiments, all doses of the dosing cycle are administered to the subject within about 8 days.
  • all doses of the dosing cycle are administered to the subject within about 7 days. In some embodiments, all doses of the dosing cycle are administered to the subject within about 6 days. In some embodiments, all doses of the dosing cycle are administered to the subject within about 5 days. In some embodiments, all doses of the dosing cycle are administered to the subject within about 4 days. In some embodiments, all doses of a dosing cycle are administered to the subject within about 13 days, within about 12 days, within about 11 days, within about 10 days, within about 9 days, within about 8 days, or within about 7 days of administration of a lymphodepleting therapy to a subject.
  • all doses of a dosing cycle are administered to the subject within about 13 days of a lymphodepleting therapy to a subject. In some embodiments, all doses of a dosing cycle are administered to the subject within about 12 days of a lymphodepleting therapy to a subject. In some embodiments, all doses of a dosing cycle are administered to the subject within about 11 days of a lymphodepleting therapy to a subject. In some embodiments, all doses of a dosing cycle are administered to the subject within about 10 days of a lymphodepleting therapy to a subject. In some embodiments, all doses of a dosing cycle are administered to the subject within about 9 days of a lymphodepleting therapy to a subject.
  • all doses of a dosing cycle are administered to the subject within about 8 days of a lymphodepleting therapy to a subject. In some embodiments, all doses of a dosing cycle are administered to the subject within about 7 days of a lymphodepleting therapy to a subject. In some embodiments, the dosing cycle consists of three doses. In some embodiments, the lymphodepleting therapy does not comprise fludarabine. In some embodiments, the lymphodepleting therapy consists of cyclophosphamide. [00234] In several embodiments, between about 1 x 109 to about 5 x 109 CAR- expressing NK cells are provided in each dose.
  • a dose comprises about 1 x 109 CAR-expressing NK cells. In several embodiments, a dose comprises about 1.5 x 109 CAR-expressing NK cells. In several embodiments, between about 2 x 109 to about 5 x 109 CAR-expressing NK cells are provided in each dose. In several embodiments, a dose comprises about 2 x 109 CAR-expressing NK cells. In several embodiments, a dose comprises about 2.5 x 109 CAR-expressing NK cells. In several embodiments, a dose comprises about 3 x 109 CAR-expressing NK cells. In several embodiments, a dose comprises about 3.5 x 109 CAR-expressing NK cells.
  • a dose comprises about 4 x 109 CAR-expressing NK cells. In several embodiments, a dose comprises about 4.5 x 109 CAR-expressing NK cells. In several embodiments, a dose comprises about 5 x 109 CAR-expressing NK cells. [00235] In several embodiments, between about 1 ⁇ 109 CAR-expressing NK cells and about 5 ⁇ 109 CAR-expressing NK cells are administered three times over a 28-day cycle. In several embodiments, between about 2 ⁇ 109 CAR-expressing NK cells and about 5 ⁇ 109 CAR-expressing NK cells are administered three times over a 28-day cycle.
  • 1 ⁇ 109 CAR-expressing NK cells are administered three times over a 28-day cycle. In several embodiments, 1.5 ⁇ 109 CAR-expressing NK cells are administered three times over a 28-day cycle. In several embodiments, 2 ⁇ 109 CAR-expressing NK cells are administered three times over a 28-day cycle. In several embodiments, 2.5 ⁇ 109 CAR- expressing NK cells are administered three times over a 28-day cycle. In several embodiments, 3 ⁇ 109 CAR-expressing NK cells are administered three times over a 28-day cycle. In several embodiments, 3.5 ⁇ 109 CAR-expressing NK cells are administered three times over a 28-day cycle.
  • 4 ⁇ 109 CAR-expressing NK cells are administered three times over a 28-day cycle. In several embodiments, 4.5 ⁇ 109 CAR-expressing NK cells are administered three times over a 28-day cycle. In several embodiments, 5 ⁇ 109 CAR-expressing NK cells are administered three times over a 28-day cycle. [00236] In several embodiments, at least about 3 x 109 CAR-expressing NK cells are administered in a dosing cycle. In several embodiments, at least about 4.5 x 109 CAR-expressing NK cells are administered in a dosing cycle. In several embodiments, at least about 6 x 109 CAR-expressing NK cells are administered in a dosing cycle.
  • each dose of NK cells is administered between about 2-4 days apart.
  • a second dose of NK cells is administered about 2-4 days after administration of the first dose.
  • a third dose of NK cells is administered about 2-4 days after administration of the second dose.
  • a second dose of NK cells is administered about 2-4 days after administration of the first dose, and a third dose of NK cells is administered about 2-4 days after administration of the second dose.
  • a second dose of NK cells is administered about 3 days after administration of the first dose. In some embodiments, a third dose of NK cells is administered about 4 days after administration of the second dose. In some embodiments, a second dose of NK cells is administered about 3 days after administration of the first dose, and a third dose of NK cells is administered about 4 days after administration of the second dose.
  • a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 2-4 days after administration of the first dose, the third dose is administered about 2-4 days after administration of the second dose, and each dose comprises about 1 x 109 CAR- expressing NK cells.
  • a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 3 days after administration of the first dose, the third dose is administered about 4 days after administration of the second dose, and each dose comprises about 1 x 109 CAR-expressing NK cells.
  • a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 2-4 days after administration of the first dose, the third dose is administered about 2-4 days after administration of the second dose, and each dose comprises about 1.5 x 109 CAR- expressing NK cells.
  • a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 3 days after administration of the first dose, the third dose is administered about 4 days after administration of the second dose, and each dose comprises about 1.5 x 109 CAR-expressing NK cells.
  • a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 2-4 days after administration of the first dose, the third dose is administered about 2-4 days after administration of the second dose, and each dose comprises about 2 x 109 CAR- expressing NK cells.
  • a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 3 days after administration of the first dose, the third dose is administered about 4 days after administration of the second dose, and each dose comprises about 2 x 109 CAR-expressing NK cells.
  • the first dose is administered on the first day of the dosing cycle (e.g., Day 0)
  • the second dose is administered about 2 days after administration of the first dose (e.g., Day 2).
  • the second dose is administered about 3 days after administration of the first dose (e.g., Day 3).
  • the second dose is administered about 4 days after administration of the first dose (e.g., Day 4). In some embodiments, the second dose is administered on Day 2 of the dosing cycle. In some embodiments, the second dose is administered on Day 3 of the dosing cycle. In some embodiments, the second dose is administered on Day 4 of the dosing cycle. In several embodiments, the third dose is administered about 2 days after administration of the second dose. In several embodiments, the third dose is administered about 3 days after administration of the second dose. In several embodiments, the third dose is administered about 4 days after administration of the second dose (e.g., Day 7). In some embodiments, the third dose is administered on Day 4 of the dosing cycle.
  • the third dose is administered on Day 5 of the dosing cycle. In some embodiments, the third dose is administered on Day 6 of the dosing cycle. In some embodiments, the third dose is administered on Day 7 of the dosing cycle. [00243] In some embodiments, each dose is separated by between about 24 hours and about 72 hours. In some embodiments, each dose is separated by at least about 24 hours. In some embodiments, each dose is separated by about 24 hours. In some embodiments, each dose is separated by at least about 48 hours. In some embodiments, each dose is separated by about 48 hours. In some embodiments, each dose is separated by at least about 72 hours. In some embodiments, each dose is separated by about 72 hours.
  • the first dose is administered on the first day of the dosing cycle (e.g., Day 0), the second dose is administered 2-4 after administration of the first dose, and the third dose is administered 2-4 days after administration of the second dose.
  • the first dose is administered on the first day of the dosing cycle (e.g., Day 0)
  • the second dose is administered about 2 days after administration of the first dose (e.g., Day 2)
  • the third dose is administered about 2 days after administration of the second dose (e.g., Day 4).
  • about 1.5 x 109 NK cells are administered on Day 0, about 1.5 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 2, and about 1.5 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 4.
  • about 2 x 109 NK cells are administered on Day 0, about 2 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 2, and about 2 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 4.
  • about 2.5 x 109 NK cells are administered on Day 0, about 2.5 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 2, and about 2.5 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 4.
  • about 3 x 109 NK cells are administered on Day 0, about 3 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 2, and about 3 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 4.
