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WO2023064872A1 - Associations de lymphocytes t car anti-bcma et d'inhibiteurs de gamma secrétase - Google Patents

Associations de lymphocytes t car anti-bcma et d'inhibiteurs de gamma secrétase Download PDF

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Publication number
WO2023064872A1
WO2023064872A1 PCT/US2022/078065 US2022078065W WO2023064872A1 WO 2023064872 A1 WO2023064872 A1 WO 2023064872A1 US 2022078065 W US2022078065 W US 2022078065W WO 2023064872 A1 WO2023064872 A1 WO 2023064872A1
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dose
pharmaceutical composition
cells
administered
seq
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PCT/US2022/078065
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Shinta CHENG
Christopher Ryan HEERY
Mark Johnson
Todd Shearer
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Precision Biosciences, Inc.
Springworks Therapeutics, Inc.
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Publication of WO2023064872A1 publication Critical patent/WO2023064872A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • 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 A61K39/46
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/417—Imidazole-alkylamines, e.g. histamine, phentolamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464416—Receptors for cytokines
    • A61K39/464417—Receptors for tumor necrosis factors [TNF], e.g. lymphotoxin receptor [LTR], CD30
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—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 NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present disclosure provides antibodies, or fragments thereof, having specificity for human B cell maturation antigen (BCMA), pharmaceutical compositions thereof, and uses thereof. Also provided are chimeric antigen receptors (CARs) comprising said antibodies or antibody fragments, genetically-modified cells comprising such CARs, pharmaceutical compositions comprising such cells, methods for making such cells, and methods of using such cells for the treatment of disorders and diseases such as cancer.
  • BCMA human B cell maturation antigen
  • CARs chimeric antigen receptors
  • MM Multiple myeloma
  • MM Multiple myeloma
  • hematopoietic stem cell transplantation along with newer drugs such as thalidomide and proteasome inhibitors often induces an initial remission, however, the tumor relapse due to chemoresistance remains a major problem.
  • BCMA B-cell maturation antigen
  • TNFR tumor necrosis family receptor
  • BCMA expression is the highest on terminally differentiated B cells. BCMA is involved in mediating the survival of plasma cells for maintaining long-term humoral immunity.
  • the expression of BCMA has been linked to a number of cancers, autoimmune disorders, and infectious diseases. Cancers with increased expression of BCMA include some hematological cancers, such as multiple myeloma, Hodgkin’s and non-Hodgkin’s lymphoma, various leukemias, and glioblastoma. Given the significant role for BCMA in diseases such as multiple myeloma, antibodies that recognize BCMA, and methods of using such agents, are desired.
  • GSI gamma secretase inhibitor
  • BCMA-specific chimeric antigen receptors a combination of a gamma secretase inhibitor (GSI) and genetically modified cells expressing BCMA-specific chimeric antigen receptors.
  • GSI gamma secretase inhibitor
  • Gamma secretase catalyzes the cleavage of membrane-bound BCMA, resulting in the release of the extracellular domain of membrane-bound BCMA as soluble BCMA.
  • Inhibition of gamma secretase activity by GSIs prevents this conversion, thereby decreasing the amount of soluble BCMA in the bloodstream and increasing the amount of membrane-bound BCMA expressed on the surface of BCMA+ cells.
  • Soluble BCMA in the bloodstream can be bound by BCMA- specific CAR T cells, preventing them from binding to BCMA+ cancer cells. Furthermore, cancer cells expressing greater amounts of membrane-bound BCMA are more efficiently recognized by anti-BCMA antibodies and BCMA-specific CAR T cells. Therefore, enhancing membrane-bound BCMA and reducing soluble BCMA through the administration of a gamma secretase inhibitor, such as PF-03084014 (Nirogacestat), enhances the efficacy of BCMA-specific CAR T cells for the treatment of BCMA+ cancers such as multiple myeloma.
  • a gamma secretase inhibitor such as PF-03084014 (Nirogacestat
  • the present disclosure provides a method of reducing the number of cancer cells in a subject, the method comprising: i) administering to the subject a lymphodepletion regimen comprising one or more chemotherapeutic lymphodepletion agents; ii) administering to the subject an effective dose of a first pharmaceutical composition comprising a population of human immune cells, wherein a plurality of the human immune cells are genetically-modified human immune cells that express a chimeric antigen receptor (CAR), wherein the lymphodepletion regimen is administered prior to administration of the first pharmaceutical composition, and wherein the CAR comprises: a) an anti-human BCMA- binding domain (i.e., a domain that specifically binds to human BCMA); b) a transmembrane domain; and c) an intracellular signaling domain; and iii) a second pharmaceutical composition comprising an effective dose of a gamma secretase inhibitor; wherein the method causes the death of one or more cancer cells in the subject,
  • the CDRH1 domain comprises an amino acid sequence set forth in SEQ ID NO: 7; the CDRH2 domain comprises an amino acid sequence set forth in SEQ ID NO: 8; the CDRH3 domain comprises an amino acid sequence set forth in SEQ ID NO: 9; the CDRL1 domain comprises an amino acid sequence set forth in SEQ ID NO: 4; the CDRL2 domain comprises an amino acid sequence set forth in SEQ ID NO: 5; and the CDRL3 domain comprises an amino acid sequence set forth in SEQ ID NO: 6.
  • the VH region comprises an amino acid sequence set forth in SEQ ID NO: 3, and the VL region comprises an amino acid sequence set forth in SEQ ID NO: 2.
  • the VH region is derived from the BCMA-20 antibody. In some embodiments, the VL region derived from the BCMA-3 antibody.
  • the antibody, or antigen-binding fragment thereof is a singlechain variable fragment (scFv).
  • the scFv comprises a linker connecting the VH region and the VL region.
  • the VH region, the VL region, and the linker have an N- terminal-to-C-terminal orientation of VH region-linker- VL region. In some embodiments, the VH region, the VL region, and the linker have an N-terminal-to-C -terminal orientation of VL region-linker- VH region.
  • the scFv comprises an amino acid sequence having at least about 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to a sequence set forth in either of SEQ ID NOs: 43 or 44.
  • the scFv comprises an amino acid sequence set forth in either of SEQ ID NOs: 43 or 44.
  • the transmembrane domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to a sequence set forth in SEQ ID NO: 30.
  • the transmembrane domain comprises an amino acid sequence set forth in SEQ ID NO: 30.
  • the intracellular signaling domain comprises a co- stimulatory domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to a sequence set forth in SEQ ID NO:
  • the intracellular signaling domain comprises a co-stimulatory domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to a sequence set forth in SEQ ID NO:
  • the intracellular signaling domain comprises a co-stimulatory domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to a sequence set forth in SEQ ID NO:
  • the intracellular signaling domain comprises a co-stimulatory domain comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to a sequence set forth in SEQ ID NO:
  • the intracellular signaling domain comprises a co-stimulatory domain comprising an amino acid sequence set forth in SEQ ID NO: 31. In some embodiments, the intracellular signaling domain comprises a co-stimulatory domain comprising an amino acid sequence set forth in SEQ ID NO: 32. In some embodiments, the intracellular signaling domain comprises a co-stimulatory domain comprising an amino acid sequence set forth in SEQ ID NO: 33. In some embodiments, the intracellular signaling domain comprises a co-stimulatory domain comprising an amino acid sequence set forth in SEQ ID NO: 34.
  • the intracellular signaling domain comprises a signaling domain having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to a sequence set forth in SEQ ID NO: 35.
  • the intracellular signaling domain comprises a signaling domain having an amino acid sequence set forth in SEQ ID NO: 35.
  • the CAR comprises a hinge domain connecting the BCMA- binding domain to the transmembrane domain.
  • the hinge domain comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to a sequence set forth in SEQ ID NO: 29.
  • the hinge domain comprises an amino acid sequence set forth in SEQ ID NO: 29.
  • the human immune cells are selected from the group consisting of T cells, natural killer (NK) cells, B cells, or macrophages. In some embodiments, the human immune cells are T cells.
  • the genetically-modified human immune cells comprise an inactivated T cell receptor (TCR) alpha gene. In some embodiments, the genetically- modified human immune cells comprise an inactivated TCR alpha constant region (TRAC) gene. In some embodiments, the genetically-modified human immune cells comprise an inactivated TCR beta gene.
  • TCR T cell receptor
  • TCR alpha constant region TCR alpha constant region
  • the genetically-modified human immune cells comprise in their genome a polynucleotide comprising a nucleic acid sequence encoding the CAR.
  • the polynucleotide is positioned within the TCR alpha gene, the TRAC gene (or TRAC region), or the TCR beta gene, such that expression of a polypeptide encoded by the TCR alpha gene, the TRAC gene, or the TCR beta gene is disrupted.
  • the polynucleotide is positioned within SEQ ID NO: 40 within the TRAC gene. In some embodiments, the polynucleotide is positioned between nucleotides 13 and 14 of SEQ ID NO: 40.
  • the human immune cells are not derived from the subject.
  • the subject is refractory to prior CAR T cell immunotherapy.
  • the lymphodepletion regimen does not include administration of a biological lymphodepletion agent during the 7-day period preceding administration of the first pharmaceutical composition.
  • the one or more chemotherapeutic agents comprises cyclophosphamide and/or fludarabine.
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 400 to about 1500 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 500 to about 1000 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 500 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering fludarabine at a dose of about 10 to about 40 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering fludarabine at a dose of about 30 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 500 mg/m 2 /day and fludarabine at a dose of about 30 mg/m 2 /day.
  • the lymphodepletion regimen is administered to the subject once daily starting 5 days and ending 3 days prior to administration of the first pharmaceutical composition.
  • the lymphodepletion regimen comprises administering cyclophosphamide once daily at a dose of about 500 mg/m 2 /day and fludarabine once daily at a dose of about 30 mg/m 2 /day, starting 5 days and ending 3 days prior to administration of the first pharmaceutical composition.
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of between about 500 and 1500 mg/m 2 /day and fludarabine at a dose of about 30 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 1000 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 1000 mg/m 2 /day and fludarabine at a dose of about 30 mg/m 2 /day.
  • the lymphodepletion regimen is administered to the subject once daily for at least one day, or for multiple days, within 7 days prior to administration of the first pharmaceutical composition.
  • the lymphodepletion regimen comprises administering cyclophosphamide once daily, starting 5 days and ending 3 days prior to administration of the pharmaceutical composition, and administering fludarabine once daily, starting 6 days and ending 3 days prior to administration of the first pharmaceutical composition.
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 1000 mg/m 2 /day once daily, starting 5 days and ending 3 days prior to administration of the pharmaceutical composition, and administering fludarabine at a dose of about 30 mg/m 2 /day once daily, starting 6 days and ending 3 days prior to administration of the first pharmaceutical composition.
  • the first pharmaceutical composition is administered at a dose of between about 0.1 x 10 6 and about 6 xlO 6 CAR T cells/kg.
  • the first pharmaceutical composition is administered at a dose of between about 0.6 x 10 6 and about 2 x 10 6 CAR T cells/kg.
  • the first pharmaceutical composition is administered at a dose of about 0.6 x 10 6 CAR T cells/kg.
  • the first pharmaceutical composition is administered at a dose of about 2 x 10 6 CAR T cells/kg.
  • the first pharmaceutical composition is administered at a dose of about 480 x 10 6 CAR T cells.
  • the first pharmaceutical composition is administered at a dose of about 960 x 10 6 CAR T cells.
  • the second pharmaceutical composition is administered at a dose of about 20 mg to about 300 mg of the gamma secretase inhibitor.
  • the second pharmaceutical composition is administered at a dose of about 50 mg to about 150 mg of the gamma secretase inhibitor.
  • the second pharmaceutical composition is administered at a dose of about 100 mg of the gamma secretase inhibitor.
  • the second pharmaceutical composition is administered orally.
  • the second pharmaceutical composition is administered once, twice, three times, or four times daily.
  • the second pharmaceutical composition is administered twice daily.
  • At least one dose of the second pharmaceutical composition is administered daily for at least one day prior to administration of the first pharmceutical composition. In some embodiments, at least one dose of the second pharmaceutical composition is administered daily for between 2 days and 7 days prior to administration of the first pharmceutical composition.
  • At least one dose of the second pharmaceutical composition is administered daily for between 3 days and 5 days prior to administration of the first pharmceutical composition.
  • At least one dose of the second pharmaceutical composition is administered daily for 3 days prior to administration of the first pharmceutical composition.
  • At least one dose of the second pharmaceutical composition is administered daily for at least 7 days after administration of the first pharmceutical composition.
  • At least one dose of the second pharmaceutical composition is administered daily for up to about 90 days after administration of the first pharmceutical composition.
  • At least one dose of the second pharmaceutical composition is administered daily for between about 7 days and about 90 days after administration of the first pharmceutical composition.
  • At least one dose of the second pharmaceutical composition is administered daily for between about 20 days and about 80 days after administration of the first pharmceutical composition.
  • At least one dose of the second pharmaceutical composition is administered daily for between about 50 days and about 70 days after administration of the first pharmceutical composition.
  • At least one dose of the second pharmaceutical composition is administered daily for about 60 days after administration of the first pharmceutical composition.
  • the second pharmaceutical composition is administered twice daily beginning 3 days prior to administration of the first pharmaceutical composition and ending about 60 days after administration of the first pharmaceutical composition, wherein the second pharmaceutical composition is administered at a dose of about 100 mg of the gamma secreatase inhibitor.
  • the gamma secretase inhibitor is selected from the group consisting of PF-03084014 (Nirogacestat), BMS-708163 (Avagacestat), RO4929097, MK0752, and LY3039478. In some embodiments, the gamma secretase inhibitor is PF- 03084014 (Nirogacestat).
  • the lymphodepletion regimen comprises administering cyclophosphamide once daily at a dose of about 500 mg/m 2 /day and fludarabine once daily at a dose of about 30 mg/m 2 /day starting 5 days and ending 3 days prior to administration of the first pharmaceutical composition, wherein the first pharmaceutical composition is administered at a dose of about 0.6 x 10 6 , 2 x 10 6 CAR T cells/kg, about 480 x 10 6 CAR T cells, or about 960 x 10 6 CAR T cells, wherein the second pharmaceutical composition is administered twice daily beginning 3 days prior to administration of the first pharmaceutical composition and ending about 60 days after administration of the first pharmaceutical composition, and wherein the second pharmaceutical composition is administered at a dose of about 100 mg of the gamma secreatase inhibitor.
  • the first pharmaceutical composition is administered at a dose of about 0.6 x 10 6 CAR T cells/kg.
  • the first pharmaceutical composition is administered at a dose of about 2 x 10 6 CAR T cells/kg.
  • the first pharmaceutical composition is administered at a dose of about 480 x 10 6 CAR T cells.
  • the first pharmaceutical composition is administered at a dose of about 960 x 10 6 CAR T cells.
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 1000 mg/m 2 /day once daily starting 5 days and ending 3 days prior to administration of the pharmaceutical composition, and administering fludarabine at a dose of about 30 mg/m 2 /day once daily starting 6 days and ending 3 days prior to administration of the first pharmaceutical composition, wherein the first pharmaceutical composition is administered at a dose of about 0.6 x 10 6 , 2 x 10 6 CAR T cells/kg, about 480 x 10 6 CAR T cells, or about 960 x 10 6 CAR T cells, wherein the second pharmaceutical composition is administered twice daily beginning 3 days prior to administration of the first pharmaceutical composition and ending about 60 days after administration of the first pharmaceutical composition, and wherein the second pharmaceutical composition is administered at a dose of about 100 mg of the gamma secreatase inhibitor.
  • the first pharmaceutical composition is administered at a dose of about 0.6 x 10 6 CAR T cells/kg. In some embodiments, the first pharmaceutical composition is administered at a dose of about 2 x 10 6 CAR T cells/kg.
  • the first pharmaceutical composition is administered at a dose of about 480 x 10 6 CAR T cells.
  • the first pharmaceutical composition is administered at a dose of about 960 x 10 6 CAR T cells.
  • the method increases an amount of membrane-bound BCMA on the surface of a cancer cell in the subject.
  • the method increases an amount of membrane-bound BCMA by a factor of at least 1.25, 1.5, 1.75, 2.00, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 15.0, or up to 20.0.
  • the method reduces the concentration of soluble BCMA in the bloodstream of the subject.
  • the method reduces the concentration of soluble BCMA in the bloodstream of the subject by a factor of at least 1.25, 1.5, 1.75, 2.00, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 15.0, or up to 20.0.
  • the method reduces a size of a tumor in a subject.
  • the method eradicates or eliminates a cancer in the subject.
  • the cancer is multiple myeloma.
  • the subject is administered an additional cancer therapy selected from the group consisting of chemotherapy, surgery, radiation, and gene therapy.
  • FIGs. 1A and IB Graph showing Nirogacestat does not affect MM. IS cell viability. MM. IS cells were cultured in the presence or absence of increasing concentrations of nirogacestat (Niro). MM. IS cells were dosed with nirogacestat either once (FIG. 1A) or once a day for 7 days (FIG. IB) and viable cell numbers were determined at 48, 96, and 168 hours.
  • FIGs. 2A and 2B Graphs showing Nirogacestat does not affect U266 cell viability.
  • U266 cells were cultured in the presence or absence of increasing concentrations of nirogacestat (Niro). U266 cells were dosed with nirogacestat either once (FIG. 2A) or once a day for 7 days (FIG. 2B) and viable cell numbers were determined at 48, 96, and 168 hours.
  • FIGs. 3A-3D Graphs showing Nirogacestat increases membrane-bound BCMA on MM cells in culture.
  • MM. IS and U266 cells were cultured in the presence or absence of increasing concentrations of nirogacestat (Niro). Cells were dosed with nirogacestat either once (FIG. 3 A, FIG. 3C) or once a day for 7 days (FIG. 3B, FIG. 3 D) and membrane-bound BCMA levels were determined at 48, 96, and 168 hours.
  • BCMA B-cell maturation antigen
  • MFI mean fluorescence intensity
  • Niro nirogacestat.
  • FIG. 4 Graph showing Nirogacestat decreases soluble BCMA in MM cell culture supernatants.
  • MM. IS cells were cultured in the presence or absence of increasing concentrations of nirogacestat. Cells were dosed with nirogacestat either once or once a day for 7 days and soluble BCMA levels were determined at 48, 96, and 168 hours.
  • FIGs. 6A and 6B Graphs showing PBCAR269A exhibits sustained MM. IS target cell cytotoxicity in the presence of nirogacestat. Two different lots of PBCAR269A CAR T cells were co-cultured with MM. IS cells in the presence or absence of nirogacestat. Culture medium was replaced daily to ensure continuous exposure to nirogacestat. Viable CD 138+ cells were quantified on Days 3, 6, 9, 13, and 16.
  • FIG. 7. Shows the study design. All study subjects who receive a dose of PBCAR269A will be followed in a separate long-term follow-up (LTFU) study (after exiting this study (due to either discontinuation or completion).
  • LTFU long-term follow-up
  • FIG. 8 Graph showing quantification of PBCAR269A cell expansion as measured in peripheral blood samples over time in subjects receiving PBCAR269A alone (Cohort A) at DL2 or DL4, or in subjects receiving PBCAR269A with nirogacestat (Cohort B) at DL2.
  • SEQ ID NO: 1 sets forth the amino acid sequence of human BCMA.
