WO2024168312A1 - Methods for treating hematopoietic malignancy - Google Patents

Methods for treating hematopoietic malignancy Download PDF

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Publication number
WO2024168312A1
WO2024168312A1 PCT/US2024/015272 US2024015272W WO2024168312A1 WO 2024168312 A1 WO2024168312 A1 WO 2024168312A1 US 2024015272 W US2024015272 W US 2024015272W WO 2024168312 A1 WO2024168312 A1 WO 2024168312A1
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subject
cells
gemtuzumab ozogamicin
administered
dose
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PCT/US2024/015272
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French (fr)
Inventor
Sritama NATH
Glen RAFFEL
Eyal Attar
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Vor Biopharma Inc.
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Publication of WO2024168312A1 publication Critical patent/WO2024168312A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A61K47/6867—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell the tumour determinant being from a cell of a blood cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered

Definitions

  • HCT Hematopoietic cell transplantation
  • AML acute myeloid leukemia
  • HCT With the reduction in transplant- related mortality over the past several decades, leukemia relapse post-HCT remains an obstacle to improved overall outcomes.
  • MRD minimal residual disease
  • HCT patients who present with evidence of persistent leukemic blasts in the bone marrow are individuals at very high risk for early relapse post HCT.
  • the present disclosure relates to a method, comprising administering to a subject an effective amount of a population of genetically engineered hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen; and administering to the subject gemtuzumab ozogamicin in a dosing regimen comprising a dosing cycle, wherein the dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin; wherein the effective amount of gemtuzumab ozogamicin is 0.1 mg/m 2 - 6.0 mg/m 2 body surface area of the subject.
  • the dosing regimen comprises at least two, at least three, or at least four dosing cycles, wherein each dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin. In some embodiments, the dosing cycle or each dosing cycle is about 4 weeks or less.
  • the effective amount of gemtuzumab ozogamicin is administered to the subject in a single dose.
  • the single dose of gemtuzumab ozogamicin is about 0.1 mg/m 2 , about 0.25 mg/m 2 , about 0.5 mg/m 2 , about 1.0 mg/m 2 , about 2.0 mg/m 2 , about 3.0 mg/m 2 , about 4.0 mg/m 2 , about 5.0 mg/m 2 , or about 6.0 mg/m 2 body surface area of the subject.
  • the single dose of gemtuzumab ozogamicin is about 0.5 mg/m 2 body surface area of the subject.
  • the single dose of gemtuzumab ozogamicin is about 0.5 mg/m 2 body surface area of the subject and the dosing cycle is about 4 weeks.
  • the effective amount of gemtuzumab ozogamicin is administered to the subject in multiple doses.
  • each of the multiple doses of gemtuzumab ozogamicin is about 0.1 mg/m 2 , about 0.25 mg/m 2 , about 0.5 mg/m 2 , about 1.0 mg/m 2 , about 2.0 mg/m 2 , about 3.0 mg/m 2 , about 4.0 mg/m 2 , about 5.0 mg/m 2 , or about 6.0 mg/m 2 body surface area of the subject.
  • each of the multiple doses of gemtuzumab ozogamicin is about 0.5 mg/m 2 body surface area of the subject.
  • the multiple doses of gemtuzumab ozogamicin comprises two doses of gemtuzumab ozogamicin.
  • a first dose is administered to the subject on day 1 of the dosing cycle and a second dose is administered in day 7 of the dosing cycle.
  • a first dose is administered to the subject on day 1 of the dosing cycle and a second dose is administered in day 14 of the dosing cycle.
  • the multiple doses of gemtuzumab ozogamicin comprises three doses of gemtuzumab ozogamicin.
  • a first dose is administered to the subject on day 1 of the dosing cycle
  • a second dose is administered in day 4 of the dosing cycle
  • a third dose is administered on day 7 of the dosing cycle.
  • a first dose is administered to the subject on day 1 of the dosing cycle
  • a second dose is administered in day 8 of the dosing cycle
  • a third dose is administered on day 16 of the dosing cycle.
  • a first dose is administered to the subject on day 1 of the dosing cycle
  • a second dose is administered to the subject once the plasma concentration of gemtuzumab ozogamicin in the subject is less than a threshold value
  • a third dose is administered to the subject once the plasma concentration of gemtuzumab ozogamicin in the subject is less than a threshold value following administration of the second dose.
  • the multiple doses of gemtuzumab ozogamicin comprises four doses of gemtuzumab ozogamicin.
  • the doses of gemtuzumab ozogamicin are administered to the subject weekly in the dosing cycle.
  • a first dose is administered to the subject on day 1 of the dosing cycle
  • a second dose is administered in day 8 of the dosing cycle
  • a third dose is administered on day 15 of the dosing cycle
  • a fourth dose is administered on day 21 of the dosing cycle.
  • the multiple doses of gemtuzumab ozogamicin are administered weekly to the subject in the dosing cycle.
  • the population of genetically engineered hematopoietic cells and the first dosing cycle are administered in temporal proximity.
  • administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen at least 60 days after administration of the population of genetically engineered hematopoietic cells.
  • administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen between 40- 60 days after administration of the population of genetically engineered hematopoietic cells, if the subject experiences early relapse.
  • the population of genetically engineered hematopoietic cells are administered prior to gemtuzumab ozogamicin.
  • the population of genetically engineered hematopoietic cells are administered in a single treatment regimen.
  • the population of genetically engineered hematopoietic cells and/or the gemtuzumab ozogamicin are administered intravenously.
  • the effective amount of the population of genetically engineered hematopoietic cells is about 10 6 cells/kilogram body weight of the subject to about 5 x 10 7 cells/kilogram body weight of the subject. In some embodiments, the effective amount of the population of genetically engineered hematopoietic cells is about 7.5 x 10 6 cells/kilogram body weight of the subject. In some embodiments, the effective amount of the population of genetically engineered hematopoietic cells is about 3 x 10 6 cells/kilogram body weight of the subject.
  • the population of genetically engineered hematopoietic cells are thawed from a cryopreserved form prior to administration.
  • the gemtuzumab ozogamicin is reconstituted from a lyophilized form prior to administration.
  • the subject has been preconditioned prior to administering the hematopoietic cells and gemtuzumab ozogamicin.
  • the method further comprises preconditioning the subject prior to administering the hematopoietic cells and gemtuzumab ozogamicin.
  • the preconditioning comprises administering one or more chemotherapeutic agents to the subject.
  • the preconditioning comprises total body irradiation of the subject.
  • the chemotherapeutic agent is selected from the group consisting of busulfan, melphalan, fludarabine, cyclophosphamide, and thiotepa.
  • the preconditioning comprises administering antibodies that bind human T cells, optionally wherein the antibodies comprise rabbit anti-thymocyte globulins (rATG).
  • the subject has, or has been diagnosed with, a hematopoietic malignancy or a hematopoietic pre-malignant disease, and wherein the hematopoietic malignancy is characterized by the presence of CD33-positive malignant cells, or wherein the hematopoietic pre-malignant disease is characterized by the presence of CD33-positive pre- malignant cells.
  • the subject has, or has been diagnosed with, CD33- positive acute myeloid leukemia.
  • the subject has, or has been diagnosed with, CD33-positive myelodysplastic syndrome.
  • the subject has, or has been diagnosed with, CD33-positive myelodysplastic syndrome and wherein the subject is at high risk of developing acute myeloid leukemia or refractory cytopenias.
  • the subject is naive to chemotherapy and/or radiation therapy, optionally wherein the subject is naive to any treatment aimed to address a hematopoietic malignancy or hematopoietic pre-malignant disease.
  • the subject has previously received chemotherapy. In some embodiments, the subject has previously received induction therapy.
  • the subject has previously entered a complete hematological remission, optionally wherein the complete hematological remission is characterized by an incomplete recovery of peripheral counts.
  • the subject has one or more risk factors associated with early leukemia relapse.
  • the one or more risk factors associated with early leukemia relapse are selected from the group consisting of: bone marrow in morphological complete remission with presence of intermediate or high-risk disease-related genetics; presence of minimal residual disease (MRD) post cyto-reductive therapy; bone marrow with persistent leukemia blasts post cyto-reductive therapy; and bone marrow blast count of about 10% or less.
  • MRD minimal residual disease
  • the subject does not have acute promyelocytic leukemia or chronic myeloid leukemia. In some embodiments, the subject has not previously received a stem cell transplantation. In some embodiments, the subject has not previously received gemtuzumab ozogamicin.
  • the method further comprises determining a percent donor chimerism and/or a level of CD33-negative myeloid hematopoiesis in a peripheral blood sample from the subject.
  • the subject has a CD33-negative absolute neutrophil count (ANC) of at least 1000 cells/pL prior to receiving the dosing regimen.
  • the subject has a CD33-negative absolute neutrophil count (ANC) of at least 500 cells/pL prior to receiving the dosing regimen.
  • the hematopoietic cells are hematopoietic stem and progenitor cells.
  • the hematopoietic stem cells are from bone marrow cells, cord blood cells, or peripheral blood mononuclear cells (PBMCs).
  • the hematopoietic stem cells are CD34 + /CD33‘.
  • the hematopoietic cells are autologous.
  • the method further comprises obtaining the autologous hematopoietic stem cells from the subject, optionally wherein the method further comprises genetically engineering the autologous stem cells to have reduced or eliminated expression of the CD33 antigen, and returning the genetically engineered hematopoietic stem cells to the subject.
  • the hematopoietic cells are allogeneic. In some embodiments, the hematopoietic cells are allogeneic hematopoietic stem cells obtained from a donor having an HLA haplotype that matches with the HLA haplotype of the subject. In some embodiments, the method further comprises obtaining hematopoietic cells from a donor having an HLA haplotype that matches with the HLA haplotype of the subject.
  • the method further comprises preparing the hematopoietic cells by modifying an endogenous gene of the hematopoietic cells encoding the CD33 antigen.
  • the whole or a portion of the endogenous gene encoding the CD33 cell-surface antigen is deleted.
  • the whole or the portion of the endogenous gene is deleted using genome editing.
  • the genome editing involves a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a CRISPR-Cas system.
  • ZFN zinc finger nuclease
  • TALEN transcription activator-like effector-based nuclease
  • CRISPR-Cas system a CRISPR-Cas system.
  • the population of genetically engineered hematopoietic cells, or descendants thereof are tremtelectogene empogeditemcel (trem-cel).
  • aspects of the present disclosure relate to methods comprising administering to a subject: gemtuzumab ozogamicin in a dosing regimen comprising a dosing cycle, wherein the dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin; wherein the effective amount of gemtuzumab ozogamicin is 0.1 mg/m 2 - 6.0 mg/m 2 body surface area of the subject; and wherein the subject is receiving or has received an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen.
  • the dosing regimen comprises at least two, at least three, or at least four dosing cycles, wherein each dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin.
  • the dosing cycle or each dosing cycle is about 4 weeks or less.
  • the effective amount of gemtuzumab ozogamicin is administered to the subject in a single dose.
  • the single dose of gemtuzumab ozogamicin is about 0.1 mg/m2, about 0.25 mg/m2, about 0.5 mg/m2, about 1.0 mg/m2, about 2.0 mg/m2, about 3.0 mg/m2, about 4.0 mg/m2, about 5.0 mg/m2, or about 6.0 mg/m2 body surface area of the subject. In some embodiments, the single dose of gemtuzumab ozogamicin is about 0.5 mg/m2 body surface area of the subject. In some embodiments, the single dose of gemtuzumab ozogamicin is about 0.5 mg/m2 body surface area of the subject and the dosing cycle is about 4 weeks.
  • the effective amount of gemtuzumab ozogamicin is administered to the subject in multiple doses.
  • each of the multiple doses of gemtuzumab ozogamicin is about 0.1 mg/m2, about 0.25 mg/m2, about 0.5 mg/m2, about 1.0 mg/m2, about 2.0 mg/m2, about 3.0 mg/m2, about 4.0 mg/m2, about 5.0 mg/m2, or about 6.0 mg/m2 body surface area of the subject.
  • each of the multiple doses of gemtuzumab ozogamicin is about 0.5 mg/m2 body surface area of the subject.
  • the multiple doses of gemtuzumab ozogamicin comprises two doses of gemtuzumab ozogamicin. In some embodiments, a first dose is administered to the subject on day 1 of the dosing cycle and a second dose is administered in day 7 of the dosing cycle. In some embodiments, a first dose is administered to the subject on day 1 of the dosing cycle and a second dose is administered in day 14 of the dosing cycle. In some embodiments, the multiple doses of gemtuzumab ozogamicin comprises three doses of gemtuzumab ozogamicin.
  • a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered in day 4 of the dosing cycle, and a third dose is administered on day 7 of the dosing cycle.
  • a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered in day 8 of the dosing cycle, and a third dose is administered on day 16 of the dosing cycle.
  • a first dose is administered to the subject on day 1 of the dosing cycle
  • a second dose is administered to the subject once the plasma concentration of gemtuzumab ozogamicin in the subject is less than a threshold value
  • a third dose is administered to the subject once the plasma concentration of gemtuzumab ozogamicin in the subject is less than a threshold value following administration of the second dose.
  • the multiple doses of gemtuzumab ozogamicin comprises four doses of gemtuzumab ozogamicin.
  • the doses of gemtuzumab ozogamicin are administered to the subject weekly in the dosing cycle.
  • a first dose is administered to the subject on day 1 of the dosing cycle
  • a second dose is administered in day 8 of the dosing cycle
  • a third dose is administered on day 15 of the dosing cycle
  • a fourth dose is administered on day 21 of the dosing cycle.
  • the population of genetically engineered hematopoietic cells and the first dosing cycle of gemtuzumab ozogamicin are administered in temporal proximity.
  • administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen at least 60 days after administration of the population of genetically engineered hematopoietic cells.
  • administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen between 40-60 days after administration of the population of genetically engineered hematopoietic cell, if the subject experiences early relapse.
  • the subject received the population of genetically engineered hematopoietic cells prior to administration of gemtuzumab ozogamicin. In some embodiments, the subject received the population of genetically engineered hematopoietic cells in a single treatment regimen.
  • the administration of gemtuzumab ozogamicin is intravenous. In some embodiments, the gemtuzumab ozogamicin is reconstituted from a lyophilized form prior to administration.
  • the effective amount of the population of genetically engineered hematopoietic cells is about 10 6 cells/kilogram body weight of the subject to about 5 x 10 7 cells/kilogram body weight of the subject. In some embodiments, the effective amount of the population of genetically engineered hematopoietic cells is about 7.5 x 10 6 cells/kilogram body weight of the subject. In some embodiments, the effective amount of the population of genetically engineered hematopoietic cells is about 3 x 10 6 cells/kilogram body weight of the subject.
  • the subject has been preconditioned prior to receiving the population of hematopoietic cells and gemtuzumab ozogamicin.
  • the method further comprises preconditioning the subject prior to administering the gemtuzumab ozogamicin.
  • the preconditioning comprises administering one or more chemotherapeutic agents to the subject.
  • the preconditioning comprises total body irradiation of the subject.
  • the chemotherapeutic agent is selected from the group consisting of busulfan, melphalan, fludarabine, cyclophosphamide, and thiotepa.
  • the preconditioning comprises administering antibodies that bind human T cells, optionally wherein the antibodies comprise rabbit anti-thymocyte globulins (rATG).
  • the subject has, or has been diagnosed with, a hematopoietic malignancy or a hematopoietic pre-malignant disease, and wherein the hematopoietic malignancy is characterized by the presence of CD33-positive malignant cells, or wherein the hematopoietic pre-malignant disease is characterized by the presence of CD33-positive pre- malignant cells.
  • the subject has, or has been diagnosed with, CD33- positive acute myeloid leukemia.
  • the subject has, or has been diagnosed with, CD33-positive myelodysplastic syndrome.
  • the subject has, or has been diagnosed with, CD33-positive myelodysplastic syndrome and wherein the subject is at high risk of developing acute myeloid leukemia or refractory cytopenias.
  • the subject is naive to chemotherapy and/or radiation therapy, optionally wherein the subject is naive to any treatment aimed to address a hematopoietic malignancy or hematopoietic pre-malignant disease.
  • the subject has previously received chemotherapy.
  • the subject has previously received induction therapy.
  • the subject has previously entered a complete hematological remission, optionally wherein the complete hematological remission is characterized by an incomplete recovery of peripheral counts.
  • the subject has one or more risk factors associated with early leukemia relapse.
  • the one or more risk factors associated with early leukemia relapse are selected from the group consisting of: bone marrow in morphological complete remission with presence of intermediate or high-risk disease-related genetics; presence of minimal residual disease (MRD) post cyto-reductive therapy; bone marrow with persistent leukemia blasts post cyto-reductive therapy; and bone marrow blast count of about 10% or less.
  • MRD minimal residual disease
  • the subject does not have acute promyelocytic leukemia or chronic myeloid leukemia. In some embodiments, the subject has not previously received a stem cell transplantation. In some embodiments, the subject has not previously received gemtuzumab ozogamicin.
  • the method further comprises determining a percent donor chimerism and/or a level of CD33-negative myeloid hematopoiesis in a peripheral blood sample from the subject.
  • the subject has a CD33-negative absolute neutrophil count (ANC) of at least 1000 cells/pL prior to receiving the dosing regimen.
  • the subject has a CD33-negative absolute neutrophil count (ANC) of at least 500 cells/pL prior to receiving the dosing regimen.
  • the hematopoietic cells are hematopoietic stem and progenitor cells.
  • the hematopoietic stem and progenitor cells are from bone marrow cells, cord blood cells, or peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the hematopoietic stem and progenitor cells are CD34+/CD33-.
  • the hematopoietic cells are autologous. In some embodiments, the hematopoietic cells are allogeneic. In some embodiments, the hematopoietic cells are allogeneic hematopoietic stem cells obtained from a donor having an HLA haplotype that matches with the HLA haplotype of the subject. In some embodiments, the method further comprises obtaining hematopoietic cells from a donor having an HLA haplotype that matches with the HLA haplotype of the subject.
  • the method further comprises preparing the population of hematopoietic cells by modifying an endogenous gene of the hematopoietic cells encoding the CD33 antigen.
  • the whole or a portion of the endogenous gene encoding the CD33 cell-surface antigen is deleted.
  • the whole or the portion of the endogenous gene is deleted using genome editing.
  • the genome editing involves a zinc finger nuclease (ZFN), a transcription activator-like effectorbased nuclease (TALEN), or a CRISPR-Cas system.
  • the population of genetically engineered hematopoietic cells, or descendants thereof are tremtelectogene empogeditemcel (trem-cel).
  • Aspects of the present disclosure relate to a method comprising administering an effective amount of gemtuzumab ozogamicin to a subject, wherein the subject is identified as having hematopoietic cells comprising a lower density of wild-type CD33 in a first biological sample relative to a second biological sample.
  • the first biological sample is obtained from the subject at a first time point and the second biological sample is obtained from the subject at a second time point; or wherein the second biological sample is obtained from a counterpart subject.
  • the method further comprises obtaining the first biological sample; and measuring the density of wild-type CD33 in the first biological sample.
  • the method further comprises obtaining the second biological sample; and measuring the density of wild-type CD33 in the second biological sample.
  • the lower density of wild-type CD33 in the first biological sample is 95% or less than the density of wild-type CD33 in the second biological sample.
  • the effective amount of gemtuzumab ozogamicin is 0.1 mg/m2 - 6.0 mg/m2 body surface area of the subject. In some embodiments, the effective amount of gemtuzumab ozogamicin is administered to the subject in a single dose or multiple doses of the effective amount.
  • the method further comprises administering an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen; or the subject is receiving or has received an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen.
  • aspects of the present disclosure provide a method comprising (a) measuring density of wild-type CD33 on a population of hematopoietic cells in a first biological sample of a subject; (b) comparing the density of wild-type CD33 on the population of hematopoietic cells in the first biological sample to the density of wild-type CD33 on a population of hematopoietic cells in a second biological sample; and (c) determining an effective amount of gemtuzumab ozogamicin for administration to the subject based on (b).
  • the method further comprises administering the effective amount of gemtuzumab ozogamicin for administration to the subject.
  • the density of wild-type CD33 on the population of hematopoietic cells in the first biological sample is lower than the density of wild-type CD33 on a population of hematopoietic cells in a second biological sample.
  • the effective amount of gemtuzumab ozogamicin is 0.1 mg/m2 - 6.0 mg/m2 body surface area of the subject.
  • the method comprises administering to the first subject an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen.
  • the first subject has received an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen.
  • FIG. 1 shows a schematic of an exemplary human clinical trial in which CD33+ acute myeloid leukemia (AML) patients who are at high risk of relapse are subjected to myeloablative hematopoietic cell transplant (HCT) with tremtelectogene empogeditemcel (trem-cel) followed by treatment with low-dose gemtuzumab ozogamicin.
  • HCT myeloablative hematopoietic cell transplant
  • trem-cel tremtelectogene empogeditemcel
  • gemtuzumab ozogamicin 0.5-2.0 mg/m 2
  • Part 1 will evaluate the safety of trem-cel and determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of gemtuzumab ozogamicin.
  • Part 2 will enroll an additional 12 patients to further evaluate the safety of trem-cel and the preliminary efficacy of the combination of trem-cel and gemtuzumab ozogamicin at the recommended phase 2 dose.
  • MTD maximum tolerated dose
  • R2D recommended phase 2 dose
  • AML refers to acute myeloid leukemia
  • G-CSE refers to granulocyte colony stimulating factor
  • GO refers to gemtuzumab ozogamicin (also known as Mylotarg®)
  • GvHD refers to graft- versus-host-disease
  • HCT refers to hematopoietic cell transplant
  • HLA refers to human leukocyte antigens
  • LTEU refers to long-term follow-up
  • MAC refers to myeloablative conditioning
  • MTD refers to maximum tolerated dose
  • PK refers to Pharmacokinetics
  • R2D refers to recommended phase 2 dose. *Conditioning prior to infusion of trem-cel consists of busulfan/melphalan/fludarabine/rATG or total body irradiation/cyclophosphamide/thiotepa/rATG.
  • FIGs. 2A and 2B show neutrophil engraftment and platelet recovery in Patient 1 after HCT with trem-cel.
  • FIG. 2A shows absolute neutrophil count at the indicated days post-HCT with trem-cel infusion.
  • the arrow indicates trem-cel neutrophil engraftment, and the vertical dotted line indicates the median neutrophil engraftment of an unmodified CD34+ graft from the clinical trial CTN1301 (Luznik et al., J Clin Oncol. (2022);40(4):356-368; ClinicalTrials.gov Identifier: NCT02345850).
  • FIG. 2B shows platelet recovery at the indicated days post-HCT with trem-cel infusion. The subject received platelet transfusions at the indicated days due to pre-existing hemorrhage risk. The arrow indicates trem-cel platelet recovery.
  • FIGs. 3A and 3B show neutrophil engraftment and platelet recovery in Patient 2 after HCT with trem-cel.
  • FIG. 3A shows absolute neutrophil count in Patient 2 at the indicated days post-HCT with trem-cel infusion.
  • the arrow indicates trem-cel neutrophil engraftment, and the vertical dotted line indicates the median neutrophil engraftment of an unmodified CD34+ graft from the clinical trial CTN1301.
  • FIG. 3B shows platelet recovery in Patient 2 at the indicated days post-HCT with trem-cel infusion.
  • the subject received platelet transfusions at the indicated days due to pre-existing hemorrhage risk.
  • the arrow indicates trem-cel platelet recovery.
  • FIGs. 4A-4D show neutrophil engraftment and platelet recovery in Patients 1-5 after HCT with trem-cel.
  • FIG. 4A shows absolute neutrophil count at the indicated days post-HCT with trem-cel infusion.
  • the arrows indicate trem-cel neutrophil engraftment
  • the vertical dotted line indicates the median neutrophil engraftment of an unmodified CD34+ graft from clinical trial CTN1301
  • the horizontal dotted line indicates an absolute neutrophil count (ANC) >500.
  • Neutrophil recovery defined as the first of three consecutive days of an absolute neutrophil count (ANC) >500 is indicated by arrows under the x-axis.
  • FIG. 4B shows platelet recovery at the indicated days post-HCT with trem-cel infusion.
  • the horizontal dotted line indicates platelet count of >20,000/pL.
  • the arrows under the x-axis indicate trem-cel platelet recovery defined as the first day of a sustained platelet count of >20,000/pL with no platelet transfusion in the preceding seven days.