  • the first dose is administered on the first day of the dosing cycle (e.g., Day 0)
  • the second dose is administered about 2 days after administration of the first dose (e.g., Day 2)
  • the third dose is administered about 3 days after administration of the second dose (e.g., Day 5).
  • about 1.5 x 109 NK cells e.g., CAR-expressing NK cells
  • about 1.5 x 109 NK cells e.g., CAR-expressing NK cells
  • about 1.5 x 109 NK cells are administered on Day 5.
  • about 2 x 109 NK cells are administered on Day 0, about 2 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 2, and about 2 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 5.
  • about 2.5 x 109 NK cells are administered on Day 0, about 2.5 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 2, and about 2.5 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 5.
  • about 3 x 109 NK cells are administered on Day 0
  • about 3 x 109 NK cells are administered on Day 2
  • about 3 x 109 NK cells are administered on Day 5.
  • the first dose is administered on the first day of the dosing cycle (e.g., Day 0)
  • the second dose is administered about 3 days after administration of the first dose (e.g., Day 3)
  • the third dose is administered about 2 days after administration of the second dose (e.g., Day 5).
  • about 1.5 x 109 NK cells are administered on Day 0, about 1.5 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 3, and about 1.5 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 5.
  • about 2 x 109 NK cells are administered on Day 0, about 2 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 3, and about 2 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 5.
  • about 2.5 x 109 NK cells are administered on Day 0, about 2.5 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 3, and about 2.5 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 5.
  • about 3 x 109 NK cells are administered on Day 0, about 3 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 3, and about 3 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 5.
  • the first dose is administered on the first day of the dosing cycle (e.g., Day 0)
  • the second dose is administered about 3 days after administration of the first dose (e.g., Day 3)
  • the third dose is administered about 3 days after administration of the second dose (e.g., Day 6).
  • about 1.5 x 109 NK cells e.g., CAR-expressing NK cells
  • about 1.5 x 109 NK cells are administered on Day 3
  • about 1.5 x 109 NK cells are administered on Day 6.
  • about 2 x 109 NK cells are administered on Day 0, about 2 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 3, and about 2 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 6.
  • about 2.5 x 109 NK cells are administered on Day 0, about 2.5 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 3, and about 2.5 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 6.
  • a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 2-4 days after administration of the first dose, the third dose is administered about 2-4 days after administration of the second dose, and each dose comprises about 2.5 x 109 CAR- expressing NK cells.
  • a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 3 days after administration of the first dose, the third dose is administered about 4 days after administration of the second dose, and each dose comprises about 2.5 x 109 CAR-expressing NK cells.
  • a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 2-4 days after administration of the first dose, the third dose is administered about 2-4 days after administration of the second dose, and each dose comprises about 3 x 109 CAR- expressing NK cells.
  • a dosing cycle comprises administration of three doses of NK cells (e.g., over a 28-day period), wherein the second dose is administered about 3 days after administration of the first dose, the third dose is administered about 4 days after administration of the second dose, and each dose comprises about 3 x 109 CAR-expressing NK cells.
  • about 1 x 109 NK cells e.g., CAR-expressing NK cells
  • about 1 x 109 NK cells are administered on Day 3
  • about 1 x 109 NK cells e.g., CAR-expressing NK cells
  • about 1.5 x 109 NK cells are administered on Day 0, about 1.5 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 3, and about 1.5 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 7.
  • about 2 x 109 NK cells are administered on Day 0, about 2 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 3, and about 2 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 7.
  • about 2.5 x 109 NK cells are administered on Day 0, about 2.5 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 3, and about 2.5 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 7.
  • about 3 x 109 NK cells are administered on Day 0, about 3 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 3, and about 3 x 109 NK cells (e.g., CAR-expressing NK cells) are administered on Day 7.
  • the first dose is administered on about Day 0 of the dosing cycle.
  • the second dose is administered on about Day 2 of the dosing cycle.
  • the second dose is administered on about Day 3 of the dosing cycle.
  • the third dose is administered on about Day 4 of the dosing cycle.
  • the third dose is administered on about Day 5 of the dosing cycle.
  • the third dose is administered on about Day 6 of the dosing cycle.
  • the third dose is administered on about Day 7 of the dosing cycle.
  • each dose is separated by between about 24 hours and about 72 hours. In some embodiments, each dose is separated by at least about 24 hours.
  • each dose is separated by about 24 hours. In some embodiments, each dose is separated by at least about 48 hours. In some embodiments, each dose is separated by about 48 hours. In some embodiments, each dose is separated by at least about 72 hours. In some embodiments, each dose is separated by about 72 hours. [00253] In some embodiments, the first dose is administered on about Day 0 of the dosing cycle, the second dose is administered on about Day 2 of the dosing cycle, and the third dose is administered on about Day 4 of the dosing cycle. In some embodiments, the first dose is administered on about Day 0 of the dosing cycle, the second dose is administered on about Day 2 of the dosing cycle, and the third dose is administered on about Day 5 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 3 of the dosing cycle
  • the third dose is administered on about Day 5 of the dosing cycle.
  • the first dose is administered on about Day 0 of the dosing cycle
  • the second dose is administered on about Day 3 of the dosing cycle
  • the third dose is administered on about Day 6 of the dosing cycle.
  • a dose of NK cells of the dosing cycle is administered on an outpatient basis.
  • two doses of NK cells of the dosing cycle are administered on an outpatient basis.
  • each dose of NK cells of the dosing cycle is administered on an outpatient basis.
  • the administration of engineered NK cells is preceded by one or more preparatory treatments.
  • the administration of engineered NK cells is preceded by lymphodepletion.
  • a subject is administered a lymphodepleting therapy prior to administration of a dosing cycle.
  • each dosing cycle is preceded by lymphodepletion.
  • a method of preparing a subject having a cancer for treatment with a composition comprising natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds to CD19, the method comprising administering a lymphodepleting therapy to the subject prior to administration of the composition to the subject.
  • NK natural killer
  • CAR chimeric antigen receptor
  • the lymphodepleting therapy comprises administration of cyclophosphamide and does not comprise administration of fludarabine.
  • a method of preparing a subject having a cancer for treatment with a composition comprising natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds to CD19 the method comprising administering a lymphodepleting therapy consisting of cyclophosphamide to the subject prior to administration of the composition to the subject.
  • NK natural killer
  • CAR chimeric antigen receptor
  • a combination of chemotherapeutic agents is used for lymphodepletion.
  • a single chemotherapeutic agent is used for lymphodepletion.
  • agents with different mechanisms of actions are optionally used.
  • different classes of agents are optionally used.
  • an antimetabolic agent is used.
  • the antimetabolic agent inhibits and/or prevents cell replication.
  • cyclophosphamide an alkylating agent that reduces tumor growth, is used in lymphodepletion.
  • the lymphodepletion comprises cyclophosphamide.
  • the lymphodepletion comprises cyclophosphamide and does not comprise fludarabine.
  • a method of preparing a subject having a cancer for treatment with a composition comprising natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds to CD19, the method comprising administering a lymphodepleting therapy consisting of cyclophosphamide to the subject prior to administration of the composition to the subject.
  • NK natural killer
  • CAR chimeric antigen receptor
  • a dose of between about 200 and 600 mg/m2 cyclophosphamide is administered, including doses of about 200 mg/m2, about 225 mg/m2, about 250 mg/m2, about 275 mg/m2, about 300 mg/m2, about 325 mg/m2, about 350 mg/m2, about 400 mg/m2, about 450 mg/m2, about 475 mg/m2, about 500 mg/m2, about 525 mg/m2, about 550 mg/m2, about 600 mg/m2, or about 700 mg/m2, or any dose between those listed.
  • a dose of about 300 mg/m2 cyclophosphamide is administered.
  • a dose of about 400 mg/m2 cyclophosphamide is administered. In several embodiments, a dose of about 500 mg/m2 cyclophosphamide is administered. In several embodiments, the dose of cyclophosphamide is given daily for days (e.g., prior to CAR-NK administration). In several embodiments, the dose of cyclophosphamide is given daily for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days (e.g., prior to CAR-NK administration). In several embodiments, the cyclophosphamide is given daily for 3 days. In several embodiments, if necessary, the dose can be split and given, for example, twice daily.