  • SEQ ID NO: 2 sets forth the amino acid sequence of the BCMA-3 antibody VL region.
  • SEQ ID NO: 3 sets forth the amino acid sequence of the BCMA-20 antibody VH region.
  • SEQ ID NO: 4 sets forth the amino acid sequence of the BCMA-3 antibody CDRL1 domain.
  • SEQ ID NO: 5 sets forth the amino acid sequence of the BCMA-3 antibody CDRL2 domain.
  • SEQ ID NO: 6 sets forth the amino acid sequence of the BCMA-3 antibody CDRL3 domain.
  • SEQ ID NO: 7 sets forth the amino acid sequence of the BCMA-20 antibody CDRH1 domain.
  • SEQ ID NO: 8 sets forth the amino acid sequence of the BCMA-20 antibody CDRH2 domain.
  • SEQ ID NO: 9 sets forth the amino acid sequence of the BCMA-20 antibody CDRH3 domain.
  • SEQ ID NO: 10 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 11 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 12 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 13 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 14 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 15 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 16 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 17 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 18 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 19 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 20 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 21 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 22 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 23 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 24 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 25 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 26 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 27 sets forth the amino acid sequence of a polypeptide linker.
  • SEQ ID NO: 28 sets forth the amino acid sequence of a spacer sequence.
  • SEQ ID NO: 29 sets forth the amino acid sequence of a CD8 hinge domain.
  • SEQ ID NO: 30 sets forth the amino acid sequence of CD8 transmembrane.
  • SEQ ID NO: 31 sets forth the amino acid sequence of an N1 co- stimulatory domain.
  • SEQ ID NO: 32 sets forth the amino acid sequence of an N6 co- stimulatory domain.
  • SEQ ID NO: 33 sets forth the amino acid sequence of a 4- IBB co-stimulatory domain.
  • SEQ ID NO: 34 sets forth the amino acid sequence of a CD28 co-stimulatory domain.
  • SEQ ID NO: 35 sets forth the amino acid sequence of a CD3 zeta signaling domain.
  • SEQ ID NO: 36 sets forth the amino acid sequence of a CD8 signal peptide.
  • SEQ ID NO: 37 sets forth the amino acid sequence of a CD8 signal peptide.
  • SEQ ID NO: 38 sets forth the nucleic acid sequence of a JeT promoter.
  • SEQ ID NO: 39 sets forth the nucleic acid sequence of an EFl alpha promoter.
  • SEQ ID NO: 40 sets forth the nucleic acid sequence of the TRC 1-2 recognition sequence (sense).
  • SEQ ID NO: 41 sets forth the nucleic acid sequence of the TRC 1-2 recognition sequence (antisense).
  • SEQ ID NO: 42 sets forth the amino acid sequence of a TRC 1-2L.1592 meganuclease.
  • SEQ ID NO: 43 sets forth the amino acid sequence of a BCMA-3L/20H scFv.
  • SEQ ID NO: 44 sets forth the amino acid sequence of a BCMA-20H/3L scFv.
  • SEQ ID NO: 45 sets forth the amino acid sequence of a BCMA-3L/20H-Spacer-CD8- CD8-N6-CD3z CAR.
  • SEQ ID NO: 46 sets forth the amino acid sequence of a BCMA-20H/3L-Spacer-CD8- CD8-N6-CD3z CAR.
  • SEQ ID NO: 47 sets forth the amino acid sequence of a CD8(+A)SP-BCMA-3L/20H- Spacer-CD8-CD8-N6-CD3z CAR.
  • SEQ ID NO: 48 sets forth the amino acid sequence of a CD8(+A)SP-BCMA-20H/3L- Spacer-CD8-CD8-N6-CD3z CAR.
  • SEQ ID NO: 49 sets forth the amino acid sequence of a CD8 signal peptide.
  • SEQ ID NO: 50 sets forth the nucleic acid sequence of a spacer sequence.
  • an “antigen-binding protein” is a protein or polypeptide that comprises an antigenbinding region or antigen-binding portion, that is, has a strong affinity to another molecule to which it binds.
  • Antigen-binding proteins encompass, for example, antibodies, chimeric antigen receptors (CARs) and fusion proteins.
  • antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant (CH) region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant (CL) region.
  • the light chain constant region is comprised of one domain, CL.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxyterminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Cl) of the classical complement system.
  • An exemplary VH is derived from the BCMA-20 antibody; and an exemplary VL is derived from the BCMA-3 antibody.
  • an exemplary scFv VH/VL pair as described in the present disclosure is referred to herein as BCMA-20H/3L scFv or BCMA-3L/20H.
  • antigen-binding portion or “antigen-binding region” of an antibody, as used herein, refers to that region or portion of the antibody that binds to the antigen and which confers antigen specificity to the antibody; fragments of antigen-binding proteins, for example, antibodies includes one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a BCMA polypeptide). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
  • antibody fragments encompassed within the term “antibody fragments” of an antibody include an antigen binding protein comprising a portion, i.e., an antigen binding region, of an intact antibody, such that the protein retains the antigen binding specificity of the antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; tandem diabodies (taDb), linear antibodies (e.g., U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng.
  • an “isolated antibody” or “isolated antigen-binding protein” is one which has been separated and/or recovered from a component of its natural environment.
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules.
  • scFv single chain Fv
  • These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • single-chain variable fragment is a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin (e.g., mouse or human) covalently linked to form a VH-VL or VL-VH heterodimer.
  • the heavy (VH) and light chains (VL) are either joined directly or joined by a pep tide-encoding linker, which connects the N-terminus of the VH with the C -terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL.
  • the linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility.
  • the linker can link the heavy chain variable region and the light chain variable region of the extracellular antigenbinding domain.
  • Non-limiting examples of linkers are disclosed in Shen et al., Anal. Chem. 80(6): 1910-1917 (2008) and WO 2014/087010, the contents of which are hereby incorporated by reference in their entireties.
  • the linker comprises amino acids having the sequence set forth in any one of SEQ ID NOs: 10-27, and variants thereof.
  • Single-chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VE-encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U.S. Pat. Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754.
  • Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hyrbidoma (Uarchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 Aug. 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J Clin Inst 2006 116(8):2252-61 ; Brocks et al., Immunotechnology 1997 3(3): 173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40).
  • F(ab) refers to a fragment of an antibody structure that binds to an antigen but is monovalent and does not have a Fc portion, for example, an antibody digested by the enzyme papain yields two F(ab) fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).
  • an antibody digested by the enzyme papain yields two F(ab) fragments and an Fc fragment (e.g., a heavy (H) chain constant region; Fc region that does not bind to an antigen).
  • F(ab')2 refers to an antibody fragment generated by pepsin digestion of whole IgG antibodies, wherein this fragment has two antigen binding (ab') (bivalent) regions, wherein each (ab') region comprises two separate amino acid chains, a part of a H chain and a light (U) chain linked by an S — S bond for binding an antigen and where the remaining H chain portions are linked together.
  • a “F(ab')2” fragment can be split into two individual Fab' fragments.
  • CDRs are defined as the complementarity determining regions of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th U.S. Department of Health and Human Services, National Institutes of Health (1987).
  • the term “hypervariable region” or “HVR” as used herein refers to each of the regions of an antibody variable domain which are hypervariable in sequence (“complementarity determining regions” or “CDRs”) and/or form structurally defined loops (“hypervariable loops”) and/or contain the antigen-contacting residues (“antigen contacts”).
  • CDRs comprise three heavy chain and three light chain CDRs or CDR regions in the variable region. CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and bind the same epitope, except for possible variant antibodies, e.g., 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.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the presently disclosed subject matter may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • recombinant antibody refers to antibodies that are prepared, expressed, created or isolated by recombinant means not existing in nature.
  • a recombinant antibody is a recombinant murine antibody.
  • Such recombinant murine antibodies have variable regions in which the framework and CDR regions are derived from murine germline immunoglobulin sequences.
  • such recombinant murine antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for murine Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to murine germline VH and VL sequences, may not naturally exist within the murine antibody germline repertoire in vivo.
  • the terms “recombinant” or “engineered,” with respect to a protein means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids that encode the protein and cells or organisms that express the protein.
  • the term “recombinant” or “engineered” means having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation, and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion. In accordance with this definition, a protein having an amino acid sequence identical to a naturally-occurring protein, but produced by cloning and expression in a heterologous host, is not considered recombinant or engineered.
  • humanized antibody is intended to refer to antibodies in which CDRs from a mammalian species (other than a human), such as a mouse, are grafted onto human framework regions. Additional framework region modifications may be made within the human framework sequences.
  • chimeric antibody is intended to refer to antibodies in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody.
  • an antibody that “specifically binds to human BCMA” is intended to refer to an antibody that binds to human BCMA with a KD of about 5xl0 -7 M or less, about IxlO -7 M or less, about 5xl0 -8 M or less, about IxlO -8 M or less, about 5xl0 -9 M or less, about IxlO -9 M or less, about 5xl0 -10 M or less, about IxlO -10 M or less, about 5xl0 -11 M or less, or about IxlO -11 M or less.
  • Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.
  • the term “does not detectably bind” refers to an antibody that does not bind a cell (e.g., a genetically-modified cell) at a level significantly greater than background, e.g., binds to the cell at a level less than 10%, 8%, 6%, 5%, or 1% above background.
  • the antibody binds to the cell at a level less than 10%, 8%, 6%, 5%, or 1% more than an isotype control antibody.
  • the binding is detected by Western blotting, flow cytometry, ELISA, antibody panning, and/or Biacore analysis.
  • “isotype” refers to the antibody class (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes.
  • BCMA and “B-cell maturation antigen” are used interchangeably, and include variants, isoforms, species homologs of human BCMA, and analogs having at least one common epitope with BCMA (e.g., human BCMA).
  • An exemplary human BCMA sequence can be found under Entrez Gene Accession No.: NP_001183.
  • BCMA may be expressed as a membrane-bound form, comprising an extracellular domain, transmembrane domain, and intracellular domain, or as a soluble form that is not associated with a particular cell.
  • the protease gamma secretase may cleave membrane -bound BCMA to release the extracellular domain, or an extracellular domain and a portion of the transmembrane domain, as soluble BCMA, with a released extracellular domain not being embedded within the membrane of any cell.
  • an “effective amount” of an antigen binding protein, e.g., an anti-BCMA antibody, or an antigen-binding fragment thereof, a pharmaceutical composition comprising thereof refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result, e.g., treating a tumor (e.g., multiple myeloma).
  • a tumor e.g., multiple myeloma
  • a “gamma secreatase inhibitor” refers to a compound, such as a small molecule, that inhibits the activity of gamma secretase.
  • “Gamma secretase” is a protease complex that cleaves membrane-bound BCMA, resulting in the release of the extracellular domain of membrane-bound BCMA as soluble BCMA.
  • “Soluble BCMA” refers to BCMA, or a fragment thereof (e.g., BCMA extaracellular domain) that is not embedded within a cell membrane. See, e.g., Laurent et al. Nat Commun. 2015. 6:7333.
  • the human gamma secretase complex comprises at least four protein subunits: presenilins (PS), nicastrin, anterior phalanx defective 1 (APH-1), and presenilin enhancer 2 (PEN-2). See, e.g., Zhang et al. Front Cell Neurosci. 2014. 8:427.
  • PS presenilins
  • APH-1 anterior phalanx defective 1
  • PEN-2 presenilin enhancer 2
  • lymphodepletion refers to the administration to a subject of one or more agents (e.g., chemotherapeutic lymphodepletion agents or biological lymphodepletion agents) capable of reducing endogenous lymphocytes in the subject for immunotherapy; e.g., a reduction of one or more lymphocytes (e.g., B cells, T cells, and/or NK cells) by at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or up to 100% relative to a control (e.g., relative to a starting amount in the subject undergoing treatment, relative to a pre-determined threshold, or relative to an untreated subject).
  • agents e.g., chemotherapeutic lymphodepletion agents or biological lymphodepletion agents
  • lymphocytes e.g., B cells, T cells, and/
  • biological lymphodepletion agent refers to a biological material, such an antibody, antibody fragment, antibody conjugate, or the like, that can be administered as part of a lymphodepletion regimen to reduce endogenous lymphocytes in the subject for immunotherapy.
  • biological lymphodepletion agents can have specificity for antigens present on lymphocytes; e.g., CD52 or CD3.
  • chemotherapeutic lymphodepletion agents refers to non- biological materials, such as small molecules, that can be administered as part of a lymphodepletion regimen to reduce endogenous lymphocytes in the subject for immunotherapy.
  • the chemotherapeutic lymphodepleting agent can be lymphodepleting but non-myeloablative.
  • mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domesticated animals e.g., cows, sheep, cats, dogs, and horses
  • primates e.g., humans and non-human primates such as monkeys
  • rabbits e.g., mice and rats
  • rodents e.g., mice and rats.
  • the individual or subject is a human.
  • treatment refers to clinical intervention in an attempt to alter the natural course of the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology.
  • Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, a partial or complete reduction in the number of cancer cells present in the subject, a partial or complete reduction in the mass or volume of a tumor present in the subject, and remission or improved prognosis.
  • compositions, engineered cells, and/or antibodies of the present disclosure are used to delay development of a disease or to slow the progression of a disease, e.g., a tumor (multiple myeloma).
  • administration of any of the pharmaceutical compositions, engineered cells, and/or antibodies of the present disclosure to a subject results in partial or complete reduction in the volume of a tumor or the number of cancer cells.
  • a “chimeric antigen receptor” or “CAR” refers to an engineered receptor that grafts specificity for an antigen (e.g., BCMA) or other ligand or molecule onto an immune effector cell (e.g., a T cell or NK cell).
  • a CAR comprises at least an extracellular ligand-binding domain or moiety, a transmembrane domain, and an intracellular domain (or moiety) that comprises one or more intracellular signaling domains and/or intracellular costimulatory domains.
  • An extracellular ligand-binding domain or moiety of a CAR can be, for example, an antibody, or antibody fragment.
  • antibody fragment may refer to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen.
  • antibody fragments include, but are not limited to, any antibody fragments described elsewhere herein and including Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VE or VH), camelid VHH domains, multi- specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody.
  • Fab fragment fragments described elsewhere herein and including Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VE or
  • An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005).
  • Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).
  • Fn3 fibronectin type III
  • the extracellular ligand-binding domain or moiety is in the form of a single-chain variable fragment (scFv) derived from a monoclonal antibody, which provides specificity for a particular epitope or antigen (e.g., an epitope or antigen preferentially present on the surface of a cell, such as a cancer cell or other disease-causing cell or particle).
  • scFv single-chain variable fragment
  • the scFv is attached via a linker sequence.
  • the scFv is murine or humanized.
  • the extracellular domain of a CAR comprises an autoantigen (see, Payne et al. (2016) Science, Vol. 353 (6295): 179- 184), which is recognized by autoantigen-specific B cell receptors on B lymphocytes, thus directing T cells to specifically target and kill autoreactive B lymphocytes in antibody-mediated autoimmune diseases.
  • CARs can be referred to as chimeric autoantibody receptors (CAARs), and are encompassed by the present disclosure.
  • the intracellular domain of a CAR can include one or more cytoplasmic signaling domains that transmit an activation signal to the T cell following antigen binding.
  • cytoplasmic signaling domains can include, without limitation, a CD3 zeta signaling domain, such as that disclosed in SEQ ID NO: 35, and variants thereof.
  • the intracellular domain of a CAR can also include one or more intracellular costimulatory domains that transmit a proliferative and/or cell- survival signal after ligand binding.
  • the co-stimulatory domain can comprise one or more TRAF-binding domains.
  • Intracellular co-stimulatory domains can be any of those known in the art and can include, without limitation, those co-stimulatory domains disclosed in WO 2018/067697 including, for example, Novel 1 (“Nl”; SEQ ID NO: 31) and Novel 6 (“N6”; SEQ ID NO: 32).
  • co-stimulatory domains include 4- IBB (SEQ ID NO: 33), CD28 (SEQ ID NO: 34), or a functional signaling domain obtained from a protein including an MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD30, CD40, CDS, ICAM-1, LFA-1 (CDl la/CD18), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma,
  • a CAR further includes additional structural elements, including a transmembrane domain that is attached to the extracellular ligand-binding domain via a hinge or spacer sequence.
  • the transmembrane domain can be derived from any membrane-bound or transmembrane protein.
  • the transmembrane polypeptide can be a subunit of the T-cell receptor (e.g., an a, polypeptide constituting CD3 complex), IL2 receptor p55 (a chain), p75 (P chain) or y chain, subunit chain of Fc receptors (e.g., Fey receptor III) or CD proteins such as the CD8 alpha chain.
  • the transmembrane domain is a CD8 alpha domain set forth in SEQ ID NO: 30, and variants thereof.
  • the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine.
  • the hinge region refers to any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain.
  • a hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • Hinge regions may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region.
  • the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence or may be an entirely synthetic hinge sequence.
  • a hinge domain can comprise a part of a human CD8 alpha chain, FcyRllla receptor or IgGl.
  • the hinge region can be a CD8 alpha domain set forth in SEQ ID NO: 29, and variants thereof.
  • modification means any insertion, deletion, or substitution of an amino acid residue in the recombinant sequence relative to a reference sequence (e.g., a wild-type or a native sequence).
  • the term “disrupted” or “disrupts” or “disrupts expression” or “disrupting a target sequence” refers to the introduction of a mutation (e.g., frameshift mutation) that interferes with the gene function and prevents expression and/or function of the polypeptide/expression product encoded thereby.
  • a mutation e.g., frameshift mutation
  • nuclease-mediated disruption of a gene can result in the expression of a truncated protein and/or expression of a protein that does not retain its wild-type function.
  • introduction of a donor template into a gene can result in no expression of an encoded protein, expression of a truncated protein, and/or expression of a protein that does not retain its wild-type function.
  • cleavage refers to the hydrolysis of phosphodiester bonds within the backbone of a recognition sequence within a target sequence that results in a double- stranded break within the target sequence, referred to herein as a “cleavage site”.
  • nuclease and “endonuclease” refers to enzymes which cleave a phosphodiester bond within a polynucleotide chain.
  • the term “meganuclease” refers to an endonuclease that binds doublestranded DNA at a recognition sequence that is greater than 12 base pairs. In some embodiments, the recognition sequence for a meganuclease of the present disclosure is 22 base pairs.
  • a meganuclease can be an endonuclease that is derived from I-Crel, and may refer to an engineered variant of I-Crel that has been modified relative to natural I-Crel with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties.
  • a meganuclease as used herein binds to double-stranded DNA as a heterodimer.
  • a meganuclease may also be a “single-chain meganuclease” in which a pair of DNA-binding domains is joined into a single polypeptide using a peptide linker.
  • homing endonuclease is synonymous with the term “meganuclease.”
  • Meganucleases of the present disclosure are substantially non-toxic when expressed in the targeted cells described herein such that cells can be transfected and maintained at 37°C without observing deleterious effects on cell viability or significant reductions in meganuclease cleavage activity when measured using the methods described herein.
  • single-chain meganuclease refers to a polypeptide comprising a pair of nuclease subunits joined by a linker.
  • a single-chain meganuclease has the organization: N-terminal subunit - Linker - C-terminal subunit.
  • the two meganuclease subunits will generally be non-identical in amino acid sequence and will bind non-identical DNA sequences.