  • FIG. 4C shows neutrophil engraftment at the indicated days post-HCT with trem-cel infusion.
  • FIG. 4D shows platelet recovery at the indicated days post-HCT with trem-cel infusion.
  • the median is shown excluding Patient 3, who experienced immune thrombocytopenia. Both neutrophil engraftment and platelet recovery were similar to unedited CD34-selected grafts. Results show full myeloid chimerism in all patients at D+28. Arrows indicate days of individual patient engraftment.
  • FIGs. 5A-5E show CD33 editing in peripheral blood and bone marrow post-HCT with trem-cel infusion.
  • FIG. 5A is a table showing CD33 editing and expression in peripheral blood across bulk cells and cells of myeloid and lymphoid lineages in Patient 1 at days 28, 60, and 100 (“D28”, “D60” and “D100,” respectively) following trem-cel infusion.
  • FIG. 5B is a table showing CD33 editing in peripheral blood cells (PB) including bulk cells and cells of myeloid and lymphoid lineages in Patients 1-4 at days 28 and 60 (“D+28” and “D+60,” respectively) post-HCT with trem-cel infusion.
  • PB peripheral blood cells
  • DC% refers to percent donor chimerism.
  • CD33 GE% refers to CD33 gene editing efficiency.
  • NC indicates sample was not collected.
  • QNS indicates quantity not sufficient.
  • Pending refers to pending collection.
  • FIG. 5C shows donor chimerism results obtained from peripheral blood analyses of monocytes in Patients 1-8 at days 28, 60, 100, and 180 (“D28”, “D60”, “D100,” and “D180,” respectively) following trem-cel infusion.
  • FIG. 5D shows the frequency of CD33 gene editing in trem-cel (“Drug Product”) and monocytes reconstituted from trem-cel in Patients 1- 8 at 28, 60, 100, and 180 days following trem-cel infusion (“D28”, “D60”, “D100,” and “DI 80,” respectively).
  • 5E shows the frequency of CD33-negative monocytes and myeloid cells in the peripheral blood or bone marrow of Patients 1-8 at 28, 60, 100, and 180 days following trem-cel infusion (“D28”, “D60”, “D100,” and “DI 80,” respectively).
  • FIGs. 6A and 6B show exemplary flow cytometry analysis of peripheral blood (FIG. 6A) and bone marrow (FIG. 6B) from Patient 1 at 28 days (D28) post-HCT with trem-cel as compared to a subject that received HCT with non-edited cells (“non-edited recipient”).
  • FIGs. 7A and 7B show analyses of CD33-negative cells in bone marrow and peripheral blood from patients transplanted with trem-cel at the indicated time points.
  • FIG. 7A is a table showing flow cytometry analysis of CD33 expression in bone marrow and peripheral blood cells from Patient 1 at days 28, 60, and 100 (“D28,” “D60,” and “D100,” respectively) following HCT infusion with trem-cel.
  • C1D1 refers to gemtuzumab ozogamicin dosing cycle 1, day 1.
  • C2D1 refers to gemtuzumab ozogamicin dosing cycle 2, day 1.
  • C3D1 refers to gemtuzumab ozogamicin dosing cycle 3, day 1.
  • FIG. 7B is a table showing flow cytometry analyses of CD33 expression in bone marrow and peripheral blood cells in Patients 1-4 at days 28 and 60 (“D+28” and “D+60,” respectively) following HCT infusion with trem-cel.
  • PB refers to peripheral blood.
  • BM refers to bone marrow.
  • NC indicates sample not collected.
  • TPD indicates to be determined.
  • FIGs. 8A-8D show pharmacokinetics associated with administration of 0.5 mg/m 2 x 1 gemtuzumab ozogamicin following HCT.
  • FIG. 8A is a table showing the pharmacokinetics associated with 0.5 mg/m 2 x 1 gemtuzumab ozogamicin administration following HCT with trem-cel (VBP101) in Patient 1 (VBP101 column) as compared to administration of 0.5 mg/m 2 x 1 gemtuzumab ozogamicin following HCT with non-edited (CD33+) cells in a reference clinical trial (0903A1-101-US; FDA Briefing Document Oncologic Drugs Advisory Committee Meeting July 11, 2017, BLA 761060 Mylotarg (gemtuzumab ozogamicin)).
  • FIG. 8B shows pharmacokinetic data associated with administration of 0.5 mg/m 2 x 1 gemtuzumab ozogamicin in Cohort 1. Results show higher gemtuzumab ozogamicin exposure in the context of CD33-negative hematopoiesis.
  • FIG. 8C is a table showing pharmacokinetics after administration of a first dose of maintenance gemtuzumab ozogamicin (GO) following HCT with trem-cel (VBP101 column) compared to pharmacokinetics observed during GO Phase 1 Study 0903A1-101-US.
  • Cmax refers to maximum concentration.
  • FIG. 8D is a graph showing the relationship between gemtuzumab ozogamicin (GO) maximum concentration (Cmax) and veno-occlusive disease in prior transplant.
  • R/R AML refers to relapsed or refractory acute myeloid leukemia.
  • FIG. 9 is a table showing absolute neutrophil and platelet counts and amount of CD33-negative cells in Patient 1 after HCT (post-HCT recovery) and after gemtuzumab ozogamicin administration in each of the three dosing cycles. indicates pre-gemtuzumab ozogamicin dosing at the initiation of the first gemtuzumab ozogamicin dosing cycle.
  • FIGs. 10A-10E show analyses of heme-protection and the percentage of CD33- negative cells in the blood of Patient 1 after administration of gemtuzumab ozogamicin (GO).
  • FIG. 10A shows neutrophil and platelet counts after administration of gemtuzumab ozogamicin at a single dose of 0.5 mg/m 2 in each dosing cycle.
  • “Cl” refers to gemtuzumab ozogamicin dosing cycle 1
  • C2 refers to gemtuzumab ozogamicin dosing cycle 2
  • C3 refers to gemtuzumab ozogamicin dosing cycle 3.
  • 10B is a table showing bone marrow measurable residual disease (MRD) at days 60, 101, and 147 (“D+60,” “D+101,” and “D+147,” respectively) following HCT infusion with trem-cel.
  • Pre-GO refers to samples obtained prior to gemtuzumab ozogamicin (GO) administration on the respective day.
  • 5d post-C2 refers to 5 days after gemtuzumab ozogamicin administration cycle 2.
  • 23 post-C3 refers to 23 days after gemtuzumab ozogamicin administration cycle 3.
  • BM MRD% refers to bone marrow measurable residual disease as defined as the percentage of AML blast cells.
  • FIG. 10C shows flow cytometry analysis of CD33 expression in peripheral blood and bone marrow cell populations (monocyte and myleloid cells) at the indicated time points following HCT infusion with trem-cel and prior to and post administration of gemtuzumab ozogamicin at a single dose of 0.5 mg/m 2 in each dosing cycle for Patient 1.
  • “Cl” refers to gemtuzumab ozogamicin dosing cycle 1
  • C2 refers to gemtuzumab ozogamicin dosing cycle 2
  • C3 refers to gemtuzumab ozogamicin dosing cycle 3.
  • PB refers to peripheral blood
  • BM refers to bone marrow.
  • FIG. 10D shows neutrophil and platelet counts after gemtuzumab ozogamicin dosing in Cohort 1 (0.5 mg/m 2 ).
  • the first gemtuzumab ozogamicin dose was given on days +68 (Patient 1), +74 (Patient 5), and +66 (Patient 6) post-hematopoietic cell transplant.
  • FIG. 10E shows an increase in CD33 negative myeloid cells during gemtuzumab ozogamicin dosing. Results show that editing of CD33 persisted over time and treatment with gemtuzumab ozogamicin selected for CD33 negative cells. indicates that Patient l’s CD33 flow was contaminated by the presence of relapsed disease after the third gemtuzumab ozogamicin dose.
  • FIG. 11 is a table showing measurable residual disease status in the bone marrow and peripheral blood of Patient 1 following gemtuzumab ozogamicin (GO) administration at a single dose of 0.5 mg/m 2 in each dosing cycle.
  • “D28,” “D60,” and “D100” refer to days 28, 60, and 100, respectively, following HCT infusion with trem-cel.
  • “D100*” indicates that the visit was 5 days post-GO infusion in Cl.
  • C1D1 refers to gemtuzumab ozogamicin dosing cycle 1, day 1.
  • C2D1 refers to gemtuzumab ozogamicin dosing cycle 2, day 1.
  • “C3D1” refers to gemtuzumab ozogamicin dosing cycle 3, day 1.
  • “C3D6” refers to gemtuzumab ozogamicin dosing cycle 3, day 6.
  • FIG. 12 is a graph showing the clinical course for Patient 2. Absolute neutrophil count (ANC) is shown on the left axis, and platelet count is shown on the right axis. The backup graft was infused on day 57 following the initial HCT infusion with trem-cel.
  • ANC Absolute neutrophil count
  • FIGs. 13A-13F show effects associated with administration of 0.5 mg/m 2 x 1 gemtuzumab ozogamicin or 1.0 mg/m 2 x 1 gemtuzumab ozogamicin following HCT with trem-cel.
  • FIG. 13A shows a graph representing pharmacokinetic data associated with administration of 0.5 mg/m 2 x 1 gemtuzumab ozogamicin in Cohort 1 (Patients 1, 5, and 6) and 1.0 mg/m 2 x 1 gemtuzumab ozogamicin in Cohort 2 (Patients 7 and 8) following HCT with trem-cel.
  • “hP67.6 Cone” refers to the concentration (in nanograms per mililiter (ng/mL)) of the antibody comprised in gemtuzumab ozogamicin.
  • FIG. 13B shows a table representing pharmacokinetic data associated with administration of 0.5 mg/m 2 x 1 gemtuzumab ozogamicin in Cohort 1 and 1.0 mg/m 2 x 1 gemtuzumab ozogamicin in Cohort 2 following HCT with trem-cel.
  • “N” refers to the number of subjects in each cohort.
  • SD refers to standard deviation;
  • “CV” refers to coefficient of variation;
  • T1/2 refers to the half-life;
  • Cmax refers to maximum concentration
  • Vz refers to the volume of distribution
  • CL refers to clearance.
  • 13C is a table showing the pharmacokinetics associated with administration of 0.5 mg/m 2 x 1 gemtuzumab ozogamicin or 1.0 mg/m 2 x 1 gemtuzumab ozogamicin following HCT with trem-cel (VBP101) 1 as compared to administration of 0.5 mg/m 2 x 1 gemtuzumab ozogamicin or 1.0 mg/m 2 x 1 gemtuzumab ozogamicin in relapse refractory CD33+ AML patients in a reference clinical trial (0903A1-101-US; FDA Briefing Document Oncologic Drugs Advisory Committee Meeting July 11, 2017, BLA 761060 My lotarg (gemtuzumab ozogamicin)).
  • FIG. 13D shows analyses of absolute neutrophil count (“ANC”) associated with administration of 0.5 mg/m 2 x 1 gemtuzumab ozogamicin in Patients 1, 5, and 6 and 1.0 mg/m 2 x 1 gemtuzumab ozogamicin in Patient 7 following HCT.
  • ANC absolute neutrophil count
  • FIG. 13E shows results obtained from analyses of CD33 editing in monocytes in peripheral blood (“PB”) samples from Cohort 1 (Patients 1, 5, and 6) after administration of multiple cycles of 0.5 mg/m 2 gemtuzumab ozogamicin and from Cohort 2 (Patient 7) after administration of 1.0 mg/m 2 x 1 gemtuzumab ozogamicin following HCT.
  • PB peripheral blood
  • 13F shows results obtained from analyses of CD33 editing in myeloid cells in peripheral blood (“PB”) samples from Cohort 1 (Patients 1, 5, and 6) after administration of multiple cycles of 0.5 mg/m 2 gemtuzumab ozogamicin and from Cohort 2 (Patient 7) after administration of 1.0 mg/m 2 x 1 gemtuzumab ozogamicin following HCT.
  • PB peripheral blood
  • the present disclosure provides targeted therapeutic approaches for use in treating hematopoietic malignancies that overcome limitations in existing therapies.
  • current CD33-targeted therapies for acute myeloid leukemia (AML) are limited by “on-target, off-leukemia” cytotoxicity directed toward normal healthy myeloid lineage cells expressing CD33.
  • the loss of the noncancerous CD33+ cells can deplete the hematopoietic system of the patient.
  • the subject can be administered rescue cells (e.g., hematopoietic cells) comprising a modification in the CD33 gene, such as tremtelectogene empogeditemcel (trem-cel).
  • CD33-modified cells can be resistant to the anti-CD33 cancer therapy and can therefore maintain or repopulate the hematopoietic system during or after anti-CD33 therapy. In this way, the normal myeloid compartment is protected from the on-target effects of CD33-targeted agents, resulting in an improved therapeutic index for these agents and better patient outcomes.
  • aspects of the present disclosure related to genetically engineered hematopoietic cells (also referred to herein as eHSCs or eHSPCs), or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen.
  • genetically engineered hematopoietic cells of the disclosure e.g., a hematopoietic stem cells (HSC) or hematopoietic progenitor cells (HPC) having a modification of the gene encoding CD33 are genetically engineered using any genetic editing methods known in the art.
  • genetically engineered hematopoietic cells of the disclosure e.g., a hematopoietic stem cells (HSC) or hematopoietic progenitor cells (HPC)
  • HSC hematopoietic stem cells
  • HPC hematopoietic progenitor cells
  • a cell e.g., HSC or HPC
  • a modification of CD33 and a modification of a second lineage- specific cell surface antigen is made using a nuclease and/or a gRNA described herein.
  • a cell described herein is capable of reconstituting the hematopoietic system of a subject.
  • a cell described herein e.g., an HSC or HPC
  • a cell described herein is capable of one or more of (e.g., all of): engrafting in a human subject, producing myeloid lineage cells, and producing and lymphoid lineage cells.
  • the cell comprises only one genetic modification.
  • the cell is only genetically modified at the CD33 locus, such as in a sequence of exon 3 of CD33. In some embodiments, the cell is genetically modified at a second locus. In some embodiments, the cell does not comprise a transgenic protein, e.g., does not comprise a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • CD33 antigen and “CD33 protein” are used interchangeably herein and refer to the CD33 protein, or a portion or fragment thereof, such as a portion that is targeted by an anti-CD33 agent, such as a cytotoxic agent comprising an anti-CD33 antigen-binding domain, such as gemtuzumab ozogamicin.
  • an anti-CD33 agent such as a cytotoxic agent comprising an anti-CD33 antigen-binding domain, such as gemtuzumab ozogamicin.
  • a genetically engineered hematopoietic cell described herein comprises substantially no CD33 protein (CD33 antigen). In some embodiments, a genetically engineered hematopoietic cell described herein comprises substantially no wildtype CD33 protein but comprises a mutant CD33 protein. In some embodiments, the mutant CD33 protein is not bound by an agent that targets CD33 for therapeutic purposes.
  • CD33KO eHSCs a genetically engineered hematopoietic cell that has been genetically engineered such that the cell has reduced or no expression of CD33
  • CD33KO eHSCs a genetically engineered hematopoietic cell that has been genetically engineered such that the cell has reduced or no expression of CD33
  • CD33KO eHSPCs a genetically engineered hematopoietic cell that has been genetically engineered such that the cell has reduced or no expression of CD33
  • CD33KO eHSCs a genetically engineered hematopoietic cell that has been genetically engineered such that the cell has reduced or no expression of CD33
  • CD33KO eHSCs a genetically engineered hematopoietic cell that has been genetically engineered such that the cell has reduced or no expression of CD33
  • CD33KO eHSCs a genetically engineered hematopoietic cell that has been genetically engineered such that the cell has reduced or no expression of
  • the cell is a circulating blood cell, e.g., a reticulocyte, megakaryocyte erythroid progenitor (MEP) cell, myeloid progenitor cell (CMP/GMP), lymphoid progenitor (LP) cell, hematopoietic stem cell (HSC), or hematopoietic progenitor cell (HPC), which may be referred to as hematopoietic stem and progenitor cells (HSPCs), or endothelial cell (EC).
  • a circulating blood cell e.g., a reticulocyte, megakaryocyte erythroid progenitor (MEP) cell, myeloid progenitor cell (CMP/GMP), lymphoid progenitor (LP) cell, hematopoietic stem cell (HSC), or hematopoietic progenitor cell (HPC), which may be referred to as hematopoietic stem and progenitor cells (
  • the cell is a bone marrow cell (e.g., a reticulocyte, an erythroid cell (e.g., erythroblast), megakaryocyte-erythroid progenitor cell (MEP cell), myeloid progenitor cell (CMP/GMP), lymphocyte predominant (LP) cell, erythroid progenitor (EP) cell, HSC, multipotent progenitor (MPP) cell, endothelial cell (EC), hemogenic endothelial (HE) cell, or mesenchymal stem cell).
  • a bone marrow cell e.g., a reticulocyte, an erythroid cell (e.g., erythroblast), megakaryocyte-erythroid progenitor cell (MEP cell), myeloid progenitor cell (CMP/GMP), lymphocyte predominant (LP) cell, erythroid progenitor (EP) cell, HSC, multipotent progenitor (MPP) cell, endothelial
  • the cell is a myeloid progenitor cell (e.g., a common myeloid progenitor (CMP) cell or granulocyte macrophage progenitor (GMP) cell).
  • CMP common myeloid progenitor
  • GMP granulocyte macrophage progenitor
  • the cell is a lymphoid progenitor cell, e.g., a common lymphoid progenitor (CLP) cell.
  • the cell is an erythroid progenitor cell (e.g., an MEP cell).
  • the cell is a hematopoietic stem/progenitor cell (e.g., a long-term HSC (LT-HSC), short term HSC (ST- HSC), MPP cell, or lineage restricted progenitor (LRP) cell).
  • a hematopoietic stem/progenitor cell e.g., a long-term HSC (LT-HSC), short term HSC (ST- HSC), MPP cell, or lineage restricted progenitor (LRP) cell.
  • the cell is a CD34+ cell, CD34+CD90+ cell, CD34+CD38+ cell, CD34+CD90+CD49+CD38+ CD45RA cell, CD105+ cell, CD31+, or CD133+ cell, or a CD34+CD90+ CD133+ cell.
  • the cell is an umbilical cord blood CD34+ HSPC, umbilical cord venous endothelial cell, umbilical cord arterial endothelial cell, amniotic fluid CD34+ cell, amniotic fluid endothelial cell, placental endothelial cell, or placental hematopoietic CD34+ cell.
  • the cell is a mobilized peripheral blood hematopoietic CD34+ cell (after the patient is treated with a mobilization agent, e.g., granulocyte colony-stimulating factor (G-CSF), etoposide, cyclophosphamide, and/or plerixafor).
  • G-CSF granulocyte colony-stimulating factor
  • the cell is a peripheral blood endothelial cell, or a population thereof.
  • the cells are hematopoietic cells, e.g., hematopoietic stem cells.
  • Hematopoietic stem cells are typically capable of giving rise to both myeloid and lymphoid progenitor cells that further give rise to myeloid cells (e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, erythrocytes, platelets, etc.) and lymphoid cells (e.g., T cells, B cells, NK cells), respectively.
  • HSCs are characterized by the expression of the cell surface marker CD34 (e.g., CD34+), which can be used for the identification and/or isolation of HSCs, and absence of cell surface markers associated with commitment to a cell lineage.
  • a population of genetically engineered hematopoietic cells described herein comprises a plurality of hematopoietic stem cells. In some embodiments, a population of genetically engineered hematopoietic cells described herein comprises a plurality of hematopoietic progenitor cells. In some embodiments, a population of genetically engineered hematopoietic cells described herein comprises a plurality of hematopoietic stem cells and a plurality of hematopoietic progenitor cells.
  • a hematopoietic stem cell refers to cells of a stem cell lineage that give rise to all the blood cell types including the erythroid (erythrocytes or red blood cells (RBCs)), myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, megakaryocytes/platelets, and dendritic cells), and lymphoid (T-cells, B-cells, NK-cells).
  • erythroid erythrocytes or red blood cells (RBCs)
  • myeloid monocytes and macrophages
  • neutrophils neutrophils
  • basophils basophils
  • eosinophils neutrophils
  • megakaryocytes/platelets basophils
  • dendritic cells dendritic cells
  • the cells used herein are selected from the group consisting of a circulating blood cell, a mobilized blood cell, a bone marrow cell, a myeloid progenitor cell, a lymphoid progenitor cell, a multipotent progenitor cell, a lineage restricted progenitor cell, an endothelial cell, or a mesenchymal stromal cell.
  • the HSC is from a non-cord blood source, an umbilical cord source, or a cord blood source.
  • the HSC is a CD34+ cell.
  • the HSC cell is capable of differentiating in vivo after transplantation into the subject.
  • the HSC cell is capable of differentiating into B cells, T cells, erythroid cells, and/or myeloid cells. In some embodiments, the HSC cell is capable of reconstituting hematopoiesis in the subject. In some embodiments, the hematopoietic stem cell has at least one of the cell surface marker characteristic of hematopoietic progenitor cells: CD34+, CD59+, Thyl/CD90+, CD381o/-, and C-kit/CDl 17+. In some embodiments, the hematopoietic progenitor are CD34+.
  • a hematopoietic stem cell is a peripheral blood stem cell obtained from a subject after the subject has been treated with granulocyte colony stimulating factor (G-CSF) (optionally in combination with plerixafor, etoposide, and/or cyclophosphamide).
  • G-CSF granulocyte colony stimulating factor
  • CD34+ cells are enriched using CliniMACS® Cell Selection System (Miltenyi Biotec).
  • CD34+ cells are stimulated in serum-free medium (e.g., CellGrow SCGM media, CellGenix) with cytokines (e.g., SCF, rhTPO, rhFLT3) before genome editing.
  • serum-free medium e.g., CellGrow SCGM media, CellGenix
  • cytokines e.g., SCF, rhTPO, rhFLT3
  • addition of SRI and dmPGE2 and/or other factors is contemplated to improve long-term en
  • a population of genetically engineered hematopoietic cells for administration in accordance with the disclosure can be allogeneic hematopoietic progenitor cells obtained from one or more donors.
  • allogeneic refers to a hematopoietic progenitor cell or biological samples comprising hematopoietic progenitor cells obtained from one or more different donors of the same species, where the genes at one or more loci are not identical.
  • a hematopoietic cell population being administered to a subject can be derived from umbilical cord blood obtained from one more unrelated donor subjects or from one or more non-identical siblings.
  • syngeneic hematopoietic cell populations can be used, such as those obtained from genetically identical animals or from identical twins.
  • the hematopoietic cells are autologous cells; that is, the hematopoietic progenitor cells are obtained or isolated from a subject and administered to the same subject (z.e., the donor and recipient are the same).
  • a population of genetically engineered hematopoietic cells described herein comprises a plurality of genetically engineered hematopoietic stem cells. In some embodiments, a population of genetically engineered hematopoietic cells described herein comprises a plurality of genetically engineered hematopoietic progenitor cells. In some embodiments, population of genetically engineered hematopoietic cells described herein comprises a plurality of genetically engineered hematopoietic stem cells and a plurality of genetically engineered hematopoietic progenitor cells. In some embodiments, the HSCs or HPCs are obtained from a subject, such as a human subject.
  • the HSCs are peripheral blood HSCs.
  • the mammalian subject is a non-human primate, a rodent (e.g., mouse or rat), a bovine, a porcine, an equine, or a domestic animal.
  • the HSCs are obtained from a human subject, such as a human subject having a hematopoietic malignancy.
  • the HSCs or HPCs are obtained from a healthy donor.
  • the HSCs or HPCs are obtained from the subject to whom the gemtuzumab ozogamicin will be subsequently administered.
  • HSCs or HPCs that are administered to the same subject from which the cells were obtained are referred to as autologous cells, whereas HSCs or HPCs that are obtained from a subject who is not the subject to whom the cells will be administered are referred to as allogeneic cells.
  • a population of genetically engineered hematopoietic cells is a heterogeneous population of cells, e.g. heterogeneous population of genetically engineered hematopoietic cells containing different CD33 mutations.
  • at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of copies of CD33 in the population of cells have a mutation effected by a genome editing approach described herein, e.g. by a CRISPR/Cas system using a gRNA described herein.
  • a population can comprise a plurality of different CD33 mutations and each mutation of the plurality contributes to the percent of copies of CD33 in the population of cells that have a mutation.