  • the additional agent inhibits one or more of DNA polymerase alpha, ribonucleotide reductase and/or DNA primase, thus inhibiting DNA synthesis.
  • the additional agent is fludarabine.
  • a dose of between about 5.0 mg/m2 – about 200 mg/m2 fludarabine is administered, including doses of about 5.0 mg/m2, about 10.0 mg/m2, about 15.0 mg/m2, about 20.0 mg/m2, about 25.0 mg/m2, about 30.0 mg/m2, about 35.0 mg/m2, about 40.0 mg/m2, about 45.0 mg/m2, about 50.0 mg/m2, about 60.0 mg/m2, about 70.0 mg/m2, about 80.0 mg/m2, about 90.0 mg/m2, about 100.0 mg/m2, about 125.0 mg/m2, about 150.0 mg/m2, about 175.0 mg/m2, about 200.0 mg/m2, or any dose between those listed.
  • a dose of about 10 mg/m2 fludarabine is administered. In several embodiments, a dose of about 20 mg/m2 fludarabine is administered. In several embodiments, a dose of about 30 mg/m2 fludarabine is administered. In several embodiments, a dose of about 40 mg/m2 fludarabine is administered. In several embodiments, a dose of about 50 mg/m2 fludarabine is administered. In several embodiments, a dose of about 100 mg/m2 fludarabine is administered. In several embodiments, the dose of fludarabine is given daily for at least about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days.
  • the dose of fludarabine is given daily for about 3 days. In several embodiments, about 30 mg/m2 fludarabine is given daily for about 3 days. In several embodiments, about 30 mg/m2 fludarabine is given daily for about 5 days. In several embodiments, if necessary, the dose can be split and given, for example, twice daily. [00261] In several embodiments, about 300 mg/m2 cyclophosphamide and about 30 mg/m2 fludarabine is each given daily for about 3 days. In several embodiments, prior to each dosing cycle, about 300 mg/m2 cyclophosphamide and about 30 mg/m2 fludarabine is each given daily for about 3 days.
  • two, three or four doses of a genetically engineered cell(s) described herein or composition thereof is administered to a subject every day, every couple of days, every third day, once a week or once every two weeks.
  • a dose(s) of a genetically engineered cell(s) described herein or composition thereof is administered for 2 days, 3 days, 5 days, 7 days, 14 days, 21 days, or 28 days.
  • a dose of a genetically engineered cell(s) described herein or composition thereof is administered for 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 months or more.
  • a dosing period is set and a certain number of doses is administered within that time period.
  • a subject receives a first dose on day 0 of the cycle, a second dose on day 3 of the cycle and a third dose on day 7 of the cycle.
  • a 28-day cycle is used with primary outcome measures evaluated at day 28.
  • lymphodepletion is performed prior to the inception of each dosing cycle, if subsequent dosing cycles are required (e.g., the subject requires further treatment).
  • a subject undergoes lymphodepletion, receives a plurality of doses of engineered cells according to a cycle, is evaluated at the end of the cycle time and, if deemed necessary undergoes a second lymphodepletion followed by a second dosing cycle.
  • a lymphodepleting therapy is only administered prior to the first dosing cycle.
  • fludarabine/cyclophosphamide is used to achieve lymphodepletion.
  • the lymphodepleting therapy comprises administration of cyclophosphamide (500 mg/m2) and fludarabine (30mg/m2), each administered daily for 5 days. Depending on the embodiment, different concentrations may be used.
  • a first and a second dosing cycle need not be the same (e.g., a first cycle may have 2 doses, while a second uses three doses).
  • the therapies and dosing regimens provided for herein provide effective anti-cancer treatment without certain CAR-T cell toxicities, such as cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS) or neurotoxicity, or graft-versus host disease.
  • CAR-T cell toxicities such as cytokine release syndrome, immune effector cell-associated neurotoxicity syndrome (ICANS) or neurotoxicity, or graft-versus host disease.
  • complete remission is achieved.
  • complete response (CR) is achieved.
  • partial response (PR) is achieved.
  • SD stable disease or limited progression of disease is accomplished.
  • Clinical outcomes can be assessed by any of the methods known in the art, including based on the Lugano classification with lymphoma response to immunomodulatory therapy criteria (LYRIC) refinement for subjects with non-Hodgkin lymphoma (NHL); the 2018 International Workshop on Chronic Lymphocytic Leukemia (iwCLL) guidelines for subjects with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL); Version 1.2020 National Comprehensive Cancer Network (NCCN) for subjects with B-cell acute lymphoblastic leukemia (B-ALL); or 6th International Workshop on Waldenström macroglobulinemia (WM) for subjects with WM.
  • NCL non-Hodgkin lymphoma
  • NCCN National Comprehensive Cancer Network
  • B-ALL B-cell acute lymphoblastic leukemia
  • WM Waldenström macroglobulinemia
  • nucleic acid and amino acid sequences that have sequence identity and/or homology of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% (and ranges therein) as compared with the respective nucleic acid or amino acid sequences of SEQ ID NOS: 1-41 (or combinations of two or more of SEQ ID NOS: 1-41) and that also exhibit one or more of the functions as compared with the respective SEQ ID NOS: 1-41 (or combinations of two or more of SEQ ID NOS: 1-41) including but not limited to, (i) enhanced proliferation, (ii) enhanced activation, (iii) enhanced cytotoxic activity against cells presenting ligands to which NK cells harboring receptors encoded by the nucleic acid and amino acid sequences bind, (iv) enhanced homing to tumor or infected sites, (v) reduced off target cytotoxic effects, (vi) enhanced secretion of immunostimulatory cytokines and
  • polynucleotides encoding the disclosed cytotoxic receptor complexes are mRNA. In some embodiments, the polynucleotide is DNA.
  • the polynucleotide is operably linked to at least one regulatory element for the expression of the cytotoxic receptor complex.
  • a vector comprising the polynucleotide encoding any of the polynucleotides provided for herein, wherein the polynucleotides are optionally operatively linked to at least one regulatory element for expression of a cytotoxic receptor complex.
  • the vector is a retrovirus.
  • engineered immune cells (such as NK and/or T cells) comprising the polynucleotide, vector, or cytotoxic receptor complexes as disclosed herein.
  • engineered NK cells comprising the polynucleotide, vector, or cytotoxic receptor complexes as disclosed herein.
  • compositions comprising a mixture of engineered immune cells (such as NK cells and/or engineered T cells), each population comprising the polynucleotide, vector, or cytotoxic receptor complexes as disclosed herein.
  • compositions comprising engineered NK cells comprising the polynucleotide, vector, or cytotoxic receptor complexes as disclosed herein.
  • the composition comprises a pharmaceutically acceptable carrier.
  • the composition comprises a cryopreservative. IV.
  • the subject has diffuse large B-cell lymphoma (DLBCL). In several embodiments, the subject has follicular lymphoma (FL). In several embodiments, the subject has high grade FL (e.g., FL grade 3b). In several embodiments, the subject has indolent lymphoma (IL). In several embodiments, the subject has grade 1, 2, or 3a FL. In several embodiments, the subject has marginal zone lymphoma (MZL). In several embodiments, the subject has mantle cell lymphoma (MCL). In several embodiments, the subject has B-cell acute lymphoblastic leukemia (B-ALL). In several embodiments, the subject has Waldenström macroglobulinemia (WM).
  • DLBCL diffuse large B-cell lymphoma
  • FL follicular lymphoma
  • FL follicular lymphoma
  • the subject has high grade FL (e.g., FL grade 3b).
  • the subject has indolent lymphoma (IL).
  • the subject has grade 1, 2,
  • the subject has Chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL). In several embodiments, the subject has CLL. In several embodiments, the subject has SLL. In several embodiments, the subject has primary mediastinal large B cell lymphoma (PMBCL). In some embodiments, the cancer is a relapsed/refractory (r/r) cancer.
  • the subject has marrow-localized disease (e.g., ⁇ 5% peripheral blasts without other evidence of extramedullary disease including lymphoblastic lymphoma). In some embodiments, the subject has ⁇ 5% peripheral blasts. In some embodiments, the subject has ⁇ 5% peripheral blasts.