  • single-chain meganucleases typically cleave pseudo-palindromic or non-palindromic recognition sequences.
  • a single-chain meganuclease may be referred to as a “single-chain heterodimer” or “single-chain heterodimeric meganuclease” although it is not, in fact, dimeric.
  • the term “meganuclease” may refer to a dimeric or single-chain meganuclease.
  • megaTAL refers to a single-chain endonuclease comprising a transcription activator-like effector (TALE) DNA binding domain with an engineered, sequence-specific homing endonuclease.
  • TALE transcription activator-like effector
  • compact TALEN refers to an endonuclease comprising a DNA-binding domain with one or more TAL domain repeats fused in any orientation to any portion of the LTevI homing endonuclease or any of the endonucleases listed in Table 2 in U.S. Application No. 20130117869 (which is incorporated by reference in its entirety), including but not limited to Mmel, EndA, Endl, I-BasI, LTevII, LTevIII, I-Twol, MspI, Mval, NucA, and NucM.
  • Compact TALENs do not require dimerization for DNA processing activity, alleviating the need for dual target sites with intervening DNA spacers.
  • the compact TALEN comprises 16-22 TAL domain repeats.
  • CRISPR CRISPR nuclease or CRISPR system nuclease refers to a CRISPR (clustered regularly interspaced short palindromic repeats)- associated (Cas) endonuclease or a variant thereof, such as Cas9, that associates with a guide RNA that directs nucleic acid cleavage by the associated endonuclease by hybridizing to a recognition site in a polynucleotide.
  • the CRISPR nuclease is a class 2 CRISPR enzyme.
  • the CRISPR nuclease is a class 2, type II enzyme, such as Cas9.
  • the CRISPR nuclease is a class 2, type V enzyme, such as Cpfl.
  • the guide RNA comprises a direct repeat and a guide sequence (often referred to as a spacer in the context of an endogenous CRISPR system), which is complementary to the target recognition site.
  • the CRISPR system further comprises a tracrRNA (trans-activating CRISPR RNA) that is complementary (fully or partially) to the direct repeat sequence (sometimes referred to as a tracr-mate sequence) present on the guide RNA.
  • the CRISPR nuclease can be mutated with respect to a corresponding wild-type enzyme such that the enzyme lacks the ability to cleave one strand of a target polynucleotide, functioning as a nickase, cleaving only a single strand of the target DNA.
  • CRISPR enzymes that function as a nickase include Cas9 enzymes with a D10A mutation within the RuvC I catalytic domain, or with a H840A, N854A, or N863A mutation.
  • recognition sequences Given a predetermined DNA locus, recognition sequences can be identified using a number of programs known in the art (Kornel Labun; Tessa G. Montague; James A. Gagnon; Summer B. Thyme; Eivind Valen. (2016).
  • CHOPCHOP v2 a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Research; doi:10.1093/nar/gkw398; Tessa G. Montague; Jose M. Cruz; James A. Gagnon; George M. Church; Eivind Valen. (2014). CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42. W401-W407).
  • TALEN refers to an endonuclease comprising a DNA- binding domain comprising a plurality of TAL domain repeats fused to a nuclease domain or an active portion thereof from an endonuclease or exonuclease, including but not limited to a restriction endonuclease, homing endonuclease, S 1 nuclease, mung bean nuclease, pancreatic DNAse I, micrococcal nuclease, and yeast HO endonuclease. See, for example, Christian et al. (2010) Genetics 186:757-761, which is incorporated by reference in its entirety.
  • Nuclease domains useful for the design of TALENs include those from a Type Ils restriction endonuclease, including but not limited to FokI, FoM, StsI, Hhal, Hindlll, Nod, BbvCI, EcoRI, Bgll, and AlwI. Additional Type Ils restriction endonucleases are described in International Publication No. WO 2007/014275, which is incorporated by reference in its entirety.
  • the nuclease domain of the TALEN is a FokI nuclease domain or an active portion thereof.
  • TAL domain repeats can be derived from the TALE (transcription activator-like effector) family of proteins used in the infection process by plant pathogens of the Xanthomonas genus.
  • TAL domain repeats are 33-34 amino acid sequences with divergent 12th and 13th amino acids. These two positions, referred to as the repeat variable dipeptide (RVD), are highly variable and show a strong correlation with specific nucleotide recognition.
  • RVD repeat variable dipeptide
  • Each base pair in the DNA target sequence is contacted by a single TAL repeat with the specificity resulting from the RVD.
  • the TALEN comprises 16-22 TAL domain repeats.
  • DNA cleavage by a TALEN requires two DNA recognition regions (i.e., “half-sites”) flanking a nonspecific central region (i.e., the “spacer”).
  • the term “spacer” in reference to a TALEN refers to the nucleic acid sequence that separates the two nucleic acid sequences recognized and bound by each monomer constituting a TALEN.
  • the TAL domain repeats can be native sequences from a naturally- occurring TALE protein or can be redesigned through rational or experimental means to produce a protein that binds to a pre-determined DNA sequence (see, for example, Boch et al.
  • each nuclease e.g., FokI
  • each nuclease monomer can be fused to a TAL effector sequence that recognizes and binds a different DNA sequence, and only when the two recognition sites are in close proximity do the inactive monomers come together to create a functional enzyme.
  • TALEN may refer to a single TALEN protein or, alternatively, a pair of TALEN proteins (i.e., a left TALEN protein and a right TALEN protein) which bind to the upstream and downstream half-sites adjacent to the TALEN spacer sequence and work in concert to generate a cleavage site within the spacer sequence.
  • upstream and downstream half-sites can be identified using a number of programs known in the art (Kornel Labun; Tessa G. Montague; James A. Gagnon; Summer B. Thyme; Eivind Valen. (2016).
  • CHOPCHOP v2 a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Research; doi:10.1093/nar/gkw398; Tessa G. Montague; Jose M. Cruz; James A. Gagnon; George M. Church; Eivind Valen. (2014). CHOPCHOP: a CRISPR/Cas9 and TALEN web tool for genome editing. Nucleic Acids Res. 42. W401-W407). It is also understood that a TALEN recognition sequence can be defined as the DNA binding sequence (i.e., half-site) of a single TALEN protein or, alternatively, a DNA sequence comprising the upstream half-site, the spacer sequence, and the downstream half- site.
  • zinc finger nuclease or “ZFN” refers to a chimeric protein comprising a zinc finger DNA-binding domain fused to a nuclease domain from an endonuclease or exonuclease, including but not limited to a restriction endonuclease, homing endonuclease, S 1 nuclease, mung bean nuclease, pancreatic DNAse I, micrococcal nuclease, and yeast HO endonuclease.
  • Nuclease domains useful for the design of zinc finger nucleases include those from a Type Ils restriction endonuclease, including but not limited to FokI, FoM, and StsI restriction enzyme. Additional Type Ils restriction endonucleases are described in International Publication No. WO 2007/014275, which is herein incorporated by reference in its entirety. The structure of a zinc finger domain is stabilized through coordination of a zinc ion. DNA binding proteins comprising one or more zinc finger domains bind DNA in a sequence-specific manner.
  • the zinc finger domain can be a native sequence or can be redesigned through rational or experimental means to produce a protein which binds to a pre-determined DNA sequence ⁇ 18 basepairs in length, comprising a pair of nine basepair half-sites separated by 2-10 basepairs. See, for example, U.S. Pat. Nos. 5,789,538, 5,925,523, 6,007,988, 6,013,453, 6,200,759, and International Publication Nos.
  • the DNA binding domains typically recognize an 18-bp recognition sequence comprising a pair of nine basepair “half-sites” separated by a 2-10 basepair “spacer sequence”, and cleavage by the nuclease creates a blunt end or a 5' overhang of variable length (frequently four basepairs).
  • zinc finger nuclease may refer to a single zinc finger protein or, alternatively, a pair of zinc finger proteins (i.e., a left ZFN protein and a right ZFN protein) that bind to the upstream and downstream half-sites adjacent to the zinc finger nuclease spacer sequence and work in concert to generate a cleavage site within the spacer sequence.
  • a pair of zinc finger proteins i.e., a left ZFN protein and a right ZFN protein
  • upstream and downstream half-sites can be identified using a number of programs known in the art (Mandell JG, Barbas CF 3rd.
  • Zinc Finger Tools custom DNA-binding domains for transcription factors and nucleases. Nucleic Acids Res.
  • a zinc finger nuclease recognition sequence can be defined as the DNA binding sequence (i.e., half-site) of a single zinc finger nuclease protein or, alternatively, a DNA sequence comprising the upstream half-site, the spacer sequence, and the downstream half-site.
  • target site or “target sequence” refers to a region of the chromosomal DNA of a cell comprising a recognition sequence for a nuclease. This term embraces chromosomal DNA duplexes as well as single- stranded chromosomal DNA.
  • the term “specificity” means the ability of a nuclease to recognize and cleave double- stranded DNA molecules only at a particular sequence of base pairs referred to as the recognition sequence, or only at a particular set of recognition sequences.
  • the set of recognition sequences will share certain conserved positions or sequence motifs, but may be degenerate at one or more positions.
  • a highly-specific nuclease is capable of cleaving only one or a very few recognition sequences. Specificity can be determined by any method known in the art.
  • a recognition sequence or “recognition site” refers to a DNA sequence that is bound and cleaved by a nuclease.
  • a recognition sequence comprises a pair of inverted, 9 basepair “half sites” which are separated by four basepairs.
  • the N-terminal domain of the protein contacts a first half-site and the C-terminal domain of the protein contacts a second half-site. Cleavage by a meganuclease produces four basepair 3' overhangs.
  • “Overhangs,” or “sticky ends” are short, single- stranded DNA segments that can be produced by endonuclease cleavage of a double-stranded DNA sequence.
  • the overhang comprises bases 10-13 of the 22 basepair recognition sequence.
  • the recognition sequence comprises a first CNNNGN sequence that is recognized by the I-TevI domain, followed by a nonspecific spacer 4-16 basepairs in length, followed by a second sequence 16-22 bp in length that is recognized by the TAL-effector domain (this sequence typically has a 5' T base).
  • Cleavage by a compact TALEN produces two basepair 3' overhangs.
  • the recognition sequence is the sequence, typically 16-24 basepairs, to which the guide RNA binds to direct cleavage. Full complementarity between the guide sequence and the recognition sequence is not necessarily required to effect cleavage.
  • Cleavage by a CRISPR nuclease can produce blunt ends (such as by a class 2, type II CRISPR nuclease) or overhanging ends (such as by a class 2, type V CRISPR nuclease), depending on the CRISPR nuclease.
  • cleavage by the CRISPR complex comprising the same will result in 5' overhangs and in certain embodiments, 5 nucleotide 5' overhangs.
  • Each CRISPR nuclease enzyme also requires the recognition of a PAM (protospacer adjacent motif) sequence that is near the recognition sequence complementary to the guide RNA.
  • PAM protospacer adjacent motif
  • the precise sequence, length requirements for the PAM, and distance from the target sequence differ depending on the CRISPR nuclease enzyme, but PAMs are typically 2-5 base pair sequences adjacent to the target/recognition sequence.
  • PAM sequences for particular CRISPR nuclease enzymes are known in the art (see, for example, U.S. Patent No.
  • PAM sequences for novel or engineered CRISPR nuclease enzymes can be identified using methods known in the art, such as a PAM depletion assay (see, for example, Karvelis et al. (2017) Methods 121-122:3-8, which is incorporated herein in its entirety).
  • the DNA binding domains typically recognize an 18-bp recognition sequence comprising a pair of nine basepair “half-sites” separated by 2-10 basepairs and cleavage by the nuclease creates a blunt end or a 5' overhang of variable length (frequently four basepairs).
  • the term “recognition half-site,” “recognition sequence half-site,” or simply “half-site” means a nucleic acid sequence in a double- stranded DNA molecule that is recognized and bound by a monomer of a homodimeric or heterodimeric meganuclease or by one subunit of a single-chain meganuclease or by one subunit of a single-chain meganuclease, or by a monomer of a TALEN or zinc finger nuclease.
  • control refers to a cell that provides a reference point for measuring changes in genotype or phenotype of a genetically-modified cell.
  • a control cell may comprise, for example: (a) a wild-type cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the genetically-modified cell; (b) a cell of the same genotype as the genetically-modified cell but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest); or, (c) a cell genetically identical to the genetically-modified cell but which is not exposed to conditions or stimuli or further genetic modifications that would induce expression of altered genotype or phenotype.
  • a “co-stimulatory domain” refers to a polypeptide domain which transmits an intracellular proliferative and/or cell-survival signal upon activation. Activation of a co-stimulatory domain may occur following homodimerization of two co-stimulatory domain polypeptides. Activation may also occur, for example, following activation of a construct comprising the co-stimulatory domain (e.g., a CAR). Generally, a co-stimulatory domain can be derived from a transmembrane co- stimulatory receptor, particularly from an intracellular portion of a co-stimulatory receptor.
  • co-stimulatory domains include, but are not limited to, those co-stimulatory domains described elsewhere herein.
  • a “co-stimulatory signal” refers to an intracellular signal induced by a co-stimulatory domain that promotes cell proliferation, expansion of a cell population in vitro and/or in vivo, promotes cell survival, modulates (e.g., upregulates or downregulates) the secretion of cytokines, and/or modulates the production and/or secretion of other immunomodulatory molecules.
  • detecttable expression of an endogenous TCR refers to the ability to detect one or more components of the TCR complex (e.g., an alpha/beta TCR complex) on the cell surface of a T cell (e.g., a CAR T cell), or a population of T cells (e.g., CAR T cells) described herein, using standard experimental methods. Such methods can include, for example, immuno staining and/or flow cytometry specific for components of the TCR itself, such as a TCR alpha or TCR beta chain, or for components of the assembled cell surface TCR complex, such as CD3. Methods for detecting cell surface expression of an endogenous TCR (e.g., an alpha/beta TCR) on an immune cell include those described in MacLeod et al. (2017) Molecular Therapy 25(4): 949-961.
  • DNA-binding affinity or “binding affinity” means the tendency of a nuclease to non-covalently associate with a reference DNA molecule (e.g., a recognition sequence or an arbitrary sequence). Binding affinity is measured by a dissociation constant, Kd. As used herein, a nuclease has “altered” binding affinity if the Kd of the nuclease for a reference recognition sequence is increased or decreased by a statistically significant percent change relative to a reference nuclease.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • An intracellular signaling domain such as CD3 zeta, can provide an activation signal to the cell in response to binding of the extracellular domain. As discussed, the activation signal can induce an effector function of the cell such as, for example, cytolytic activity or cytokine secretion.
  • an effective amount or “therapeutically effective amount”, as it relates to CARs of the invention and genetically-modified cells comprising such CARs refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
  • the amount will vary depending on the therapeutic (e.g., a genetically-modified cell such as a CAR T cell, CAR NK cell) formulation or composition, the disease and its severity, and the age, weight, physical condition and responsiveness of the subject to be treated.
  • an effective amount of a cell comprising a CAR described herein, or pharmaceutical compositions described herein reduces at least one symptom or the progression of a disease (e.g., cancer).
  • an effective amount of the pharmaceutical compositions or genetically-modified cells described herein reduces the level of proliferation or metastasis of cancer, causes a partial or full response or remission of cancer, or reduces at least one symptom of cancer in a subject.
  • emulsion refers to, without limitation, any oil-in-water, water-in-oil, water-in-oil-in-water, or oil-in-water-in-oil dispersions or droplets, including lipid structures that can form as a result of hydrophobic forces that drive apolar residues (e.g., long hydrocarbon chains) away from water and polar head groups toward water, when a water immiscible phase is mixed with an aqueous phase.
  • lipid structures that can form as a result of hydrophobic forces that drive apolar residues (e.g., long hydrocarbon chains) away from water and polar head groups toward water, when a water immiscible phase is mixed with an aqueous phase.
  • a genetically-modified cell refers to a cell or organism in which, or in an ancestor of which, a genomic DNA sequence has been deliberately modified by recombinant technology.
  • the term “genetically-modified” encompasses the term “transgenic.”
  • a genetically-modified cell is an immune cell, such as, for example, a genetically-modified human T cell, NK cell, B cell, and others. Exemplary genetically-modified immune cells of the present disclosure are described in U.S. Publication No. US 2020/0370014, published November 26, 2020, herein incorporated by reference.
  • homologous recombination refers to the natural, cellular process in which a double- stranded DNA-break is repaired using a homologous DNA sequence as the repair template (see, e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976).
  • the homologous DNA sequence may be an endogenous chromosomal sequence or an exogenous nucleic acid that was delivered to the cell.
  • non-homologous end-joining refers to the natural, cellular process in which a double-stranded DNA-break is repaired by the direct joining of two non-homologous DNA segments (see, e.g. Cahill et al. (2006), Front. Biosci. 11:1958-1976). DNA repair by non-homologous end-joining is error-prone and frequently results in the untemplated addition or deletion of DNA sequences at the site of repair. In some instances, cleavage at a target recognition sequence results in NHEJ at a target recognition site.
  • Nuclease-induced cleavage of a target site in the coding sequence of a gene followed by DNA repair by NHEJ can introduce mutations into the coding sequence, such as frameshift mutations, that disrupt gene function.
  • engineered nucleases can be used to effectively knock-out a gene in a population of cells.
  • a “human T cell” or “T cell” refers to a T cell isolated from a human donor.
  • the human donor is not the subject treated according to the method (i.e., the T cells are allogeneic), but instead a healthy human donor.
  • the human donor is the subject treated according to the method.
  • T cells, and cells derived therefrom can include, for example, isolated T cells that have not been passaged in culture, or T cells that have been passaged and maintained under cell culture conditions without immortalization.
  • human natural killer cell or “human NK cell” or “natural killer cell” or “NK cell” refers to a type of cytotoxic lymphocyte critical to the innate immune system.
  • the role NK cells play is analogous to that of cytotoxic T-cells in the vertebrate adaptive immune response.
  • NK cells provide rapid responses to virally infected cells and respond to tumor formation, acting at around 3 days after infection.
  • Human NK cells, and cells derived therefrom, include isolated NK cells that have not been passaged in culture, NK cells that have been passaged and maintained under cell culture conditions without immortalization, and NK cells that have been immortalized and can be maintained under cell culture conditions indefinitely.
  • linker refers to a peptide or a short oligopeptide sequence used to join two subunits into a single polypeptide.
  • a linker may have a sequence that is found in natural proteins or may be an artificial sequence that is not found in any natural protein.
  • a linker may be flexible and lacking in secondary structure or may have a propensity to form a specific three-dimensional structure under physiological conditions.
  • a linker may have a length of about 2 to 10 amino acids.
  • a linker may have a length of about 10 to 80 amino acids.
  • a linker may have a length of more than 80 amino acids.
  • a linker may be arranged between antibody VH and VL regions.
  • linkers may have an amino acid sequence as set forth in any one of SEQ ID NOs: 10-27, and variants thereof.
  • a linker may have an amino acid sequence as set forth in SEQ ID NO: 10, and variants thereof.
  • a linker may be arranged between the transmembrane domain and the intracellular domain of a CAR.
  • a linker also referred to herein as a “spacer” may be positioned between an anti-BCMA binding domain and the transmembrane domain of a CAR.