  • the expression of CD33 on the genetically engineered hematopoietic cell is compared to the expression of CD33 on a naturally occurring hematopoietic cell (e.g., a wild-type counterpart).
  • the genetic engineering results in a reduction in the expression level of CD33 by at least 50%, at least 60%, at least 70%, at least 80%, 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%, or at least 99% as compared to the expression of CD33 on a naturally occurring hematopoietic cell (e.g., a wild-type counterpart).
  • the genetically engineered hematopoietic cell expresses less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of CD33 as compared to a naturally occurring hematopoietic cell (e.g., a wild-type counterpart).
  • a naturally occurring hematopoietic cell e.g., a wild-type counterpart
  • the genetic engineering results in a reduction in the expression level of wild-type CD33 by at least 50%, at least 60%, at least 70%, at least 80%, 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%, or at least 99% as compared to the expression of the level of wild-type CD33 on a naturally occurring hematopoietic cell (e.g., a wild-type counterpart).
  • a naturally occurring hematopoietic cell e.g., a wild-type counterpart
  • the genetically engineered hematopoietic cell expresses less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of CD33 as compared to a naturally occurring hematopoietic cell (e.g., a wildtype counterpart).
  • a naturally occurring hematopoietic cell e.g., a wildtype counterpart
  • the genetic engineering results in a reduction in the expression level of wild-type lineage- specific cell surface antigen (e.g., CD33) by at least 50%, at least 60%, at least 70%, at least 80%, 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%, or at least 99% as compared to a suitable control (e.g., a cell or plurality of cells).
  • the suitable control comprises the level of the wild-type lineage- specific cell surface antigen measured or expected in a plurality of non-engineered cells from the same subject.
  • the suitable control comprises the level of the wild-type lineage- specific cell surface antigen measured or expected in a plurality of cells from a healthy subject. In some embodiments, the suitable control comprises the level of the wild-type lineage- specific cell surface antigen measured or expected in a population of cells from a pool of healthy individuals (e.g., 10, 20, 50, or 100 individuals). In some embodiments, the suitable control comprises the level of the wild-type lineage- specific cell surface antigen measured or expected in a subject in need of a treatment described herein, e.g., an anti-CD33 therapy, e.g., wherein the subject has a cancer, wherein cells of the cancer express CD33.
  • an anti-CD33 therapy e.g., wherein the subject has a cancer, wherein cells of the cancer express CD33.
  • the suitable control comprises the level of the wild-type lineage- specific cell surface antigen measured in the cells prior to being subjected to genetic engineering to reduce or eliminate expression of CD33.
  • a population of genetically engineered hematopoietic cells is referred to as “tremtelectogene empogeditemcel” or “trem-cel.” Accordingly, trem-cel is a product comprising genome-edited hematopoietic stem and progenitor allogeneic donor cells wherein CD33 has been deleted using genome engineering.
  • At least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of copies of CD33 in of the cells in trem-cel have a mutation effected by a genome editing approach described herein, e.g. by a CRISPR/Cas system using a gRNA described herein.
  • trem-cel is manufactured with a CD33 editing efficacy of at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.
  • trem-cel is manufactured with 88% CD33 editing efficacy.
  • the cells in trem-cel comprises reduction in the expression level of CD33 by at least 50%, at least 60%, at least 70%, at least 80%, 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%, or at least 99% as compared to the expression of CD33 on a naturally occurring hematopoietic cell (e.g., a wild-type counterpart).
  • hematopoietic cell e.g., a wild-type counterpart
  • the genetically engineered hematopoietic cells of trem-cel express less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of CD33 as compared to a naturally occurring hematopoietic cell (e.g., a wild-type counterpart that has not be genetically engineered).
  • a naturally occurring hematopoietic cell e.g., a wild-type counterpart that has not be genetically engineered.
  • At least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cells in trem-cel are CD34+ cells. In some embodiments, 97% of the cells in trem-cel are CD34+.
  • a method of making the genetically engineered hematopoietic cells described herein comprises a step of providing a wild-type cell, e.g., a wild-type hematopoietic stem or progenitor cell.
  • the wild-type cell is an unedited cell comprising (e.g., expressing) two functional copies of the lineage- specific cell surface antigen (e.g., CD33).
  • the cell comprises a CD33 gene sequence according to SEQ ID NO: 16.
  • the cell comprises a CD33 gene sequence encoding a CD33 protein that is encoded in SEQ ID NO: 16, e.g., the CD33 gene sequence may comprise one or more silent mutations relative to SEQ ID NO: 16.
  • the wild-type cell expresses the lineage- specific cell surface antigen (e.g., CD33), or gives rise to a more differentiated cell that expresses the lineage- specific cell surface antigen at a level comparable to (or within 90%-110%, 80%-120%, 70%-130%, 60- 140%, or 50%-150% of) HL60 or MOLM-13 cells.
  • the wild-type cell binds an antibody that binds the lineage- specific cell surface antigen (e.g., an anti-CD33 antibody, e.g., P67.6), or gives rise to a more differentiated cell that binds the antibody at a level comparable to (or within 90%-110%, 80%-120%, 70%-130%, 60-140%, or 50%-150% of) binding of the antibody to HL60 or MOLM-13 cells.
  • Antibody binding may be measured, for example, by flow cytometry.
  • the genetically engineered hematopoietic stem or progenitor cell comprises a genetic mutation in the exon 3 of an endogenous CD33 gene, wherein the genetic mutation is at a site described herein (see, Table 1).
  • One aspect of the present disclosure provides a genetically engineered hematopoietic stem and/or progenitor cell comprises a genetic mutation in exon 3 of an endogenous CD33 gene, wherein the genetic mutation is at a site targeted by a gRNA, such as any of the gRNAs presented in Table 1.
  • an engineered cell described herein comprises two mutations, the first mutation being in CD33 and the second mutation being in a second lineage- specific cell surface antigen.
  • a cell can, in some embodiments, be resistant to two agents: an anti-CD33 agent (e.g., gemtuzumab ozogamicin) and an agent targeting the second lineagespecific cell surface antigen.
  • an anti-CD33 agent e.g., gemtuzumab ozogamicin
  • an agent targeting the second lineagespecific cell surface antigen e.g., gemtuzumab ozogamicin
  • such a cell can be produced using two or more gRNAs described herein, e.g., a gRNA of Table 3 and a second gRNA.
  • the cell can be produced using, e.g. , a ZFN or TALEN.
  • the disclosure also provides populations comprising cells described herein.
  • the second mutation is at a gene encoding a lineage- specific cell-surface antigen, such as any of the lineage- specific cell-surface antigens described herein.
  • a mutation effected by the methods and compositions provided herein results in a loss of function of a gene product encoded by the target gene, e.g., in the case of a mutation in the CD33 gene, in a loss of function of a CD33 protein.
  • the loss of function is a reduction in the level of expression of the gene product, e.g., reduction to a lower level of expression, or a complete abolishment of expression of the gene product.
  • the mutation results in the expression of a non-functional variant of the gene product.
  • a truncated gene product in the case of the mutation generating a premature stop codon in the encoding sequence, a truncated gene product, or, in the case of the mutation generating a nonsense or mis sense mutation, a gene product characterized by an altered amino acid sequence, which renders the gene product non-functional.
  • the function of a gene product is binding or recognition of a binding partner.
  • the reduction in expression of the gene product, e.g., of CD33, of the second lineage-specific cell-surface antigen, or both is to less than or equal to 50%, less than or equal to 40%, less than or equal to 30%, less than or equal to 20%, less than or equal to 10%, less than or equal to 5%, less than or equal to 2%, or less than or equal to 1% of the level in a wild-type or non-engineered counterpart cell.
  • At least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or more of copies of CD33 in the population of genetically engineered hematopoietic cells generated by the methods and/or using the compositions provided herein have a mutation.
  • at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of copies of the second lineage- specific cell surface antigen in the population of genetically engineered hematopoietic cells have a mutation.
  • At least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of copies of CD33 and of the second lineagespecific cell surface antigen in the population of genetically engineered hematopoietic cells have a mutation.
  • the population comprises one or more wild-type cells.
  • the population comprises one or more cells that comprise one wild-type copy of CD33.
  • the population comprises one or more cells that comprise one wild-type copy of the second lineage- specific cell surface antigen.
  • Gemtuzumab ozogamicin is a recombinant, humanized anti- CD33 monoclonal antibody (IgG4 K antibody hP67.6) linked with (covalently attached to) the cytotoxic antitumor antibiotic calicheamicin (N-acetyl-y-calicheamicin) via a bifunctional linker (4-(4-acetylphenoxy) butanoic acid).
  • Gemtuzumab ozogamicin is available commercially as Mylotarg® (Wyeth Pharmaceuticals, Philadelphia, Pa.).
  • the antibody portion of gemtuzumab ozogamicin referred to as hP67.6, binds specifically to the CD33 antigen.
  • Gemtuzumab ozogamicin contains amino acid sequences of which approximately 98.3% are of human origin. The constant region and framework regions contain human sequences while the complementarity-determining regions are derived from a murine antibody (P67.6) that binds CD33. This antibody is linked to N-acetyl-gamma calicheamicin via a bifunctional linker. Gemtuzumab ozogamicin has approximately 50% of the antibody loaded with 4-6 moles calicheamicin per mole of antibody. The remaining 50% of the antibody is not linked to the calicheamicin derivative. Gemtuzumab ozogamicin has a molecular weight of 151 to 153 kDa.
  • Gemtuzumab ozogamicin is an antibody-drug conjugate (ADC) comprising an anti- CD33 antibody conjugated to a toxin or drug molecule. Binding of the antibody or fragment thereof to the corresponding antigen allows for delivery of the toxin or drug molecule to a cell that presents CD33 on its cell surface (e.g., target cell), thereby resulting in death of the target cell.
  • ADC antibody-drug conjugate
  • gemtuzumab ozogamicin interacts with and induces cytotoxicity of cells expressing CD33, however as described herein, administration of gemtuzumab ozogamicin may induce cytotoxicity of not only cancer cells expressing CD33 but also normal, healthy cells that also express CD33, e.g. i “on-target, off-leukemia” effects.
  • aspects of the present disclosure provide methods involving administering to a subject an effective amount of a population of genetically engineered hematopoietic cells (also referred to herein as eHSCs or eHSPCs), as described herein, and gemtuzumab ozogamicin in a dosing regimen comprising at least one dosing cycle, wherein the dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin.
  • eHSCs genetically engineered hematopoietic cells
  • the subject is diagnosed with a hematopoietic malignancy and is directed to undergo a combination treatment involving administration of a population of genetically engineered hematopoietic cells, as described herein, and gemtuzumab ozogamicin in a dosing regimen comprising at least one dosing cycle, e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more dosing cycles, wherein each dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin.
  • a dosing regimen comprising at least one dosing cycle, e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more dosing cycles, wherein each dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin.
  • a combination treatment involves more than one aspect of the treatment that may be performed together (e.g., administered at the same time or in a single composition) but also encompasses more than one treatment within a treatment regimen aimed to treat the malignancy, or any symptom or manifestation thereof.
  • some methods described herein involve the combination treatment to treat a hematopoietic malignancy (e.g., acute myeloid leukemia) involving administering an effective amount of a population of genetically engineered hematopoietic cells, as described herein, and gemtuzumab ozogamicin in a dosing regimen comprising at least one dosing cycle (e.g., one, two, three, four dosing cycles), wherein each dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin.
  • a hematopoietic malignancy e.g., acute myeloid leukemia
  • gemtuzumab ozogamicin e.g., one, two, three, four dosing cycles
  • the methods described herein involve the combination treatment to treat a premalignant stage of a hematopoietic malignancy (e.g., myelodysplastic syndrome (MDS)) involving administering an effective amount of a population of genetically engineered hematopoietic cells, as described herein, and gemtuzumab ozogamicin in a dosing regimen comprising at least one dosing cycle (e.g., one, two, three, four dosing cycles), wherein each dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin.
  • MDS myelodysplastic syndrome
  • Some combination treatment methods provided herein comprise sequential administration of a population of genetically engineered hematopoietic cells (e.g., CD33KO eHSPCs) and gemtuzumab ozogamicin/Mylotarg®, including, for example, administration of the dosing regimen of gemtuzumab ozogamicin/Mylotarg® after administration of the genetically engineered hematopoietic cells.
  • a population of genetically engineered hematopoietic cells e.g., CD33KO eHSPCs
  • gemtuzumab ozogamicin/Mylotarg® including, for example, administration of the dosing regimen of gemtuzumab ozogamicin/Mylotarg® after administration of the genetically engineered hematopoietic cells.
  • an effective number of genetically engineered hematopoietic stem cells e.g., C 33-modil'icd hematopoietic stem cells described herein, is administered in combination with a dosing regimen of gemtuzumab ozogamicin.
  • an effective number of cells comprising a modified CD33 and a modified second lineagespecific cell surface antigen are administered in combination with a dosing regimen of gemtuzumab ozogamicin.
  • an effective amount of a population of genetically engineered hematopoietic stem cells comprises about 10 2 cells/kilogram to about IO 10 cells/kilogram body weight of a subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, e.g., C 33-modil'icd hematopoietic stem cells described herein, comprises about 10 4 cells/kilogram to about 10 8 cells/kilogram body weight of a subject.
  • an effective amount of a population of genetically engineered hematopoietic stem cells comprises about 10 6 cells/kilogram to about 10 8 cells/kilogram body weight of a subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, e.g., C 55-modil'icd hematopoietic stem cells described herein, comprises about 10 5 cells/kilogram to about 10 7 cells/kilogram body weight of a subject.
  • an effective amount of a population of genetically engineered hematopoietic stem cells comprises about 10 6 cells/kilogram to about 10 7 cells/kilogram body weight of a subject.
  • an effective amount of a population of genetically engineered hematopoietic stem cells comprises about 10 5 cells/kilogram, about 10 6 cells/kilogram, about 10 7 cells/kilogram, or about 10 8 cells/kilogram body weight of a subject.
  • an effective amount a population of genetically engineered hematopoietic stem cells comprises at least 10 2 cells, at least 10 3 cells, at least 10 4 cells, at least 10 5 cells, at least 2 x 10 5 cells, at least 3 x 10 5 cells, at least 4 x 10 5 cells, at least 5 x 10 5 cells, at least 6 x 10 5 cells, at least 7 x 10 5 cells, at least 8 x 10 5 cells, at least 9 x 10 5 cells, at least 10 6 cells, at least 2 x 10 6 cells, at least 3 x 10 6 cells, at least 4 x 10 6 cells, at least 5 x 10 6 cells, at least 6 x 10 6 cells, at least 7 x 10 6 cells, at least 8 x 10 6 cells, at least 9 x 10 6 cells, at least 1 x 10 7 cells, or multiples thereof.
  • an effective amount of a population of genetically engineered hematopoietic stem cells comprises about 1.0 x 10 5 , about 2.0 x 10 5 , about 3.0 x 10 5 , about 4.0 x 10 5 , about 5.0 x 10 5 , about 6.0 x 10 5 , about 7.0 x 10 5 , about 8.0 x 10 5 , about 9.0 x 10 5 , about 1.0 x 10 6 , about 2.0 x 10 6 , about 3.0 x 10 6 , about 4.0 x 10 6 , about 5.0 x 10 6 , about 6.0 x 10 6 , about 7.0 x 10 6 , about 8.0 x 10 6 , about 9.0 x 10 6 , about 1.0 x 10 7 , about 2.0 x 10 7 , about 3.0 x 10 7 , about 4.0 x 10 7 , 5.0 x 10 7 ,
  • an effective amount of a population of genetically engineered hematopoietic stem cells comprises about 3.0 x 10 6 cells/kilogram body weight of a subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, e.g., CD33-modified hematopoietic stem cells described herein, comprises between about 6 x 10 6 - 8 x 10 6 cells/kilogram body weight of a subject.
  • an effective amount of a population of genetically engineered hematopoietic stem cells comprises about 7.6 x 10 6 cells/kilogram body weight of a subject.
  • an effective amount of trem-cel comprises about 3 x 10 6 cells/kilogram body weight of a subject.
  • an effective amount of trem-cel comprises about 10 6 cells/kilogram body weight of a subject.
  • an effective amount of trem-cel comprises about 5 x 10 5 cells/kilogram body weight of a subject.
  • an effective amount of trem-cel comprises about 10 5 cells/kilogram body weight of a subject.
  • an effective amount of trem-cel comprises about 1.0 x 10 5 , about 2.0 x 10 5 , about 3.0 x 10 5 , about 4.0 x 10 5 , about 5.0 x 10 5 , about 6.0 x 10 5 , about 7.0 x 10 5 , about 8.0 x 10 5 , about 9.0 x 10 5 , about 1.0 x 10 6 , about 2.0 x 10 6 , about 3.0 x 10 6 , about 4.0 x 10 6 , about 5.0 x 10 6 , about 6.0 x 10 6 , about 7.0 x 10 6 , about 8.0 x 10 6 , about 9.0 x 10 6 , about 1.0 x 10 7 , about 2.0 x 10 7 , about 3.0 x 10 7 , about 4.0 x 10 7 , 5.0 x 10 7 , about 6.0 x 10 7 , about 7.0 x 10 7 , about 8.0 x 10 7 , about 9.0 x 10 7 , or
  • an effective amount of trem-cel comprises between about 6 x 10 6 - 8 x 10 6 cells/kilogram body weight of a subject. In some embodiments, an effective amount of trem-cel comprises about 7.6 x 10 6 cells/kilogram body weight of a subject. In some embodiments, an effective amount of trem-cel comprises about 3 x 10 6 cells/kilogram body weight of a subject. In some embodiments, an effective amount of trem-cel comprises less than about 3 x 10 6 cells/kilogram body weight of a subject.
  • Hematopoietic stem cells e.g., CD34+ hematopoietic stem cells
  • that can, at least in some embodiments, serve as the starting material for generating the genetically engineered hematopoietic stem cells, e.g., CD33-modified hematopoietic stem cells described herein, can be derived from one or more donors or can be obtained from an autologous source.
  • the genetically engineered hematopoietic stem cells, e.g., CD33-modified hematopoietic stem cells described herein are expanded in culture prior to administration to a subject in need thereof.
  • a typical number of cells, e.g., immune cells or hematopoietic cells, administered to a mammal can be, for example, in the range of one million to 100 billion cells; however, amounts below or above this exemplary range are also within the scope of the present disclosure.
  • gemtuzumab ozogamicin is administered in a dosing regimen comprising multiple dosing cycles (e.g., 2, 3, 4 dosing cycles) each comprising administration of an effective amount of gemtuzumab ozogamicin, e.g., in combination with the population of genetically engineered hematopoietic stem cells, e.g., CDAi-modil'icd hematopoietic stem cells described herein.
  • an effective amount of a gemtuzumab ozogamicin is about 0.01 mg/m 2 to about 6.0 mg/m 2 body surface area of a subject.
  • an effective amount of a gemtuzumab ozogamicin is about 0.01 mg/m 2 to about 9.0 mg/m 2 body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin (e.g.
  • each dosing cycle is about 0.05 mg/m 2 to about 2.5 mg/m 2 , about 0.1 mg/m 2 to about 2.0 mg/m 2 , about 0.1 mg/m 2 to about 1.0 mg/m 2 , about 1.0 mg/m 2 to about 2.0 mg/m 2 , or about 1.5 mg/m 2 to about 2.5 mg/m 2 , 0.05 mg/m 2 to about 6.0 mg/m 2 , about 0.1 mg/m 2 to about 6.0 mg/m 2 , about 0.1 mg/m 2 to about 3.0 mg/m 2 , about 1.0 mg/m 2 to about 6.0 mg/m 2 , about 1.5 mg/m 2 to about 2.5 mg/m 2 , about 3.0 mg/m 2 to about 6.0 mg/m 2 body surface area of a subject.
  • an effective amount of gemtuzumab ozogamicin is about 0.05 mg/m 2 , about 0.1 mg/m 2 , about 0.25 mg/m 2 , about 0.5 mg/m 2 , about 0.6 mg/m 2 , about 0.7 mg/m 2 , about 0.8 mg/m 2 , about 0.9 mg/m 2 , about 1.0 mg/m 2 , about 1.1 mg/m 2 , about 1.2 mg/m 2 , about 1.3 mg/m 2 , about 1.4 mg/m 2 , about 1.5 mg/m 2 , about 1.6 mg/m 2 , about 1.7 mg/m 2 , about 1.8 mg/m 2 , about 1.9 mg/m 2 , about 2.0 mg/m 2 , about 2.1 mg/m 2 , about 2.2 mg/m 2 , about 2.3 mg/m 2 , about
  • an effective amount of gemtuzumab ozogamicin is about 0.5 mg/m 2 body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 1.0 mg/m 2 body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 2.0 mg/m 2 body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 3.0 mg/m 2 body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 4.0 mg/m 2 body surface area of a subject.
  • an effective amount of gemtuzumab ozogamicin is about 5.0 mg/m 2 body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 6.0 mg/m 2 body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 7.0 mg/m 2 body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 8.0 mg/m 2 body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 9.0 mg/m 2 body surface area of a subject.
  • an effective amount of a population of genetically engineered hematopoietic cells is about 10 4 cells/kilogram to about 10 8 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 0.1 mg/m 2 to about 3.0 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells e.g.
  • CD33-modified hematopoietic stem cells described herein is about 10 6 cells/kilogram to about 10 7 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 0.1 mg/m 2 to about 6.0 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells is about 3.0 x 10 6 cells/kilogram body weight of a subject
  • an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 0.1 mg/m 2 , about 0.25 mg/m 2 , about 0.5 mg/m 2 , about 1.0 mg/m 2 , about 2.0 mg/m 2 , about 3.0 mg/m 2 , about 4.0 mg/m 2 , about 5.0 mg/m 2 , or about 6.0 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells is about 3.0 x 10 6 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 0.5 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells e.g. CD33- modified hematopoietic stem cells described herein.
  • CD33- modified hematopoietic stem cells described herein is about 3.0 x 10 6 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 1.0 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein is about 3.0 x 10 6 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 2.0 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells is about 3.0 x 10 6 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 3.0 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells e.g. CD33- modified hematopoietic stem cells described herein.
  • CD33- modified hematopoietic stem cells described herein is about 3.0 x 10 6 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 4.0 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein is about 3.0 x 10 6 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 5.0 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells is about 3.0 x 10 6 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 6.0 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells is about 7.6 x 10 6 cells/kilogram body weight of a subject
  • an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 0.1 mg/m 2 , about 0.25 mg/m 2 , about 0.5 mg/m 2 , about 1.0 mg/m 2 , about 2.0 mg/m 2 , about 3.0 mg/m 2 , about 4.0 mg/m 2 , about 5.0 mg/m 2 , or about 6.0 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells is about 7.6 x 10 6 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 0.5 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells e.g. CD33- modified hematopoietic stem cells described herein.
  • CD33- modified hematopoietic stem cells described herein is about 7.6 x 10 6 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 1.0 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein is about 7.6 x 10 6 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 2.0 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells is about 7.6 x 10 6 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 3.0 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells e.g. CD33- modified hematopoietic stem cells described herein.
  • CD33- modified hematopoietic stem cells described herein is about 7.6 x 10 6 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 4.0 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein is about 7.6 x 10 6 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 5.0 mg/m 2 body surface area of the subject.
  • an effective amount of a population of genetically engineered hematopoietic cells is about 7.6 x 10 6 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 6.0 mg/m 2 body surface area of the subject.
  • a population of genetically engineered hematopoietic cells and the dosing regimen of gemtuzumab ozogamicin are administered in temporal proximity.
  • temporal proximity refers to the timing of the administration of the population of genetically engineered hematopoietic cells relative to the administration of gemtuzumab ozogamicin.
  • administration of the population of genetically engineered hematopoietic cells and a gemtuzumab ozogamicin in temporal proximity can include administration of the hematopoietic cells prior to, following, or at approximately the same time as the administration of the dosing regimen of gemtuzumab ozogamicin.
  • the treatments may be admixed or in separate volumes.
  • administration in combination includes administration in the same course of treatment, e.g., in the course of treating a cancer with an anti-CD33 therapy, the subject may be administered an effective number of C -modil'icd cells concurrently or sequentially, e.g., before, during, or after the treatment, with gemtuzumab ozogamicin.