  • the subject has r/r B-ALL. In some embodiments, the subject has r/r B-ALL with ⁇ 5% peripheral blasts. In some embodiments, the subject has r/r B-ALL with ⁇ 5% peripheral blasts. In some embodiments, the subject does not have evidence of extramedullary disease. In some embodiments, the subject does not have evidence of extramedullary disease. In some embodiments, the subject does not have evidence of extramedullary disease including lymphoblastic lymphoma. In some embodiments, the subject does not have other evidence of extramedullary disease including lymphoblastic lymphoma.
  • the subject has been treated with a previous line of therapy.
  • the subject is relapsed/refractory (R/R) to a previous line of therapy.
  • the previous line of therapy comprises one previous line of therapy.
  • the subject has MCL, the previous line of therapy is one previous line of therapy, and the one previous line of therapy is not CAR T cells.
  • the subject has WM, and the previous line of therapy is one previous line of therapy.
  • the previous line of therapy comprises two previous lines of therapy.
  • the previous line of therapy comprises three previous lines of therapy.
  • the previous line of therapy comprises four previous lines of therapy.
  • the subject did not respond to or relapsed within 12 months of completion of the prior line of therapy.
  • the subject did not respond to the prior line of therapy.
  • the subject relapsed within 12 months of completion of the prior line of therapy.
  • the subject has been treated with at least two lines of prior therapy.
  • the subject if the subject (i) has MCL, (ii) has not been previously treated with CAR T cells; or (iii) has WM, the subject has been previously treated with at least one prior line of therapy.
  • the previous line of therapy comprises an inhibitor of Bruton’s tyrosine kinase (BTKi).
  • the subject has been previously treated with a BTKi.
  • the subject has a cancer for which a BTKi is approved, and the previous line of therapy comprises a BTKi.
  • the subject is R/R to a BTKi.
  • the BTKi comprises ibrutinib.
  • the BTKi is ibrutinib. In some embodiments, the subject has been previously treated with ibrutinib. In some embodiments, the subject is R/R to ibrutinib. [00279] In some embodiments, the previous line of therapy comprises a tyrosine kinase inhibitor. In some embodiments, the subject has Philadelphia chromosome (Ph+) B- ALL and the previous line of therapy comprises a tyrosine kinase inhibitor. [00280] In some embodiments, the previous line of therapy comprises a Bcl-2 inhibitor. In some embodiments, the subject has been previously treated with a Bcl-2 inhibitor.
  • the subject has CLL or SLL, and the previous line of therapy comprises a Bcl-2 inhibitor.
  • the subject is R/R to a Bcl-2 inhibitor.
  • the Bcl-2 inhibitor comprises venetoclax.
  • the Bcl-2 inhibitor is venetoclax.
  • the subject has been previously treated with venetoclax.
  • the subject is R/R to venetoclax.
  • the previous line of therapy comprises a BTKi and a Bcl-2 inhibitor.
  • the subject has been previously treated with a BTKi and a Bcl-2 inhibitor.
  • the subject is R/R to a BTKi and a Bcl-2 inhibitor.
  • the BTKi is ibrutinib.
  • the Bcl-2 inhibitor is venetoclax.
  • the previous line of therapy comprises a CD20- targeted therapy and a cytotoxic chemotherapy (e.g., anthracycline).
  • the CD20-targeted therapy is an anti-CD20 antibody.
  • the anti-CD20 antibody is an anti-CD20 monoclonal antibody.
  • the anti-CD20 antibody comprises rituximab.
  • the anti-CD20 antibody is rituximab.
  • the cytotoxic chemotherapy comprises anthracycline. In some embodiments, the cytotoxic chemotherapy is anthracycline. In some embodiments, the subject has been previously treated with an anti-CD20 monoclonal antibody and a cytotoxic therapy (e.g., anthracycline). In some embodiments, the subject is R/R to an anti-CD20 monoclonal antibody and a cytotoxic therapy (e.g., anthracycline). In some embodiments, if the previous line of therapy comprises a CD20-targeted therapy, cells of the cancer are CD20+ (e.g., as assessed locally). [00283] In some embodiments, the previous line of therapy comprises a CD19- directed therapy.
  • the subject has been previously treated with a CD19- directed therapy.
  • the previous line of therapy comprises chimeric antigen receptor (CAR) T cells.
  • CAR CAR T cells
  • the subject has been previously treated with CAR T cells (CAR T exposed).
  • the subject has been previously treated with anti-CD19 CAR T cells.
  • the CAR T cells are autologous CAR T cells.
  • the subject has been previously treated with autologous CAR T cells.
  • the subject has been previously treated with autologous anti-CD19 CAR T cells.
  • if the previous line of therapy comprises a CD19-directed therapy cells of the cancer are CD19+ (e.g., as assessed locally).
  • the previous line of therapy does not comprise a CD19-directed therapy. In some embodiments, the subject has not been previously treated with a CD19-directed therapy. In some embodiments, the previous line of therapy does not comprise chimeric antigen receptor (CAR) T cells. In some embodiments, the subject has not been previously treated with CAR T cells (CAR T na ⁇ ve). In some embodiments, the subject has not been previously treated with autologous CAR T cells. In some embodiments, the subject has not been previously treated with anti-CD19 CAR T cells. In some embodiments, the subject has not been previously treated with autologous anti-CD19 CART T cells. [00285] In some embodiments, the subject is a human.
  • the subject is an adult. In some embodiments, the subject is at least 18 years of age. [00286] In some embodiments, the subject has an Eastern Cooperative Oncology Group Performance Status (ECOG) of 0, 1, or 2. In some embodiments, the subject has an Eastern Cooperative Oncology Group Performance Status (ECOG) of 0 or 1. In some embodiments, the subject has an ECOG of 0. In some embodiments, the subject has an ECOG of 1. In some embodiments, the subject has an ECOG of 2. [00287] In some embodiments, the subject has adequate organ function. In some embodiments, adequate organ function comprises a platelet count ⁇ 30,000/ ⁇ L. In some embodiments, the subject has a white blood cell count of less than or equal to 109/L.
  • adequate organ function comprises serum creatinine value ⁇ 1.5 ⁇ upper limit of normal (ULN). In some embodiments, adequate organ function comprises total bilirubin value ⁇ 1.5 ⁇ ULN or ⁇ 3.0 ⁇ ULN for subjects with hereditary benign hyperbilirubinemia. In some embodiments, adequate organ function comprises aspartate aminotransferase (AST)/serum glutamic-oxaloacetic transaminase (SGOT) value ⁇ 3 ⁇ ULN and alanine aminotransferase (ALT)/serum glutamic pyruvic transaminase (SGPT) value ⁇ 3 ⁇ ULN.
  • AST aspartate aminotransferase
  • SGOT aspartate aminotransferase
  • ALT alanine aminotransferase
  • SGPT alanine aminotransferase
  • adequate organ function comprises baseline international normalized ratio (INR) ⁇ 2 or activated partial thromboplastin time (aPTT) of ⁇ 2 times ULN. In some embodiments, adequate organ function comprises, the subject does not require oxygen therapy. [00288] In some embodiments, the subject does not have Burkitt lymphoma. In some embodiments, the subject does not have primary central nervous system (CNS) lymphoma. In some embodiments, the subject does not have Richter’s transformation. In some embodiments, the subject does not have Richter’s transformation to Hodgkin lymphoma. In some embodiments, the subject does not have any evidence of active CNS malignancy. V.
  • compositions and methods described herein relate to administering immune cells comprising a tumor-directed chimeric antigen receptor and/or tumor-directed chimeric receptor to a subject with cancer.
  • Cancers derived from B-cell lineages are a worldwide healthcare burden.
  • the cancer is a hematologic malignancy.
  • the cancer is a leukemia or a lymphoma.
  • the lymphoma is a double hit/expressor lymphoma.
  • the lymphoma is a triple hit/expressor lymphoma.
  • the cancer comprises Richter’s transformation.
  • various embodiments provided for herein include treatment or prevention of various malignancies, such as non-Hodgkin lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), mantle cell lymphoma, marginal zone lymphomas, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, primary central nervous system lymphoma, primary intraocular lymphoma.
  • the cancer is non- Hodgkin lymphoma.