  • spacers can include, for example, a spacer set forth in SEQ ID NO: 28, and variants thereof.
  • the spacer set forth in SEQ ID NO: 28 is encoded by a nucleic acid sequence comprising SEQ ID NO: 50.
  • operably linked is intended to mean a functional linkage between two or more elements.
  • an operable linkage between a nucleic acid sequence encoding a nuclease described herein and a regulatory sequence is a functional link that allows for expression of the nucleic acid sequence encoding the nuclease.
  • Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame.
  • recombinant DNA construct As used herein, the term “recombinant DNA construct,” “recombinant construct,” “expression cassette,” “expression construct,” “chimeric construct,” “construct,” and “recombinant DNA fragment” are used interchangeably herein and are single or doublestranded polynucleotides.
  • a recombinant construct comprises an artificial combination of nucleic acid fragments, including, without limitation, regulatory and coding sequences that are not found together in nature.
  • a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source and arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector.
  • the terms “recombinant” or “engineered,” with respect to a protein means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids that encode the protein and cells or organisms that express the protein.
  • the term “recombinant” or “engineered” means having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation, and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion.
  • Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation, and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion.
  • a protein having an amino acid sequence identical to a naturally-occurring protein, but produced by cloning and expression in a heterologous host is not considered recombinant or engineered.
  • a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source and arranged in a manner different than that found in nature.
  • a construct may be used by itself or may be used in conjunction with a vector.
  • the terms “reducing,” “reduces”, or “reduced” each refer to a reduction in the proliferation or number of cancer cells; or the mass or volume of a tumor. Such a reduction may be up to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or up to 100%.
  • the term “reduced” may refer to a reduction in the percentage of cells in a population of cells that express an endogenous polypeptide (i.e., an endogenous alpha/beta T cell receptor or CD3) at the cell surface when compared to a population of control cells.
  • a reduction in the proliferation or number of cancer cells may be determined by standard experimental methods known in the art.
  • a reduction in the number of cancer cells may be determined by immunohistochemistry (IHC) followed by flow cytometry, e.g., flow cytometry analysis of cells expressing characteristic tumor surface antigens in a blood sample withdrawn from the subject; followed by cell counting.
  • a reduction in the mass or volume of a tumor may be determined by any method known in the art.
  • a reduction in the mass or volume of a tumor may be determined by histopathology, immunohistochemistry, flow cytometry, physical examination, imaging, e.g. magnetic resonance imaging, computed tomography, biopsy, or DNA or RNA sequencing.
  • a reduction in the mass or volume of a tumor may be determined by measuring the number of circulating cancer cells in the subject by immunohistochemistry and flow cytometry.
  • the terms “reduces” and “reduced” as used herein may refer to a reduction in the symptoms or severity of a disease, such as a cancer. Accordingly, the term “reduced” encompasses both a partial reduction and a complete reduction of a disease state.
  • a “chimeric antigen receptor T cell” or “CAR T cell” refers to a human T cell modified to comprise a transgene encoding a CAR, wherein the CAR is expressed on the cell surface of the T cell.
  • the CAR T cell may be derived from any “human T cell,” as that term is used herein.
  • a CAR T cell may be derived from a human T cell that has been isolated from a human donor, e.g., a human donor that is not the subject treated according to the method (i.e., the CAR T cells are allogeneic), but instead a healthy human donor.
  • CAR T cells, and cells derived therefrom may be derived from, for example, isolated T cells that have not been passaged in culture, or T cells that have been passaged and maintained under cell culture conditions without immortalization.
  • T cell receptor alpha gene or “TCR alpha gene” refer to the locus in a T cell which encodes the T cell receptor alpha subunit.
  • the T cell receptor alpha gene may refer to NCBI Gene ID number 6955, before or after rearrangement. Following rearrangement, the T cell receptor alpha gene comprises an endogenous promoter, rearranged V and J segments, the endogenous splice donor site, an intron, the endogenous splice acceptor site, and the T cell receptor alpha constant region locus, which comprises the subunit coding exons.
  • T cell receptor alpha constant region or “TCR alpha constant region” or “TRAC” refers to a coding sequence of the T cell receptor alpha gene.
  • the TCR alpha constant region includes the wild-type sequence, and functional variants thereof (i.e., naturally-occurring variants), identified by NCBI Gene ID NO. 28755.
  • the terms “response,” “complete response,” “complete response with incomplete blood count recovery,” “refractory disease,” “partial response,” “disease progression” or “progressive disease,” “refractory disease,” “relapse” or “relapsed disease” each refer to assessments of disease state and response in subjects following treatment according to the methods disclosed herein.
  • vector or “recombinant DNA vector” may be a construct that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art.
  • Vectors can include, without limitation, plasmid vectors and recombinant AAV vectors, or any other vector known in the art suitable for delivering a gene to a target cell. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleotides or nucleic acid sequences of the invention.
  • a “vector” also refers to a viral vector.
  • Viral vectors can include, without limitation, retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors (AAV).
  • wild-type refers to the most common naturally occurring allele (i.e., polynucleotide sequence) in the allele population of the same type of gene, wherein a polypeptide encoded by the wild-type allele has its original functions.
  • wild-type also refers to a polypeptide encoded by a wild-type allele. Wild-type alleles (i.e., polynucleotides) and polypeptides are distinguishable from mutant or variant alleles and polypeptides, which comprise one or more mutations and/or substitutions relative to the wildtype sequence(s).
  • Wild-type nucleases are distinguishable from recombinant or non- naturally-occurring nucleases.
  • the term “wild-type” can also refer to a cell, an organism, and/or a subject which possesses a wild-type allele of a particular gene, or a cell, an organism, and/or a subject used for comparative purposes.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
  • any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • the antibodies and antigen-binding fragments of the presently disclosed subject matter are characterized by particular functional features or properties of the antibodies.
  • the antibodies and antigen-binding fragments bind specifically to BCMA (e.g., bind to human BCMA).
  • BCMA e.g., bind to human BCMA
  • the antibodies and antigen-binding fragments bind specifically to a human BCMA having an amino acid sequence of SEQ ID NO: 1.
  • an antibody or fragment of the presently disclosed subject matter binds to BCMA with high affinity, for example with a KD of IxlO -6 M or less, e.g., about IxlO -7 M or less, about IxlO -8 M or less, about IxlO -9 M or less, about IxlO -10 M or less, or about IxlO -11 M or less.
  • a presently disclosed anti-BCMA antibody or fragment binds to BCMA (e.g., human BCMA) with a KD of from about IxlO -11 M to about IxlO -6 M, e.g., from about IxlO -11 M to about IxlO -9 M, from about IxlO -10 M to about IxlO -9 M, from IxlO -9 M to about IxlO -8 M, from about IxlO -8 M to about IxlO -7 M, or from about IxlO -7 M to about IxlO -6 M.
  • BCMA e.g., human BCMA
  • KD KD of from about IxlO -11 M to about IxlO -6 M, e.g., from about IxlO -11 M to about IxlO -9 M, from about IxlO -10 M to about IxlO -9 M, from IxlO -9 M to about IxlO -8 M
  • a presently disclosed anti-BCMA antibody or fragment binds to BCMA (e.g., human BCMA) with a KD of about IxlO -8 M or less. In certain embodiments, a presently disclosed anti-BCMA antibody or fragment binds to BCMA (e.g., human BCMA) with a KD of from about IxlO -9 M to about IxlO -10 M. In certain embodiments, a presently disclosed anti-BCMA antibody or fragment binds to BCMA (e.g., human BCMA) with a KD of from about IxlO -9 M to about 2.5xl0 -9 M.
  • a presently disclosed anti-BCMA antibody or fragment binds to BCMA (e.g., human BCMA) with a KD of from about 1.38xl0 -9 M to about 2.14xl0 -9 M. In certain embodiments, a presently disclosed anti-BCMA antibody or fragment binds to BCMA (e.g., human BCMA) with a KD of about 1.38xl0 -9 M. In certain embodiments, a presently disclosed anti-BCMA antibody or fragment binds to BCMA (e.g., human BCMA) with a KD of about 2.14x10 -9 M.
  • the VH region amino acid sequence of an anti-BCMA antibody or fragment is given by SEQ ID NO: 3.
  • the VL region amino acid sequence of an anti-BCMA antibody or fragment is given by SEQ ID NO: 2.
  • an anti-BCMA antibody or fragment comprises i) a VH region comprising the amino acid sequence of SEQ ID NO: 3, and ii) a VL region comprising the amino acid sequence of SEQ ID NO: 2, which are reproduced below.
  • VL region DIVLTQSPPSLAMSLGKRATISCRASESVTIPGQHLINWYQQKPGQPPKLLIQRASNVE SGVPARFSGSGSRTDFTLTIDPVEEDDVAVYYCLQTRGIPRTFGGGTKLEIK (SEQ ID NO: 2)
  • VH and VL region of an anti-BCMA antibody or fragment may be combined in either orientation, with the C-terminus of the VH region linked to the N-terminus of the VL region, or the C-terminus of the VL region linked to the N-terminus of the VH region.
  • BCMA binding of such antibodies or fragments can be tested using the binding assays known in the art, including for example, ELISAs, Western blots, RIAs, and Biacore analysis.
  • the presently disclosed subject matter provides antibodies or fragments that comprise the heavy chain CDRs (CDRH1, CDRH2, and CDRH3) of the VH region of SEQ ID NO: 3, and light chain CDRs (CDRL1, CDRL2, and CDRL3) of the VL region of SEQ ID NO: 2.
  • CDRH1, CDRH2, and CDRH3 heavy chain CDRs
  • CDRL1, CDRL2, and CDRL3 light chain CDRs
  • the CDR sequences of the VH and VL regions are identified by the Kabat numbering scheme.
  • the CDR sequences of the VH and VL regions are identified by the Chothia numbering scheme.
  • the amino acid sequence of the CDRH1 domain of the anti- BCMA antibody or fragment, as determined by the Kabat numbering scheme is set forth as SEQ ID NO: 7.
  • the amino acid sequence of the CDRH2 domain of the anti-BCMA antibody or fragment, as determined by the Kabat numbering scheme is set forth as SEQ ID NO: 8.
  • the amino acid sequence of the CDRH3 domain of the anti-BCMA antibody or fragment, as determined by the Kabat numbering scheme is set forth as SEQ ID NO: 9.
  • the amino acid sequence of the CDRL1 domain of the anti-BCMA antibody or fragment, as determined by the Kabat numbering scheme is set forth as SEQ ID NO: 4.
  • amino acid sequence of the CDRL2 domain of the anti-BCMA antibody or fragment, as determined by the Kabat numbering scheme is set forth as SEQ ID NO: 5.
  • amino acid sequence of the CDRL3 domain of the anti-BCMA antibody or fragment, as determined by the Kabat numbering scheme is set forth as SEQ ID NO: 6.
  • amino acid sequences of the CDR domains of an antibody or fragment are summarized as follows:
  • CDRL1 RASES VTIPGQHLIN (SEQ ID NO: 4)
  • CDRL2 RASNVES (SEQ ID NO: 5)
  • CDRL3 LQTRGIPRT (SEQ ID NO: 6)
  • CDRH1 HYSIN (SEQ ID NO: 7)
  • CDRH2 WINTETRESTYAYDFKG (SEQ ID NO: 8)
  • CDRH3 DYKQAMDY (SEQ ID NO: 9)
  • the constant region/framework region of the anti-BCMA antibodies or fragments disclosed herein can be altered, for example, by amino acid substitution, to modify the properties of the antibody (e.g., to increase or decrease one or more of: antigen binding affinity, Fc receptor binding, antibody carbohydrate, for example, glycosylation, fucosylation etc, the number of cysteine residues, effector cell function, effector cell function, complement function or introduction of a conjugation site).
  • Antibodies or fragments of the presently disclosed subject matter can be tested for binding to BCMA by, for example, standard EEISA.
  • each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, Ill.). Competition studies using unlabeled monoclonal antibodies and biotinylated monoclonal antibodies can be performed using BCMA coated-ELISA plates as described above. Biotinylated mAb binding can be detected with a strep-avidin-alkaline phosphatase probe.
  • isotype ELIS can be performed using reagents specific for antibodies of a particular isotype.
  • Anti-BCMA human IgGs can be further tested for reactivity with BCMA antigen by Western blotting.
  • KD is measured by a radiolabeled antigen binding assay (RIA).
  • RIA radiolabeled antigen binding assay
  • an RIA is performed with the Fab version of an antibody of interest and its antigen.
  • solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (125I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)).
  • KD is measured using a BIACORE® surface plasmon resonance assay.
  • a BIACORE® surface plasmon resonance assay For example, an assay using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.).
  • the antibodies or fragments of the present disclosure may be prepared and purified using known methods in the art.
  • cDNA sequences encoding a heavy chain and a light chain may be cloned and engineered into an expression vector.
  • the engineered immunoglobulin expression vector may then be stably transfected into a mammalian host cell, such as a Chinese Hamster Ovary (CHO) cells (e.g., GS-CHO) or NS0 cells.
  • Stable clones may be verified for expression of an antibody specifically binding to human BCMA.
  • Positive clones may be expanded into serum-free culture medium for antibody production in bioreactors.
  • Media, into which an antibody has been secreted may be purified by conventional techniques.
  • the medium may be conveniently applied to a Protein A column that has been equilibrated with a compatible buffer, such as phosphate buffered saline.
  • a compatible buffer such as phosphate buffered saline.
  • the column is washed to remove nonspecific binding components.
  • the bound antibody is eluted, for example, by pH gradient and antibody fractions are detected, such as by SDS-PAGE, and then pooled.
  • the antibody may be further purified, concentrated and/or sterile filtered using common techniques. Soluble aggregate and multimers may be effectively removed by common techniques, including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography.
  • the product may subsequently be processed for use, for example, in a pharmaceutical formulation.
  • the anti-BCMA antibodies described herein can be in the form of an anti-BCMA single-domain antibody (sdAb) fragment comprising the CDRH1, CDRH2, and CDRH3 domains, or comprising a VH region, or variants thereof, of any antibody described herein.
  • sdAb single-domain antibody
  • An anti-BCMA antibody described herein can also be in the form of an anti-BCMA single-chain variable fragment (scFv).
  • scFv is a fusion protein of the variable regions of the VH region and VL region of any antibody described herein or variants thereof, that are covalently linked to form a VH-VL or VL-VH heterodimer.
  • the VH region and VL region are either joined directly or joined by a peptide-encoding linker, which connects the N- terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N- terminus of the VL.
  • the linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility.
  • Examples of linkers useful for connecting a VH region and VL region in an scFv include those set forth in any one of SEQ ID NOs: 10-27, and variants thereof.
  • the linker comprises an amino acid sequence set forth in SEQ ID NO: 10, or variants thereof.
  • the invention encompasses scFvs, either with or without a linker, generated from the VH and VL regions, and variants thereof, of any antibody described herein, or of any antibody comprising the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3 domains described herein.
  • the invention further encompasses scFvs, either with or without a linker, that are prepared by mixing and matching the VH region and VL regions, and variants thereof, of any antibody disclosed herein, or of any antibody comprising the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3 domains described herein.
  • an scFv encompassed by the invention can have a 5' to 3' orientation of, for example, VH-VL, VL-VH, VH-linker-VL, or VL-linker-VH.
  • an scFv encompassed by the invention is an scFv comprising the amino acid sequence of any one of SEQ ID NOs: 43 and 44, and variants thereof.
  • an antibody or fragment of the presently disclosed subject matter comprises heavy and light chain variable regions comprising amino acid sequences that are homologous to the amino acid sequences of the antibodies described herein, and wherein the antibodies or fragments retain the desired functional properties of the anti- BCMA antibodies or fragments of the presently disclosed subject matter.
  • the presently disclosed subject matter provides an isolated antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein: (a) the heavy chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous to an amino acid sequence set forth inSEQ ID NO: 3; and/or (b) the light chain variable region comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homolog
  • the VH and/or VL amino acid sequences can be at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% homologous to the sequences set forth above.
  • An antibody or fragment having VH and VL regions having high (i.e., 80% or greater) homology to the VH and VL regions of the sequences set forth above can be obtained by mutagenesis (e.g., site-directed or PCR-mediated mutagenesis), followed by testing of the encoded altered antibody for retained function (i.e., the binding affinity) using the binding assays described herein.
  • mutagenesis e.g., site-directed or PCR-mediated mutagenesis
  • the encoded altered antibody for retained function i.e., the binding affinity
  • the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.
  • the percent homology between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent homology between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol.
  • the protein sequences of the presently disclosed subject matter can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences.
  • Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • compositions and Methods of Use relate to methods of reducing the number of cancer cells in a subject.
  • the methods generally comprise: i) administering to the subject a lymphodepletion regimen comprising one or more chemotherapeutic lymphodepletion agents; ii) administering to the subject an effective dose of a first pharmaceutical composition comprising a population of human immune cells, wherein a plurality of the human immune cells are genetically-modified human immune cells that express a chimeric antigen receptor (CAR), wherein the lymphodepletion regimen is administered prior to administration of the first pharmaceutical composition, and wherein the CAR comprises: a) an anti-human BCMA- binding domain that specifically binds to human BCMA; b) a transmembrane domain; and c) an intracellular signaling domain; and iii) a second pharmaceutical composition comprising an effective dose of a gamma secretase inhibitor; wherein the method causes the death of one or more cancer cells in the subject, and wherein
  • CAR chimeric
  • lymphodepletion is accomplished by administering one or more chemotherapeutic lymphodepletion agents to the subject. Lymphodepletion reduces the number of lymphocytes in a subject and is performed prior to the administration of a cell therapy (e.g., CAR T cell therapy).
  • a cell therapy e.g., CAR T cell therapy
  • lymphodepletion includes reducing the size of a tumor due to killing of intratumoral lymphocytes; reducing the number of T regulatory cells in the subject, particularly the tumor, thereby making the environment more conducive to CAR T cell activity; removing lymphocytes that can absorb pro-inflammatory cytokines such as IL-2, IL-7, and IL- 15; and removing CD8+ T cells that may inhibit or kill engrafted CAR T cells.
  • lymphodepletion agents include, for example, cyclophosphamide, fludarabine, busulfan, treosulfan, and melphalan. See, e.g., Shaw et al. Front Pediatr. 2019. 7:434.
  • the dosing regimen of one or more lymphodepletion agents is measured in units of mass / area / time (e.g., mg/m 2 /day), wherein the mass refers to the mass of the administered lymphodepletion agent, the area refers to the body surface area of the subject to which the lymphodepletion agent is administered, and the time refers to the duration of time over which a given dose, in terms of mass / area (e.g., mg/m 2 ), is administered.
  • Formulae for calculating the body surface area of a subject are known in the art, and generally depend on a subject’s height and weight. See, e.g., Du Bois and Du Bois. Arch Intern Med. 1916. 17:863-871.
  • a dosage refers to a given amount of a composition per day
  • the dosage is satisfied if that amount of the composition is administered in a given period of 24 hours (e.g., 1 lam Monday to 1 lam Tuesday), or in a given calendar day (e.g., Monday).