  • administering in temporal proximity comprises administering a population of genetically engineered hematopoietic stem cells and gemtuzumab ozogamicin within a single treatment regimen. In some embodiments, administering in temporal proximity comprises administering a population of genetically engineered hematopoietic stem cells and a gemtuzumab ozogamicin dosing regimen simultaneously or concurrently. In some embodiments, administering in temporal proximity comprises administering a population of genetically engineered hematopoietic stem cells and a gemtuzumab ozogamicin dosing regimen sequentially (e.g., administering either treatment before the other treatment).
  • a population of genetically engineered hematopoietic stem cells is administered prior to a gemtuzumab ozogamicin dosing regimen.
  • administering in temporal proximity comprises administering a population of genetically engineered hematopoietic stem cells within 120 days (e.g., within 90 days, within 60 days, within 30 days, within 20 days, within 10 days, within 7 days, or within 1 day) of administering a dosing regimen of gemtuzumab ozogamicin.
  • administering in temporal proximity comprises administering a population of genetically engineered hematopoietic stem cells to a subject within 120 days (e.g., within 90 days, within 60 days, within 30 days, within 20 days, within 10 days, within 7 days, or within 1 day) prior to administering a dosing regimen of gemtuzumab ozogamicin to the subject.
  • administering in temporal proximity comprises administering a population of genetically engineered hematopoietic stem cells within 365 days (e.g., within 300 days, within 250 days, within 200 days, within 150 days, or within 100 days) of administering a dosing regimen of gemtuzumab ozogamicin.
  • administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen at least 60 days after administration of the population of genetically engineered hematopoietic cells. In some embodiments, administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen between 20-60 days after administration of the population of genetically engineered hematopoietic cells, if the subject experiences early relapse. In some embodiments, administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen between 30-60 days after administration of the population of genetically engineered hematopoietic cells, if the subject experiences early relapse.
  • administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen between 40-60 days after administration of the population of genetically engineered hematopoietic cells, if the subject experiences early relapse. In some embodiments, administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen between 50-60 days after administration of the population of genetically engineered hematopoietic cells, if the subject experiences early relapse.
  • the subject is evaluated based on one or more parameters, such as level of engraftment, following administration of the population of genetically engineered hematopoietic cells, and described herein, prior to administration of gemtuzumab ozogamicin.
  • the subject has a CD33-negative absolute neutrophil count (ANC) above a threshold value (e.g., at least 500 cells/pL, at least 1000 cells/pL) prior to administration of the gemtuzumab ozogamicin.
  • ANC CD33-negative absolute neutrophil count
  • the dosing regimen of gemtuzumab ozogamicin comprises dosing cycles, each of which involves administering an effective amount of gemtuzumab ozogamicin.
  • the effective amount of gemtuzumab ozogamicin is administered in multiple doses, for example at a regular interval (e.g., every day, every two days, every three days, every four days, every five days, every six days, every week (e.g., every 7 days), every two weeks (e.g., every 14 days), every three weeks (e.g., every 21 days), every four weeks (e.g., every 28 days), every month, every two months, every three months, every four months, every five months, or every six months).
  • a regular interval e.g., every day, every two days, every three days, every four days, every five days, every six days, every week (e.g., every 7 days), every two weeks (e.g., every 14 days), every three weeks (e.g., every 21 days), every
  • each dosing cycle of gemtuzumab ozogamicin is administered in multiple doses of the effective amount every four weeks.
  • an effective amount of gemtuzumab ozogamicin is administered in a first dosing cycle, which may be followed by one or more subsequent dosing cycles of the effective amount, where each dosing cycle is separated by approximately four weeks (e.g., 28 days).
  • each dosing cycle is separated by about two weeks to about six weeks (e.g., about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about three weeks to about five weeks, or about four weeks to about six weeks).
  • an effective amount of gemtuzumab ozogamicin is administered to a subject in at least one dose, at least two doses, at least three doses, at least four doses, between one and six doses, between one and four doses, or between one and three doses.
  • the doses of gemtuzumab ozogamicin in a dosing cycle are administered to a subject daily for the duration of the dosing cycle.
  • the doses of gemtuzumab ozogamicin in a dosing cycle are administered to a subject weekly for the duration of the dosing cycle.
  • the effective amount of gemtuzumab ozogamicin is about 0.5 mg/m 2 .
  • the effective amount of gemtuzumab ozogamicin is administered in multiple doses of about 0.5 mg/m 2 every four weeks. In some embodiments, the effective amount of gemtuzumab ozogamicin is 0.5 mg/m 2 and is administered to the subject in a single dose per dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 0.5 mg/m 2 and is administered to the subject in a multiple, fractionated dose per dosing cycle, such that the total gemtuzumab ozogamicin administered to the subject per dosing cycle is 0.5 mg/m 2 .
  • the effective amount of gemtuzumab ozogamicin is about 1.0 mg/m 2 . In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in multiple doses of about 1.0 mg/m 2 every four weeks. In some embodiments, the effective amount of gemtuzumab ozogamicin is 1.0 mg/m 2 and is administered to the subject in a single dose per dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 1.0 mg/m 2 and is administered to the subject in a multiple, fractionated dose per dosing cycle, such that the total gemtuzumab ozogamicin administered to the subject per dosing cycle is 1.0 mg/m 2 .
  • the effective amount of gemtuzumab ozogamicin is about 2.0 mg/m 2 . In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in multiple doses of about 2.0 mg/m 2 every four weeks. In some embodiments, the effective amount of gemtuzumab ozogamicin is 2.0 mg/m 2 and is administered to the subject in a single dose per dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 2.0 mg/m 2 and is administered to the subject in a multiple, fractionated dose per dosing cycle, such that the total gemtuzumab ozogamicin administered to the subject per dosing cycle is 2.0 mg/m 2 .
  • the effective amount of gemtuzumab ozogamicin is about 3.0 mg/m 2 . In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in multiple doses of about 3.0 mg/m 2 every four weeks. In some embodiments, the effective amount of gemtuzumab ozogamicin is 3.0 mg/m 2 and is administered to the subject in a single dose per dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 3.0 mg/m 2 and is administered to the subject in a multiple, fractionated dose per dosing cycle, such that the total gemtuzumab ozogamicin administered to the subject per dosing cycle is 3.0 mg/m 2 .
  • the effective amount of gemtuzumab ozogamicin is about 4.0 mg/m 2 . In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in multiple doses of about 4.0 mg/m 2 every four weeks. In some embodiments, the effective amount of gemtuzumab ozogamicin is 4.0 mg/m 2 and is administered to the subject in a single dose per dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 4.0 mg/m 2 and is administered to the subject in a multiple, fractionated dose per dosing cycle, such that the total gemtuzumab ozogamicin administered to the subject per dosing cycle is 4.0 mg/m 2 .
  • the effective amount of gemtuzumab ozogamicin is about 5.0 mg/m 2 . In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in multiple doses of about 5.0 mg/m 2 every four weeks. In some embodiments, the effective amount of gemtuzumab ozogamicin is 5.0 mg/m 2 and is administered to the subject in a single dose per dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 5.0 mg/m 2 and is administered to the subject in a multiple, fractionated dose per dosing cycle, such that the total gemtuzumab ozogamicin administered to the subject per dosing cycle is 5.0 mg/m 2 .
  • the effective amount of gemtuzumab ozogamicin is about 6.0 mg/m 2 . In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in multiple doses of about 6.0 mg/m 2 every four weeks. In some embodiments, the effective amount of gemtuzumab ozogamicin is 6.0 mg/m 2 and is administered to the subject in a single dose per dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 6.0 mg/m 2 and is administered to the subject in a multiple, fractionated dose per dosing cycle, such that the total gemtuzumab ozogamicin administered to the subject per dosing cycle is 6.0 mg/m 2 .
  • the effective amount of gemtuzumab ozogamicin is about 9.0 mg/m 2 . In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in three doses of about 3.0 mg/m 2 . In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in two doses, e.g., one dose of about 6.0 mg/m 2 and one dose of about 3.0 mg/m 2 .
  • the effective amount of gemtuzumab ozogamicin is 9.0 mg/m 2 and is administered to the subject in a multiple, fractionated dose per dosing cycle, such that the total gemtuzumab ozogamicin administered to the subject per dosing cycle is 9.0 mg/m 2 .
  • a subject in need of treatment in accordance with the present disclosure has been identified as having newly-diagnosed, de novo CD33-positive AML.
  • the recommended treatment course including gemtuzumab ozogamicin consists of 1 induction cycle and 2 consolidation cycles.
  • the recommended dose of gemtuzumab ozogamicin is 3 mg/m 2 (up to one 4.5 mg vial) on days 1, 4, and 7 in combination with daunorubicin and cytarabine.
  • gemtuzumab ozogamicin is not administered during the second induction cycle.
  • the recommended dose of gemtuzumab ozogamicin is 3 mg/m 2 on day 1 (up to one 4.5 mg vial) in combination with daunorubicin and cytarabine.
  • the dosing regimen of gemtuzumab ozogamicin comprises 2, 3, or 4 dosing cycles, each comprising administration of an effective amount of gemtuzumab ozogamicin, wherein the effective amount is 0.1 mg/m 2 - 3.0 mg/m 2 body surface area of the subject.
  • the effective amount is administered in a single dose, e.g. t that is administered on day 1 of each dosing cycle.
  • the effective amount is administered in multiple doses, e.g. t that are administered on prescribed days of each dosing cycle.
  • the effective amount is administered in multiple doses, e.g. t at least 2, 3, 4, 5, or more doses, which may be referred to as fractionated dosing.
  • the effective amount of gemtuzumab ozogamicin is administered in two doses in a dosing cycle.
  • a first dose is administered to the subject on day 1 of the dosing cycle and a second dose is administered on any of days 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 of the dosing cycle.
  • the effective amount of gemtuzumab ozogamicin is administered in three doses in a dosing cycle.
  • the effective amount is administered in multiple doses, and a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered on any of days 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 of the dosing cycle, and a third dose is administered on any one of days 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28, such that administration of the third dose is after administration of the second dose.
  • the effective amount is administered in multiple doses, and a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered in day 4 of the dosing cycle, and a third dose is administered on day 7 of the dosing cycle. In some embodiments, the effective amount is administered in multiple doses, and a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered in day 7 of the dosing cycle, and a third dose is administered on day 14 of the dosing cycle.
  • the effective amount of gemtuzumab ozogamicin is administered in four doses in a dosing cycle.
  • the effective amount is administered in multiple doses, and a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered on any of days 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 of the dosing cycle, a third dose is administered on any one of days 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28, such that administration of the third dose is after administration of the second dose, and a fourth dose is administered on any one of days 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28, such that administration of the fourth dose is after administration of the third dose.
  • the effective amount is administered in multiple doses, and a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered in day 7 of the dosing cycle, a third dose is administered on day 14 of the dosing cycle, and a fourth dose is administered on day 21 of the dosing cycle.
  • each of the doses of the multiple doses of a dosing cycle may be of the same or different amounts of gemtuzumab ozogamicin.
  • the effective amount of gemtuzumab ozogamicin is 9 mg/m 2 and is administered in multiple doses of 3 mg/m 2 , wherein a first dose of 3 mg/m 2 is administered to the subject on day 1, a second dose of 3 mg/m 2 is administered to the subject day 4 of the dosing cycle, and a third dose of 3 mg/m 2 is administered to the subject day 7 of the dosing cycle.
  • the effective amount of gemtuzumab ozogamicin is 9 mg/m 2 and is administered in multiple doses, wherein a first dose of 6 mg/m 2 is administered to the subject and a second dose of 3 mg/m 2 is administered on a subsequent day of the dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 9 mg/m 2 and is administered in multiple doses, wherein a first dose of 6 mg/m 2 is administered to the subject on day 1 of the dosing cycle, a second dose of 3 mg/m 2 is administered on day 8 of the dosing cycle.
  • the effective amount is administered in multiple doses, and a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered to the subject once the plasma concentration of gemtuzumab ozogamicin in the subject is less than a threshold value, and a third dose is administered to the subject once the plasma concentration of gemtuzumab ozogamicin in the subject is less than a threshold value following administration of the second dose.
  • the methods further comprise monitoring the plasma concentration of gemtuzumab ozogamicin. In some embodiments, if the plasma concentration of gemtuzumab ozogamicin is below a threshold value, one or more additional doses of gemtuzumab ozogamicin are administered.
  • a threshold value of gemtuzumab ozogamicin in the plasma of a subject comprises an AUC of 60,000 nanograms (ng) of gemtuzumab ozogamicin x hours (h) since the last measurable time point that gemtuzumab ozogamicin was administered to the subject per mililiter (mL) of plasma sample (ng x h/mL) or less.
  • the subject is monitored following administration of one or more doses of gemtuzumab ozogamicin in a dosing cycle and may be administered one or more additional doses, based on factors such as the presence of minimal residual disease (MRD), pharmacokinetic measures, toxicity, and/or other functional tests.
  • MRD minimal residual disease
  • a subject is pretreated with one or more of a corticosteroid, antihistamine, and acetaminophen prior to administration of gemtuzumab ozogamicin.
  • the subject is pretreated approximately 1 hour (e.g., about 30 minutes to 1.5 hours, about 45 minutes to 1.5 hours, about 1 to 2 hours, or about 45 minutes to 1 hour) prior to administration of gemtuzumab ozogamicin.
  • a subject is pretreated with approximately 650 mg acetaminophen (e.g., orally) and approximately 50 mg diphenhydramine (e.g., orally or intravenously) 1 hour prior to administration of gemtuzumab ozogamicin.
  • a subject is pretreated with approximately 1 mg/kg methylprednisolone or an equivalent dose of an alternative corticosteroid within 30 minutes prior to administration of gemtuzumab ozogamicin.
  • Pediatric subjects may be pretreated with acetaminophen 15 mg/kg (maximum of 650 mg), diphenhydramine 1 mg/kg (maximum of 50 mg), and 1 mg/kg methylprednisolone orally or intravenously; additional doses of acetaminophen and diphenhydramine may be administered every 4 hours after the initial pretreatment dose.
  • Pretreatment may be repeated with the same dose of methylprednisolone or an equivalent corticosteroid for any sign of an infusion reaction, such as fever, chills, hypotension, or dyspnea during the infusion or within 4 hours afterwards.
  • the subject does not have a homozygous dominant genotype for CD33 single nucleotide polymorphism (SNP) rs 12459419.
  • the subject does not have acute promyelocytic leukemia or chronic myeloid leukemia.
  • the subject does not have a genetic translocation associated with acute promyelocytic leukemia or chronic myeloid leukemia, optionally wherein the genetic translocation is t(15; 17)(q22; q21) or t(9; 22)(q34; ql l).
  • the subject has not previously received autologous or allogeneic stem cell transplantation.
  • the subject has not previously received gemtuzumab ozogamicin.
  • the method further comprises determining a percent donor chimerism and/or a level of CD33-negative myeloid hematopoiesis in a peripheral blood sample from the subject.
  • gemtuzumab ozogamicin is reconstituted from a lyophilized form prior to administration.
  • the lyophilized form comprises approximately 4.5 mg of a lyophilized cake or powder.
  • the lyophilized form comprises a lyophilized cake or powder in a single-dose vial for reconstitution and/or dilution.
  • a subject has been preconditioned prior to administration of gemtuzumab ozogamicin and/or a population of genetically engineered hematopoietic stem cells.
  • preconditioning of a subject comprises administering one or more chemotherapeutic agents to the subject.
  • chemotherapeutic agents include, without limitation, busulfan, melphalan, fludarabine, cyclophosphamide, and thiotepa.
  • preconditioning comprises total body irradiation of a subject.
  • preconditioning comprises administering antibodies that bind human T cells (e.g., rabbit anti-thymocyte globulins (rATG), equine anti-thymocyte globulins (eATG)).
  • preconditioning occurs within two weeks (e.g., within 14 days, within 12 days, within 10 days, within 9 days, within 7 days) prior to administration of gemtuzumab ozogamicin and/or hematopoietic cells.
  • preconditioning occurs over a period of about one day to about ten days. In some embodiments, preconditioning occurs over a period of about nine days.
  • cells or tissues thereof having higher a higher density of CD33 antigens relative to cells with reduced or eliminated expression of wild-type CD33 are capable of binding to higher amounts of gemtuzumab ozogamicin and, therefore, may exhibit higher levels of gemtuzumab ozogamicin-dependent cytotoxic effects.
  • the CD33 antigen density on hematopoietic cells in a subject from subject who has received hematopoietic cell transplant with a population of genetically engineered hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen, would be expected to be lower than the CD33 antigen density on hematopoietic cells that express wild-type CD33 and/or have not been genetically engineered.
  • the effective amount of gemtuzumab ozogamicin administered to the subject is 0.1 mg/m 2 - 6.0 mg/m 2 body surface area of the subject.
  • a method comprises administering an effective amount of gemtuzumab ozogamicin administering and an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen.
  • the effective amount of the population of genetically modified hematopoietic cells, or descendants thereof is 5 x 10 7 cells/kilogram body weight of the subject.
  • Administration of gemtuzumab ozogamicin and/or the population of genetically modified hematopoietic cells, or descendants thereof can be performed using any of the methods provided by the disclosure.
  • Non-limiting examples of biological samples include whole blood samples, plasma samples, blood samples processed to enrich for white blood cells, bone marrow samples, or peripheral blood samples.
  • the first biological sample can be obtained from the subject at a first time point and the second biological sample can be obtained from the subject at a second time point occurring after the first time point.
  • the second biological sample is obtained from a counterpart subject (e.g., a sample obtained directly from the second subject or obtained from a banked sample collected from the second subject).
  • Methods for detecting CD33 antigen density can involve, without limitation, quantitative protein expression analyses (e.g., flow cytometry, enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence assays, such as meso-scale detection (MSD) assays), western blot), quantitative RNA analyses (e.g., real-time quantitative polymerase chain reaction (RT-qPCR)), or any combination thereof.
  • detecting CD33 antigen density comprises flow cytometry.
  • flow cytometry is performed using an antibody or other labeling agent comprising an antigen-binding fragment that binds to the same regions of CD33 that is targeted by gemtuzumab ozogamicin.
  • the antibody is hP67.6 or the labeling agent comprising antigen-binding fragment thereof.
  • a first biological sample obtained from a subject is identified as having hematopoietic cells comprising a lower density of CD33 (e.g., wild-type CD33) relative to a second biological sample.
  • the lower density of CD33 (e.g., wild-type CD33) in the first biological sample relative to the second biological sample is characterized by an expression level of CD33 (e.g., wild-type CD33) in the second biological sample which is 95% or less than an expression level of CD33 (e.g., wild-type CD33) in the first biological sample.
  • the lower density of CD33 in the first biological sample relative to the second biological sample is characterized by an expression level of CD33 in the first biological sample that is 90-95%, 85-90%, 80-85%, 75- 80%, 70-75%, 60-70%, 50-60%, 40-50%, 30-40%, 20-30%, or less than 20% than the expression level of CD33 in the second biological sample.
  • the lower density of CD33 (e.g., wild-type CD33) in the first biological sample relative to the second biological sample is characterized by an expression level of CD33 in the first biological sample that is 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or less than 10% than the expression level of CD33 in the second biological sample.
  • a lower density of CD33 (e.g., wild-type CD33) is measured in a first biological sample which was obtained from a subject experiencing remission from a hematopoietic malignancy and a second biological sample is obtained from the subject after the subject has experienced or is suspected of experiencing a relapse of the hematopoietic malignancy.
  • the effective amount of gemtuzumab ozogamicin administered to the subject is 2.0 mg/m 2 or less (e.g., 0.1-0.25 mg/m 2 , 0.25-0.5 mg/m 2 , 0.5-1.0 mg/m 2 , or 1.0-2.0 mg/m 2 ) when the expression level of CD33 in the first biological sample is 75% or less than the expression level of CD33 in the second biological sample.
  • the effective amount of gemtuzumab ozogamicin administered to the subject is about 0.1 mg/m 2 , 0.25 mg/m 2 , about 0.5 mg/m 2 , about 1.0 mg/m 2 , or about 2.0 mg/m 2 when the expression level of CD33 in the first biological sample is 50% or less than the expression level of CD33 in the second biological sample.
  • the effective amount of gemtuzumab ozogamicin administered to the subject is more than 2.0 mg/m 2 (e.g., 2.0- 3.0 mg/m 2 , 3.0-4.0 mg/m 2 , 4.0-5.0 mg/m 2 , or 5.0-6.0 mg/m 2 ) when the expression level of CD33 in the first biological sample is more than 75% of the expression level of CD33 in the second biological sample.
  • a composition of the disclosure may be administered via a route such as, but not limited to, enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritone
  • administration of the population of genetically engineered hematopoietic cells and the gemtuzumab ozogamicin may be performed by the same administration route (e.g., intravenous infusion) or by different administration routes.
  • Modes of administration include injection, infusion, instillation, and/or ingestion.
  • injection includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracap sular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion.
  • the route is intravenous.
  • administration by injection or infusion can be made.
  • a population of genetically engineered hematopoietic stem cells can be administered systemically.
  • systemic administration refers to the administration of a population of progenitor cells other than directly into a target site, tissue, or organ, such that it enters, instead, the subject's circulatory system and, thus, is subject to metabolism and other like processes.
  • a treatment having a composition for the treatment of a hematopoietic malignancy can be determined by the skilled clinician. However, a treatment is considered “effective treatment,” if any one or all of the signs or symptoms of the hematopoietic malignancy are altered in a beneficial manner, or other clinically accepted symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein.
  • Treatment includes any treatment of a disease in an individual or an animal (some nonlimiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.
  • compositions comprising populations of cells comprising any of the genetically modified hematopoietic cells, or descendants thereof, described herein.
  • the composition comprises a population of at least 1 x 10 6 cells per milliliter (mL) in a medium, wherein the population of cells comprise genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen.
  • the population comprises at least 2 x 10 6 cells per mL, at least 3 x 10 6 cells per mL, at least 4 x 10 6 cells per mL, at least 5 x 10 6 cells per mL, at least 6 x 10 6 cells per mL, at least at least 7 x 10 6 cells per mL, at least 8 x 10 6 cells per mL, or at least 9 x 10 6 cells per mL.
  • the medium has a volume between about 5-150 mL. In some embodiments, the medium has a volume between about 10-100 mL. In some embodiments, the medium has a volume between about 25-75 mL. In some embodiments, the medium has a volume between about 30-70 mL. In some embodiments, the medium has a volume between about 40-60 mL. In some embodiments, the medium has a volume of about 45 mL. In some embodiments, the medium has a volume of about 30 mL. In some embodiments, the medium has a volume of about 35 mL. In some embodiments, the medium has a volume of about 40 mL. In some embodiments, the medium has a volume of about 50 mL.
  • the medium has a volume of about 55 mL. In some embodiments, the medium has a volume of about 60 mL. In some embodiments, the medium has a volume of about 70 mL. In some embodiments, the medium has a volume between about 40-50 mL. In some embodiments, the medium has a volume of about 40 mL, 41 mL, 42 mL, 43 mL, 44 mL, 45 mL, 46 mL, 47 mL, 48 mL, 49 mL, or about 50 mL. In some embodiments, the medium has a volume of about 45 mL.
  • the composition comprises a population of between about 1 x 10 6 - 1 x 10 8 cells total in the medium. In some embodiments, the composition comprises a population of about 1 x 10 7 , 2 x 10 7 , 3 x 10 7 , 4 x 10 7 , 5 x 10 7 , 6 x 10 7 , 7 x 10 7 , 8 x 10 7 , 9 x 10 7 , or 1 x 10 8 cells total in the medium.
  • the population comprises at least 0.5 x 10 6 cells per mL, at least 1 x 10 6 cells per mL, at least 2 x 10 6 cells per mL, at least 3 x 10 6 cells per mL, at least 4 x 10 6 cells per mL, at least 5 x 10 6 cells per mL, at least 6 x 10 6 cells per mL, at least at least 7 x 10 6 cells per mL, at least 8 x 10 6 cells per mL, or at least 9 x 10 6 cells per mL.
  • the population comprises at least 0.5 x 10 6 cells per mL, at least 1 x 10 6 cells per mL, at least 2 x 10 6 cells per mL, at least 3 x 10 6 cells per mL, at least 4 x 10 6 cells per mL, at least 5 x 10 6 cells per mL, at least 6 x 10 6 cells per mL, at least at least 7 x 10 6 cells per mL, at least 8 x 10 6 cells per mL, or at least 9 x 10 6 cells per mL.