  • the cancer is B-cell lymphoma. In some embodiments, the cancer is diffuse large B-cell lymphoma. In some embodiments, the cancer is follicular lymphoma. In some embodiments, the cancer is chronic lymphocytic leukemia. In some embodiments, the cancer is chronic myelogenous leukemia. In some embodiments, the cancer is mantle cell lymphoma. In some embodiments, the cancer is marginal zone lymphoma.
  • Additional types of cancer include, but are not limited to, Hodgkin lymphoma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), adrenocortical carcinoma, Kaposi sarcoma, lymphoma, gastrointestinal cancer, appendix cancer, central nervous system cancer, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain tumors (including but not limited to astrocytomas, spinal cord tumors, brain stem glioma, craniopharyngioma, ependymoblastoma, ependymoma, medulloblastoma, medulloepithelioma), breast cancer, bronchial tumors, cervical cancer, colon cancer, chronic myeloproliferative disorders, ductal carcinoma, endometrial cancer, esophageal cancer, gastric cancer, renal cell cancer, leukemia, oral cancer, nasopharyngeal cancer, liver cancer, lung cancer (
  • the cancer is a B cell-derived NHL, such as an aggressive large B cell lymphoma (LBCL).
  • the LBCL is diffuse large B cell lymphoma (DLBCL) not otherwise specified; high grade B cell lymphoma; DLBCL derived from follicular lymphoma (FL) (FL grade 3b); DLBCL derived from Richter’s transformation to DLBCL from chronic lymphocytic leukemia (CLL); primary mediastinal LBCL; and DLBCL derived from Waldenström macroglobulinemia (WM).
  • the cancer is a NHL.
  • the cancer is a LBCL.
  • the cancer is an aggressive LBCL.
  • the cancer is DLBCL. In some embodiments, the cancer is FL grade 3b. [00294] In some embodiments, the cancer is an indolent lymphoma (IL). In some embodiments, the IL is a low grade FL (FL grades 1, 2, and 3a), MCL, or MZL. In some embodiments, the IL is a low grade FL (FL grades 1, 2, and 3a). In some embodiments, the IL is MCL. In some embodiments, the IL is MZL. In some embodiments, the cancer is a low grade FL (FL grades 1, 2, and 3a). In some embodiments, the cancer is FL grade 1. In some embodiments, the cancer is FL grade 2. In some embodiments, the cancer is FL grade 3a.
  • IL indolent lymphoma
  • the IL is a low grade FL (FL grades 1, 2, and 3a), MCL, or MZL. In some embodiments, the IL is a low grade FL (FL grades 1, 2, and 3a). In some embodiments, the cancer is FL grade
  • the cancer is MCL. In some embodiments, the cancer is MZL. [00295] In some embodiments, the cancer is CLL or SLL. In some embodiments, the cancer is CLL. In some embodiments, the cancer is SLL. In some embodiments, the cancer is B-ALL. [00296] In some embodiments, the cancer is relapsed/refractory (R/R). In some embodiments, the cancer is R/R NHL. In some embodiments, the cancer is R/R LBCL. In some embodiments, the cancer is R/R CLL. In some embodiments, the cancer is R/R SLL. In some embodiments, the cancer is R/R B-ALL. [00297] In some embodiments, cells of the cancer express CD19.
  • cells of the cancer express CD19 at the time of administration of a dose (e.g., the first dose) of genetically engineered NK cells. Expression of CD19 can be determined by any methods known in the art, including by flow cytometry.
  • cells of the cancer express CD20.
  • cells of the cancer express CD20 at the time of administration of a dose (e.g., the first dose) of genetically engineered NK cells. Expression of CD20 can be determined by any methods known in the art, including by flow cytometry.
  • the cancer has been previously treated with CAR T cells (CAR T exposed). In some embodiments, the cancer is relapsed/refractory to CAR T cells.
  • the CAR T cells are anti-CD19 CAR T cells. In some embodiments, the CAR T cells are autologous CAR T cells. In some embodiments, the CAR T cells are autologous anti-CD19 CAR T cells. Thus, in some embodiments, the cancer has been previously treated with autologous anti-CD19 CAR T cells. In some embodiments, the cancer is R/R to autologous anti-CD19 CAR T cells. In some embodiments, the cancer has not been previously treated with CAR T cells (CAR T na ⁇ ve). In some embodiments, the cancer has not been previously treated with anti-CD19 CAR T cells, optionally autologous anti-CD19 CAR T cells.
  • the cancer is not R/R to anti-CD19 CAR T cells.
  • the cancer is aggressive LBCL that has not been previously treated with CAR T cells (CAR T na ⁇ ve), optionally autologous CAR T cells.
  • the cancer is aggressive LBCL that has not been previously treated with anti- CD19 CAR T cells (CAR T na ⁇ ve), optionally autologous anti-CD19 CAR T cells.
  • the cancer is a R/R LBCL that has not been previously treated with CAR T cells (CAR T na ⁇ ve), optionally autologous CAR T cells.
  • the cancer is a R/R LBCL that has not been previously treated with anti-CD19 CAR T cells (CAR T na ⁇ ve), optionally autologous anti-CD19 CAR T cells.
  • the cancer is a R/R NHL that has not been previously treated with CAR T cells (CAR T na ⁇ ve), optionally autologous CAR T cells.
  • the cancer is a R/R NHL that has not been previously treated with anti-CD19 CAR T cells (CAR T na ⁇ ve), optionally autologous anti-CD19 CAR T cells.
  • the cancer is MCL that has not been previously treated with CAR T cells (CAR T na ⁇ ve), optionally autologous CAR T cells. In some embodiments, the cancer is MCL that has not been previously treated with anti-CD19 CAR T cells (CAR T na ⁇ ve), optionally autologous anti-CD19 CAR T cells. In some embodiments, the cancer is an IL that has not been previously treated with CAR T cells (CAR T na ⁇ ve), optionally autologous CAR T cells. In some embodiments, the cancer is an IL that has not been previously treated with anti-CD19 CAR T cells (CAR T na ⁇ ve), optionally autologous anti-CD19 CAR T cells.
  • the cancer is B-ALL that has not been previously treated with CAR T cells (CAR T na ⁇ ve), optionally autologous CAR T cells. In some embodiments, the cancer is B-ALL that has not been previously treated with anti-CD19 CAR T cells (CAR T na ⁇ ve), optionally autologous anti-CD19 CAR T cells. [00301] In some embodiments, the cancer is aggressive LBCL that has been previously treated with CAR T cells (CAR T exposed), optionally autologous CAR T cells. In some embodiments, the cancer is aggressive LBCL that has been previously treated with anti- CD19 CAR T cells (CAR T exposed), optionally autologous anti-CD19 CAR T cells.
  • the cancer is a R/R LBCL that has been previously treated with CAR T cells (CAR T exposed), optionally autologous CAR T cells. In some embodiments, the cancer is a R/R LBCL that has been previously treated with anti-CD19 CAR T cells (CAR T exposed), optionally autologous anti-CD19 CAR T cells. In some embodiments, the cancer is a R/R NHL that has been previously treated with CAR T cells (CAR T exposed), optionally autologous CAR T cells. In some embodiments, the cancer is a R/R NHL that has been previously treated with anti-CD19 CAR T cells (CAR T exposed), optionally autologous anti-CD19 CAR T cells.
  • the cancer is MCL that has been previously treated with CAR T cells (CAR T exposed), optionally autologous CAR T cells. In some embodiments, the cancer is MCL that has been previously treated with anti-CD19 CAR T cells (CAR T exposed), optionally autologous anti-CD19 CAR T cells. In some embodiments, the cancer is an IL that has been previously treated with CAR T cells (CAR T exposed), optionally autologous CAR T cells. In some embodiments, the cancer is an IL that has been previously treated with anti-CD19 CAR T cells (CAR T exposed), optionally autologous anti-CD19 CAR T cells.
  • the cancer is B-ALL that has been previously treated with CAR T cells (CAR T exposed), optionally autologous CAR T cells. In some embodiments, the cancer is B-ALL that has been previously treated with anti-CD19 CAR T cells (CAR T exposed), optionally autologous anti- CD19 CAR T cells. [00302] In some embodiments, the cancer is a LBCL (e.g., DLBCL) that has been previously treated with an anti-CD20 monoclonal antibody and a cytotoxic chemotherapy (e.g., anthracycline).