  • the lymphodepletion regimen comprises administering cyclophosphamide to the subject. In some embodiments, the lymphodepletion regimen comprises administering cyclophosphamide to the subject at a dose of about 100 to about 2000 mg/m 2 /day. In some embodiments, the lymphodepletion regimen comprises administering cyclophosphamide to the subject at a dose of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering cyclophosphamide to the subject at a dose of about 100-400, 400-800, 800-1200, 1200- 1600, or 1600-2000 mg/m 2 /day. In some embodiments, the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 400 to about 1500 mg/m 2 /day. In some embodiments, the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 500 to about 1000 mg/m 2 /day. In some embodiments, the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 500 mg/m 2 /day. In some embodiments, the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 1000 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering fludarabine to the subject. In some embodiments, the lymphodepletion regimen comprises administering fludarabine to the subject at a dose of about 5 to about 80 mg/m 2 /day. In some embodiments, the lymphodepletion regimen comprises administering fludarabine to the subject at a dose of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg/m 2 /day. In some embodiments, the lymphodepletion regimen comprises administering fludarabine to the subject at a dose of about 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60- 70, or 70-80 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering fludarabine at a dose of about 10 to about 40 mg/m 2 /day. In some embodiments, the lymphodepletion regimen comprises administering fludarabine at a dose of about 30 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering cyclophosphamide and fludarabine to the subject. In some embodiments, the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 500 to about 1500 mg/m 2 /day and administering fludarabine at a dose of about 10 to about 40 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 mg/m 2 /day, and administering fludarabine at a dose of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 100-400, 400-800, 800-1200, 1200- 1600, or 1600-2000 mg/m 2 /day, and administering fludarabine at a dose of about 5-10, 10- 20, 20-30, 30-40, 40-50, 50-60, 60-70, or 70-80 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of between about 500 and 1500 mg/m 2 /day and fludarabine at a dose of about 30 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 500 mg/m 2 /day and fludarabine at a dose of about 30 mg/m 2 /day. In some embodiments, the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 1000 mg/m 2 /day and fludarabine at a dose of about 30 mg/m 2 /day.
  • the lymphodepletion regimen is administered to the subject 1, 2, 3, 4, 5, or 6, 7, 8, 9, 10, 11, or 12 times daily. In some embodiments, the lymphodepletion regimen is administered to the subject once daily. In some embodiments, cyclophosphamide is administered separately from fludarabine. In some embodiments, cyclophosphamide and fludarabine are comprised in separate compositions that are administered at about the same time. In some embodiments, cyclophosphamide and fludarabine are comprised in separate compositions that are administered at different times. In other embodiments, cyclophosphamide and fludarabine are comprised in a single composition that is administered to the subject.
  • compositions comprising cyclophosphamide but not fludarabine, fludarabine but not cyclophosphamide, cyclophosphamide and fludarabine, or other lymphodepletion agent(s) may be administered over the course of a dosing regimen to achieve a desired dose of one or more lymphodepletion agents.
  • the lymphodepletion regimen is administered parenterally, intramuscularly, intravenously, intradermally, sublingually, buccally, ocularly, intranasally, subcutaneously, intrathecally, intratumorally, orally, vaginally, or rectally. In some embodiments, the lymphodepletion regimen is administered orally. In some embodiments, the lymphodepletion regimen is administered intravenously.
  • compositions comprising cyclophosphamide but not fludarabine, fludarabine but not cyclophosphamide, cyclophosphamide and fludarabine, or other lymphodepletion agent(s) may be administered through a combination of routes to achieve a desired dose of one or more lymphodepletion agents.
  • the lymphodepletion regimen is administered to the subject starting on a first day and ending on a last day.
  • the first day is 3, 4, 5, 6, 7, 8, 9, or 10, 11, 12, 13, or 14 days prior to the administration of the first pharmaceutical composition.
  • the last day is 0 (i.e., the last day is the same day as the first day), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days after the first day of lymphodepletion regimen administration.
  • the last day occurs no more than 7 days prior to the administration of the first pharmaceutical composition.
  • the last day occurs 1, 2, 3, 4, 5, 6, or 7 days prior to the administration of the first pharmaceutical composition.
  • the lymphodepletion regimen is administered for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days.
  • a lymphodepletion regimen is said to be administered for 1 day if the first day and the last day are the same day of lymphodepletion regimen administration.
  • the lymphodepletion regimen is administered to the subject once daily starting 5 days and ending 3 days prior to administration of the first pharmaceutical composition. In some embodiments, the lymphodepletion regimen is administered to the subject once daily for at least one day, or for multiple days, within 7 days prior to administration of the first pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises administering cyclophosphamide once daily, starting 5 days and ending 3 days prior to administration of the pharmaceutical composition, and administering fludarabine once daily, starting 6 days and ending 3 days prior to administration of the first pharmaceutical composition.
  • the lymphodepletion regimen comprises administering cyclophosphamide once daily at a dose of about 500 mg/m 2 /day and fludarabine once daily at a dose of about 30 mg/m 2 /day, starting 5 days and ending 3 days prior to administration of the first pharmaceutical composition.
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 1000 mg/m 2 /day once daily, starting 5 days and ending 3 days prior to administration of the pharmaceutical composition, and administering fludarabine at a dose of about 30 mg/m 2 /day once daily, starting 6 days and ending 3 days prior to administration of the first pharmaceutical composition.
  • the lymphodepletion regimen does not include administration of a biological lymphodepletion agent during the 7-day period preceding administration of the first pharmaceutical composition comprising genetically modified human immune cells. In some embodiments, at least 168 hours elapse between the administration of any lymphodepletion agents and the administration of the first pharmaceutical composition comprising genetically modified human immune cells.
  • the inclusion of a period of time between the administration of any lymphodepletion agents and the administration of CAR T cells allows for lymphodepletion agents to be metabolized by the subject, reducing their concentration in the bloodstream. Providing a “washout” period after the lymphodepletion regimen prevents residual lymphodepletion agents in the subject from killing administered immune cells, thereby maintaining the efficacy of the administered immune cells.
  • aspects of the present disclosure relate to methods comprising administering a first pharmaceutical composition comprising genetically modified human immune cells comprsing a chimeric antigen receptor (CAR), in conjunction with a second pharmaceutical composition comprising a gamma secretase inhibitor.
  • the human immune cells are selected from the group consisting of T cells, natural killer (NK) cells, B cells, or macrophages.
  • the human immune cells are T cells (e.g., CAR T cells).
  • the human immune cells are not derived from the subject. Human immune cells that are not derived from the subject are said to be allogeneic, while human immune cells that are derived from the subject are said to be autologous.
  • the genetically-modified human immune cells comprise an inactivated T cell receptor (TCR) alpha gene, an inactivated TCR alpha constant region (TRAC) gene, and/or an inactivated TCR beta gene.
  • the genetically- modified human immune cells comprise in their genome a polynucleotide comprising a nucleic acid sequence encoding the CAR.
  • the polynucleotide is positioned within the TCR alpha gene, the TRAC gene, or the TCR beta gene, such that expression of a polypeptide encoded by the TCR alpha gene, the TRAC gene, or the TCR beta gene is disrupted.
  • the polynucleotide can be inserted, for example, within a nuclease recognition sequence (e.g., within a meganuclease, CRISPR system nuclease, TALEN, zinc finger nuclease, or megaTAL recognition sequence).
  • a nuclease recognition sequence e.g., within a meganuclease, CRISPR system nuclease, TALEN, zinc finger nuclease, or megaTAL recognition sequence.
  • the polynucleotide is positioned within SEQ ID NO: 40 (i.e., the TRC 1-2 recognition sequence) within the TRAC gene.
  • the polynucleotide is positioned between nucleotides 13 and 14 of SEQ ID NO: 40.
  • the first pharmaceutical composition is administered at a dose of about 0.1 x 10 6 to about 6 x 10 6 CAR T cells/kg of subject body mass. In some embodiments, the first pharmaceutical composition is administered at a dose of about 0.1 x 10 6 , 0.2 x 10 6 , 0.3 x 10 6 , 0.4 x 10 6 , 0.5 x 10 6 , 0.6 x 10 6 , 0.7 x 10 6 , 0.8 x 10 6 , 0.9 x 10 6 , 1.0 x
  • the first pharmaceutical composition is administered at a dose of about 0.1 x 10 6 to about 0.5 xlO 6 , about 0.5 x 10 6 to about 1.0 xlO 6 , about 1.0 x 10 6 to about 1.5 xlO 6 , about 1.5 x 10 6 to about 2.0 xlO 6 , about 2.0 x 10 6 to about 2.5 xlO 6 , about 2.5 x 10 6 to about 3.0 xlO 6 , about 3.0 x 10 6 to about 3.5 xlO 6 , about 3.5 x 10 6 to about 4.0 xlO 6 , about 4.0 x 10 6 to about 4.5 xlO 6 , about 4.5 x 10 6 to about 5.0 xlO 6 , about 5.0 x 10 6 to about 5.5 xlO
  • the first pharmaceutical composition is administered at a dose of about 0.6 x 10 6 to about 2 x 10 6 CAR T cells/kg. In some embodiments, the first pharmaceutical composition is administered at a dose of about 0.6 x 10 6 , 0.7 x 10 6 , 0.8 x 10 6 , 0.9 x 10 6 , 1.0 x 10 6 , 1.1 x 10 6 , 1.2 x 10 6 , 1.3 x 10 6 , 1.4 x 10 6 , 1.5 x 10 6 , 1.6 x 10 6 , 1.7 x 10 6 , 1.8 x 10 6 , 1.9 x 10 6 , or 2.0 x 10 6 CAR T cells/kg.
  • the first pharmaceutical composition is administered at a dose of about 0.6 x 10 6 to about 0.8 xlO 6 , about 0.8 x 10 6 to about 1.2 xlO 6 , about 1.2 x 10 6 to about 1.6 xlO 6 , or about 1.6 x 10 6 to about 2.0 x 10 6 CAR T cells/kg.
  • the first pharmaceutical composition is administered at a dose of about 0.6 x 10 6 CAR T cells/kg. In some embodiments, the first pharmaceutical composition is administered at a dose of about 1 x 10 6 CAR T cells/kg. In some embodiments, the first pharmaceutical composition is administered at a dose of about 2 x 10 6 CAR T cells/kg. In some embodiments, the first pharmaceutical composition is administered at a dose of about 480 x 10 6 CAR T cells. In some embodiments, the first pharmaceutical composition is administered at a dose of about 960 x 10 6 CAR T cells. In some embodiments, the first pharmaceutical composition is administered in a fixed (or flat) dose. In some embodiments, the first pharmaceutical composition is administered in multiple doses, e.g., multiple fixed doses.
  • the first pharmaceutical composition is administered in split doses, i.e., as 2 administrations of a fixed dose. For instance, a fixed dose of 6.0 x 10 6 cells/kg may be “split” into two doses of 3.0 x 10 6 cells/kg. In some embodiments, the first pharmaceutical composition is administered in an escalating dose.
  • aspects of the present disclosure relate to methods comprising administering a second pharmaceutical composition comprising a gamma secretase inhibitor in conjunction with the administration of a first pharmaceutical composition comprising anti-BCMA CAR cells.
  • Gamma secreatase is a protease complex known to cleave BCMA.
  • the use of gamma secretase inhibitors has been proposed to prevent the cleavage of BCMA and the subsequent generation of soluble BCMA protein in the serum, which may bind the cells of the invention and potentially reduce their efficacy.
  • BCMA CAR T cells BCMA CAR T cells
  • gamma secretase inhibitors useful with the invention include, without limitation, nirogacestat, crenigacastat (LY3039478), LY411575, avagacestat (BMS-708163), AL101 (BMS-906024), AL102 (B MS-986115), RO492087 (RG-4733), MK-0752, and CPX-POM.
  • the gamma secretase inhibitor is selected from the group consisting of PF- 03084014 (Nirogacestat), BMS-708163 (Avagacestat), RO4929097, MK0752, and LY3039478.
  • the gamma secretase inhibitor is PF-03084014 (Nirogacestat).
  • Nirogacestat is a small molecule that selectively and reversibly binds to the gamma secretase complex, preventing proteolytic cleavage of multiple cell surface receptors, such as BCMA and Notch. The administration of Nirogacestat thus inhibits gamma secretase- mediated cleavage of BCMA, thereby increasing the abundance of BCMA on cancer cell surfaces and preventing release of soluble BCMA that can detract BCMA-specific CAR T cells from binding to BCMA on cancer cells.
  • an effective dose of a gamma secretase inhibitor can be administered to a subject in combination with a genetically-modified cell or pharmaceutical composition described herein.
  • the gamma secretase inhibitor can be administered prior to administration of the genetically-modified cell or pharmaceutical composition described herein.
  • the gamma secretase inhibitor can be admininstered concurrently with the genetically-modified cell or pharmaceutical composition described herein.
  • the gamma secretase inhibitor can be admininstered after administration of the genetically-modified cell or pharmaceutical composition described herein.
  • the second pharmaceutical composition is administered at a dose of about 10 mg to about 400 mg of the gamma secretase inhibitor. In some embodiments, the second pharmaceutical composition is administered at a dose of about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 mg of the gamma secretase inhibitor. In some embodiments, the second pharmaceutical composition is administered at a dose of about 20- 50, 50-100, 100-150, 150-200, 200-250, or 250-300 mg of the gamma secretase inhibitor.
  • the second pharmaceutical composition is administered at a dose of about 20 mg to about 300 mg of the gamma secretase inhibitor. In some embodiments, the second pharmaceutical composition is administered at a dose of about 50 mg to about 150 mg of the gamma secretase inhibitor. In some embodiments, the second pharmaceutical composition is administered at a dose of about 100 mg of the gamma secretase inhibitor.
  • the second pharmaceutical composition comprising a gamma secretase inhibitor is administered parenterally, intramuscularly, intravenously, intradermally, sublingually, buccally, ocularly, intranasally, subcutaneously, intrathecally, intratumorally, orally, vaginally, or rectally. In some particular embodiments, the second pharmaceutical composition comprising a gamma secretase inhibitor is administered orally.
  • the second pharmaceutical composition comprising a gamma secretase inhibitor is administered once, twice, three times, or four times daily.
  • the number of times a pharmaceutical composition is administered “daily” may refer to the number of times the composition is administered in a calendar day, or to the number of times the composition is administered in a given period of 24 hours.
  • the second pharmaceutical composition is administered once daily.
  • the second pharmaceutical composition is administered twice daily.
  • the second pharmaceutical composition is administered three times daily.
  • the second pharmaceutical composition is administered four times daily.
  • at least one dose of the second pharmaceutical composition is administered daily for at least one day prior to administration of the first pharmceutical composition.
  • at least one dose of the second pharmaceutical composition is administered daily for between 2 days and 7 days prior to administration of the first pharmceutical composition.
  • At least one dose of the second pharmaceutical composition is administered daily for between 3 days and 5 days prior to administration of the first pharmceutical composition. In some embodiments, at least one dose of the second pharmaceutical composition is administered daily for 3 days prior to administration of the first pharmceutical composition.
  • At least one dose of the second pharmaceutical composition is administered daily for at least 7 days after administration of the first pharmceutical composition (e.g., after administration of a fixed dose of first pharmceutical composition, or after administration of the first dose of a multiple-dose regimen of the first pharmceutical composition).
  • At least one dose of the second pharmaceutical composition is administered daily for up to about 90 days after administration of the first pharmceutical composition. In some embodiments, at least one dose of the second pharmaceutical composition is administered daily for between about 7 days and about 90 days after administration of the first pharmceutical composition. In some embodiments, at least one dose of the second pharmaceutical composition is administered daily for between about 20 days and about 80 days after administration of the first pharmceutical composition. In some embodiments, at least one dose of the second pharmaceutical composition is administered daily for between about 50 days and about 70 days after administration of the first pharmceutical composition. In some embodiments, at least one dose of the second pharmaceutical composition is administered daily for about 60 days after administration of the first pharmceutical composition.
  • the second pharmaceutical composition is administered twice daily beginning 3 days prior to administration of the first pharmaceutical composition and ending about 60 days after administration of the first pharmaceutical composition, wherein the second pharmaceutical composition is administered at a dose of about 100 mg of the gamma secreatase inhibitor.
  • the lymphodepletion regimen comprises administering cyclophosphamide once daily at a dose of about 500 mg/m 2 /day and fludarabine once daily at a dose of about 30 mg/m 2 /day starting 5 days and ending 3 days prior to administration of the first pharmaceutical composition, wherein the first pharmaceutical composition is administered at a dose of about 0.6 x 10 6 CAR T cells/kg, about 2 x 10 6 CAR T cells/kg, about 480 x 10 6 CAR T cells, or about 960 x 10 6 CAR T cells, and wherein the second pharmaceutical composition is administered twice daily beginning 3 days prior to administration of the first pharmaceutical composition and ending about 60 days after administration of the first pharmaceutical composition, wherein the second pharmaceutical composition is administered at a dose of about 100 mg of the gamma secreatase inhibitor.
  • the gamma secretase inhibitor is PF-03084014 (Nirogacestat).
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 1000 mg/m 2 /day once daily starting 5 days and ending 3 days prior to administration of the pharmaceutical composition, and administering fludarabine at a dose of about 30 mg/m 2 /day once daily starting 6 days and ending 3 days prior to administration of the first pharmaceutical composition, wherein the first pharmaceutical composition is administered at a dose of about 0.6 x 10 6 CAR T cells/kg, about 2 x 10 6 CAR T cells/kg, about 480 x 10 6 CAR T cells, or about 960 x 10 6 CAR T cells, and wherein the second pharmaceutical composition is administered twice daily beginning 3 days prior to administration of the first pharmaceutical composition and ending about 60 days after administration of the first pharmaceutical composition, wherein the second pharmaceutical composition is administered at a dose of about 100 mg of the gamma secreatase inhibitor.
  • the gamma secretase inhibitor is PF-03084014 (Ni
  • the method results in a reduction of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the volume of a tumor in the subject.
  • the method results in a reduction of at least 10% of the volume of the tumor in the subject.
  • the method results in a reduction of at least 30% of the volume of the tumor in the subject.
  • reduction in the volume (or mass) of a tumor is determined using a standard experimental method known in the art, such as biopsy of an affected organ, histopathology, immunohistochemistry, flow cytometry, physical examination, imaging, and DNA or RNA sequencing.
  • a reduction in the mass or volume of a tumor may be determined by measuring the number of circulating cancer cells in the subject by immunohistochemistry and flow cytometry and cell counting.
  • the method results in a reduction of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the mass of one or more tumors in the subject. In some embodiments, the method results in a reduction of at least 30% of the mass of one or more tumors in the subject.
  • the method results in a reduction of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the mass of a primary tumor in the subject. In some embodiments, the method results in a reduction of at least 30% of the mass of a primary tumor in the subject.
  • the method results in a reduction of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the volume of a primary tumor in the subject.
  • the method results in a reduction of at least 30% of the volume of a primary tumor in the subject.
  • a primary tumor refers to the largest tumor in a subject, by mass or volume.
  • a primary tumor refers to a tumor in which the largest percentage of cells are cancer cells.
  • the method results in a reduction of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% in the number of circulating cancer cells in the subject.
  • the method results in a reduction of at least 10% of the number of circulating cancer cells in the subject.
  • the method results in a reduction of at least 30% of the number of circulating cancer cells in the subject.
  • Measuring the number of circulating cancer cells in the subject may be used to evaluate the effect of a method as an addition, or alternative, to measuring the size or growth rate of a tumor.