  • the cell population comprises at least 1 x 10 9 viable cells, at least 2 x 10 9 viable cells, at least 3 x 10 9 viable cells, at least 4 x 10 9 viable cells, at least 5 x 10 9 viable cells, at least 6 x 10 9 viable cells, at least 7 x 10 9 viable cells, at least 8 x 10 9 viable cells, at least 9 x 10 9 viable cells, at least 1 x IO 10 viable cells, at least 2 x IO 10 viable cells, at least 3 x IO 10 viable cells, at least 4 x IO 10 viable cells, at least 5 x IO 10 viable cells, at least 6 x IO 10 viable cells, at least 7 x IO 10 viable cells, at least 8 x IO 10 viable cells, at least 9 x IO 10 viable cells, or at least 1 x 10 11 viable cells, wherein, in some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least
  • At least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells of population are genetically modified hematopoietic cells, or descendants thereof, having reduced or eliminated expression of a CD33 antigen.
  • the medium is a cryopreservation medium comprising a cryoprotectant.
  • cryoprotects include cetamide, agarose, alginate, 1-analine, albumin, ammonium acetate, butanediol, chondroitin sulfate, chloroform, choline, diethylene glycol, dimethyl acetamide, dimethyl formamide, dimethylsulfoxide (DMSO), erythritol, ethanol, ethylene glycol, formamide, glucose, glycerol, a-glycerol phosphate, glycerol monoacetate, glycine, hydroxyethyl starch, inositol, lactose, magnesium chloride, magnesium sulfate, maltose, mannitol, mannose, methanol, methyl acetamide, methylformamide, methyl urea, phenol, pluronic polyol, polyethylene glycol, polyvinylpyrrol
  • the composition is in a frozen state.
  • the composition has been subjected to a cryopreservation process.
  • cryopreservation processes are methods aimed, for example, to preserve (viable) cells by cooling and storing a sample comprising the cells at a low temperature (e.g., at or below -80 °C).
  • the cryopreservation process is controlled-rate freezing.
  • aspects of the present disclosure relate to populations of genetically engineered hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen.
  • the gene encoding CD33 may be engineered by any means known in the art such that the cell has reduced or eliminated expression of a CD33 antigen.
  • the complex may comprise two strands forming a duplex structure, or three or more strands forming a multi-stranded complex.
  • the binding may constitute a step in a more extensive process, such as the cleavage of the target domain by a Cas endonuclease.
  • the gRNA binds to the target domain with perfect complementarity, and in other embodiments, the gRNA binds to the target domain with partial complementarity, e.g., with one or more mismatches.
  • the full targeting domain of the gRNA base pairs with the targeting domain. In other embodiments, only a portion of the target domain and/or only a portion of the targeting domain base pairs with the other. In an embodiment, the interaction is sufficient to mediate a target domain-mediated cleavage event.
  • Cas9 molecule refers to a molecule or polypeptide that can interact with a gRNA and, in concert with the gRNA, home or localize to a site which comprises a target domain.
  • Cas9 molecules include naturally occurring Cas9 molecules and engineered, altered, or modified Cas9 molecules that differ, e.g., by at least one amino acid residue, from a naturally occurring Cas9 molecule.
  • gRNA and “guide RNA” are used interchangeably throughout and refer to a nucleic acid that promotes the specific targeting or homing of a gRNA/Cas9 molecule complex to a target nucleic acid.
  • a gRNA can be unimolecular (having a single RNA molecule), sometimes referred to herein as sgRNAs, or modular (comprising more than one, and typically two, separate RNA molecules).
  • a gRNA may bind to a target domain in the genome of a host cell.
  • the gRNA e.g., the targeting domain thereof
  • the gRNA may be partially or completely complementary to the target domain.
  • the gRNA may also comprise a “scaffold sequence,” (e.g., a tracrRNA sequence), that recruits a Cas9 molecule to a target domain bound to a gRNA sequence (e.g., by the targeting domain of the gRNA sequence).
  • the scaffold sequence may comprise at least one stem loop structure and recruits an endonuclease.
  • Exemplary scaffold sequences can be found, for example, in Jinek, et al. Science (2012) 337(6096):816-821, Ran, et al. Nature Protocols (2013) 8:2281-2308, PCT Publication No. WO2014/093694, and PCT Publication No. WO2013/176772.
  • mutation is used herein to refer to a genetic change (e.g., insertion, deletion, or substitution) in a nucleic acid compared to a reference sequence, e.g., the corresponding wild-type nucleic acid.
  • a mutation to a gene detargetizes the protein produced by the gene.
  • the term “detargetizes” refers to mutating a gene such that the protein produced by the gene is no longer recognized (“targeted”), or is recognized to a lesser extent, by an agent that binds to the corresponding wildtype protein that has not been mutated.
  • the gene is mutated such that is produces a protein that lacks an epitope that is bound/recognized by an antigen that targets the protein.
  • a detargetized CD33 protein is not bound by, or is bound at a lower level by, an agent that targets CD33.
  • the “targeting domain” of the gRNA is complementary to the “target domain” on the target nucleic acid.
  • the strand of the target nucleic acid comprising the nucleotide sequence complementary to the core domain of the gRNA is referred to herein as the “complementary strand” of the target nucleic acid.
  • Guidance on the selection of targeting domains can be found, e.g., in Fu Y et al, Nat Biotechnol (2014) 32: 279-284 (doi: 10.1038/nbt.2808) and Sternberg SH et al., Nature (2014) 507(7490): 62-7 (doi: 10.1038/naturel3011).
  • a cell e.g., HSC or HPC
  • a nuclease described herein is made using a nuclease described herein.
  • Exemplary nucleases include Cas molecules (e.g., Cas9 or Casl2a), TALENs, ZFNs, and meganucleases.
  • a nuclease is used in combination with a CD33 gRNA described herein (e.g. according to Table 3).
  • a CD33 gRNA described herein is complexed with a Cas9 molecule.
  • Various Cas9 molecules can be used.
  • a Cas9 molecule is selected that has the desired PAM specificity to target the gRNA/Cas9 molecule complex to the target domain in CD33.
  • genetically engineering a cell also comprises introducing one or more (e.g., 1, 2, 3 or more) Cas9 molecules into the cell.
  • Cas9 molecules of a variety of species can be used in the methods and compositions described herein.
  • the Cas9 molecule is of, or derived from, .S'. pyogenes (SpCas9), .S', aureus (SaCas9) or .S', thermophilus.
  • Cas9 molecules include those of, or derived from, Staphylococcus aureus, Neisseria meningitidis (NmCas9), Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., Cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Brady rhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni (CjCas9), Campylobacter lari, Candidatus punic eispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium acco
  • the Cas9 molecule is a naturally occurring Cas9 molecule.
  • the Cas9 molecule is an engineered, altered, or modified Cas9 molecule that differs, e.g., by at least one amino acid residue, from a reference sequence, e.g., the most similar naturally occurring Cas9 molecule or a sequence of Table 50 of PCT Publication No. W02015/157070, which is herein incorporated by reference in its entirety.
  • a naturally occurring Cas9 molecule typically comprises two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each of which further comprises domains described, e.g., in PCT Publication No. WO 2015/157070, e.g., in Figs. 9A-9B therein, which is incorporated herein by reference in its entirety.
  • REC recognition
  • NUC nuclease
  • the REC lobe comprises the arginine-rich bridge helix (BH), the RECI domain, and the REC2 domain.
  • the REC lobe appears to be a Cas9-specific functional domain.
  • the BH domain is a long alpha helix and arginine rich region and comprises amino acids 60-93 of the sequence of .S'. pyogenes Cas9.
  • the RECI domain is involved in recognition of the repeat: anti-repeat duplex, e.g., of a gRNA or a tracrRNA.
  • the RECI domain comprises two RECI motifs at amino acids 94 to 179 and 308 to 717 of the sequence of .S', pyogenes Cas9.
  • the REC2 domain comprises amino acids 180-307 of the sequence of .S', pyogenes Cas9.
  • the NUC lobe comprises the RuvC domain (also referred to herein as RuvC-like domain), the HNH domain (also referred to herein as HNH-like domain), and the PAM- interacting (PI) domain.
  • RuvC domain shares structural similarity to retroviral integrase superfamily members and cleaves a single strand, e.g., the non-complementary strand of the target nucleic acid molecule.
  • the RuvC domain is assembled from the three split RuvC motifs (RuvC I, RuvCII, and RuvCIII, which are often commonly referred to in the art as RuvCI domain, or N-terminal RuvC domain, RuvCII domain, and RuvCIII domain) at amino acids 1-59, 718-769, and 909-1098, respectively, of the sequence of .S'. pyogenes Cas9. Similar to the RECI domain, the three RuvC motifs are linearly separated by other domains in the primary structure, however in the tertiary structure, the three RuvC motifs assemble and form the RuvC domain.
  • the HNH domain shares structural similarity with HNH endonucleases, and cleaves a single strand, e.g., the complementary strand of the target nucleic acid molecule.
  • the HNH domain lies between the RuvC II-III motifs and comprises amino acids 775-908 of the sequence of .S', pyogenes Cas9.
  • the PI domain interacts with the PAM of the target nucleic acid molecule and comprises amino acids 1099-1368 of the sequence of .S'. pyogenes Cas9.
  • Crystal structures have been determined for naturally occurring bacterial Cas9 molecules (Jinek et al., Science (2014) 343(6176): 1247997) and for .S'. pyogenes Cas9 with a guide RNA (e.g., a synthetic fusion of crRNA and tracrRNA) (Nishimasu et al., Cell (2014) 156:935-949; and Anders et al., Nature (2014) doi: 10.1038/naturel3579).
  • a guide RNA e.g., a synthetic fusion of crRNA and tracrRNA
  • a Cas9 molecule described herein has nuclease activity, e.g., double strand break activity.
  • the Cas9 molecule has been modified to inactivate one of the catalytic residues of the endonuclease.
  • the Cas9 molecule is a nickase and produces a single stranded break. See, e.g., Dabrowska et al. Frontiers in Neuroscience (2016) 12(75). It has been shown that one or more mutations in the RuvC and HNH catalytic domains of the enzyme may improve Cas9 efficiency. See, e.g., Sarai et al. Currently Pharma. Biotechnol. (2017) 18(13).
  • the Cas9 molecule is fused to a second domain, e.g., a domain that modifies DNA or chromatin, e.g., a deaminase or demethylase domain.
  • the Cas9 molecule is modified to eliminate its endonuclease activity.
  • a Cas9 molecule described herein is administered together with a template for homology directed repair (HDR). In some embodiments, a Cas9 molecule described herein is administered without an HDR template.
  • HDR homology directed repair
  • the Cas9 molecule is modified to enhance specificity of the enzyme (e.g., reduce off-target effects, maintain robust on-target cleavage).
  • the Cas9 molecule is an enhanced specificity Cas9 variant (e.g., eSPCas9). See, e.g., Slaymaker et al. Science (2016) 351 (6268): 84-88.
  • the Cas9 molecule is a high fidelity Cas9 variant (e.g., SpCas9-HFl). See, e.g., Kleinstiver et al. Nature (2016) 529: 490-495.
  • Cas9 molecules are known in the art and may be obtained from various sources and/or engineered/modified to modulate one or more activities or specificities of the enzymes.
  • the Cas9 molecule has been engineered/modified to recognize one or more PAM sequence.
  • the Cas9 molecule has been engineered/modified to recognize one or more PAM sequence that is different than the PAM sequence the Cas9 molecule recognizes without engineering/modification.
  • the Cas9 molecule has been engineered/modified to reduce off-target activity of the enzyme.
  • the nucleotide sequence encoding the Cas9 molecule is modified further to alter the specificity of the endonuclease activity (e.g., reduce off-target cleavage, decrease the endonuclease activity or lifetime in cells, increase homology-directed recombination and reduce non-homologous end joining). See, e.g., Komor et al. Cell (2017) 168: 20-36.
  • the nucleotide sequence encoding the Cas9 molecule is modified to alter the PAM recognition of the endonuclease.
  • the Cas9 molecule SpCas9 recognizes PAM sequence NGG
  • relaxed variants of the SpCas9 comprising one or more modifications of the endonuclease e.g., VQR SpCas9, EQR SpCas9, VRER SpCas9
  • PAM recognition of a modified Cas9 molecule is considered “relaxed” if the Cas9 molecule recognizes more potential PAM sequences as compared to the Cas9 molecule that has not been modified.
  • the Cas9 molecule SaCas9 recognizes PAM sequence NNGRRT, whereas a relaxed variant of the SaCas9 comprising one or more modifications (e.g., KKH SaCas9) may recognize the PAM sequence NNNRRT.
  • the Cas9 molecule FnCas9 recognizes PAM sequence NNG, whereas a relaxed variant of the FnCas9 comprising one or more modifications of the endonuclease (e.g., RHA FnCas9) may recognize the PAM sequence YG.
  • the Cas9 molecule is a Cpfl endonuclease comprising substitution mutations S542R and K607R and recognize the PAM sequence TYCV.
  • the Cas9 molecule is a Cpfl endonuclease comprising substitution mutations S542R, K607R, and N552R and recognize the PAM sequence TATV. See, e.g., Gao et al. Nat. Biotechnol. (2017) 35(8): 789-792.
  • more than one (e.g., 2, 3, or more) Cas molecules are used.
  • at least one of the Cas9 molecule is a Cas9 enzyme.
  • at least one of the Cas molecules is a Cpfl enzyme.
  • at least one of the Cas9 molecules is derived from Streptococcus pyogenes.
  • at least one of the Cas9 molecules is derived from Streptococcus pyogenes and at least one Cas9 molecules is derived from an organism that is not Streptococcus pyogenes.
  • the Cas9 molecule is a base editor.
  • Base editor endonuclease generally comprises a catalytically inactive Cas9 molecule fused to a function domain. See, e.g., Eid et al. Biochem. J. (2016) 475(11): 1955-1964; Rees et al. Nature Reviews Genetics (2016) 19:770-788.
  • the catalytically inactive Cas9 molecule is dCas9.
  • the catalytically inactive Cas9 molecule (dCas9) is fused to one or more uracil glycosylase inhibitor (UGI) domains.
  • UBI uracil glycosylase inhibitor
  • the endonuclease comprises a dCas9 fused to an adenine base editor (ABE), for example an ABE evolved from the RNA adenine deaminase TadA.
  • ABE adenine base editor
  • the endonuclease comprises a dCas9 fused to cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDAl, activation- induced cytidine deaminase (AID)).
  • the catalytically inactive Cas9 molecule has reduced activity and is nCas9.
  • the Cas9 molecule comprises a nCas9 fused to one or more uracil glycosylase inhibitor (UGI) domains.
  • the Cas9 molecule comprises a nCas9 fused to an adenine base editor (ABE), for example an ABE evolved from the RNA adenine deaminase TadA.
  • the Cas9 molecule comprises a nCas9 fused to cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDAl, activation-induced cytidine deaminase (AID)).
  • base editors include, without limitation, BE1, BE2, BE3, HF-BE3, BE4, BE4max, BE4-Gam, YE1-BE3, EE-BE3, YE2-BE3, YEE-CE3, VQR-BE3, VRER-BE3, SaBE3, SaBE4, SaBE4-Gam, Sa(KKH)-BE3, Target-AID, Target-AID-NG, xBE3, eA3A- BE3, BE-PLUS, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR- ABE, VRER-ABE, Sa(KKH)-ABE, and CRISPR-SKIP.
  • the base editor has been further modified to inhibit base excision repair at the target site and induce cellular mismatch repair.
  • Any of the Cas9 molecules described herein may be fused to a Gam domain (bacteriophage Mu protein) to protect the Cas9 molecule from degradation and exonuclease activity. See, e.g., Eid et al. Biochem. J. (2016) 475(11): 1955-1964.
  • the Cas9 molecule belongs to class 2 type V of Cas endonuclease.
  • Class 2 type V Cas endonucleases can be further categorized as type V-A, type V-B, type V-C, and type V-U. See, e.g., Stella et al. Nature Structural & Molecular Biology (2017).
  • the Cas molecule is a type V-A Cas endonuclease, such as a Cpfl nuclease.
  • the Cas9 molecule is a type V-B Cas endonuclease, such as a C2cl endonuclease.
  • the Cas molecule is Mad7TM (from Inscripta).
  • the Cas9 molecule is a Cpfl nuclease or a variant thereof.
  • the Cpfl nuclease may also be referred to as Cas 12a. See, e.g., Strohkendl et al. Mol. Cell (2016) 71: 1-9.
  • a composition or method described herein involves, or a host cell expresses, a Cpfl nuclease derived from Provetella spp.
  • the nucleotide sequence encoding the Cpfl nuclease may be codon optimized for expression in a host cell. In some embodiments, the nucleotide sequence encoding the Cpfl endonuclease is further modified to alter the activity of the protein. In some embodiments, catalytically inactive variants of Cas molecules (e.g., of Cas9 or Cas 12a) are used according to the methods described herein.
  • a catalytically inactive variant of Cpfl may be referred to dCasl2a.
  • catalytically inactive variants of Cpfl maybe fused to a function domain to form a base editor. See, e.g., Rees et al. Nature Reviews Genetics (2016) 19:770-788.
  • the catalytically inactive Cas9 molecule is dCas9.
  • the endonuclease comprises a dCasl2a fused to one or more uracil glycosylase inhibitor (UGI) domains.
  • UFI uracil glycosylase inhibitor
  • the Cas9 molecule comprises a dCasl2a fused to an adenine base editor (ABE), for example an ABE evolved from the RNA adenine deaminase TadA.
  • ABE adenine base editor
  • the Cas molecule comprises a dCasl2a fused to cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDAl, activation-induced cytidine deaminase (AID)).
  • the Cas9 molecule may be a Cas 14 endonuclease or variant thereof.
  • Cas 14 endonucleases are derived from archaea and tend to be smaller in size (e.g., 400-700 amino acids). Additionally, Casl4 endonucleases do not require a PAM sequence. See, e.g., Harrington et al. Science (2016) 362 (6416).
  • any of the Cas9 molecules described herein may be modulated to regulate levels of expression and/or activity of the Cas9 molecule at a desired time.
  • it may be advantageous to increase levels of expression and/or activity of the Cas9 molecule during particular phase(s) of the cell cycle.
  • levels of homology- directed repair are reduced during the Gi phase of the cell cycle, therefore increasing levels of expression and/or activity of the Cas9 molecule during the S phase, G2 phase, and/or M phase may increase homology-directed repair following the Cas endonuclease editing.
  • levels of expression and/or activity of the Cas9 molecule are increased during the S phase, G2 phase, and/or M phase of the cell cycle.
  • the Cas9 molecule fused to the N-terminal region of human Geminin. See, e.g., Gutschner et al. Cell Rep. (2016) 14(6): 1555-1566.
  • levels of expression and/or activity of the Cas9 molecule are reduced during the Gi phase.
  • the Cas9 molecule is modified such that it has reduced activity during the Gi phase. See, e.g., Lomova et al. Stem Cells (2016) 37(2): 284-294.
  • any of the Cas9 molecules described herein may be fused to an epigenetic modifier (e.g., a chromatin-modifying enzyme, e.g., DNA methylase, histone deacetylase).
  • an epigenetic modifier e.g., a chromatin-modifying enzyme, e.g., DNA methylase, histone deacetylase.
  • Cas9 molecule fused to an epigenetic modifier may be referred to as “epieffectors” and may allow for temporal and/or transient endonuclease activity.
  • the Cas9 molecule is a dCas9 fused to a chromatin-modifying enzyme.
  • a cell or cell population described herein is produced using zinc finger (ZFN) technology.
  • the ZFN recognizes a target domain described herein, e.g., in Table 1.
  • zinc finger mediated genomic editing involves use of a zinc finger nuclease, which typically comprises a zinc finger DNA binding domain and a nuclease domain.
  • the zinc finger binding domain may be engineered to recognize and bind to any target domain of interest, e.g., may be designed to recognize a DNA sequence ranging from about 3 nucleotides to about 21 nucleotides in length, or from about 8 to about 19 nucleotides in length.
  • Zinc finger binding domains typically comprise at least three zinc finger recognition regions e.g., zinc fingers).
  • Restriction endonucleases capable of sequence- specific binding to DNA (at a recognition site) and cleaving DNA at or near the site of binding are known in the art and may be used to form ZFN for use in genomic editing.
  • Type IIS restriction endonucleases cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains.
  • the DNA cleavage domain may be derived from the FokI endonuclease.
  • a cell or cell population described herein is produced using TALEN technology.
  • the TALEN recognizes a target domain described herein, e.g., in Table 1.
  • TALENs are engineered restriction enzymes that can specifically bind and cleave a desired target DNA molecule.
  • a TALEN typically contains a Transcriptional Activator-Like Effector (TALE) DNA-binding domain fused to a DNA cleavage domain.
  • TALE Transcriptional Activator-Like Effector
  • the DNA binding domain may contain a highly conserved 33-34 amino acid sequence with a divergent 2 amino acid RVD (repeat variable dipeptide motif) at positions 12 and 13.
  • the RVD motif determines binding specificity to a nucleic acid sequence and can be engineered to specifically bind a desired DNA sequence.
  • the DNA cleavage domain may be derived from the FokI endonuclease.
  • the FokI domain functions as a dimer, using two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing.
  • a TALEN specific to a target gene of interest can be used inside a cell to produce a double-stranded break (DSB).
  • a mutation can be introduced at the break site if the repair mechanisms improperly repair the break via non-homologous end joining. For example, improper repair may introduce a frame shift mutation.
  • a foreign DNA molecule having a desired sequence can be introduced into the cell along with the TALEN.
  • this process can be used to correct a defect or introduce a DNA fragment into a target gene of interest, or introduce such a defect into the endogenous gene, thus decreasing expression of the target gene.
  • a gRNA can comprise a number of domains.
  • a unimolecular, sgRNA, or chimeric, gRNA comprises, e.g., from 5' to 3': a targeting domain (which is complementary to a target nucleic acid in the CD33 gene; a first complementarity domain; a linking domain; a second complementarity domain (which is complementary to the first complementarity domain); a proximal domain; and optionally, a tail domain.
  • the targeting domain may comprise a nucleotide sequence that is complementary, e.g., at least 80, 85, 90, or 95% complementary, e.g., fully complementary, to the target sequence on the target nucleic acid.
  • the targeting domain is part of an RNA molecule and will therefore comprise the base uracil (U), while any DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, in an embodiment, it is believed that the complementarity of the targeting domain with the target sequence contributes to specificity of the interaction of the gRNA/Cas9 molecule complex with a target nucleic acid.
  • the uracil bases in the targeting domain will pair with the adenine bases in the target sequence.
  • the target domain itself comprises in the 5' to 3' direction, an optional secondary domain, and a core domain.
  • the core domain is fully complementary with the target sequence.
  • the targeting domain is 5 to 50 nucleotides in length.
  • the targeting domain may be between 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length.
  • the targeting domain is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
  • the targeting domain is between 10-30, or between 15-25, nucleotides in length.
  • a targeting domain comprises a core domain and a secondary targeting domain, e.g., as described in PCT Publication No. WO 2015/157070, which is incorporated by reference in its entirety.
  • the core domain comprises about 8 to about 13 nucleotides from the 3' end of the targeting domain (e.g., the most 3' 8 to 13 nucleotides of the targeting domain).
  • the secondary domain is positioned 5' to the core domain.
  • the core domain has exact complementarity with the corresponding region of the target sequence.
  • the core domain can have 1 or more nucleotides that are not complementary with the corresponding nucleotide of the target sequence.
  • the first complementarity domain is complementary with the second complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions.
  • the first complementarity domain is 5 to 30 nucleotides in length.
  • the first complementarity domain comprises 3 subdomains, which, in the 5' to 3' direction are: a 5' subdomain, a central subdomain, and a 3' subdomain.
  • the 5' subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length.
  • the central subdomain is 1, 2, or 3, e.g., 1, nucleotide in length.
  • the 3' subdomain is 3 to 25, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
  • the first complementarity domain can share homology with, or be derived from, a naturally occurring first complementarity domain. In an embodiment, it has at least 50% homology with a .S'. pyogenes, S. aureus or .S', thermophilus, first complementarity domain.
  • a linking domain serves to link the first complementarity domain with the second complementarity domain of a unimolecular gRNA.
  • the linking domain can link the first and second complementarity domains covalently or non-covalently.
  • the linkage is covalent.
  • the linking domain is, or comprises, a covalent bond interposed between the first complementarity domain and the second complementarity domain.