  • a LBCL e.g., DLBCL
  • cytotoxic chemotherapy e.g., anthracycline
  • the cancer is a LBCL (e.g., DLBCL) that has been previously treated with an anti-CD20 monoclonal antibody and anthracycline.
  • the cancer is an IL that has been previously treated with an anti-CD20 monoclonal antibody and a cytotoxic chemotherapy (e.g., anthracycline).
  • the cancer is an IL that has been previously treated with an anti-CD20 monoclonal antibody and anthracycline.
  • the cancer is a MCL, CLL, SLL, or WM that has been previously treated with an inhibitor of Bruton’s tyrosine kinase (BTKi) (e.g., ibrutinib).
  • BTKi tyrosine kinase
  • the cancer is a MCL that has been previously treated with a BTKi (e.g., ibrutinib).
  • the cancer is a MCL that has been previously treated with a BTKi (e.g., ibrutinib) and anti-CD19 CAR T cells.
  • the cancer is a CLL that has been previously treated with a BTKi (e.g., ibrutinib).
  • the cancer is a SLL that has been previously treated with a BTKi (e.g., ibrutinib). In some embodiments, the cancer is a WM that has been previously treated with a BTKi (e.g., ibrutinib). [00304] In some embodiments, the cancer is a CLL or SLL that has been previously treated with a Bcl-2 inhibitor (e.g., venetoclax). In some embodiments, the cancer is a CLL that has been previously treated with a Bcl -2 inhibitor (e.g., venetoclax). In some embodiments, the cancer is a SLL that has been previously treated with a Bcl-2 inhibitor (e.g., venetoclax).
  • a Bcl-2 inhibitor e.g., venetoclax
  • the cancer is a CLL or SLL that has been previously treated with a Bcl-2 inhibitor (e.g., venetoclax) and a BTKi (e.g., ibrutinib).
  • a Bcl-2 inhibitor e.g., venetoclax
  • a BTKi e.g., ibrutinib
  • the cancer is a CLL that has been previously treated with a Bcl-2 inhibitor (e.g., venetoclax) and a BTKi (e.g., ibrutinib).
  • the cancer is a SLL that has been previously treated with a Bcl-2 inhibitor (e.g., venetoclax) and a BTKi (e.g., ibrutinib).
  • Fc region herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • the C-terminal lysine (Lys447) of the Fc region may or may not be present.
  • numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.
  • full length antibody is one which has been separated from a component of its natural environment.
  • an antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoretic (e.g. , SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g. , ion exchange or reverse phase HPLC).
  • electrophoretic e.g. , SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatographic e.g. , ion exchange or reverse phase HPLC.
  • An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.
  • An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.
  • isolated nucleic acid encoding an anti-CD19 antibody refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
  • host cell refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such nucleic acid molecule(s) in a single vector or separate vectors, and such nucleic acid molecule(s) present at one or more locations in a host cell.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages.
  • Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • 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 antibodies and antibody chains and other peptides, e.g. , linkers and CD19- binding peptides, may include amino acid residues including natural and/or non-natural amino acid residues.
  • 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.
  • percent (%) amino acid sequence identity and “percent identity” and “sequence identity” when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g.
  • an amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid.
  • Amino acid substitutions may be introduced into a binding molecule, e.g. , antibody, of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, or decreased immunogenicity.
  • Amino acids generally can be grouped according to the following common side- chain properties.
  • водород [00316] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; [00317] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; [00318] (3) acidic: Asp, Glu; [00319] (4) basic: His, Lys, Arg; [00320] (5) residues that influence chain orientation: Gly, Pro; [00321] (6) aromatic: Trp, Tyr, Phe. [00322] Non-conservative amino acid substitutions will involve exchanging a membrane of one of these classes for another class. [00323] 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.”
  • the term "package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
  • a “subject” is a mammal, such as a human or other animal, and typically is human.
  • the subject e.g., patient, to whom the agent or agents, cells, cell populations, or compositions are administered, is a mammal, typically a primate, such as a human.
  • 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.
  • the subject is a non-primate mammal, such as a rodent.
  • treatment 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, 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.
  • Preventing 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.
  • the provided cells and compositions are used to delay development of a disease or to slow the progression of a disease.
  • a “therapeutically effective amount” of an agent 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.
  • the provided methods involve administering the cells and/or compositions at effective amounts, e.g., therapeutically effective amounts.
  • 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, non-aqueous or any combination thereof.
  • 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.
  • 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 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.
  • 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.
  • a surface marker 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 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.
  • a method of treating a subject having a cancer comprising administering to the subject a first dosing cycle comprising a first dose of natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: the second dose is administered to the subject between 2-4 days after administration of the first dose to the subject; the third dose is administered to the subject between 2-4 days after administration of the second dose to the subject; and each of the first, second and third doses comprises at least about 1 ⁇ 10 9 CAR- expressing NK cells.
  • NK natural killer
  • CAR chimeric antigen receptor
  • a method of treating a subject having a cancer comprising administering to the subject a first dosing cycle comprising a first dose of natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: all three doses are administered to the subject within between about 4 days and about 10 days; and each of the first, second and third doses comprises at least about 1 ⁇ 10 9 CAR- expressing NK cells.
  • NK natural killer
  • CAR chimeric antigen receptor
  • a method of treating a subject having a cancer comprising administering to the subject a first dosing cycle comprising a first dose of natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: all three doses are administered to the subject within between about 4 days and about 10 days; the subject is administered a lymphodepleting therapy prior to administration of the first dosing cycle; and the lymphodepleting therapy comprises cyclophosphamide and does not comprise fludarabine. 4.
  • NK natural killer
  • CAR chimeric antigen receptor
  • a method of preparing a subject having a cancer for treatment with natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19 comprising administering a lymphodepleting therapy to the subject prior to administration of a first dosing cycle of the genetically engineered NK cells to the subject, wherein: the first dosing cycle comprises a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, each of the first, second and third doses comprises at least about 1 ⁇ 10 9 CAR- expressing NK cells; and all three doses are administered to the subject within between about 4 days and about 10 days. 5.
  • NK natural killer
  • CAR chimeric antigen receptor
  • a method of treating a subject having a cancer comprising administering to the subject a first dosing cycle comprising a first dose of natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: the second dose is administered to the subject between 2-4 days after administration of the first dose to the subject; the third dose is administered to the subject between 2-4 days after administration of the second dose to the subject; and each of the first, second and third doses comprises at least about 2 ⁇ 10 9 CAR- expressing NK cells.
  • each of the first, second and third doses comprises about 2 ⁇ 10 9 CAR-expressing NK cells. 10. The method of any one of Embodiments 1-6 and Embodiment 8, wherein each of the first, second and third doses comprises about 2.5 ⁇ 10 9 CAR-expressing NK cells. 11. The method of any one of Embodiments 1, 2, and 5-10, wherein the subject is administered a lymphodepleting therapy prior to administration of the first dosing cycle. 12. The method of any one of Embodiments 1-11 wherein the first dosing cycle is followed by an additional dosing cycle. 13.
  • the method of any one of Embodiments 1-12 wherein, if the subject exhibits a clinical response, optionally a complete response (CR), following the first dosing cycle, the method comprises an additional dosing cycle as consolidation treatment. 14.
  • the method of any one of Embodiments 1-13 wherein, if the subject exhibits a clinical response following a dosing cycle and subsequently exhibits disease progression, the method comprises an additional dosing cycle as retreatment.
  • the method of any one of Embodiments 1-14 wherein the method comprises between one dosing cycle and five dosing cycles. 16.
  • the method of any one of Embodiments 1-15 wherein the subject is administered a lymphodepleting therapy prior to each dosing cycle. 17.
  • Embodiment 20 wherein the single dose of cyclophosphamide is administered to the subject about 3 days prior to administration of the dosing cycle.
  • 22 The method of Embodiment 20 or Embodiment 21, wherein the single dose of cyclophosphamide is about 1000 mg/m 2 .
  • 23 The method of any one of Embodiments 3-7 and 9-19, wherein the lymphodepleting therapy comprises three doses of cyclophosphamide.
  • 24 The method of Embodiment 23, wherein each dose of cyclophosphamide is about 500 mg/m 2 . 25.