  • cancers of hematopoietic cells such as leukemias and lymphomas may not necessarily form tumors, and so tracking the number or proportion of circulating cells that are cancer cells allows for evaluation of a method’s effectiveness when measuring tumor size is not feasible.
  • circulating cancer cells may be cells that are shed from a tumor and may form secondary growths (metastases) at other anatomical sites. Metastases are associated with poor prognoses and increased risk of death in cancer, and so reducing the number of circulating cancer cells in a subject is a desirable outcome even when a tumor is present in the subject. See, e.g., Zhong et al., Mol Cancer. 2020. 19(1):15.
  • the method results in a reduction of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the tumor growth rate in the subject.
  • the method results in a reduction of at least 10% of the tumor growth rate in the subject.
  • the method results in a reduction of at least 30% of the tumor growth rate in the subject. See, e.g., Hather et al., Cancer Inform. 2014. 13(Suppl 4): 65-72.
  • the method results in a reduction of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% in the number of tumor cells in a tissue in the subject.
  • tumor cells are identified as containing a tumor antigen based on immunohistochemistry analysis.
  • Immunohistochemistry refers to staining cells with an antibody or other reagent that selectively binds to or interacts with a target antigen, such as a tumor antigen, and visualizing stained cells, such as by microscopy.
  • a target antigen such as a tumor antigen
  • Cells that are stained are identified as containing an antigen and/or being a tumor cell, while cells that are not stained are classified as not containing an antigen and/or not being a tumor cell.
  • tumor cells are identified using histology.
  • the method results in a reduction of at least 10% in the number of tumor cells in a tissue in the subject. In some embodiments, the method results in a reduction of at least 30% of a number of tumor cells in a tissue in the subject.
  • the method prevents or reduces the frequency of metastasis in the subject for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 15 months, at least 18 months, at least 21 months, at least 24 months, at least 30 months, at least 36 months, at least 42 months, at least 48 months, at least 54 months, at least 60 months, at least 66 months, at least 72 months, at least 78 months, at least 84 months, at least 90 months, at least 96 months, at least 102 months, at least 108 months, at least 114 months, or at least 120 months.
  • Metastasis refers to the development of a secondary tumor or mass of cancer cells that is in a separate anatomical site from a primary tumor. Generally, metastasis occurs when a cancer cell is released from a tumor, circulates through the body, and begins replicating in a distinct anatomical site that is separate from the tumor that released it.
  • the method prevents or reduces the frequency of metastasis for at least 6 months. In some embodiments, the method prevents or reduces the frequency of metastasis for at least 12 months. In some embodiments, the method prevents or reduces the frequency of metastasis for at least 24 months. In some embodiments, the method prevents or reduces the frequency of metastasis for at least 36 months.
  • the method prevents or reduces the frequency of metastasis for at least 48 months. In some embodiments, the method prevents or reduces the frequency of metastasis for at least 60 months. In some embodiments, the method prevents or reduces the frequency of metastasis for at least 72 months. In some embodiments, the method prevents or reduces the frequency of metastasis for at least 84 months. In some embodiments, the method prevents or reduces the frequency of metastasis for at least 96 months. In some embodiments, the method prevents or reduces the frequency of metastasis for at least 108 months. In some embodiments, the method prevents or reduces the frequency of metastasis for at least 120 months.
  • the method prevents the recurrence of cancer for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 15 months, at least 18 months, at least 21 months, at least 24 months, at least 30 months, at least 36 months, at least 42 months, at least 48 months, at least 54 months, at least 60 months, at least 66 months, at least 72 months, at least 78 months, at least 84 months, at least 90 months, at least 96 months, at least 102 months, at least 108 months, at least 114 months, or at least 120 months.
  • the method prevents the recurrence of cancer for at least 6 months. In some embodiments, the method prevents the recurrence of cancer for at least 12 months. In some embodiments, the method prevents the recurrence of cancer for at least 24 months. In some embodiments, the method prevents the recurrence of cancer for at least 36 months. In some embodiments, the method prevents the recurrence of cancer for at least 48 months. In some embodiments, the method prevents the recurrence of cancer for at least 60 months. In some embodiments, the method prevents the recurrence of cancer for at least 72 months. In some embodiments, the method prevents the recurrence of cancer for at least 84 months.
  • the method prevents the recurrence of cancer for at least 96 months. In some embodiments, the method prevents the recurrence of cancer for at least 108 months. In some embodiments, the method prevents the recurrence of cancer for at least 120 months.
  • Recurrence of cancer can be determined by any one of multiple methods known in the art, including biopsy of an affected organ, histopathology, immunohistochemistry, flow cytometry, physical examination, imaging, and DNA or RNA sequencing.
  • the method eradicates the cancer in the subject.
  • Eradication of a cancer in a subject refers to the reduction of cancer cell numbers to the extent that no cells of the cancer can be detected.
  • following performance of the method cancer cells cannot be detected in the subject by one or more methods selected from the group consisting of flow cytometry, immunohistochemistry, biopsy, magnetic resonance imaging, and computed tomography.
  • the method increases an amount of membrane-bound BCMA on the surface of a cancer cell in the subject. In some embodiments, the method increases an amount of membrane-bound BCMA by a factor of at least 1.25, 1.5, 1.75, 2.00, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 15.0, or up to 20.0.
  • Methods of quantifying membrane-bound BCMA on cell surfaces are known in the art, and include microscopy, flow cytometry, and ELISPOT assays. Cells may be stained by incubation with antibodies that comprise a fluorescent marker, such as a fluorophore, and that bind specifically to an epitope of BCMA, such as the extracellular domain of BCMA.
  • Stained cells may then be visualized by microscopy to quantify the fluorescence intensity corresponding to the fluorophore of the anti-BCMA antibody, with the fluorescence intensity of stained cancer cells being compared to that of control cells stained by incubation with the same antibody.
  • fluorescence may be quantified by flow cytometry, in which one or more populations of cells are stained, and the mean or median fluorescence intensity of cancer cells can be calculated and compared to the mean or median fluorescence intensity of control cells, such as cells from a subject that was not administered a gamma secretase inhibitor.
  • the method reduces the concentration of soluble BCMA in the bloodstream of the subject. In some embodiments, the method reduces the concentration of soluble BCMA in the bloodstream of the subject by a factor of at least 1.25, 1.5, 1.75, 2.00, 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 15.0, or up to 20.0.
  • Methods of measuring the concentration of soluble BCMA in the bloodstream are known in the art, and include ELISA, western blotting, and chromatography.
  • the subject is administered an additional cancer therapy selected from the group consisting of chemotherapy, surgery, radiation, and gene therapy.
  • the method further comprises administering an additional anti-cancer agent to the subject.
  • the anti-cancer agent is an antibody or antigenbinding fragment thereof.
  • the antibody or antigen-binding fragment thereof is an immune checkpoint inhibitor.
  • An immune checkpoint inhibitor refers to an agent that blocks the activity of one or more immune checkpoints, including PD-1, PD-L1, and CTLA-4.
  • the antibody or antigen-binding fragment thereof is an anti- PD-1 antibody, anti-PD-Ll antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-TIM3 antibody, anti-LAG3 antibody, anti-CD200 antibody, or anti-CD200R antibody.
  • the anti-cancer agent is a chemotherapeutic agent. In some embodiments, the anti-cancer agent is an agonist of a positive immune checkpoint.
  • a positive immune checkpoint refers to a receptor on a cell that, when stimulated, transduces an activating signal to the cell.
  • the positive immune checkpoint is selected from the group consisting of CD27, CD28, CD40, CD122, 4-1BB, 0X40, GITR, and ICOS. In some embodiments, the positive immune checkpoint is selected from the group consisting of CD40, 4-1BB, and GITR.
  • the anti-cancer agent is an antibody or antigenbinding fragment thereof that binds to CD27, CD28, CD40, CD122, 4-1BB, 0X40, GITR, or ICOS. In some embodiments, the anti-cancer agent is an antibody or antigen-binding fragment thereof that binds to CD40, 4- IBB, or GITR.
  • the anti-cancer agent is a chemotherapeutic agent.
  • a chemotherapeutic agent is a chemical compound useful in the treatment of cancer.
  • examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL); beta-lapachone; lapachol; colchicines; betulinic acid;
  • calicheamicin especially calicheamicin gammall and calicheamicin omegall (see, e.g., Nicolaou et al., Angew. Chem Inti. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, doxorubicin (including ADRIAMYCIN, morpholino-doxor
  • Chemotherapeutic agents also include anti-hormonal agents that act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer, and are often in the form of systemic, or whole-body treatment. They may be hormones themselves. Examples include anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX tamoxifen), raloxifene (EVISTA), droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); anti-progesterones; estrogen receptor down-regulators (ERDs); estrogen receptor antagonists such as fulvestrant (FASLODEX); agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as leuprolide acetate (LUPRON and ELIGARD
  • chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS or OSTAC), etidronate (DIDROCAL), NE-58095, zoledronic acid/zoledronate (ZOMETA), alendronate (FOSAMAX), pamidronate (AREDIA), tiludronate (SKELID), or risedronate (ACTONEL); as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); anti-sense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE vaccine and gene therapy vaccines, for example, ALLOVECTIN vaccine, LEUVECTIN vaccine, and VAXID vaccine; topoisomerase 1 inhibitor (e.g.,
  • the subject is refractory to prior CAR T cell immunotherapy.
  • the subject is a human.
  • the subject has or is at risk of developing a cancer that is characterized by the presence of one or more BCMA+ cells.
  • the cancer is multiple myeloma, Non-Hodgkin Lymphoma, Hodgkin Lymphoma, Chronic Lymphocytic Leukemia (CLL), glioblastoma, and Waldenstrom’s Macroglobulinemia.
  • the cancer is multiple myeloma, such as relapsing or refractory multiple myeloma.
  • administration of any of the disclosed pharmaceutical compositions, or any of the disclosed methods of immunotherapy results in an achievement of a partial response or a complete response to the immunotherapy.
  • the partial response or the complete response is maintained through at least 28 days after administration of the pharmaceutical composition.
  • compositions used in the methods provided herein may be formulated with one or more pharmaceutically acceptable carriers.
  • suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
  • Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the binding proteins.
  • the compositions of the injection can, as is well known in the art, be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the mammal.
  • kits for the treatment or prevention of a tumor e.g., multiple myeloma
  • the kit comprises a therapeutic composition containing an effective amount of BCMA-specific CAR T cells, a gamma secretase inhibitor, and/or lymphodepletion agent(s) in unit dosage form.
  • the kit comprises a sterile container which contains a therapeutic or prophylactic vaccine; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • the BCMA-specific CAR T cells, gamma secretase inhibitor, and/or lymphodepletion agent(s) are provided together with instructions for administering the cells, inhibitors, and agents to a subject having or at risk of developing a tumor (e.g., multiple myeloma).
  • the instructions will generally include information about the use of the composition for the treatment or prevention of a tumor (e.g., multiple myeloma).
  • the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of a neoplasia (e.g., multiple myeloma) or symptoms thereof; precautions; warnings; indications; counter- indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references.
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • a CAR comprises at least an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the intracellular domain, or cytoplasmic domain can comprise, for example, at least one co- stimulatory domain and one or more signaling domains.
  • the extracellular domain of a CAR can comprise, for example, a target- specific binding element (e.g., an antibody or antibody fragment that specifically binds to BCMA) otherwise referred to herein as an extracellular ligand-binding domain or anti- BCMA binding domain.
  • the CAR of the present disclosure is engineered to specifically bind to human BCMA, an antigen that is expressed on the surface of certain human cancers.
  • the amino acid sequence of human BCMA is provided in SEQ ID NO: 1.
  • the extracellular ligand-binding domain has specificity for BCMA.
  • the extracellular ligand-binding domain is a single-chain variable fragment (scFv) comprising: (a) a heavy chain variable domain (VH) derived from the BCMA-20 antibody, and a light chain variable domain (VL) derived from the BCMA-3 antibody.
  • scFv single-chain variable fragment
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the extracellular ligand-binding domain or moiety of a CAR can be, for example, an antibody or antibody fragment, particularly any anti- BCMA antibody, or antigen-binding fragment thereof, described herein.
  • An antibody fragment can, for example, be at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, singledomain antibodies (sdAbs), camelid VHH domains, multi- specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody.
  • An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005).
  • Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).
  • Fn3 fibronectin type III
  • the extracellular ligand-binding domain or moiety of a CAR is in the form of a single-chain variable fragment (scFv) derived from an anti-BCMA antibody, or antigen-binding fragment thereof, described herein, which provides specificity for human BCMA.
  • scFv single-chain variable fragment
  • the VH and VL regions of an scFv can be arranged such that the VH region is the 5' domain and the VL region is the 3' domain, or they can be arranged such that the VL region is the 5' domain and the VH region is the 3' domain.
  • the VH region and VL region are connected by a polypeptide by a linker such as, for examples, those linkers described elsewhere hererin.
  • the scFv is murine or humanized.
  • the anti-BCMA binding domain of the CAR can comprise any scFv described herein such as, for example, scFvs comprising an amino acid sequence set forth in any one of SEQ ID NOs: 43 and 44, and variants thereof.
  • the extracellular ligand-binding domain of a CAR can also comprise an autoantigen (see, Payne et al. (2016), Science 353 (6295): 179-184), that can be recognized by autoantigen-specific B cell receptors on B lymphocytes, thus directing T cells to specifically target and kill autoreactive B lymphocytes in antibody-mediated autoimmune diseases.
  • Such CARs can be referred to as chimeric autoantibody receptors (CAARs), and their use is encompassed by the invention.
  • CAARs chimeric autoantibody receptors
  • the extracellular ligand-binding domain of a CAR can also comprise a naturally-occurring ligand for an antigen of interest, or a fragment of a naturally- occurring ligand which retains the ability to bind the antigen of interest.
  • a CAR comprises a transmembrane domain which links the extracellular ligandbinding domain with the intracellular signaling and co- stimulatory domains via a hinge region or spacer sequence.
  • the transmembrane domain can be derived from any membranebound or transmembrane protein.
  • the transmembrane polypeptide can be a subunit of the T-cell receptor (e.g., polypeptide constituting CD3 complex), IL2 receptor p55 (a chain), p75 (P chain) or y chain, subunit chain of Fc receptors (e.g., Fey receptor III) or CD proteins such as the CD8 alpha chain.
  • transmembrane domains of particular use in this invention may be derived from TCRa, TCRp, TCR ⁇ , CD3 ⁇ , CD3s, CD3y, CD38, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD32, CD33, CD34, CD37, CD45, CD64, CD80, CD86, CD134, CD137, and CD154.
  • any transmembrane domain is contemplated for use herein as long as the domain is capable of anchoring a CAR comprising the extracellular domain to a cell membrane.
  • Transmembrane domains can also be identified using any method known in the art or described herein.
  • the transmembrane domain of the CAR is a CD8 transmembrane domain comprising an amino acid sequence set forth in SEQ ID NO: 30, and variants thereof.
  • a CAR disclosed herein further comprises a hinge region.
  • the hinge region refers to any oligo- or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain.
  • a hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • Hinge regions may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region.
  • the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence or may be an entirely synthetic hinge sequence.
  • a hinge domain can comprise a part of a human CD8 alpha chain, FcyRllla receptor or IgGl.
  • the hinge region of the CAR is a CD8 hinge region comprising an amino acid sequence set forth in SEQ ID NO: 29, and variants thereof.
  • Intracellular signaling domains of a CAR are responsible for activation of at least one of the normal effector functions of the cell in which the CAR has been placed and/or activation of proliferative and cell survival pathways.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • the intracellular signaling domain can include one or more cytoplasmic signaling domains that transmit an activation signal to the T cell following antigen binding.
  • Such cytoplasmic signaling domains can include, without limitation, a CD3 zeta signaling domain comprising an amino acid sequence set forth in SEQ ID NO: 35, and variants thereof.
  • the intracellular domain of a CAR can also include one or more intracellular costimulatory domains that transmit a proliferative and/or cell- survival signal after ligand binding.
  • the co-stimulatory domain can comprise one or more TRAF-binding domains.
  • Intracellular co-stimulatory domains can be any of those known in the art and can include, without limitation, those co-stimulatory domains disclosed in WO 2018/067697 including, for example, Novel 1 (“Nl”; SEQ ID NO: 31), Novel 6 (“N6”; SEQ ID NO: 32), 4- IBB (SEQ ID NO: 33), CD28 (SEQ ID NO: 34), or variants thereof.
  • co-stimulatory domains can include a functional signaling domain obtained from a protein including an MHC class I molecule, TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), activating NK cell receptors, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD30, CD40, CDS, ICAM-1, LFA-1 (CDlla/CD18), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, VLA
  • the intracellular domains of a CAR described herein may be linked to each other in a specified or random order.
  • the co-stimulatory domain is proximal to the transmembrane domain relative to the intracellular signaling domain.
  • the intracellular domain of a CAR described herein may contain short polypeptide linker or spacer regions, between 2 to 30 amino acids in length.
  • the intracellular domain of a CAR described herein may contain short polypeptide linker or spacer regions, between 2 to 10 amino acids in length.
  • the linker or spacer regions may include an amino acid sequence that substantially comprises glycine and serine.
  • CARs of the invention can, in some examples, further comprise a spacer sequence that is positioned between the extracellular hinge domain and the anti-BCMA binding domain.
  • the spacer can comprise an amino acid sequence set forth in SEQ ID NO: 28, or variants thereof.
  • the spacer of SEQ ID NO: 28 is encoded by a nucleic acid sequence comprising SEQ ID NO: 50. GLSGL (SEQ ID NO: 28)
  • CARs of the invention can also comprise a signal peptide.
  • Such signal peptides can be positioned at the 5' end of the polypeptide, typically connected to the anti-BCMA binding domain.
  • the CAR comprises a signal peptide comprising an amino acid sequence set forth in SEQ ID NO: 36, or variants thereof.
  • the signal peptide can comprise an amino acid sequence set forth in SEQ ID NO: 37, and variants thereof.
  • the signal peptide can comprise an amino acid sequence set forth in SEQ ID NO: 49, and variants thereof.
  • MALPVTALLLPLALLLHAAQP SEQ ID NO: 36
  • MALPVTALLLPLALLLHAAQPA SEQ ID NO: 37
  • MALPVTALLLPLALLLHAARP SEQ ID NO: 49
  • An exemplary CAR of the present disclosure may be selected from a BCMA-3L/20H- Spacer-CD8-CD8-N6-CD3z CAR, a BCMA-20H/3L-Spacer-CD8-CD8-N6-CD3z CAR, a BCMA-3L/20H-Spacer-CD8-CD8-N6-CD3z CAR, a BCMA-20H/3L-Spacer-CD8-CD8-N6- CD3z CAR, a CD8(+A)SP-BCMA-3L/20H-Spacer-CD8-CD8-N6-CD3z CAR, a CD8(+A)SP-BCMA-20H/3L-Spacer-CD8-CD8-N6-CD3z CAR, a CD8(+A)SP-BCMA- 3L/20H-Spacer-CD8-CD8-N6-CD3z CAR, a CD8(+A)SP-BCMA- 3L/20H-Spacer-CD8-CD8-
  • An exemplary CAR of the present disclosure may comprise the amino acid sequence of any of SEQ ID NOs: 45-48.