  • the linking domain comprises one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides.
  • the linking domain comprises at least one non-nucleotide bond, e.g., as disclosed in PCT Publication No. WO 2018/126176, the entire contents of which are incorporated herein by reference.
  • the second complementarity domain is complementary, at least in part, with the first complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions.
  • the second complementarity domain can include a sequence that lacks complementarity with the first complementarity domain, e.g., a sequence that loops out from the duplexed region.
  • the second complementarity domain is 5 to 27 nucleotides in length. In some embodiments, the second complementarity domain is longer than the first complementarity region.
  • the complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.
  • the second complementarity domain comprises 3 subdomains, which, in the 5' to 3' direction are: a 5' subdomain, a central subdomain, and a 3' subdomain.
  • the 5' subdomain is 3 to 25, e.g., 4 to
  • the central subdomain is 1, 2, 3, 4, or 5 nucleotides in length.
  • the 3' subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9, nucleotides in length.
  • the 5' subdomain and the 3' subdomain of the first complementarity domain are respectively, complementary, e.g., fully complementary, with the 3' subdomain and the 5' subdomain of the second complementarity domain.
  • the proximal domain is 5 to 20 nucleotides in length. In some embodiments, the proximal domain can share homology with or be derived from a naturally occurring proximal domain. In some embodiments, it has at least 50% homology with a proximal domain from 5. pyogenes, S. aureus, or 5. thermophilus.
  • tail domains are suitable for use in gRNAs.
  • the tail domain is 0 (absent), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length.
  • the tail domain nucleotides are from or share homology with a sequence from the 5' end of a naturally occurring tail domain.
  • the tail domain includes sequences that are complementary to each other and which, under at least some physiological conditions, form a duplexed region.
  • the tail domain is absent or is 1 to 50 nucleotides in length.
  • the tail domain can share homology with or be derived from a naturally occurring proximal tail domain.
  • the tail domain includes nucleotides at the 3' end that are related to the method of in vitro or in vivo transcription.
  • a modular gRNA comprises: a first strand comprising, e.g., from 5' to 3': a targeting domain (which is complementary to a target nucleic acid in the CD33 gene), and a first complementarity domain; and a second strand, comprising, preferably from 5' to 3': optionally, a 5' extension domain, a second complementarity domain, a proximal domain, and optionally, a tail domain.
  • the gRNA is chemically modified.
  • the gRNA may comprise one or more modification chosen from phosphorothioate backbone modification, 2'-O-Me-modified sugars (e.g., at one or both of the 3’ and 5’ termini), 2’F- modified sugar, replacement of the ribose sugar with the bicyclic nucleotide-cEt, 3 'thioPACE (MSP), or any combination thereof.
  • MSP 3 'thioPACE
  • Suitable gRNA modifications are described, e.g., in Rahdar et al. PNAS (2015) 112 (51) E7110-E7117 and Hendel et al., Nat Biotechnol.
  • a gRNA described herein comprises one or more 2'-O-methyl-3'- phosphorothioate nucleotides, e.g., at least 2, 3, 4, 5, or 6 2'-O-methyl-3 '-phosphorothioate nucleotides.
  • a gRNA described herein comprises modified nucleotides (e.g., 2'-O-methyl-3'-phosphorothioate nucleotides) at the three terminal positions and the 5’ end and/or at the three terminal positions and the 3’ end.
  • the gRNA may comprise one or more modified nucleotides, e.g., as described in PCT Publication Nos. WO/2017/214460, WO/2017/089433, and WO/2017/164356, which are incorporated by reference their entirety.
  • a gRNA described herein is chemically modified.
  • the gRNA may comprise one or more 2’-0 modified nucleotide, e.g., 2’-O-methyl nucleotide.
  • the gRNA comprises a 2’-0 modified nucleotide, e.g., 2’- O-methyl nucleotide at the 5’ end of the gRNA.
  • the gRNA comprises a 2’-0 modified nucleotide, e.g., 2’-O-methyl nucleotide at the 3’ end of the gRNA.
  • the gRNA comprises a 2’-O-modified nucleotide, e.g., 2’-O-methyl nucleotide at both the 5’ and 3’ ends of the gRNA.
  • the gRNA is 2’-O-modified, e.g. 2’-O-methyl-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, and the third nucleotide from the 5’ end of the gRNA.
  • the gRNA is 2’-O-modified, e.g.
  • the gRNA is 2’-O-modified, e.g.
  • the gRNA is 2’-O-modified, e.g.
  • the nucleotide at the 3’ end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3’ end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2’-O- modified, e.g.
  • the 2’-O-methyl nucleotide comprises a phosphate linkage to an adjacent nucleotide.
  • the 2’-O-methyl nucleotide comprises a phosphorothioate linkage to an adjacent nucleotide.
  • the 2’-O-methyl nucleotide comprises a thioPACE linkage to an adjacent nucleotide.
  • the gRNA may comprise one or more 2’-O-modified and 3’phosphorous-modified nucleotide, e.g., a 2’-O-methyl 3’phosphorothioate nucleotide.
  • the gRNA comprises a 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’phosphorothioate nucleotide at the 5’ end of the gRNA.
  • the gRNA comprises a 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O- methyl 3’phosphorothioate nucleotide at the 3’ end of the gRNA.
  • the gRNA comprises a 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’phosphorothioate nucleotide at the 5’ and 3’ ends of the gRNA.
  • the gRNA comprises a backbone in which one or more non-bridging oxygen atoms has been replaced with a sulfur atom.
  • the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g.
  • the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g. 2’-O-methyl 3’phosphorothioate-modified at the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA.
  • the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g. 2’-O-methyl 3’phosphorothioate-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA.
  • the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g.
  • the nucleotide at the 3’ end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3’ end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g.
  • the gRNA may comprise one or more 2’-O-modified and 3’- phosphorous-modified, e.g., 2’-O-methyl 3’thioPACE nucleotide.
  • the gRNA comprises a 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’thioPACE nucleotide at the 5’ end of the gRNA.
  • the gRNA comprises a 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’thioPACE nucleotide at the 3’ end of the gRNA.
  • the gRNA comprises a 2’-O- modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’thioPACE nucleotide at the 5’ and 3’ ends of the gRNA.
  • the gRNA comprises a backbone in which one or more non-bridging oxygen atoms have been replaced with a sulfur atom and one or more non-bridging oxygen atoms have been replaced with an acetate group.
  • the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g.
  • the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g. 2’-O-methyl 3 ’thioPACE-modified at the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA.
  • the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g. 2’-O-methyl 3 ’thioPACE-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA.
  • the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g.
  • the nucleotide at the 3’ end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3’ end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g.
  • the gRNA comprises a chemically modified backbone. In some embodiments, the gRNA comprises a phosphorothioate linkage. In some embodiments, one or more non-bridging oxygen atoms have been replaced with a sulfur atom. In some embodiments, the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, and the third nucleotide from the 5’ end of the gRNA each comprise a phosphorothioate linkage.
  • the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA each comprise a phosphorothioate linkage.
  • the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA each comprise a phosphorothioate linkage.
  • the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and at the fourth nucleotide from the 3’ end of the gRNA each comprise a phosphorothioate linkage.
  • the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA each comprise a phosphorothioate linkage.
  • the gRNA comprises a thioPACE linkage.
  • the gRNA comprises a backbone in which one or more non-bridging oxygen atoms have been replaced with a sulfur atom and one or more non-bridging oxygen atoms have been replaced with an acetate group.
  • the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, and the third nucleotide from the 5’ end of the gRNA each comprise a thioPACE linkage.
  • the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA each comprise a thioPACE linkage.
  • the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA each comprise a thioPACE linkage.
  • the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and at the fourth nucleotide from the 3’ end of the gRNA each comprise a thioPACE linkage.
  • the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA each comprise a thioPACE linkage.
  • the present disclosure provides a number of useful gRNAs that can target an endonuclease to human CD33.
  • the gRNA used in the methods described herein target a sequence in exon 3 of CD33.
  • Table 1 below illustrates target domains in human endogenous CD33 that can be bound by gRNAs described herein.
  • Table 1 Exemplary Cas9 target site sequences of human CD33 are provided, as are exemplary targeting domain sequences useful for targeting such sites.
  • the first sequence represents the DNA target domain sequence
  • the second sequence represents the reverse complement thereof
  • the third sequence represents an exemplary targeting domain sequence of a gRNA that can be used to target the respective target site.
  • the CD33 (CCDS33084.1) cDNA sequence is provided below as SEQ ID NO: 16.
  • Exon 3 of CD33 is provided separately below as SEQ ID NO: 17. Underlining indicates the regions complementary to gRNA A, gRNA B, gRNA C, gRNA D (or the reverse complement thereof). Note that the target regions for gRNA A, gRNA B, and gRNA D partially overlap.
  • a gRNA described herein can be used in combination with a second gRNA, e.g., for directing nucleases to two sites in a genome.
  • a second gRNA e.g., for directing nucleases to two sites in a genome.
  • kits described herein e.g., a kit comprising one or more gRNAs according to Table 1 also comprises a Cas9 molecule, or a nucleic acid encoding the Cas9 molecule.
  • the first and second gRNAs are gRNAs according to Table 1 or variants thereof.
  • the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence of Table 1 or a variant thereof) and the second gRNA targets a gene encoding a lineage- specific cell-surface antigen chosen from: CD5, CD6, CD7, BCMA, CD19, CD20, CD30, R0R1, B7H6, B7H3, CD23, CD38, C-type lectin like molecule-1, CS1, IL-5, Ll-CAM, PSCA, PSMA, CD138, CD133, CD70, CD7, CD13, NKG2D, NKG2D ligand, CLEC12A, CD11, CD123, CD56, CD34, CD14, CD66b, CD41, CD61, CD62, CD235a, CD 146, CD326, LMP2, CD22, CD52, CD 10, CD3/TCR, CD79/BCR, and CD26.
  • a lineage- specific cell-surface antigen chosen from: CD5, CD6, CD7
  • the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding a lineage- specific cell-surface antigen associated with a specific type of cancer, such as, without limitation, CD20, CD22 (Non-Hodgkin's lymphoma, B-cell lymphoma, chronic lymphocytic leukemia (CLL)), CD52 (B-cell CLL), CD33 (acute myeloid leukemia (AML)), CD 10 (gplOO) (Common (pre-B) acute lymphocytic leukemia and malignant melanoma), CD3/T-cell receptor (TCR) (T-cell lymphoma and leukemia), CD79/B-cell receptor (BCR) (B-cell lymphoma and leukemia), CD26 (epithelial and lymphoid malignancies), human leukocyte antigen associated with
  • the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding a lineage- specific cell-surface antigen chosen from: CD5, CD6, CD7, CD13, CD19, CD22, CD20, CD25, CD30, CD32, CD38, CD44, CD45, CD47, CD56, 96, CD117, CD123, CD135, CD174, CLL-1, BCMA, folate receptor 0, IL1RAP, MUC1, NKG2D/NKG2DL, TIM-3, or WT1.
  • a lineage- specific cell-surface antigen chosen from: CD5, CD6, CD7, CD13, CD19, CD22, CD20, CD25, CD30, CD32, CD38, CD44, CD45, CD47, CD56, 96, CD117, CD123, CD135, CD174, CLL-1, BCMA, folate receptor 0, IL1R
  • the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding a lineage- specific cell-surface antigen chosen from: CD la, CD lb, CDlc, CD Id, CDle, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CDl la, CDl lb, CDl lc, CDl ld, CDwl2, CD13, CD14, CD15, CD16, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32a, CD32b, CD32c, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41,
  • the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence of according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding a lineage- specific cell-surface antigen chosen from: CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLECL1); epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (CD2); ganglioside GD3 (aNeu5Ac(2- 8)aNeu5Ac(2-3)bDGalp(l-4)bDGlep(l-l)Cer); TNF receptor family member B cell maturation (BCMA), Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)); prostate-specific membrane antigen (PSMA), Tn anti
  • the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding a lineage- specific cell-surface antigen chosen from: CDl la, CD18, CD19, CD20, CD31, CD34, CD44, CD45, CD47, CD51, CD58, CD59, CD63, CD97, CD99, CD100, CD102, CD123, CD127, CD133, CD135, CD157, CD172b, CD217, CD300a, CD305, CD317, CD321, and CLL1.
  • a lineage- specific cell-surface antigen chosen from: CDl la, CD18, CD19, CD20, CD31, CD34, CD44, CD45, CD47, CD51, CD58, CD59, CD63, CD97, CD99, CD100, CD102, CD123, CD127, CD133, CD135, CD157, CD172b, CD217, CD
  • the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding a lineage- specific cell-surface antigen chosen from: CD 123, CLL1, CD38, CD135 (FLT3), CD56 (NCAM1), CD117 (c-KIT), FRp (FOLR2), CD47, CD82, TNFRSF1B (CD120B), CD191, CD96, PTPRJ (CD148), CD70, LILRB2 (CD85D), CD25 (IL2Ralpha), CD44, CD96, NKG2D Ligand, CD45, CD7, CD15, CD19, CD20, CD22, CD37, CD82, CD312 (EMR2).
  • a lineage- specific cell-surface antigen chosen from: CD 123, CLL1, CD38, CD135 (FLT3), CD56 (NCAM1), CD117 (c-KIT
  • the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding a lineage- specific cell-surface antigen chosen from: CD7, CDl la, CD15, CD18, CD19, CD20, CD22, CD25, CD31, CD34, CD37, CD38, CD44, CD45, CD47, CD51, CD56, CD58, CD59, CD63, CD70, CD82, CD85D, CD96, CD97, CD99, CD100, CD102, CD117, CD120B, CD123, CD127, CD133, CD135, CD148, CD157, CD172b, CD191, CD217, CD300a, CD305, CD317, CD321, CLL1, FRp (FOLR2), or NKG2D Ligand.
  • a lineage-specific cell-surface antigen chosen from: CD7, CDl la, CD15, CD18, CD
  • the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding CLL-1.
  • the first gRNA is a CD33 gRNA described herein (e.g., a gRNA according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding CD 123.
  • Example 1 Generation of genetically engineered hematopoietic cells comprising a modified gene encoding CD33
  • the Cas9 sgRNAs indicated in Table 1 were designed based on the SpCas9 PAM (5'- NGG-3') with close proximity to the target region and evaluated for predicted specificity by minimizing potential off-target sites in the human genome with an online search algorithm (e.g., the Benchling algorithm, Doench et al 2016, Hsu et al 2013).
  • Cas9 sgRNAs are synthesized using the gRNA targeting domains provided below and the Cas9 sgRNA scaffold sequence 5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
  • nucleotide sequence of sgRNA A is
  • nucleotide sequence of sgRNA B is
  • nucleotide sequence of sgRNA C is
  • nucleotide sequence of sgRNA D is
  • nucleotide sequence of sgRNA E is
  • All designed synthetic sgRNAs are produced with chemically modified nucleotides at the three terminal positions at both the 5' and 3' ends.
  • the modified nucleotides contained 2'- O-methyl-3'-phosphorothioate (abbreviated as “ms”) and the ms-sgRNAs are HPLC -purified.
  • Cas9 protein is purchased from Synthego.
  • nucleotide sequence of sgRNA A is 5'-CmsCmsCmsCAGGACGACGCACGCCGGUUUUAGAGCUAGAAAUAGCAAGUUAA AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmsU msUmsU-3' (SEQ ID NO: 24, targeting domain sequence in bold).
  • nucleotide sequence of sgRNA E showing the modified nucleotides
  • Peripheral blood mononuclear cells are collected from healthy donor subject by apheresis following hematopoietic stem cell mobilization.
  • the donor CD34+ cells are electroporated with Cas9 protein and any of the indicated CD33 -targeting Cas9 gRNAs disclosed herein, e.g., having the targeting domain sequences provided in Tables 1 and 3, e.g., gRNA A, gRNA, B, gRNA C, gRNA D, or gRNA E.
  • the edited cells are cultured for less than 48 hours. Upon harvest, the cells are washed, resuspended in the final formulation, and cryopreserved.
  • a representative sample of the edited HSCs is evaluated for viability and expression of CD33, or absence thereof, by staining for CD33 using an anti-CD33 antibody e.g., P67.7) and analyzed by flow cytometry.
  • Edited CD33KO eHSPC populations exhibiting at least 70% cell viability and at least 45% CD33 editing efficiency (i.e., absence of CD33 expression in at least 45% of the cells in the cell population) at 48 hours after electroporation are used for HCT.
  • Example 2 Treatment of a subject having AML with CD33KO eHSPC generated using gRNA A, and the CD33-targeted ADC gemtuzumab ozogamicin/Mylotarg®
  • a subject having CD33-positive AML is treated with an allogeneic HCT comprising CD33KO eHSPCs and with the ADC gemtuzumab ozogamicin/Mylotarg®.
  • a population of cells comprising CD34+ hematopoietic stem cells is obtained from a healthy donor who is HLA matched at 8/8 loci (HLA-A, -B, -C, DRB1) to the subject.
  • apheresis procedures are performed in order to obtain a target of 10 x 10 6 viable cells/kg (where kg refers to recipient subject weight) from the donor for processing and subsequent administration to the recipient subject.
  • kg refers to recipient subject weight
  • From this apheresis product at least 3.0 x 10 6 viable cells/kg (recipient weight) undergo minimal manipulation and are cryopreserved to serve as a back-up stem cell source, e.g., for use as a rescue dose.
  • the remainder of the apheresis product is used for processing and preparation of the CD33KO eHSPC population for HCT.
  • the CD33KO eHSPC population for HCT is prepared by enriching the apheresis product for CD34+ cells, followed by electroporation and editing with a CD33 gRNA/Cas9 complex using any of the CD33 gRNAs as described in Example 1.
  • the edited cells are subsequently placed in culture for ⁇ 48 hours. Upon harvest, after the culture duration is finished, cells are washed, resuspended in the final formulation, and cryopreserved. Cell viability and editing efficiency are confirmed using a representative sample as described in Example 1, and CD33KO eHSPC populations meeting the criteria set forth in Example 1 (at least 70% viability and at least 45% CD33 editing efficiency) are used for HCT.
  • a population for administration to a subject comprises a CD33KO eHSPC population satisfying these viability and editing efficiency criteria of at least 3 x 10 6 cells/kg body weight of the recipient subject, and preferably comprises at least 4 x 10 6 cells/kg, 5 x 10 6 cells/kg, 6 x 10 6 cells/kg, or 7 x 10 6 cells/kg of the recipient subject.
  • the subject After completion of the conditioning regimen, the subject receives an HCT comprising the thawed CD33KO eHSPCs via an intravenous (IV) infusion.
  • the day of the HCT is day 0 of the treatment regimen.
  • the subject is assessed for CD33KO eHSPC engraftment at day 28 by measuring the absolute peripheral neutrophil count (ANC) for CD33KO (CD33-) neutrophils in the subject.
  • the subject is deemed to exhibit neutrophil recovery (also referred to as successful CD33KO neutrophil engraftment) if the subject exhibits an absolute peripheral CD33KO neutrophil count of >500 cells/pL CD33- ANC at 28 days after CD33KO eHSPC HCT.
  • a bone marrow biopsy is obtained from the subject on day 60 in order to assess disease status and hematopoietic recovery.
  • percent donor chimerism and CD33- negative (CD33-) myeloid hematopoiesis are determined from the peripheral blood at this time. If the subject exhibits successful CD33- HSC engraftment and CD33- hematopoiesis at day 60, the subject is subsequently administered gemtuzumab ozogamicin/My lotarg®.
  • the CD33- ANC is monitored in the subject prior to administration of gemtuzumab ozogamicin/My lotarg®, and the subject should preferably have >1000/dL CD33- ANC prior to receiving gemtuzumab ozogamicin/My lotarg®.
  • gemtuzumab ozogamicin/Mylotarg® is preferably initiated within 30 days of the bone marrow biopsy at day 60, i.e., is preferably initiated by day 90.
  • initiation of gemtuzumab ozogamicin/Mylotarg® may be delayed up to day 120 or later if a subject’s clinical status, e.g., in view of comorbidities, including, for example, HCT- related comorbidities, necessitate such a delay, or in order to allow attainment of >1000/dL CD33- ANC in a subject.
  • gemtuzumab ozogamicin/Mylotarg® is initiated more than 30 days after the Day 60 bone marrow biopsy, a repeat bone marrow biopsy is completed prior to starting gemtuzumab ozogamicin/Mylotarg®.
  • Gemtuzumab ozogamicin/Mylotarg® is administered to the subject at a dose within the range of 0.1mg/m 2 to 6 mg/m 2 , e.g., at a dose of 0.1mg/m 2 , 0.25 mg/m 2 , 0.5 mg/m 2 , lmg/m 2 , 2 mg/m 2 , 3 mg/m 2 , 4 mg/m 2 , 5 mg/m 2 , or 6 mg/m 2 .
  • Gemtuzumab ozogamicin/Mylotarg® is administered to the subject in a regimen of 4- week (28d) treatment cycles, wherein the subject receives the entire amount of a respective treatment cycle, e.g., at 0.5mg/m 2 of gemtuzumab ozogamicin/Mylotarg®, on day 1 of the respective 4-week (28d) treatment cycle.
  • the subject may receive the amount of gemtuzumab ozogamicin/Mylotarg® fractionated between multiple doses administered on separate prescribed days, e.g., on days 1, 4, and 7 of the respective 4-week (28d) dosing cycle, or one days 1, 8, and 16 of the 4-week dosing cycle.
  • the subject may receive the amount of gemtuzumab ozogamicin/Mylotarg® fractionated between multiple doses each administered once the plasma concentration of gemtuzumab ozogamicin falls below a threshold level.
  • the subject At completion of the last gemtuzumab ozogamicin/Mylotarg® treatment cycle, the subject is monitored for disease status and hematopoietic chimerism and is monitored for these parameters at six months, 1 year, 2 years, and then annually for up to 15 years after completion of the final treatment cycle.
  • Example 3 Clinical scale manufacturing of human CD33KO eHSPC
  • HSPCs allogeneic CRISPR/Cas9 genome edited hematopoietic stem/progenitor cells lacking the CD33 protein were manufactured for the treatment of human leukocyte antigen (HLA)-matched patients with high-risk CD33+ acute myeloid leukemia (AML).
  • HLA human leukocyte antigen
  • AML acute myeloid leukemia
  • the resulting HSPC populations are suitable for infusion into HLA- matched human patients with AML undergoing hematopoietic cell transplant, e.g., patients who are known to be at high-risk for leukemia relapse and mortality post-transplantation.
  • the final HSPC populations were formulated at a volume of 45mL in cryopreservation media ready for cryopreservation, storage in the vapor phase of liquid nitrogen, subsequent thawing and administration via intravenous (IV) infusion to a recipient patient.
  • IV intravenous
  • Each batch was manufactured from leukapheresis starting material obtained from a single donor to generate a one-donor-to-one -recipient HLA matched product, allowing for the manufacture of the product for a specifically matched patient.
  • a healthy donor was subjected to a leukapheresis procedure.
  • Leukapheresis starting material was collected and stored at 2-8 °C before initiation of cell manipulation.
  • Cell number and viability of the leukapheresis starting material was tested by flow cytometry. Cell viability was confirmed to be >80%. A sample was removed for cell analysis and other assessments.
  • a leukapheresis rescue dose was removed from the leukapheresis material to obtain a volume comprising 3xl0 6 CD34+ cells/kg of patient weight.
  • the rescue dose material was processed and cryopreserved and then stored in the vapor phase of liquid nitrogen at ⁇ -140 °C.
  • the leukapheresis starting material was processed to remove red blood cells, platelets, and plasma.
  • the processed material was then enriched for CD34-positive cells and then transferred into 250 mL conical tubes.
  • a Cas9/gRNA ribonucleoprotein (RNP) complex was prepared prior to electroporation by mixing Cas9 protein and gRNA E under sterile conditions. Cells in the 250 mL conical tube were spun down at 200 xg, resuspended in electroporation buffer, mixed with the prepared RNP complex, and electroporated in a single-use sterile electroporation cassette.