  • Embodiment 23 or Embodiment 24 wherein a dose of cyclophosphamide is administered to the subject on each of about 5 days prior, 4 days prior, and 3 days prior to administration of the first dose of the dosing cycle.
  • 26 The method of any one of Embodiments 4-7 and 9-25, wherein the lymphodepleting therapy does not comprise fludarabine.
  • 27 The method of any one of Embodiments 4-7 and 9-25, wherein the lymphodepleting therapy comprises fludarabine, optionally wherein a dose of fludarabine is between about 20 mg/m 2 and about 40 mg/m 2 .
  • the lymphodepleting therapy comprises three doses of fludarabine. 29.
  • each dose of fludarabine is about 30 mg/m 2 .
  • 30. The method of any one of Embodiments 27-29, wherein a dose of fludarabine is administered to the subject on each of about 5 days prior, 4 days prior, and 3 days prior to administration of the first dose of the dosing cycle.
  • 31. The method of any one of Embodiments 1-30, further comprising administration of a therapeutic agent that targets CD20.
  • 32. The method of any one of Embodiments 1-31, wherein the subject is administered a therapeutic agent that targets CD20.
  • 33. The method of Embodiment 31 or Embodiment 32, wherein the therapeutic agent is an anti-CD20 monoclonal antibody. 34.
  • Embodiment 33 wherein the anti-CD20 antibody is rituximab.
  • the therapeutic agent that targets CD20 is administered in an amount between about 150 mg/m 2 and about 500 mg/m 2 .
  • 36 The method of any one of Embodiments 31-35, wherein the therapeutic agent that targets CD20 is administered in an amount of about 375 mg/m 2 .
  • 37 The method of any one of Embodiments 31-36, wherein the therapeutic agent is administered to the subject at least one time and the at least one time is at least 2 days prior to administration of the first dose of the dosing cycle. 38.
  • a method of treating a subject having a cancer comprising administering to the subject a first dosing cycle comprising a first dose of natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: the second dose is administered to the subject about 3 days after administration of the first dose to the subject, the third dose is administered to the subject about 4 days after administration of the second dose to the subject; each of the first, second and third doses comprises between about 1.0 ⁇ 10 9 CAR- expressing NK cells and about 2 ⁇ 10 9 CAR-expressing NK cells; the subject is administered a lymphodepleting therapy prior to administration of the first dosing cycle; and the lymphodepleting therapy consists of a dose of about 500 mg/m 2 of cyclophosphamide on each of 5 days, 4 days, and 3 days prior to administration of the first dose of the dos
  • a method of treating a subject having a cancer comprising administering to the subject a first dosing cycle comprising a first dose of natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: the second dose is administered to the subject about 3 days after administration of the first dose to the subject, the third dose is administered to the subject about 4 days after administration of the second dose to the subject; each of the first, second and third doses comprises about 1.5 ⁇ 10 9 CAR-expressing NK cells; the subject is administered a lymphodepleting therapy comprising (i) a dose of about 500 mg/m 2 of cyclophosphamide on each of 5 days, 4 days, and 3 days prior to administration of the first dose of the dosing cycle and (ii) a dose of about 30 mg/m 2 of fludarabine on each of 5 days, 4 days, and 3
  • a method of treating a subject having a cancer comprising administering to the subject a first dosing cycle comprising a first dose of natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: the second dose is administered to the subject about 3 days after administration of the first dose to the subject, the third dose is administered to the subject about 4 days after administration of the second dose to the subject; each of the first, second and third doses comprises about 2 ⁇ 10 9 CAR-expressing NK cells or about 2.5 ⁇ 10 9 CAR-expressing NK cells; the subject is administered a lymphodepleting therapy comprising (i) a dose of about 500 mg/m 2 of cyclophosphamide on each of 5 days, 4 days, and 3 days prior to administration of the first dose of the dosing cycle and (ii) a dose of about 30 mg/m 2 of fluor
  • a method of preparing a subject having a cancer for treatment with a composition comprising natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds to CD19, the method comprising administering a lymphodepleting therapy to the subject prior to administration of the composition to the subject, wherein the lymphodepleting therapy consists of cyclophosphamide.
  • NK natural killer
  • CAR chimeric antigen receptor
  • LBCL large B-cell lymphoma
  • DLBCL diffuse large B-cell lymphoma
  • FL follicular lymphoma
  • MZL marginal zone lymphoma
  • MCL mantle cell lymphoma
  • WM Waldenström macroglobulinemia
  • B-ALL B-cell acute lymphoblastic leukemia
  • B-ALL B-cell acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • a line of previous therapy comprises an anti-CD20 monoclonal antibody and a cytotoxic chemotherapy, optionally wherein the cytotoxic therapy is anthracycline.
  • a line of previous therapy comprises chimeric antigen receptor-expressing T (CAR T) cells, optionally wherein a line of previous therapy comprises autologous anti-CD19 CAR T cells.
  • CAR T chimeric antigen receptor-expressing T
  • a line of previous therapy comprises autologous anti-CD19 CAR T cells.
  • a line of previous therapy does not comprise CAR T cells, optionally wherein a line of previous therapy does not comprise autologous anti-CD19 CAR T cells.
  • a line of previous therapy comprises an inhibitor of Bruton’s tyrosine kinase (BTKi), optionally wherein the BTKi is ibrutinib.
  • BTKi tyrosine kinase
  • a line of previous therapy comprises an inhibitor of Bcl-2, optionally wherein the Bcl-2 inhibitor is venetoclax.
  • the CAR comprises: (a) an antigen-binding moiety that targets CD19; (b) a transmembrane domain; and (c) an intracellular signaling domain comprising an OX40 domain and a CD3zeta domain.
  • the antigen-binding moiety comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the VH comprises a CDR-1, a CDR-2, and a CDR-3 comprising the amino acid sequences set forth in SEQ ID NOS: 24, 25, and 26, respectively; and the VL comprises a CDR-1, a CDR-2, and a CDR-3 comprising the amino acid sequences set forth in SEQ ID NOS: 27, 28, and 29, respectively; the VH comprises a CDR-1, a CDR-2, and a CDR-3 comprising the amino acid sequences set forth in SEQ ID NOS: 30, 31, and 32, respectively; and the VL comprises a CDR-1, a CDR-2, and a CDR-3 comprising the amino acid sequences set forth in SEQ ID NOS: 33, 34, and 29, respectively; the VH comprises the amino acid sequence set forth in SEQ ID NO: 35 and/or the VL comprises the amino acid
  • NK cells are genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19; and the NK cells are for administration as a first dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: the second dose is for administration to the subject between 2-4 days after administration of the first dose to the subject, the third dose is for administration to the subject between 2-4 days after administration of the second dose to the subject; and each of the first, second and third doses comprises at least about 1 ⁇ 10 9 CAR-expressing NK cells.
  • CAR chimeric antigen receptor
  • NK cells are genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19; and the NK cells are for administration as a first dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein all three doses are administered to the subject within between about 4 days and about 10 days; and each of the first, second and third doses comprises at least about 1 ⁇ 10 9 CAR- expressing NK cells.
  • CAR chimeric antigen receptor
  • NK cells are genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19; the NK cells are for administration as a first dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein all three doses are administered to the subject within between about 4 days and about 10 days; and the subject is administered a lymphodepleting therapy prior to administration of the first dosing cycle, wherein the lymphodepleting therapy comprises cyclophosphamide and does not comprise fludarabine. 78.
  • CAR chimeric antigen receptor
  • a lymphodepleting therapy in the manufacture of a medication for preparing a subject having a cancer for treatment with natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19
  • the lymphodepleting therapy is for administration to the subject prior to administration of a first dosing cycle of the genetically engineered NK cells to the subject, wherein: the first dosing cycle comprises a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, each of the first, second and third doses comprises at least about 1 ⁇ 10 9 CAR- expressing NK cells; and all three doses are for administration to the subject within between about 4 days and about 10 days.
  • NK natural killer
  • CAR chimeric antigen receptor
  • Embodiments 76-78 wherein the second dose is for administration to the subject between 2-4 days after administration of the first dose to the subject and the third dose is for administration to the subject between 2-4 days after administration of the second dose to the subject.