  • the CAR comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or more, sequence identity to any of SEQ ID NOs: 45-48 and has specificity for BCMA.
  • the CAR comprises an amino acid sequence that differs from the sequence of any of SEQ ID NOs: 45-48 by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 amino acids.
  • CARs of the invention comprise an amino acid sequence set forth in SEQ ID NOs: 45 or 46, and variants thereof.
  • Such CARs comprise: (a) scFvs described herein, which include the VH of the BCMA-20 antibody and VL region of the BCMA-3 antibody, which are connected by a linker set forth in SEQ ID NO: 10; (b) a spacer set forth in SEQ ID NO: 28 (e.g., encoded by SEQ ID NO: 50); (c) a CD8 hinge domain set forth in SEQ ID NO: 29; (d) a CD8 transmembrane domain set forth in SEQ ID NO: 30; (e) an N6 co- stimulatory domain set forth in SEQ ID NO: 32; and (f) a CD3 zeta signaling domain set forth in SEQ ID NO: 35.
  • CARs of the invention comprise an amino acid sequence set forth in SEQ ID NOs: 47 or 48, and variants thereof. These CARs comprise the same elements as those of SEQ ID NOs: 45 or 46, and further comprise a 5' signal peptide set forth in SEQ ID NO: 37.
  • any of the polynucleotides described herein that encode a CAR can be prepared by a routine method, such as recombinant technology.
  • Methods for preparing a CAR described herein may involve, in some embodiments, the generation of a polynucleotide that encodes a polypeptide comprising each of the domains of the CAR (e.g., at least an extracellular domain, a transmembrane domain, and an intracellular domain).
  • a genetically-modified cell of the invention comprises a polynucleotide encoding a CAR described herein.
  • a polynucleotide or expression cassette which encodes a CAR described herein is present (i.e., integrated) within the genome of the genetically-modified cell or, alternatively, is not integrated into the genome of the cell.
  • the polynucleotide or expression cassette is present in the genetically-modified cell in a recombinant DNA construct, in an mRNA, in a viral genome, or in another polynucleotide which is not integrated into the genome of the cell.
  • genetically-modified cells of the invention can contain a polynucleotide encoding a CAR described herein, positioned within the genome of the cell.
  • genetically-modified cells contain a polynucleotide encoding a CAR described herein, positioned within the endogenous T cell receptor alpha gene, the endogenous T cell receptor alpha gene, or the T cell receptor beta gene of the cell.
  • a polynucleotide encoding a CAR described herein is positioned within the endogenous T cell receptor alpha constant region gene, such as within exon 1 of the T cell receptor alpha constant region gene.
  • a polynucleotide encoding a CAR described herein is positioned specifically within SEQ ID NO: 40 (i.e., the TRC 1-2 recognition sequence) within the T cell receptor alpha constant region (i.e., TRAC) gene.
  • a polynucleotide encoding a CAR described herein is positioned between positions 13 and 14 of SEQ ID NO: 40 (i.e., the TRC 1-2 recognition sequence) within the TRAC gene.
  • Immune cells of the present disclosure can be obtained from any source, such as peripheral blood mononuclear cells (PBMCs), cord blood, tissue from site of an infection, ascites, pleural effusion, bone marrow, tissues such as spleen, lymph node, thymus, or tumor tissue.
  • PBMCs peripheral blood mononuclear cells
  • the population of immune cells is derived from PBMCs.
  • Immune cells useful for the invention may also be derived from pluripotent stem cells (e.g., induced pluripotent stem cells) that have been differentiated into an immune cell.
  • the immune cells of the invention are T cells or NK cells, particularly human T cells or human NK cells, or cells derived therefrom.
  • Such cells can be, for example, primary T cells or primary NK cells.
  • T cells or NK cells are obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as those described herein above.
  • cells from the circulating blood of an individual are obtained by apheresis. Methods of preparing cells capable of expressing a CAR described herein may comprise expanding isolated cells ex vivo.
  • Expanding cells may involve any method that results in an increase in the number of cells capable of expressing a CAR described herein, for example, by allowing the cells to proliferate or stimulating the cells to proliferate. Methods for stimulating expansion of cells will depend on the type of cell used for expression of a CAR and will be evident to one of skill in the art.
  • the cells expressing a CAR described herein are expanded ex vivo prior to administration to a subject.
  • Genetically-modified immune cells comprising a CAR described herein can exhibit increased proliferation when compared to appropriate control cells that do not comprise a CAR.
  • cells comprising a CAR described herein further exhibit increased activation and proliferation in vitro or in vivo following stimulation with an appropriate antigen.
  • proliferate refers to an expansion in the number of CAR T cells described herein, in a subject following administration during immunotherapy.
  • Such proliferation or expansion can be determined by methods known in the art and those shown in the examples herein, which include, for example, utilizing PCR analysis to determine the number of copies of a CAR transgene per pg of DNA isolated from peripheral blood mononuclear cells over a time course following administration of the pharmaceutical composition comprising CAR T cells, or using flow cytometry to determine the number of CAR-positive T cells in blood.
  • cells such as CAR T cells and CAR NK cells, can exhibit increased activation, proliferation, and/or increased cytokine secretion compared to a control cell lacking the CARs described herein.
  • Increased cytokine secretion can include the increased secretion of IFN-y, IL-2, TNF-a, among others.
  • Methods for measuring cell activation and cytokine production are well known in the art, and some suitable methods are provided in the examples herein.
  • a suicide gene can encode a cytotoxic polypeptide, a polypeptide that has the ability to convert a nontoxic pro-drug into a cytotoxic drug, and/or a polypeptide that activates a cytotoxic gene pathway within the cell. That is, a suicide gene is a nucleic acid that encodes a product that causes cell death by itself or in the presence of other compounds. A representative example of such a suicide gene is one that encodes thymidine kinase of herpes simplex virus.
  • genes that encode thymidine kinase of varicella zoster virus and the bacterial gene cytosine deaminase that can convert 5-fluorocytosine to the highly toxic compound 5- fluorouracil are also include as non-limiting examples genes that encode caspase- 9, caspase-8, or cytosine deaminase. In some examples, caspase-9 can be activated using a specific chemical inducer of dimerization (CID).
  • a suicide gene can also encode a polypeptide that is expressed at the surface of the cell that makes the cells sensitive to therapeutic and/or cytotoxic monoclonal antibodies.
  • a suicide gene can encode recombinant antigenic polypeptide comprising an antigenic motif recognized by the anti-CD20 mAb Rituximab and an epitope that allows for selection of cells expressing the suicide gene.
  • a suicide gene can encode recombinant antigenic polypeptide comprising an antigenic motif recognized by the anti-CD20 mAb Rituximab and an epitope that allows for selection of cells expressing the suicide gene.
  • the RQR8 polypeptide described in WO2013153391 which comprises two Rituximab-binding epitopes and a QBEndlO-binding epitope.
  • Rituximab can be administered to a subject to induce cell depletion when needed.
  • a suicide gene may include a QBEndlO-binding epitope expressed in combination with a truncated EGFR polypeptide.
  • Cells modified by the methods and compositions described herein can express a CAR described herein and further lack expression of an endogenous T cell receptor (e.g., an alpha/beta T cell receptor) due to inactivation of the TCR alpha gene, the TRAC gene, and/or the TCR beta region gene.
  • an endogenous T cell receptor e.g., an alpha/beta T cell receptor
  • the T cell alpha chain and TCR beta chain are required for assembly of the endogenous alpha/beta T cell receptor; therefore, disrupted expression of one or both of these chains also disrupts assembly of the endogenous alpha/beta T cell receptor on the cell surface. This further results in a lack of detectable expression of CD3 on the cell surface, because CD3 is also a component of the endogenous alpha/beta T cell receptor.
  • CAR T cells of the present invention can comprise an inactivated TCR alpha gene and/or an inactivated TCR beta gene.
  • Inactivation of the TCR alpha gene and/or TCR beta gene to generate the CAR T cells of the present invention occurs in at least one or both alleles where the TCR alpha gene and/or TCR beta gene is being expressed.
  • inactivation may occur by disruption of either one of the alleles of the TCR alpha or TCR beta gene. Accordingly, inactivation of one or both alleles prevents expression of the endogenous TCR alpha chain or the endogenous TCR beta chain protein.
  • Inactivation of the TCR alpha gene and/or the TCR beta gene results in CAR T cells that have no detectable cell surface expression of the endogenous alpha/beta TCR.
  • the endogenous alpha/beta TCR incorporates CD3. Therefore, cells with an inactivated TCR alpha gene and/or TCR beta chain can have no detectable cell surface expression of CD3, e.g., as determined by flow cytometry specific for CD3.
  • the inactivated gene is a TCR alpha constant region (TRAC) of the TCR alpha gene.
  • the TCR alpha gene, the TRAC region, or the TCR beta gene is inactivated by insertion of a transgene encoding the CAR. In some examples, one or both alleles of the TCR alpha gene is inactivated by insertion of a transgene encoding the CAR. In some embodiments, one or both alleles of the TRAC region is inactivated by insertion of a transgene encoding the CAR. In some examples, one or both alleles of the TCR beta gene is inactivated by insertion of a transgene encoding the CAR.
  • Insertion of the CAR transgene disrupts expression of the endogenous TCR alpha chain or TCR beta chain and, therefore, prevents assembly of an endogenous alpha/beta TCR on the T cell surface.
  • the CAR transgene can be inserted, for example, within an engineered nuclease recognition sequence (e.g., a meganuclease, CRISPR system nuclease, TALEN, zinc finger nuclease, or megaTAL recognition sequence).
  • the CAR transgene is inserted into the TRAC gene.
  • a CAR transgene is inserted into the TRAC gene at an engineered meganuclease recognition sequence comprising SEQ ID NO: 40.
  • the CAR transgene is inserted into SEQ ID NO: 40 between nucleotide positions 13 and 14. Insertion of the CAR transgene within SEQ ID NO: 1 can be mediated by an engineered meganuclease, such as the TRC 1-2L.1592 meganuclease set forth in SEQ ID NO: 42.
  • an engineered meganuclease such as the TRC 1-2L.1592 meganuclease set forth in SEQ ID NO: 42.
  • the polynucleotide encoding the CAR can further comprise additional control sequences.
  • the sequence can include homologous recombination enhancer sequences, Kozak sequences, poly adenylation sequences, transcriptional termination sequences, selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, and the like.
  • Sequences encoding engineered nucleases can also include at least one nuclear localization signal. Examples of nuclear localization signals are known in the art (see, e.g., Lange et al., J. Biol. Chem., 2007, 282:5101-5105).
  • the polynucleotide encoding the CAR can further comprise a promoter that is operably linked to the CAR coding sequence.
  • the polynucleotide includes a promoter comprising an amino acid sequence set forth in SEQ ID NO: 38 (i.e., a JeT promoter). In some examples, the polynucleotide includes a promoter comprising an amino acid sequence set forth in SEQ ID NO: 39 (i.e., an EFl alpha promoter).
  • compositions Comprising Genetically-Modified Immune Cells
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising genetically-modified immune cells described herein, or a population of genetically-modified immune cells described herein, and a pharmaceutically-acceptable carrier.
  • Such pharmaceutical compositions can be prepared in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (21st ed. 2005).
  • cells are typically admixed with a pharmaceutically acceptable carrier and the resulting composition is administered to a subject (e.g., a human).
  • the pharmaceutically acceptable carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject.
  • the pharmaceutical compositions of the present disclosure further comprise one or more additional agents useful in the treatment of a disease (e.g., cancer) in a subject.
  • a disease e.g., cancer
  • pharmaceutical compositions of the present disclosure can further include biological molecules, such as cytokines (e.g., IL- 2, IL-7, IL-15, and/or IL-21), which promote in vivo cell proliferation and engraftment.
  • cytokines e.g., IL- 2, IL-7, IL-15, and/or IL-21
  • Pharmaceutical compositions comprising genetically-modified cells of the present disclosure can be administered in the same composition as an additional agent or biological molecule or, alternatively, can be co-administered in separate compositions.
  • the present disclosure also provides genetically-modified immune cells, or populations thereof, described herein for use as a medicament.
  • the present disclosure further provides the use of genetically-modified immune cells, or populations thereof, described herein in the manufacture of a medicament for treating a disease in a subject in need thereof.
  • the medicament is useful for cancer immunotherapy in subjects in need thereof.
  • the pharmaceutical compositions and medicaments of the present disclosure are useful for treating any disease state that can be targeted by adoptive immunotherapy.
  • the pharmaceutical compositions and medicaments of the present disclosure are useful as immunotherapy in the treatment of cancer.
  • the pharmaceutical composition is useful for treating a BCMA-related disease by killing a BCMA expressing (i.e., BCMA-positive) target cell.
  • the pharmaceutical composition is useful for treating multiple myeloma (MM), Non-Hodgkin Lymphoma, Hodgkin Lymphoma, Chronic Lymphocytic Leukemia (CLL), glioblastoma, and Waldenstrom’s Macroglobulinemia.
  • the cancer is relapsed or refractory multiple myelome (r/r MM).
  • the pharmaceutical composition comprising a population of human T cells is referred to as PBCAR269A, which comprises a plurality of CAR T cells expressing a BCMA-specific CAR having a sequence set forth in SEQ ID NO: 47.
  • a pharmaceutical composition described herein that comprises a population of genetically-modified immune cells (e.g., CAR T cells) is administered to a subject.
  • a population of genetically-modified immune cells e.g., CAR T cells
  • an effective amount of such a pharmaceutical composition can be administered to a subject having a disease or disorder.
  • the population of genetically- modified immune cells administered to the subject facilitate the reduction of the proliferation, reduce the number, or kill target cells (e.g., cancer cells) in the recipient.
  • target cells e.g., cancer cells
  • genetically-modified immune cells of the present disclosure are able to replicate and expand in vivo, resulting in long-term persistence that can lead to sustained control of a disease.
  • the genetically-modified immune cells are selected from the group consisting of T cells, natural killer (NK) cells, B cells, or macrophages.
  • the genetically-modified immune cells are T cells (CAR T cells).
  • the immune cells are not derived from the subject. Immune cells that are not derived from the subject are said to be allogeneic, while immune cells that are derived from the subject are said to be autologous.
  • parenteral e.g., intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC), or infusion
  • IV intravenous
  • IM intramuscular
  • SC subcutaneous
  • infusion administration
  • the administration may be by continuous infusion or by single or multiple boluses.
  • the agent is infused over a period of less than about 12 hours, less than about 10 hours, less than about 8 hours, less than about 6 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, or less than about 1 hour.
  • the infusion occurs slowly at first and then is increased over time.
  • the subject is further administered an additional therapeutic agent or treatment, including, but not limited to gene therapy, radiation, surgery, or a chemotherapeutic agent(s) (i.e., chemotherapy).
  • an additional therapeutic agent or treatment including, but not limited to gene therapy, radiation, surgery, or a chemotherapeutic agent(s) (i.e., chemotherapy).
  • compositions of the present disclosure to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size (if present), extent of infection or metastasis, and condition of the patient (subject).
  • a pharmaceutical composition comprising the genetically-modified immune cells described herein is administered at a dosage of 10 4 to 10 9 cells/kg body weight, including all integer values within those ranges.
  • the dosage is 10 5 to 10 7 cells/kg body weight, including all integer values within those ranges.
  • the pharmaceutical composition comprising the genetically-modified immune cells is administered at a dose of about 0.1 x 10 6 to about 6 x 10 6 cells/kg of subject body mass. In some embodiments, the pharmaceutical composition comprising the genetically-modified immune cells is administered at a dose of about 0.1 x 10 6 , 0.2 x 10 6 , 0.3 x 10 6 , 0.4 x 10 6 , 0.5 x 10 6 , 0.6 x 10 6 , 0.7 x 10 6 , 0.8 x 10 6 , 0.9 x 10 6 , 1.0 x 10 6 , 1.1 x 10 6 , 1.2 x 10 6 , 1.3 x 10 6 , 1.4 x
  • the pharmaceutical composition comprising the genetically-modified immune cells is administered at a dose of about 0.1 x 10 6 to about 0.5 xlO 6 , about 0.5 x 10 6 to about 1.0 xlO 6 , about 1.0 x 10 6 to about 1.5 xlO 6 , about 1.5 x 10 6 to about 2.0 xlO 6 , about 2.0 x 10 6 to about 2.5 xlO 6 , about 2.5 x 10 6 to about 3.0 xlO 6 , about 3.0 x 10 6 to about 3.5 xlO 6 , about 3.5 x 10 6 to about 4.0 xlO 6 , about 4.0 x 10 6 to about 4.5 xlO 6 , about 4.5 x 10 6 to about 5.0 xlO 6 , about 5.0 x 10 6 to about 5.5 xlO 6 , or about 5.5 x 10 6 to about 6.0 x 10 6 cells/kg.
  • the pharmaceutical composition comprising the genetically-modified immune cells is administered at a dose of about 0.6 x 10 6 to about 2 x 10 6 cells/kg. In some embodiments, the pharmaceutical composition comprising the genetically-modified immune cells is administered at a dose of about 0.6 x 10 6 , 0.7 X 10 6 , 0.8 X 10 6 , 0.9 x 10 6 , 1.0 x 10 6 , 1.1 x 10 6 , 1.2 x 10 6 , 1.3 x 10 6 , 1.4 x 10 6 , 1.5 x 10 6 , 1.6 x 10 6 , 1.7 x 10 6 , 1.8 x 10 6 , 1.9 x 10 6 , or 2.0 x 10 6 cells/kg.
  • the pharmaceutical composition comprising the genetically-modified immune cells is administered at a dose of about 0.6 x 10 6 to about 0.8 xlO 6 , about 0.8 x 10 6 to about 1.2 xlO 6 , about 1.2 x 10 6 to about 1.6 xlO 6 , or about 1.6 x 10 6 to about 2.0 x 10 6 cells/kg.
  • the pharmaceutical composition comprising the genetically-modified immune cells is administered at a dose of about 0.6 x 10 6 cells/kg.
  • the pharmaceutical composition comprising the genetically-modified immune cells is administered at a dose of about 1 x 10 6 cells/kg.
  • the pharmaceutical composition comprising the genetically-modified immune cells is administered at a dose of about 2 x 10 6 cells/kg. In some embodiments, the pharmaceutical composition comprising the genetically-modified immune cells is administered at a dose of about 480 x 10 6 cells. In some embodiments, the pharmaceutical composition comprising the genetically-modified immune cells is administered at a dose of about 960 x 10 6 cells.
  • cell compositions are administered multiple times at these dosages.
  • the cell composition is administered in a fixed (or flat) dose.
  • the cell composition is administered in multiple doses, e.g., multiple fixed doses.
  • the first pharmaceutical composition is administered in split doses, i.e., as 2 administrations of a fixed dose. For instance, a fixed dose of 6.0 x 10 6 cells/kg may be “split” into two doses of 3.0 x 10 6 cells/kg.
  • the first cell composition is administered in an escalating dose.
  • the genetically-modified immune cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988).
  • the optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • the administration of genetically-modified immune cells of the present disclosure reduces at least one symptom of a target disease or condition.
  • administration of genetically-modified immune cells of the present disclosure can reduce at least one symptom of a cancer, such as multiple myeloma or other BCMA-related cancers.
  • Symptoms of cancers, such as BCMA-related cancers are well known in the art and can be determined by known techniques. 12.