  • RNP gRNA ribonucleoprotein
  • the cells were removed from the cassette, transferred to culture media, and incubated in suspension culture at 37 °C and 5% CO2. Cell cultures were monitored for cell count and viability. Once cells recovered from electroporation (determined by cell viability being > 80%), the cells were washed to reduce cellular debris and other residuals, and resuspended in serum-free, animal component-free, and defined cryopreservation medium containing 10% DMSO. The cells were formulated at a volume of 45 mL and cryopreserved in a controlled rate freezer (CRF). Samples were taken to determine cell counts, viability, percentage of cells expressing particular markers (e.g., CD34, CD3, CD19, CD14, CD56). editing efficiency, and residual Cas9 as shown in Table 4 below. Table 4. Analysis of exemplary cell preparations
  • Example 4 CD33-Deleted Hematopoietic Stem and Progenitor Cells Display Engraftment after Hematopoietic Cell Transplant (HCT) and Tolerate Post-HCT Gemtuzumab Ozogamicin (GO) Treatment Without Cytopenias.
  • This example describes an evaluation of the safety of infusion of engineered CD33- deficient hematopoietic cells (referred to as trem-cel; formerly known as VOR33) and gemtuzumab ozogamicin (GO; tradename My lotarg®) in human acute myeloid leukemia (AML) patients who are at a high risk of relapse post-hematopoietic cell transplant (HCT).
  • trem-cel engineered CD33- deficient hematopoietic cells
  • GO gemtuzumab ozogamicin
  • VBP101 open-label first-in-human trial referred to as VBP101 evaluates CD33-positive AML patients who are at high risk of relapse undergo myeloablative HCT with trem-cel followed by treatment with low-dose gemtuzumab ozogamicin.
  • Part 1 of the study will enroll 9-18 patients in 3 cohorts and who were treated with escalating doses of gemtuzumab ozogamicin (0.5-2.0 mg/m 2 ) in 28-day dosing cycles for up to 4 cycles.
  • the safety of trem-cel will be assessed and the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of gemtuzumab ozogamicin will be determined.
  • MTD maximum tolerated dose
  • RP2D recommended phase 2 dose
  • an additional 12 patients will be enrolled to further evaluate the safety of trem- cel and the preliminary efficacy of trem-cel and gemtuzumab ozogamicin at RP2D.
  • eligible subjects are between 18-70 years of age with CD33+ AML and be candidates for myeloablative conditioning (MAC) HCT.
  • Subjects are also considered at a high risk of relapse but have had previously been subjected to autologous or allogeneic HCT and have not previously been treated with GO (GO-naive).
  • Subjects are subjected to a 9 day conditioning regimen consisting of busulfan/melphalan/fludarabine/rATG or total body irradiation/cyclophosphamide/ thiotepa/rATG; Following infusion with an CD33-deleted allograft (e.g., trem-cel), cells are allowed to recover and engraft for 60 days and then are subjected to anti-CD33 maintenance therapy involving a dosing regimen of gemtuzumab ozogamicin comprising up to 4 dosing cycles (each 28 days) of gemtuzumab ozogamicin in escalating doses (an effective amount of 0.5-2.0 mg/m 2 ).
  • a 9 day conditioning regimen consisting of busulfan/melphalan/fludarabine/rATG or total body irradiation/cyclophosphamide/ thiotepa/rATG
  • an CD33-deleted allograft e.
  • the primary endpoint of the study is successful neutrophil engraftment at 28 days post-HCT.
  • Key secondary endpoints include, platelet engraftment, persistence of CD33- negative engraftment, primary and secondary graft failure, graft- versus-host disease, transplant-related mortality, and GO pharmacokinetic assessment.
  • the first patient was a 64-year-old female patient (weight 69.9 kg) diagnosed with AML including the following high-risk features: highly complex (adverse) cytogenetics, myelodysplastic syndrome (MDS)-related changes (MRC), and TP53 mutation.
  • the patient required 2 courses of cytarabine/daunorubicin to achieve complete response (CR).
  • CR complete response
  • HiDAC high dose cytarabine
  • Donor cells were mobilized using granulocyte colony stimulating factor (G-CSF) and plerixafor.
  • G-CSF granulocyte colony stimulating factor
  • a CD33 CRISPR/Cas9 gene-edited donor allograft was developed to enable post-HCT CD33- directed therapies while protecting healthy donor cells from on-target myelosuppression.
  • Hematopoietic cells deficient in CD33 e.g., gemtuzumab ozogamicin were manufactured with 97% CD34+ cells and 88% gene editing efficacy.
  • the patient received myeloablative conditioning (MAC) with busulfan/melphalan/fludarabine/rabbit anti-thymocyte globulin (ATG) prior to trem-cel infusion of 7.6 x 10 6 cells/kg.
  • MAC myeloablative conditioning
  • the second patient was a 32-year-old male (120.7 kg) diagnosed with AML after myeloid sarcoma partially resected from the small bowel and omentum.
  • Initial cytogenetics were Inv 16 and +22.
  • Subsequent adverse risk t(3;3) was identified.
  • Initial treatment consisted of 7+3 cytarabine/daunorubicin with complete response (CR)
  • Minimal residual disease was less than 0.1%.
  • the patient then received 3 cycles of HiDAC, remaining in CR with persistence of abdominal disease by positron emission tomography (PET).
  • PET positron emission tomography
  • a 10/10 HLA matched unrelated donor (MUD) was identified and consented.
  • Donor cells were mobilized with G-CSF and plerixafor.
  • Trem-cel was manufactured with 87% CD33 gene editing efficacy; the patient’s trem-cel dose was 3.2 x 10 6 CD34+ cells/kg.
  • the patient received MAC with busulfan/melphalan/fludarabine/rabbit ATG prior to trem-cel infusion.
  • the third patient (Patient 3) was a 55-year-old female (114.1 kg) with AML with myelodysplastic syndrome (MDS)-related changes (MRC). Cytogenetics were normal. DNMT3A, IDH2, and SMC1A mutations were detected. Minimal residual disease was less than 0.1%.
  • a 10/10 HLA matched unrelated donor (MUD) was identified and consented.
  • Donor cells were mobilized using granulocyte colony stimulating factor (G-CSF) and plerixafor.
  • Trem-cel was manufactured with 80% CD33 gene editing efficacy; the patient’s trem-cel dose was 2.6 x 10 6 CD34+ cells/kg.
  • the patient received MAC with busulfan/melphalan/fludarabine/equine ATG prior to trem-cel infusion.
  • the fourth patient was a 68-year-old male (72.4 kg) with AML with myelodysplastic syndrome (MDS)-related changes (MRC).
  • Patient 4 exhibited complex cytogenetics and active disease. NRAS, ZRSR2, and TET2 mutations were detected. Minimal residual disease was 16%.
  • a 10/10 HLA matched unrelated donor (MUD) was identified and consented.
  • Donor cells were mobilized using granulocyte colony stimulating factor (G-CSF) and plerixafor.
  • Trem-cel was manufactured with 89% CD33 gene editing efficacy; the patient’s trem-cel dose was 5.8 x 10 6 CD34+ cells/kg.
  • the patient received MAC with busulfan/melphalan/fludarabine/rabbit ATG prior to trem-cel infusion.
  • the fifth patient was a 66-year-old male (102.1 kg) with secondary AML.
  • the patient exhibited normal cytogenetics. KIT D816V, CBL, SRSF2, RUNX1/2, and BCORL1 mutations were detected. Minimal residual disease was 0.1% MRD.
  • a 10/10 HLA matched unrelated donor (MUD) was identified and consented.
  • Donor cells were mobilized using granulocyte colony stimulating factor (G-CSF) and plerixafor.
  • Trem-cel was manufactured with 85% CD33 gene editing efficacy; the patient’s trem-cel dose was 4.6 x 10 6 CD34+ cells/kg.
  • the patient received MAC with busulfan/melphalan/fludarabine/rabbit ATG prior to trem-cel infusion.
  • neutrophil engraftment was observed between days 10- 11 post HCT (z.e., at 10 days post HCT in Patients 1 and 3-5 and at day 11 in Patient 2). Platelet recovery was observed at 22 days post HCT in Patient 1, 15 days post HCT in Patient 4, and 17 days post HCT in Patient 2 (FIGs. 2A-4D).
  • Donor chimerism analysis in Patient 1 indicated whole blood and myeloid cells were 100% of donor origin, which was maintained through day 100 (D100) of the assessment. Donor chimerism of 97% was observed in T cells at the D100 assessment. Indel analysis of whole blood showed 95.2%, 95.9%, and 99.4% gene editing efficiency at D28, D60, and D100, respectively, in bulk cells (FIG. 5A).
  • CD33-deficient cells administered to Patient 1 e.g., trem-cel
  • CD33 editing was largely consistent across bulk cells and cells of myeloid and lymphoid lineages (FIG. 5A).
  • CD33 editing (-3%) in bulk cells, monocytes, and B cells at D100 reflects the elimination of CD33+ cells and enrichment of CD33-negative cells after 1 dosing cycle of 0.5 mg/m 2 and 1.0 mg/m 2 of gemtuzumab ozogamicin. Enrichment of B cells (95.6% at D60 to 98.5% at D100) was unexpected as they are not high- CD33 expressing cells.
  • CD33 editing correlates well with CD33 expression in monocytes (FIG. 5A).
  • donor chimerism analysis of Patients 3-4 indicated whole blood, monocytes, NK cells, and B cells were 100% of donor origin which was maintained, in Patient 3, through day 60 (D+60). Similar analyses in Patient 2 indicated whole blood cells were 94% of donor origin at D+28, and myeloid cells were 99-100% of donor origin. Donor chimerism of 4%, 99%, and 52% were observed in the T cell population at D+28 in Patients 2-4, respectively. Indel analysis of whole blood showed -86-96% gene editing efficiency at D+28 post HCT in Patients 2-4. By D+60 post HCT, 87.9% gene editing efficiency was detected in Patient 3 (FIG. 5B).
  • PB samples from Patient 1 approximately 94% of monocytes and approximately 95% of myeloid cells were found to be CD33-negative (FIG. 7A).
  • PB samples from Patient 2 approximately 93% of monocytes and 99% of myeloid cells were found to be CD33-negative.
  • PB samples from Patient 3 approximately 82% of monocytes and approximately 86% of myeloid cells were found to be CD33-negative.
  • PB samples of Patient 4 approximately 90% of monocytes and approximately 95% of myeloid cells were found to be CD33-negative.
  • BM from Patient 1 approximately 95% of maturing myeloid, approximately 92% of maturing monocyte, and approximately 94% of CD34+ myeloblasts were found to be CD33- negative with development patterns comparable to non-edited cells at day 28 post-HCT.
  • BM from Patient 2 approximately 98% of maturing myeloid cells and approximately 91% of monocytes that were found to be CD33-negative.
  • BM of Patient 3 approximately 86% of myeloid cells and approximately 80% of monocytes were found to be CD33-negative.
  • BM of Patient 4 approximately 94% of maturing myeloid cells and 90% of monocytes were found to be CD33-negative (FIGs. 6A-6B and 7A-7B).
  • Patient 1 received gemtuzumab ozogamicin at 0.5 mg/m 2 at 68 days post-HCT during the first dosing cycle of the anti-CD33 maintenance therapy period.
  • Plasma samples were collected pre-GO dosing and post-GO dosing on day 1 of the first gemtuzumab ozogamicin dosing cycle (C1D1) (1, 2, 3, 4, and 6 hours) and on day 8 of the first gemtuzumab ozogamicin dosing cycle (C1D8) and analyzed for hP67.6 (the anti-CD33 portion of gemtuzumab ozogamicin) by ELISA.
  • AEs Serious adverse events after trem-cel dosing in Patient 1 included Grade 3 renal colic attributable to nephrolithiasis and Grade 3 deep venous thrombosis.
  • Infectious AEs included Grade 1 and Grade 2 skin infection, Grade 2 CMV reactivation, and Grade 2 BK virus (urine) which resolved or, in other instances, showed signs of resolving.
  • Hepatic AEs included Grade 1 and Grade 2 aspartate aminotransferase (AST)/alanine transaminase (ALT) elevations and both were attributed to anti-fungal therapy and ultimately resolved. No trem- cel- or GO-related AEs were reported for up to 3 cycles. Prior to HCT, the patient had 1.8% MRD in the BM and none was observed post-HCT at day 28 and day 60. MRD in the BM was detected at D101 (0.3%) and D129 (2.2%) (FIG. 10B).
  • Patient 3 had neutrophil engraftment at D+10 did not have platelet recovery as of D+l 17.
  • the D+60 BM biopsy was hypocellular and showed decreased megakaryocytes.
  • a positive platelet reactive antibody was identified on D+53 and characterized as an anti-HLA Class I Ab.
  • D+100 BM biopsy was normocellular with normal megakaryocytes. As of D+l 17 the platelet count was 15,000/pL without transfusion in prior 11 days.
  • trem-cel was found to be well-tolerated. No related AEs were reported in Patients 4 and 5, and no unexpected AEs were reported. All 5 patients transplanted with trem-cel demonstrated primary neutrophil engraftment (days 10-11), which was similar to patients who received non-edited CD34 selected grafts. All patients achieved high levels of myeloid donor chimerism by day +28. A high level of CD33-negative hematopoiesis was achieved (>80%) in the 4 patients evaluable to day +28.
  • gemtuzumab ozogamicin demonstrated a Cmax and AUC comparable to doses of 1-2 mg/m 2 and 4-5 mg/m 2 , respectively, in an AML patient.
  • gemtuzumab ozogamicin dose 0.5 mg/m 2
  • neutrophil and platelet counts remained stable through multiple cycles of GO, including in the presence of MRD. This suggests protection from gemtuzumab ozogamicin hemato toxicity.
  • CD33 antigen density refers to the number of CD33 molecules present on a cell. This Example specifically relates to the density of CD33 antigens, e.g., CD33 antigens capable of binding gemtuzumab ozogamicin, as well as diagnostic and therapeutic methods related to the same.
  • CD33 antigen density can be used to refer to the number of CD33 molecules expressed at the cell surface of an individual cell (e.g., a hematopoietic cell). However, CD33 antigen density can also be used to refer to the number of cells in a tissue, such as a peripheral blood or bone marrow, expressing CD33 antigens capable of binding to gemtuzumab ozogamicin.
  • CD33 antigen density may be used to inform gemtuzumab ozogamicin dosing regimens in subjects (e.g., human subjects).
  • Methods for determining CD33 antigen density will involve quantitative analyses of CD33 protein levels at the cell surface. For example, cells (e.g., hematopoietic cells expressing wild-type CD33) are contacted with an anti-CD33 antibody, such as hP67.6 (the antibody component of gemtuzumab ozogamicin), which is fluorescently labeled or contacted with an anti-CD33 antibody which is not fluorescently labeled prior to being contacted with a fluorescently labeled secondary antibody. Flow cytometry analyses are performed to measure the amount of fluorescent signal corresponding to antibody-bound cells.
  • an anti-CD33 antibody such as hP67.6 (the antibody component of gemtuzumab ozogamicin)
  • Control samples are used to normalize fluorescent signals.
  • Genetically engineered cells comprising reduced or eliminated expression of CD33 can be further assessed by DNA sequencing analyses to correlate DNA editing efficiency to the fluorescent signals corresponding to antibody -bound cells measured by flow cytometry.
  • Estimations of CD33 antigen density can be further compared with pharmacokinetic and/or pharmacodynamic data via methods described herein to determine correlations between CD33 antigen density and response to gemtuzumab ozogamicin treatment.
  • the overall antigen density of CD33 may be influential in the expected PK and PD of a given dose of gemtuzumab ozogamicin, consistent with the principles of target mediated drug disposition.
  • the large reservoir of CD33 expression can be assumed from both malignant and normal myeloid cells in relapsed and refractor AML patients, requiring a sufficient dose of gemtuzumab ozogamicin (z.e., > 2 mg/m2) for efficacy.
  • Total CD33+ receptor expression per cell, as well as the total CD33+ cell counts can both drive the relative antigen density within a given population.
  • the dose of gemtuzumab ozogamicin required to reach an effective outcome may be lower than that used in R/R AML patients with higher disease burden and a CD33+ normal myeloid compartment.
  • Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context.
  • the disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.
  • any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.
  • the disclosure contemplates all combinations of any one or more of the foregoing embodiments, as well as combinations with any one or more of the embodiments set forth in the detailed description and examples.
  • sequence database reference numbers e.g., sequence database reference numbers
  • GenBank, Unigene, and Entrez sequences referred to herein, e.g., in any Table herein are incorporated by reference.
  • sequence accession numbers specified herein, including in any Table herein refer to the database entries current as of May 23, 2019.

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Abstract

Aspects of the disclosure provide methods for treating a hematopoietic malignancy (e.g., acute myeloid leukemia). In some aspects, the disclosure provides methods of treatment using a population of genetically engineered CD33-deficient hematopoietic cells and gemtuzumab ozogamicin.

Description

METHODS FOR TREATING HEMATOPOIETIC MALIGNANCY
RELATED APPLICATIONS
The application claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application number 63/484,173 filed February 9, 2023, U.S. Provisional Application number 63/507,052 filed June 8, 2023, and U.S. Provisional Application number 63/597,494 filed November 9, 2023, each of which are incorporated by reference in its entirety.
REFERENCE TO AN ELECTRONIC SEQUENCE LISTING
The contents of the electronic sequence listing (V029170043WO00-SEQ-CEW.xml; Size: 64,504 bytes; and Date of Creation: February 9, 2024) is herein incorporated by reference in its entirety.
BACKGROUND
Hematopoietic cell transplantation (HCT) can be used to treat patients with acute myeloid leukemia (AML) in complete remission but at high risk of relapse, especially those with intermediate or adverse disease-related genetics. With the reduction in transplant- related mortality over the past several decades, leukemia relapse post-HCT remains an obstacle to improved overall outcomes. Several recent studies have revealed that the presence of minimal residual disease (MRD) while in morphological complete remission at the time of HCT is independently associated with a significantly increased risk of leukemia relapse and death. In addition, patients who present with evidence of persistent leukemic blasts in the bone marrow are individuals at very high risk for early relapse post HCT.
SUMMARY
The methods described herein provide new approaches to reduce the risk of leukemia relapse and improve overall outcomes in patients with acute myeloid leukemia. In some aspects, the present disclosure relates to a method, comprising administering to a subject an effective amount of a population of genetically engineered hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen; and administering to the subject gemtuzumab ozogamicin in a dosing regimen comprising a dosing cycle, wherein the dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin; wherein the effective amount of gemtuzumab ozogamicin is 0.1 mg/m2 - 6.0 mg/m2 body surface area of the subject.
In some embodiments, the dosing regimen comprises at least two, at least three, or at least four dosing cycles, wherein each dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin. In some embodiments, the dosing cycle or each dosing cycle is about 4 weeks or less.
In some embodiments, the effective amount of gemtuzumab ozogamicin is administered to the subject in a single dose. In some embodiments, the single dose of gemtuzumab ozogamicin is about 0.1 mg/m2, about 0.25 mg/m2, about 0.5 mg/m2, about 1.0 mg/m2, about 2.0 mg/m2, about 3.0 mg/m2, about 4.0 mg/m2, about 5.0 mg/m2, or about 6.0 mg/m2 body surface area of the subject. In some embodiments, the single dose of gemtuzumab ozogamicin is about 0.5 mg/m2 body surface area of the subject. In some embodiments, the single dose of gemtuzumab ozogamicin is about 0.5 mg/m2 body surface area of the subject and the dosing cycle is about 4 weeks.
In some embodiments, the effective amount of gemtuzumab ozogamicin is administered to the subject in multiple doses. In some embodiments, each of the multiple doses of gemtuzumab ozogamicin is about 0.1 mg/m2, about 0.25 mg/m2, about 0.5 mg/m2, about 1.0 mg/m2, about 2.0 mg/m2, about 3.0 mg/m2, about 4.0 mg/m2, about 5.0 mg/m2, or about 6.0 mg/m2 body surface area of the subject. In some embodiments, each of the multiple doses of gemtuzumab ozogamicin is about 0.5 mg/m2 body surface area of the subject.
In some embodiments, the multiple doses of gemtuzumab ozogamicin comprises two doses of gemtuzumab ozogamicin. In some embodiments, a first dose is administered to the subject on day 1 of the dosing cycle and a second dose is administered in day 7 of the dosing cycle. In some embodiments, a first dose is administered to the subject on day 1 of the dosing cycle and a second dose is administered in day 14 of the dosing cycle.
In some embodiments, the multiple doses of gemtuzumab ozogamicin comprises three doses of gemtuzumab ozogamicin. In some embodiments, a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered in day 4 of the dosing cycle, and a third dose is administered on day 7 of the dosing cycle. In some embodiments, a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered in day 8 of the dosing cycle, and a third dose is administered on day 16 of the dosing cycle. In some embodiments, a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered to the subject once the plasma concentration of gemtuzumab ozogamicin in the subject is less than a threshold value, and a third dose is administered to the subject once the plasma concentration of gemtuzumab ozogamicin in the subject is less than a threshold value following administration of the second dose.
In some embodiments, the multiple doses of gemtuzumab ozogamicin comprises four doses of gemtuzumab ozogamicin. In some embodiments, the doses of gemtuzumab ozogamicin are administered to the subject weekly in the dosing cycle. In some embodiments, a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered in day 8 of the dosing cycle, a third dose is administered on day 15 of the dosing cycle, and a fourth dose is administered on day 21 of the dosing cycle.
In some embodiments, the multiple doses of gemtuzumab ozogamicin are administered weekly to the subject in the dosing cycle.
In some embodiments, the population of genetically engineered hematopoietic cells and the first dosing cycle are administered in temporal proximity. In some embodiments, administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen at least 60 days after administration of the population of genetically engineered hematopoietic cells. In some embodiments, administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen between 40- 60 days after administration of the population of genetically engineered hematopoietic cells, if the subject experiences early relapse. In some embodiments, the population of genetically engineered hematopoietic cells are administered prior to gemtuzumab ozogamicin.
In some embodiments, the population of genetically engineered hematopoietic cells are administered in a single treatment regimen.
In some embodiments, the population of genetically engineered hematopoietic cells and/or the gemtuzumab ozogamicin are administered intravenously.
In some embodiments, the effective amount of the population of genetically engineered hematopoietic cells is about 106 cells/kilogram body weight of the subject to about 5 x 107 cells/kilogram body weight of the subject. In some embodiments, the effective amount of the population of genetically engineered hematopoietic cells is about 7.5 x 106 cells/kilogram body weight of the subject. In some embodiments, the effective amount of the population of genetically engineered hematopoietic cells is about 3 x 106 cells/kilogram body weight of the subject.
In some embodiments, the population of genetically engineered hematopoietic cells are thawed from a cryopreserved form prior to administration.
In some embodiments, the gemtuzumab ozogamicin is reconstituted from a lyophilized form prior to administration. In some embodiments, the subject has been preconditioned prior to administering the hematopoietic cells and gemtuzumab ozogamicin. In some embodiments, the method further comprises preconditioning the subject prior to administering the hematopoietic cells and gemtuzumab ozogamicin. In some embodiments, the preconditioning comprises administering one or more chemotherapeutic agents to the subject. In some embodiments, the preconditioning comprises total body irradiation of the subject. In some embodiments, the chemotherapeutic agent is selected from the group consisting of busulfan, melphalan, fludarabine, cyclophosphamide, and thiotepa. In some embodiments, the preconditioning comprises administering antibodies that bind human T cells, optionally wherein the antibodies comprise rabbit anti-thymocyte globulins (rATG).
In some embodiments, the subject has, or has been diagnosed with, a hematopoietic malignancy or a hematopoietic pre-malignant disease, and wherein the hematopoietic malignancy is characterized by the presence of CD33-positive malignant cells, or wherein the hematopoietic pre-malignant disease is characterized by the presence of CD33-positive pre- malignant cells. In some embodiments, the subject has, or has been diagnosed with, CD33- positive acute myeloid leukemia. In some embodiments, the subject has, or has been diagnosed with, CD33-positive myelodysplastic syndrome. In some embodiments, the subject has, or has been diagnosed with, CD33-positive myelodysplastic syndrome and wherein the subject is at high risk of developing acute myeloid leukemia or refractory cytopenias.
In some embodiments, the subject is naive to chemotherapy and/or radiation therapy, optionally wherein the subject is naive to any treatment aimed to address a hematopoietic malignancy or hematopoietic pre-malignant disease.
In some embodiments, the subject has previously received chemotherapy. In some embodiments, the subject has previously received induction therapy.
In some embodiments, the subject has previously entered a complete hematological remission, optionally wherein the complete hematological remission is characterized by an incomplete recovery of peripheral counts.