  • a lymphodepleting therapy for preparing a subject having a cancer for treatment with a composition comprising natural killer (NK) cells genetically engineered to express a chimeric antigen receptor (CAR) that binds to CD19, wherein the lymphodepleting therapy consists of cyclophosphamide.
  • NK natural killer
  • CAR chimeric antigen receptor
  • each of the first, second and third doses comprises at least about 1 ⁇ 10 9 CAR-expressing NK cells.
  • each of the first, second and third doses comprises at least about 1 ⁇ 10 9 CAR-expressing NK cells.
  • each of the first, second and third doses comprises about 1.5 ⁇ 10 9 CAR-expressing NK cells.
  • NK cells are genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19; and the NK cells are for administration as a first dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: the second dose is for administration to the subject between 2-4 days after administration of the first dose to the subject, the third dose is for administration to the subject between 2-4 days after administration of the second dose to the subject; and each of the first, second and third doses comprises at least about 2 ⁇ 10 9 CAR-expressing NK cells.
  • CAR chimeric antigen receptor
  • each of the first, second and third doses comprises about 2 ⁇ 10 9 CAR-expressing NK cells.
  • each of the first, second and third doses comprises about 2.5 ⁇ 10 9 CAR-expressing NK cells.
  • NK cells are genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19; and the NK cells are for administration as a first dosing cycle comprising a first dose of the genetically engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: the second dose is for administration to the subject between 2-4 days after administration of the first dose to the subject, the third dose is for administration to the subject between 2-4 days after administration of the second dose to the subject, and each of the first, second and third doses comprises between about 1.0 ⁇ 10 9 CAR-expressing NK cells and about 2 ⁇ 10 9 CAR-expressing NK cells; the subject is administered a lymphodepleting therapy prior to administration of the first dosing cycle; and the lymphodepleting therapy consists of a dose of about 500 mg/m 2 of
  • NK cells are genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19; the NK cells are for administration as a first dosing cycle comprising a first dose of the generally engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: the second dose is for administration to the subject about 3 days after administration of the first dose to the subject, the third dose is for administration to the subject about 4 days after administration of the second dose to the subject; each of the first, second and third doses comprises at least about 1 ⁇ 10 9 CAR-expressing NK cells; the subject was administered a lymphodepleting therapy comprising (i) a dose of about 500 mg/m 2 of cyclophosphamide on each of 5 days, 4 days, and 3 days prior to administration of the first dose of the dosing cycle and
  • CAR chimeric antigen receptor
  • each of the first, second and third doses comprises about 1.5 ⁇ 10 9 CAR-expressing NK cells.
  • NK natural killer
  • the NK cells are genetically engineered to express a chimeric antigen receptor (CAR) that binds CD19; the NK cells are for administration as a first dosing cycle comprising a first dose of the generally engineered NK cells, a second dose of the genetically engineered NK cells, and a third dose of genetically engineered NK cells, wherein: the second dose is for administration to the subject about 3 days after administration of the first dose to the subject, the third dose is for administration to the subject about 4 days after administration of the second dose to the subject; each of the first, second and third doses comprises about 2 ⁇ 10 9 CAR- expressing NK cells or about 2.5 ⁇ 10 9 CAR-expressing NK cells; the subject
  • Example 1 Administration of anti-CD19 CAR-expressing NK cells to subjects with cancer
  • Therapeutic anti-CD19 CAR-expressing NK cell compositions (CD19 CAR NK cells) were administered to subjects with cancer (e.g., B-cell malignancies) in accordance with a non-limiting dosing regimen.
  • Primary NK cells were isolated by immunoaffinity-based selection from leukapheresis samples from healthy donors and cultured in the presence of a stimulatory cell line. Isolated NK cells were subsequently transduced with a viral vector (e.g., retroviral vector) encoding a non-limiting example of a CD19-directed CAR (see Figure 1), expanded in culture, and cryopreserved.
  • a viral vector e.g., retroviral vector
  • the CD19-directed CAR contains an extracellular anti-CD19 scFv (e.g., SEQ ID NO:37), a CD8alpha hinge (e.g., SEQ ID NO: 6) and transmembrane domain (e.g., SEQ ID NO:8), and an intracellular signaling domain containing an OX40 co-stimulatory signaling region (e.g., SEQ ID NO:14) and a CD3zeta signaling domain (e.g., SEQ ID NO:18).
  • SEQ ID NO:37 extracellular anti-CD19 scFv
  • a CD8alpha hinge e.g., SEQ ID NO: 6
  • transmembrane domain e.g., SEQ ID NO:8
  • an intracellular signaling domain containing an OX40 co-stimulatory signaling region e.g., SEQ ID NO:14
  • CD3zeta signaling domain e.g., SEQ ID NO:18
  • the viral vector further contains a sequence encoding a membrane-bound interleukin-15 (mbIL15; e.g., SEQ ID NO:40), which is separated from the CAR-encoding sequence by a sequence encoding a T2A ribosomal skip sequence (e.g., SEQ ID NO:20, encoded by SEQ ID NO:19).
  • mbIL15 membrane-bound interleukin-15
  • T2A ribosomal skip sequence e.g., SEQ ID NO:20, encoded by SEQ ID NO:19.
  • B-cell malignancies including large B-cell lymphoma (LBCL, including diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma grade 3b (FL3b)), mantle cell lymphoma (MCL), follicular lymphoma (FL) grades 1, 2, and 3a, and marginal zone lymphoma (MZL); have received two or more prior lines of therapy; have an ECOG status of 0-2; and are either na ⁇ ve or exposed to CD19 CAR T cell therapy.
  • Subjects were administered a lymphodepleting (LD) therapy prior to administration of the dosing cycle.
  • LD lymphodepleting
  • the LD therapy consists of either 500 mg/m2 Cy on each of Days -3, -4, and -5 ( Figure 2A) or 1000 mg/m2 Cy on Day -3 ( Figure 2B), the Cy alone or in combination with 30 mg/m2 fludarabine (Flu) on each of Days -5, -4, and -3.
  • the CD19 CAR NK cells were administered beginning on Day 0 of a 28-day dosing cycle.
  • subjects are administered a dose of 1.5 x 109, 2 x 109, or 2.5 x 109 CD19 CAR NK cells on each of Days 0, 3, and 7.
  • subjects are administered a single dose of 375 mg/m2 rituximab on Day -3.
  • Primary endpoints may include any of the following: (1) incidence, nature, and severity of treatment related adverse events will be evaluated with an adverse event defined as any unfavorable and unintended sign including clinically significant abnormal laboratory findings, symptom or disease measured (e.g., 30 days) after the last dose of the NK cells; and (2) proportion of subjects experiencing dose-limiting toxicities of the NK cells.
  • Secondary outcome measures may include any of the following: (1) pharmacokinetic parameters in the context of the immune system, including but not limited to maximum concentration (Cmax), time to reach maximum concentration (Tmax), area under the concentration-time curve (AUC), half-life (t1/2), and duration of persistence of the CD19 CAR-NK cells in the peripheral blood and other target tissues such as bone marrow; (2) humoral and cellular immunogenicity against the CD19 CAR NK cells; (3) changes in serum cytokine levels such as interferon-gamma (IFN- ⁇ ) and other host responses to CD19 CAR NK cells in peripheral; (4) best overall response rates in dose finding and safety lead-in cohorts; and/or (5) other antitumor measurements, which may include duration of response (DOR), time-to-first response, time-to-best response, bridge-to-transplant rate, event-free survival (EFS), progression free survival (PFS), and overall survival (OS) using standard disease specific response assessment criteria.
  • Cmax maximum concentration
  • Tmax time to reach maximum concentration

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Abstract

Plusieurs modes de réalisation de la présente divulgation concernent des méthodes et des compositions comprenant des cellules génétiquement modifiées pour l'immunothérapie anticancéreuse. Dans plusieurs modes de réalisation, la présente divulgation concerne des cellules modifiées pour exprimer des récepteurs antigéniques chimériques ciblant CD19, ainsi que l'administration de telles cellules conformément à certains schémas posologiques.
PCT/US2024/035555 2023-06-27 2024-06-26 Méthodes de traitement utilisant des immunothérapies ciblant cd19 Pending WO2025006561A2 (fr)

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