  • variants are intended to mean substantially similar sequences.
  • a “variant” polypeptide is intended to mean a polypeptide derived from the “native” polypeptide by deletion or addition of one or more amino acids at one or more internal sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native polypeptide.
  • a “native” polynucleotide or polypeptide comprises a parental sequence from which variants are derived.
  • Variant polypeptides encompassed by the embodiments are biologically active. That is, they continue to possess the desired biological activity of the native protein.
  • Such variants may result, for example, from human manipulation.
  • Biologically active variants of polypeptides described herein will have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, sequence identity to the amino acid sequence of the native polypeptide, as determined by sequence alignment programs and parameters described elsewhere herein.
  • a biologically active variant of a polypeptide may differ from that polypeptide or subunit by as few as about 1-40 amino acid residues, as few as about 1-20, as few as about 1-10, as few as about 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds.
  • a “variant” comprises a deletion and/or addition of one or more nucleotides at one or more sites within the native polynucleotide.
  • variants of the nucleic acids of the embodiments will be constructed such that the open reading frame is maintained.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the embodiments.
  • Variant polynucleotides include synthetically derived polynucleotides, such as those generated, for example, by using site- directed mutagenesis but which still encode a polypeptide or RNA.
  • variants of a particular polynucleotide of the embodiments will have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.
  • Variants of a particular polynucleotide can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide.
  • deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the polypeptide. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by screening the polypeptide for its biological activity.
  • PBCAR269A is a chimeric antigen receptor (CAR)-T cell composition expressing a CAR construct with a B cell maturation antigen (BCMA)-targeting moiety designed to be specifically activated in the presence of BCMA+ target cells that results in target cell killing.
  • Nirogacestat is a small molecule gamma secretase inhibitor (GSI) that prevents the cleavage of BCMA from cellular membranes and is therefore hypothesized to increase PBCAR269A activity against MM target cells.
  • GSI small molecule gamma secretase inhibitor
  • This study was designed to analyze the activity of PBCAR269A CAR T cells generated against BCMA+ multiple myeloma (MM) target cell lines in the presence and absence of nirogacestat through in vitro proliferation and cytotoxicity assays. Additionally, the effect of nirogacestat on MM target cell viability and membrane-bound BCMA expression levels were assessed.
  • cryopreserved PBCAR269A CAR T cells were thawed and added to Xuri T cell expansion medium supplemented with 5% fetal bovine serum (FBS). The cell suspension was then centrifuged, the supernatant decanted, and the cells resuspended in Xuri medium supplemented with 5% FBS, and 10 ng/mL of IL- 15 and IL-21 and incubated overnight in a humidified incubator at 37 °C.
  • FBS fetal bovine serum
  • Nirogacestat was reconstituted to 10 mM in DMSO.
  • BCMA + target (MM. IS and U266) cells were used to evaluate the response of PBCAR269A CAR T cells to antigen.
  • Target cells and were thawed and passaged prior to assay setup, then washed and resuspeneded in fresh complete growth medium prior to coculture.
  • PBCAR269A CAR T cells were cocultured with MM. IS or U266 cells at the effector:target (E:T) ratios described below in total volumes of 200 pL. On the days indicated below, proliferation and/or cytotoxicity was assessed. Assessment of nirogacestat on MM cell viability
  • nirogacestat was plated in 96-well round bottom culture plates at 1 x 10 5 cells/well in triplicate. Cells were then treated with increasing concentrations of either nirogacestat (0 nM - 2000 nM) or DMSO diluted in cell culture medium. Nirogacestat or DMSO control was added either once or at daily intervals for seven consecutive days. Media containing nirogacestat or DMSO were replaced daily in those conditions. The concentration range selected for nirogacestat was based on observed peak plasma concentrations of nirogacestat in clinical studies (up to -450 ng/mL [-1000 nM]). Total viable cell numbers were determined at fixed intervals of 48, 96, and 168 hours using the CellTiter-Glo system per the manufacturer’s instructions.
  • nirogacestat reduced the levels of soluble BCMA shed from MM.
  • IS and U266 cells supernatant from these cultures was collected at 48, 96, and 168 hours to evaluate levels of soluble BCMA via ELISA (enzyme-linked immunosorbent assay) per the manufacturer’s instructions. Briefly, plates were coated with human BCMA capture antibody and incubated overnight at room temperature. After blocking and washing the plates, supernatant from cultures of MM. IS or U266 treated with or without nirogacestat was added to the blocked plates and incubated for 2 hours at room temperature.
  • PBCAR269A CAR T cells and MM were co-cultured with or without nirogacestat in a repeated antigen encounter assay. Briefly, two different lots of PBCAR269A were cocultured with MM. IS target cells at a fixed E:T (1:10) in the presence or absence of 2000 nM nirogacestat in 12-well plates. Media in all conditions was refreshed daily. At set time points during the assay, fresh MM. IS cells were added to experimental wells based on calculated PBCAR269A cell expansion during the previous incubation period and overall target cytolysis to re-establish the starting E:T ratio.
  • the viability of the MM cell lines MM. IS and U266 was evaluated after 48-, 96-, and 168-hour time points after a single dose, or after continuous daily dosing, of nirogacestat at increasing concentrations. As shown in FIGs. 1A-1B, MM. IS cell viability was not altered by a single dose (FIG. 1A) or continuous daily dosing (FIG. IB) of increasing concentrations of nirogacestat at any time point. Similarly, as shown in FIGs. 2A-2B, U266 cell viability was not altered by a single dose (FIG. 2A) or continuous daily dosing (FIG. 2B) of increasing concentrations of nirogacestat at any time point. Although subtle differences in target cell numbers were seen after 168 hours between nirogacestat dosing conditions (single v daily addition), this was attributed to differences in proliferation because of daily replenishment of cell media.
  • MM To determine if nirogacestat altered the expression of membrane -bound BCMA on target cells, MM. IS and U266 cell lines were treated with increasing concentrations of nirogacestat, ranging from 20 nM to 2000 nM. Target cells were subsequently analyzed for fluctuations in membrane bound BCMA at fixed intervals of 48, 96, and 168 hours post- nirogacestat addition. For flow cytometric analysis, MM cell lines were stained with an antiCD 138 antibody (Sun et al, 2019) and an anti-BCMA antibody, as noted above.
  • nirogacestat had on membrane-bound BCMA
  • cells were first gated for expression of CD138 and mean fluorescence intensity (MFI) of membrane-bound BCMA was then calculated on the CD 138+ cell population.
  • MFI mean fluorescence intensity
  • Both single and continuous daily treatment with nirogacestat resulted in an increased MFI of BCMA on both cell lines (FIG. 3A-3D).
  • an ELISA performed with cell culture supernatant from these samples demonstrated a marked decrease in soluble BCMA that inversely correlated with nirogacestat dose and expression of membrane-bound BCMA (FIG. 4).
  • nirogacestat had any effect on PBCAR269A cell survival and proliferation after engaging BCMA+ cells.
  • IS targets were added with or without nirogacestat in a repeated antigen encounter assay. Briefly, two different lots of PBCAR269A were co-cultured with MM. IS target cells at a fixed effector: target ratio (E:T) in the presence or absence of 2000 nM nirogacestat dosed daily. At various time points during the assay, fresh MM. IS cells were added to experimental wells based on calculated PBCAR269A cell expansion during the previous incubation period and overall target cytolysis to re-establish the starting E:T ratio.
  • E:T target ratio
  • MM To determine if nirogacestat treatment impacts PBCAR269A target-cell cytolysis, MM. IS cell numbers were measured at fixed time points after co-culture with PBCAR269A CAR T cells. At early time points, cytolysis of MM. IS targets, as determined by detectable CD138+ cells during flow cytometric analysis, was comparable in all conditions regardless of the CAR T cell lot or experimental condition ( Figure 6A). At late time points, however, PBCAR269A CAR T cells cultured with nirogacestat showed superior MM. IS target cell clearing compared with PBCAR269A CAR T cells alone ( Figure 6 A, grey bars; expanded for clarity in Figure 6B). This indicates that nirogacestat may increase longitudinal cytolytic activity by driving higher antigen expression on target cell lines.
  • PBCAR269A is an allogeneic anti-B-cell maturation antigen (BCMA) chimeric antigen receptor (CAR) T-cell composition derived from qualified donor T cells that have been genetically edited to remove the expression of the endogenous T-cell receptor (TCR). Insertion and expression of the anti-BCMA CAR provides the ability to specifically target and bind BCMA-positive (BCMA+) cells, and removal of the TCR significantly reduces the risk of graft- versus-host disease (GvHD) when it is administered to human leukocyte antigen (HLA)-mismatched patients.
  • PBCAR269A is being developed as an off-the-shelf treatment for multiple myeloma (MM).
  • the primary objective of this study is to evaluate the safety and tolerability of PBCAR269A with or without nirogacestat in study subjects with r/r MM.
  • the primary endpoint is the identification of the maximum tolerated dose (MTD) for PBCAR269A based on the incidence of any dose limiting toxicities (DLT)s.
  • MTD maximum tolerated dose
  • lymphodepletion chemotherapy Before initiating treatment with PBCAR269A, all study subjects will be administered lymphodepletion chemotherapy. Study subjects in Cohort B will also receive nirogacestat 100 mg oral BID prior to lymphodepletion, starting on Day -3 and ending on Day 60.
  • the initial lymphodepletion chemotherapy regimen will be composed of fludarabine (30 mg/m 2 /day) and cyclophosphamide (500 mg/m 2 /day) from Days -5 to -3.
  • IV intravenous
  • Phase 1 4 escalating dose groups in each cohort will be enrolled and treated sequentially, with the possibility of a single de-escalation dose group. Within each dose group, at least 3 and at most 6 study subjects will be treated with a single dose of PBCAR269A using a standard 3 + 3 design.
  • the starting dose of PBCAR269A will be 0.6 x 10 6 CAR T cells/kg body weight.
  • a DLT is defined as any treatment-emergent adverse event (TEAE) that meets the criteria specified. If 0 of 3 study subjects experience a DLT, the dose may be increased to the next dose level. If a DLT is observed in 1 of 3 study subjects at a given dose level, up to 3 additional study subjects (for a total of up to 6 study subjects) may be enrolled and treated at that dose level. When 3 additional study subjects are added to the dose group, the dose will be increased to the next dose level if ⁇ 1 of 6 study subjects experiences a DLT. If >2 of the 3 to 6 study subjects in a dose group experience a DLT, the MTD has been exceeded, and up to 3 additional study subjects (for a total of 6) will be treated at the previous (lower) dose level.
  • TEAE treatment-emergent adverse event
  • a dose level with established safety (based on 0 of 3 or 1 of 6 subjects experiencing a DLT)
  • up to 12 total subjects may be enrolled at the sponsors discretion to better characterize pharmacokinetics and pharmacodynamics as well as preliminary indications of efficacy. Data from additional subjects enrolled at a given dose level for further characterization will not contribute to the MTD determination.
  • the MTD in this study is defined as the highest dose at which ⁇ 1 of 6 study subjects experiences a DLT.
  • the MTD is most likely to be used for the planned expansion part of the study.
  • the MTD may not be selected as the recommended Phase 2a dose if a lower dose achieves similar anti-tumor effect with a similar or better safety profile.
  • MTD will be determined independently in cohorts A and B. Selection of the recommended Phase 2 dose (RP2D) will be based on both the safety and efficacy profile of the MTD in each cohort.
  • the first treated study subject in each dose group (including the dose de- escalation group, i.e., Dose -1) will be observed for 28 days for safety before any subsequent study subject receives any study treatment to provide an adequate safety monitoring window. There will be a staggered interval of 14 days between the second and third study participants; if the first study participant experiences a DLT, then there will be a 28-day interval between the second and third study participants. If no DLTs are observed, then subsequent study participants at that dose level can be treated without a formal stagger delay.
  • the first, second, and third treated study subject in each dose group (including the dose de-escalation group, i.e., Dose -1) will be observed for 28 days for safety before any subsequent study subject receives any study treatment to provide an adequate safety monitoring window. If no DLTs are observed, no waiting period is required for additional subjects. Subsequent study subjects (i.e., in a dose expansion) at that dose level can be treated without a formal stagger delay.
  • Dose escalation may proceed once safety of the current dose level has been established, which is dependent upon the last study subject enrolled in the current dose level completing the 28-day DLT evaluation period.
  • Cohort A and Cohort B will proceed with dose escalation separately. However, the last study subject in a given dose level in Cohort A must complete the 28-day DLT evaluation period before the first study subject in that dose level in Cohort B may be treated (see Figure 7). Study subjects will be sequentially enrolled in cohorts open at the same time (i.e., first subject enrolled in dose level 2 of Cohort A, second subject in dose level 1 of Cohort B). Based on the lack of DLTs in Cohort A DL1 and DL2, Cohort B will initiate enrollment in DL2 (2 x 10 6 cells/kg).
  • Study subjects are monitored for DLTs and AEs during the Treatment Period through Day 28. Study subjects will continue to be monitored for safety after Day 28 and will be followed until death, disease progression, subsequent systemic therapy, withdrawal due to intolerable toxicity, withdrawal of consent, or Day 360, whichever occurs first.
  • Phase 2a will use the dose level(s) at or below the MTD established in Phase 1.
  • Phase 2a dose includes PBCAR269A with nirogacestat, inclusion of a second cohort treated with the same dose of PBCAR269A may be warranted for safety and efficacy comparison.
  • the initial lymphodepletion regimen (fludarabine [30 mg/m 2 /day] and cyclophosphamide [500 mg/m 2 /day]) in this study is a conditioning treatment before CAR T- cell therapy.
  • Alternative lymphodepletion regimens may be considered in the future to account for the potential for allogeneic rejection to limit PBCAR269A persistence and efficacy.
  • Observations of toxicity or efficacy based on doses of human CAR T cells administered to immunodeficient mice bearing human tumors are useful as qualitative indicators of the likelihood of toxicity or efficacy in humans, but those data cannot be quantitatively translated (i.e., allometrically scaled) in selecting a safe starting dose in humans.
  • the proposed starting dose at 0.6 x 10 6 CAR T cells/kg may provide an opportunity for meaningful efficacy while preserving a significant margin for safety.
  • nirogacestat used in this trial will be 100 mg BID (“bis in die”, twice per day) PO (“per os”, by mouth or orally).
  • BID bis in die
  • PO per os
  • PBCAR269A at DL2 (2 x 10 6 cells/kg) will be used initially in combination with nirogacestat.
  • Phase 1 (dose escalation): Approximately 48 subjects are planned to be enrolled between the two Cohorts.
  • Evaluable subjects for the primary variable assessment of safety and tolerability are defined as subjects who receive a dose of PBCAR269A and either complete the Treatment Period through Day 28, discontinue early due to disease progression, or experience a DLT. Non-evaluable subjects will be replaced. All enrolled subjects will be evaluable for all other endpoints, including overall assessment of adverse events and evidence of clinical benefit.
  • Phase 2a dose expansion: Approximately 20 to 30 additional subjects are planned to be enrolled to further understand the safety profile and potential clinical activity-efficacy of the treatment regimen identified in Phase 1. If the Phase 2a dose includes PBCAR269A with nirogacestat and a separate cohort of subjects treated with PBCAR269A without nirogacestat is warranted, an additional 20 to 30 subjects will be enrolled, for a total of 40 to 60 study subjects.
  • the 3 dose groups will be sequentially enrolled with the possibility of a single de- escalation- dose group (Table 2). Note that dose levels -1, 1, 2, 3 and 4 are not to exceed a total dose of 6 x 10 A 6, 60 x 10 A 6, 200 x 10 A 6, 480 x 10 A 6 and 960x 10 A 6 cells, respectively.
  • BID twice daily
  • CAR chimeric antigen receptor
  • max maximum
  • Phase 2a will use the MTD determined in Phase 1. All subjects will receive a single dose of the study treatment and follow the same administration and assessment procedures. Study subjects will be treated with a single dose of PBCAR269A with or without nirogacestat and followed for safety and efficacy in the same manner as Phase 1 of the study. All study subjects will also receive the lymphodepletion regimen that was established in Phase 1. If the recommended phase 2 dose includes PBCAR269A with nirogacestat, inclusion of a second cohort treated with the same dose of PBCAR269A alone may be warranted for safety and efficacy comparison.
  • peripheral blood samples were obtained at pre-determined time points from subjects dosed with either PBCAR269A alone (Cohort A) at DL2 (2 x 10 6 cells/kg) or DL4, (960 x 10 6 cells)), or PBCAR269A with nirogacestat (Cohort B) at DL2 (2 x 10 6 cells/kg)).
  • PBCAR269A alone Cohort A
  • DL4 DL4
  • PBCAR269A with nirogacestat (Cohort B) at DL2 (2 x 10 6 cells/kg)
  • peripheral blood samples were analyzed for PBCAR269A cells by flow cytometric analysis. Briefly, a fixed volume of sample was stained with an antibody cocktail to determine counts of relevant cell types, including lymphocytes, in acquired peripheral blood.
  • the remaining sample volume was separately stained with a cocktail of antibodies, including CD3, CD4, CD8, and an anti-idiotype PBCAR269A antibody, to determine the frequency of PBCAR269A cells, among others, in analyzed samples.
  • the relevant cell count and cell frequency measurements are then multiplied to determine the total number of PBCAR269A cells in subject samples over time.
  • the number of PBCAR269A cells were quantified in peripheral blood samples over time in subjects receiving PBCAR269A alone (Cohort A) at DL2 or DL4, or in subjects receiving PBCAR269A with nirogacestat (Cohort B) at DL2.
  • subjects receiving PBCAR269A alone at DL2 peak expansion of administered CAR T cells was observed at Day 7 post-PBCAR dosing, but the overall magnitude and persistence of PBCAR269A in those subjects was limited.
  • subjects dosed with PBCAR269A cells alone at DL4 (960 x 10 6 cells), which is roughly a ⁇ 5-fold increase in starting CAR T dose, showed marked improvements in detectable peak expansion and subsequent kinetics.

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Abstract

La présente divulgation concerne des anticorps et des fragments de ceux-ci ayant une spécificité pour l'antigène de maturation des cellules B humaines (BCMA, « B cell maturation antigen »), ainsi que des récepteurs antigéniques chimériques (CAR, « chimeric antigen receptor ») comprenant de tels anticorps et fragments de liaison à l'antigène, ainsi que des cellules génétiquement modifiées comprenant de tels CAR. La divulgation concerne également des méthodes d'utilisation de ces cellules en association avec des inhibiteurs de gamma secrétase pour le traitement de troubles et de maladies associés au BCMA, tels que le cancer.
PCT/US2022/078065 2021-10-14 2022-10-13 Associations de lymphocytes t car anti-bcma et d'inhibiteurs de gamma secrétase WO2023064872A1 (fr)

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US11872211B2 (en) 2022-05-20 2024-01-16 Springworks Therapeutics, Inc. Treatments with nirogacestat
US11951096B2 (en) 2022-05-20 2024-04-09 Springworks Therapeutics, Inc. Treatments with nirogacestat
US12011435B2 (en) 2022-05-20 2024-06-18 Springworks Therapeutics, Inc. Treatments with nirogacestat
US12138246B2 (en) 2024-04-02 2024-11-12 Springworks Therapeutics, Inc. Treatments with nirogacestat

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