In some embodiments, the subject has one or more risk factors associated with early leukemia relapse. In some embodiments, the one or more risk factors associated with early leukemia relapse are selected from the group consisting of: bone marrow in morphological complete remission with presence of intermediate or high-risk disease-related genetics; presence of minimal residual disease (MRD) post cyto-reductive therapy; bone marrow with persistent leukemia blasts post cyto-reductive therapy; and bone marrow blast count of about 10% or less.
In some embodiments, the subject does not have acute promyelocytic leukemia or chronic myeloid leukemia. In some embodiments, the subject has not previously received a stem cell transplantation. In some embodiments, the subject has not previously received gemtuzumab ozogamicin.
In some embodiments, the method further comprises determining a percent donor chimerism and/or a level of CD33-negative myeloid hematopoiesis in a peripheral blood sample from the subject. In some embodiments, the subject has a CD33-negative absolute neutrophil count (ANC) of at least 1000 cells/pL prior to receiving the dosing regimen. In some embodiments, the subject has a CD33-negative absolute neutrophil count (ANC) of at least 500 cells/pL prior to receiving the dosing regimen.
In some embodiments, the hematopoietic cells are hematopoietic stem and progenitor cells. In some embodiments, the hematopoietic stem cells are from bone marrow cells, cord blood cells, or peripheral blood mononuclear cells (PBMCs). In some embodiments, the hematopoietic stem cells are CD34+/CD33‘.
In some embodiments, the hematopoietic cells are autologous. In some embodiments, the method further comprises obtaining the autologous hematopoietic stem cells from the subject, optionally wherein the method further comprises genetically engineering the autologous stem cells to have reduced or eliminated expression of the CD33 antigen, and returning the genetically engineered hematopoietic stem cells to the subject.
In some embodiments, the hematopoietic cells are allogeneic. In some embodiments, the hematopoietic cells are allogeneic hematopoietic stem cells obtained from a donor having an HLA haplotype that matches with the HLA haplotype of the subject. In some embodiments, the method further comprises obtaining hematopoietic cells from a donor having an HLA haplotype that matches with the HLA haplotype of the subject.
In some embodiments, the method further comprises preparing the hematopoietic cells by modifying an endogenous gene of the hematopoietic cells encoding the CD33 antigen. In some embodiments, the whole or a portion of the endogenous gene encoding the CD33 cell-surface antigen is deleted. In some embodiments, the whole or the portion of the endogenous gene is deleted using genome editing. In some embodiments, the genome editing involves a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a CRISPR-Cas system. In some embodiments, the population of genetically engineered hematopoietic cells, or descendants thereof, are tremtelectogene empogeditemcel (trem-cel).
Aspects of the present disclosure relate to methods comprising administering to a subject: gemtuzumab ozogamicin in a dosing regimen comprising a dosing cycle, wherein the dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin; wherein the effective amount of gemtuzumab ozogamicin is 0.1 mg/m2 - 6.0 mg/m2 body surface area of the subject; and wherein the subject is receiving or has received an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen.
In some embodiments, the dosing regimen comprises at least two, at least three, or at least four dosing cycles, wherein each dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin. In some embodiments, the dosing cycle or each dosing cycle is about 4 weeks or less. In some embodiments, the effective amount of gemtuzumab ozogamicin is administered to the subject in a single dose. In some embodiments, the single dose of gemtuzumab ozogamicin is about 0.1 mg/m2, about 0.25 mg/m2, about 0.5 mg/m2, about 1.0 mg/m2, about 2.0 mg/m2, about 3.0 mg/m2, about 4.0 mg/m2, about 5.0 mg/m2, or about 6.0 mg/m2 body surface area of the subject. In some embodiments, the single dose of gemtuzumab ozogamicin is about 0.5 mg/m2 body surface area of the subject. In some embodiments, the single dose of gemtuzumab ozogamicin is about 0.5 mg/m2 body surface area of the subject and the dosing cycle is about 4 weeks.
In some embodiments, the effective amount of gemtuzumab ozogamicin is administered to the subject in multiple doses. In some embodiments, each of the multiple doses of gemtuzumab ozogamicin is about 0.1 mg/m2, about 0.25 mg/m2, about 0.5 mg/m2, about 1.0 mg/m2, about 2.0 mg/m2, about 3.0 mg/m2, about 4.0 mg/m2, about 5.0 mg/m2, or about 6.0 mg/m2 body surface area of the subject. In some embodiments, each of the multiple doses of gemtuzumab ozogamicin is about 0.5 mg/m2 body surface area of the subject.
In some embodiments, the multiple doses of gemtuzumab ozogamicin comprises two doses of gemtuzumab ozogamicin. In some embodiments, a first dose is administered to the subject on day 1 of the dosing cycle and a second dose is administered in day 7 of the dosing cycle. In some embodiments, a first dose is administered to the subject on day 1 of the dosing cycle and a second dose is administered in day 14 of the dosing cycle. In some embodiments, the multiple doses of gemtuzumab ozogamicin comprises three doses of gemtuzumab ozogamicin. In some embodiments, a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered in day 4 of the dosing cycle, and a third dose is administered on day 7 of the dosing cycle. In some embodiments, a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered in day 8 of the dosing cycle, and a third dose is administered on day 16 of the dosing cycle. In some embodiments, a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered to the subject once the plasma concentration of gemtuzumab ozogamicin in the subject is less than a threshold value, and a third dose is administered to the subject once the plasma concentration of gemtuzumab ozogamicin in the subject is less than a threshold value following administration of the second dose.
In some embodiments, the multiple doses of gemtuzumab ozogamicin comprises four doses of gemtuzumab ozogamicin. In some embodiments, the doses of gemtuzumab ozogamicin are administered to the subject weekly in the dosing cycle. In some embodiments, a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered in day 8 of the dosing cycle, a third dose is administered on day 15 of the dosing cycle, and a fourth dose is administered on day 21 of the dosing cycle.
In some embodiments, the population of genetically engineered hematopoietic cells and the first dosing cycle of gemtuzumab ozogamicin are administered in temporal proximity. In some embodiments, administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen at least 60 days after administration of the population of genetically engineered hematopoietic cells. In some embodiments, administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen between 40-60 days after administration of the population of genetically engineered hematopoietic cell, if the subject experiences early relapse.
In some embodiments, the subject received the population of genetically engineered hematopoietic cells prior to administration of gemtuzumab ozogamicin. In some embodiments, the subject received the population of genetically engineered hematopoietic cells in a single treatment regimen.
In some embodiments, the administration of gemtuzumab ozogamicin is intravenous. In some embodiments, the gemtuzumab ozogamicin is reconstituted from a lyophilized form prior to administration.
In some embodiments, the effective amount of the population of genetically engineered hematopoietic cells is about 106 cells/kilogram body weight of the subject to about 5 x 107 cells/kilogram body weight of the subject. In some embodiments, the effective amount of the population of genetically engineered hematopoietic cells is about 7.5 x 106 cells/kilogram body weight of the subject. In some embodiments, the effective amount of the population of genetically engineered hematopoietic cells is about 3 x 106 cells/kilogram body weight of the subject.
In some embodiments, the subject has been preconditioned prior to receiving the population of hematopoietic cells and gemtuzumab ozogamicin. In some embodiments, the method further comprises preconditioning the subject prior to administering the gemtuzumab ozogamicin. In some embodiments, the preconditioning comprises administering one or more chemotherapeutic agents to the subject. In some embodiments, the preconditioning comprises total body irradiation of the subject. In some embodiments, the chemotherapeutic agent is selected from the group consisting of busulfan, melphalan, fludarabine, cyclophosphamide, and thiotepa. In some embodiments, the preconditioning comprises administering antibodies that bind human T cells, optionally wherein the antibodies comprise rabbit anti-thymocyte globulins (rATG).
In some embodiments, the subject has, or has been diagnosed with, a hematopoietic malignancy or a hematopoietic pre-malignant disease, and wherein the hematopoietic malignancy is characterized by the presence of CD33-positive malignant cells, or wherein the hematopoietic pre-malignant disease is characterized by the presence of CD33-positive pre- malignant cells. In some embodiments, the subject has, or has been diagnosed with, CD33- positive acute myeloid leukemia. In some embodiments, the subject has, or has been diagnosed with, CD33-positive myelodysplastic syndrome. In some embodiments, the subject has, or has been diagnosed with, CD33-positive myelodysplastic syndrome and wherein the subject is at high risk of developing acute myeloid leukemia or refractory cytopenias.
In some embodiments, the subject is naive to chemotherapy and/or radiation therapy, optionally wherein the subject is naive to any treatment aimed to address a hematopoietic malignancy or hematopoietic pre-malignant disease. In some embodiments, the subject has previously received chemotherapy. In some embodiments, the subject has previously received induction therapy.
In some embodiments, the subject has previously entered a complete hematological remission, optionally wherein the complete hematological remission is characterized by an incomplete recovery of peripheral counts. In some embodiments, the subject has one or more risk factors associated with early leukemia relapse. In some embodiments, the one or more risk factors associated with early leukemia relapse are selected from the group consisting of: bone marrow in morphological complete remission with presence of intermediate or high-risk disease-related genetics; presence of minimal residual disease (MRD) post cyto-reductive therapy; bone marrow with persistent leukemia blasts post cyto-reductive therapy; and bone marrow blast count of about 10% or less.
In some embodiments, the subject does not have acute promyelocytic leukemia or chronic myeloid leukemia. In some embodiments, the subject has not previously received a stem cell transplantation. In some embodiments, the subject has not previously received gemtuzumab ozogamicin.
In some embodiments, the method further comprises determining a percent donor chimerism and/or a level of CD33-negative myeloid hematopoiesis in a peripheral blood sample from the subject. In some embodiments, the subject has a CD33-negative absolute neutrophil count (ANC) of at least 1000 cells/pL prior to receiving the dosing regimen. In some embodiments, the subject has a CD33-negative absolute neutrophil count (ANC) of at least 500 cells/pL prior to receiving the dosing regimen.
In some embodiments, the hematopoietic cells are hematopoietic stem and progenitor cells. In some embodiments, the hematopoietic stem and progenitor cells are from bone marrow cells, cord blood cells, or peripheral blood mononuclear cells (PBMCs). In some embodiments, the hematopoietic stem and progenitor cells are CD34+/CD33-.
In some embodiments, the hematopoietic cells are autologous. In some embodiments, the hematopoietic cells are allogeneic. In some embodiments, the hematopoietic cells are allogeneic hematopoietic stem cells obtained from a donor having an HLA haplotype that matches with the HLA haplotype of the subject. In some embodiments, the method further comprises obtaining hematopoietic cells from a donor having an HLA haplotype that matches with the HLA haplotype of the subject.
In some embodiments, the method further comprises preparing the population of hematopoietic cells by modifying an endogenous gene of the hematopoietic cells encoding the CD33 antigen. In some embodiments, the whole or a portion of the endogenous gene encoding the CD33 cell-surface antigen is deleted. In some embodiments, the whole or the portion of the endogenous gene is deleted using genome editing. In some embodiments, the genome editing involves a zinc finger nuclease (ZFN), a transcription activator-like effectorbased nuclease (TALEN), or a CRISPR-Cas system. In some embodiments, the population of genetically engineered hematopoietic cells, or descendants thereof, are tremtelectogene empogeditemcel (trem-cel). Aspects of the present disclosure relate to a method comprising administering an effective amount of gemtuzumab ozogamicin to a subject, wherein the subject is identified as having hematopoietic cells comprising a lower density of wild-type CD33 in a first biological sample relative to a second biological sample.
In some embodiments, the first biological sample is obtained from the subject at a first time point and the second biological sample is obtained from the subject at a second time point; or wherein the second biological sample is obtained from a counterpart subject. In some embodiments, the method further comprises obtaining the first biological sample; and measuring the density of wild-type CD33 in the first biological sample. In some embodiments, the method further comprises obtaining the second biological sample; and measuring the density of wild-type CD33 in the second biological sample.
In some embodiments, the lower density of wild-type CD33 in the first biological sample is 95% or less than the density of wild-type CD33 in the second biological sample. In some embodiments, the effective amount of gemtuzumab ozogamicin is 0.1 mg/m2 - 6.0 mg/m2 body surface area of the subject. In some embodiments, the effective amount of gemtuzumab ozogamicin is administered to the subject in a single dose or multiple doses of the effective amount.
In some embodiments, the method further comprises administering an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen; or the subject is receiving or has received an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen.
Aspects of the present disclosure provide a method comprising (a) measuring density of wild-type CD33 on a population of hematopoietic cells in a first biological sample of a subject; (b) comparing the density of wild-type CD33 on the population of hematopoietic cells in the first biological sample to the density of wild-type CD33 on a population of hematopoietic cells in a second biological sample; and (c) determining an effective amount of gemtuzumab ozogamicin for administration to the subject based on (b).
In some embodiments, the method further comprises administering the effective amount of gemtuzumab ozogamicin for administration to the subject. In some embodiments, the density of wild-type CD33 on the population of hematopoietic cells in the first biological sample is lower than the density of wild-type CD33 on a population of hematopoietic cells in a second biological sample.
In some embodiments, the effective amount of gemtuzumab ozogamicin is 0.1 mg/m2 - 6.0 mg/m2 body surface area of the subject. In some embodiments, the method comprises administering to the first subject an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen. In some embodiments, the first subject has received an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen.
The summary above is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the Detailed Description, the Drawings, the Examples, and the Claims.
BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which constitute a part of this specification, illustrate several embodiments of the invention and together with the description, serve to explain the principles of the invention.
FIG. 1 shows a schematic of an exemplary human clinical trial in which CD33+ acute myeloid leukemia (AML) patients who are at high risk of relapse are subjected to myeloablative hematopoietic cell transplant (HCT) with tremtelectogene empogeditemcel (trem-cel) followed by treatment with low-dose gemtuzumab ozogamicin. In Part 1 of the study, patients are enrolled in 3 cohorts who are treated with escalating doses of gemtuzumab ozogamicin (0.5-2.0 mg/m2) in a 28-day treatment cycle for up to 4 cycles. Part 1 will evaluate the safety of trem-cel and determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of gemtuzumab ozogamicin. Part 2 will enroll an additional 12 patients to further evaluate the safety of trem-cel and the preliminary efficacy of the combination of trem-cel and gemtuzumab ozogamicin at the recommended phase 2 dose. “AML” refers to acute myeloid leukemia; “G-CSE” refers to granulocyte colony stimulating factor; “GO” refers to gemtuzumab ozogamicin (also known as Mylotarg®); “GvHD” refers to graft- versus-host-disease; “HCT” refers to hematopoietic cell transplant; “HLA” refers to human leukocyte antigens; “LTEU” refers to long-term follow-up; “MAC” refers to myeloablative conditioning; “MTD” refers to maximum tolerated dose; “PK” refers to Pharmacokinetics; “RP2D” refers to recommended phase 2 dose. *Conditioning prior to infusion of trem-cel consists of busulfan/melphalan/fludarabine/rATG or total body irradiation/cyclophosphamide/thiotepa/rATG.
FIGs. 2A and 2B show neutrophil engraftment and platelet recovery in Patient 1 after HCT with trem-cel. FIG. 2A shows absolute neutrophil count at the indicated days post-HCT with trem-cel infusion. The arrow indicates trem-cel neutrophil engraftment, and the vertical dotted line indicates the median neutrophil engraftment of an unmodified CD34+ graft from the clinical trial CTN1301 (Luznik et al., J Clin Oncol. (2022);40(4):356-368; ClinicalTrials.gov Identifier: NCT02345850). FIG. 2B shows platelet recovery at the indicated days post-HCT with trem-cel infusion. The subject received platelet transfusions at the indicated days due to pre-existing hemorrhage risk. The arrow indicates trem-cel platelet recovery.
FIGs. 3A and 3B show neutrophil engraftment and platelet recovery in Patient 2 after HCT with trem-cel. FIG. 3A shows absolute neutrophil count in Patient 2 at the indicated days post-HCT with trem-cel infusion. The arrow indicates trem-cel neutrophil engraftment, and the vertical dotted line indicates the median neutrophil engraftment of an unmodified CD34+ graft from the clinical trial CTN1301. FIG. 3B shows platelet recovery in Patient 2 at the indicated days post-HCT with trem-cel infusion. The subject received platelet transfusions at the indicated days due to pre-existing hemorrhage risk. The arrow indicates trem-cel platelet recovery.
FIGs. 4A-4D show neutrophil engraftment and platelet recovery in Patients 1-5 after HCT with trem-cel. FIG. 4A shows absolute neutrophil count at the indicated days post-HCT with trem-cel infusion. The arrows indicate trem-cel neutrophil engraftment, the vertical dotted line indicates the median neutrophil engraftment of an unmodified CD34+ graft from clinical trial CTN1301, and the horizontal dotted line indicates an absolute neutrophil count (ANC) >500. Neutrophil recovery defined as the first of three consecutive days of an absolute neutrophil count (ANC) >500 is indicated by arrows under the x-axis. FIG. 4B shows platelet recovery at the indicated days post-HCT with trem-cel infusion. The horizontal dotted line indicates platelet count of >20,000/pL. The arrows under the x-axis indicate trem-cel platelet recovery defined as the first day of a sustained platelet count of >20,000/pL with no platelet transfusion in the preceding seven days. FIG. 4C shows neutrophil engraftment at the indicated days post-HCT with trem-cel infusion. FIG. 4D shows platelet recovery at the indicated days post-HCT with trem-cel infusion. For the “Platelet Recovery,” the median is shown excluding Patient 3, who experienced immune thrombocytopenia. Both neutrophil engraftment and platelet recovery were similar to unedited CD34-selected grafts. Results show full myeloid chimerism in all patients at D+28. Arrows indicate days of individual patient engraftment.
FIGs. 5A-5E show CD33 editing in peripheral blood and bone marrow post-HCT with trem-cel infusion. FIG. 5A is a table showing CD33 editing and expression in peripheral blood across bulk cells and cells of myeloid and lymphoid lineages in Patient 1 at days 28, 60, and 100 (“D28”, “D60” and “D100,” respectively) following trem-cel infusion. FIG. 5B is a table showing CD33 editing in peripheral blood cells (PB) including bulk cells and cells of myeloid and lymphoid lineages in Patients 1-4 at days 28 and 60 (“D+28” and “D+60,” respectively) post-HCT with trem-cel infusion. “DC%” refers to percent donor chimerism. “CD33 GE%” refers to CD33 gene editing efficiency. “NC” indicates sample was not collected. “QNS” indicates quantity not sufficient. “Pending” refers to pending collection. FIG. 5C shows donor chimerism results obtained from peripheral blood analyses of monocytes in Patients 1-8 at days 28, 60, 100, and 180 (“D28”, “D60”, “D100,” and “D180,” respectively) following trem-cel infusion. FIG. 5D shows the frequency of CD33 gene editing in trem-cel (“Drug Product”) and monocytes reconstituted from trem-cel in Patients 1- 8 at 28, 60, 100, and 180 days following trem-cel infusion (“D28”, “D60”, “D100,” and “DI 80,” respectively). FIG. 5E shows the frequency of CD33-negative monocytes and myeloid cells in the peripheral blood or bone marrow of Patients 1-8 at 28, 60, 100, and 180 days following trem-cel infusion (“D28”, “D60”, “D100,” and “DI 80,” respectively).
FIGs. 6A and 6B show exemplary flow cytometry analysis of peripheral blood (FIG. 6A) and bone marrow (FIG. 6B) from Patient 1 at 28 days (D28) post-HCT with trem-cel as compared to a subject that received HCT with non-edited cells (“non-edited recipient”).
FIGs. 7A and 7B show analyses of CD33-negative cells in bone marrow and peripheral blood from patients transplanted with trem-cel at the indicated time points. FIG. 7A is a table showing flow cytometry analysis of CD33 expression in bone marrow and peripheral blood cells from Patient 1 at days 28, 60, and 100 (“D28,” “D60,” and “D100,” respectively) following HCT infusion with trem-cel. “C1D1” refers to gemtuzumab ozogamicin dosing cycle 1, day 1. “C2D1” refers to gemtuzumab ozogamicin dosing cycle 2, day 1. “C3D1” refers to gemtuzumab ozogamicin dosing cycle 3, day 1. “Pre-dose” refers to samples obtained prior to gemtuzumab ozogamicin administration on the respective day, and “post-dose” refers to samples obtained after gemtuzumab ozogamicin administration. FIG. 7B is a table showing flow cytometry analyses of CD33 expression in bone marrow and peripheral blood cells in Patients 1-4 at days 28 and 60 (“D+28” and “D+60,” respectively) following HCT infusion with trem-cel. “PB” refers to peripheral blood. “BM” refers to bone marrow. “NC” indicates sample not collected. “TBD” indicates to be determined.
FIGs. 8A-8D show pharmacokinetics associated with administration of 0.5 mg/m2 x 1 gemtuzumab ozogamicin following HCT. FIG. 8A is a table showing the pharmacokinetics associated with 0.5 mg/m2 x 1 gemtuzumab ozogamicin administration following HCT with trem-cel (VBP101) in Patient 1 (VBP101 column) as compared to administration of 0.5 mg/m2 x 1 gemtuzumab ozogamicin following HCT with non-edited (CD33+) cells in a reference clinical trial (0903A1-101-US; FDA Briefing Document Oncologic Drugs Advisory Committee Meeting July 11, 2017, BLA 761060 Mylotarg (gemtuzumab ozogamicin)). “Cmax” refers to maximum concentration; “Tm x” refers to the time taken to reach the maximal concentration or time to Cmax; “AUCiast” refers to the area under the curve from time = 0 to the last measurable time point; “AUCinf” refers to the area under the curve from time = 0 extrapolated to infinity; “T1/2” refers to the half-life; “CL” refers to clearance; “Vz” refers to the volume of distribution. FIG. 8B shows pharmacokinetic data associated with administration of 0.5 mg/m2 x 1 gemtuzumab ozogamicin in Cohort 1. Results show higher gemtuzumab ozogamicin exposure in the context of CD33-negative hematopoiesis. Data from 3.0 mg/m2 mean (+/- CI) are data digitized from simulations presented in Hibma el al, 2019. FIG. 8C is a table showing pharmacokinetics after administration of a first dose of maintenance gemtuzumab ozogamicin (GO) following HCT with trem-cel (VBP101 column) compared to pharmacokinetics observed during GO Phase 1 Study 0903A1-101-US. “Cmax” refers to maximum concentration. “AUCinf” refers to the area under the curve from time = 0 extrapolated to infinity. FIG. 8D is a graph showing the relationship between gemtuzumab ozogamicin (GO) maximum concentration (Cmax) and veno-occlusive disease in prior transplant. “R/R AML” refers to relapsed or refractory acute myeloid leukemia.
FIG. 9 is a table showing absolute neutrophil and platelet counts and amount of CD33-negative cells in Patient 1 after HCT (post-HCT recovery) and after gemtuzumab ozogamicin administration in each of the three dosing cycles.
Figure imgf000015_0001
indicates pre-gemtuzumab ozogamicin dosing at the initiation of the first gemtuzumab ozogamicin dosing cycle.
FIGs. 10A-10E show analyses of heme-protection and the percentage of CD33- negative cells in the blood of Patient 1 after administration of gemtuzumab ozogamicin (GO). FIG. 10A shows neutrophil and platelet counts after administration of gemtuzumab ozogamicin at a single dose of 0.5 mg/m2 in each dosing cycle. “Cl” refers to gemtuzumab ozogamicin dosing cycle 1; “C2” refers to gemtuzumab ozogamicin dosing cycle 2; and “C3” refers to gemtuzumab ozogamicin dosing cycle 3. FIG. 10B is a table showing bone marrow measurable residual disease (MRD) at days 60, 101, and 147 (“D+60,” “D+101,” and “D+147,” respectively) following HCT infusion with trem-cel. “Pre-GO” refers to samples obtained prior to gemtuzumab ozogamicin (GO) administration on the respective day. “5d post-C2” refers to 5 days after gemtuzumab ozogamicin administration cycle 2. “23 post-C3” refers to 23 days after gemtuzumab ozogamicin administration cycle 3. “BM MRD%” refers to bone marrow measurable residual disease as defined as the percentage of AML blast cells. FIG. 10C shows flow cytometry analysis of CD33 expression in peripheral blood and bone marrow cell populations (monocyte and myleloid cells) at the indicated time points following HCT infusion with trem-cel and prior to and post administration of gemtuzumab ozogamicin at a single dose of 0.5 mg/m2 in each dosing cycle for Patient 1. “Cl” refers to gemtuzumab ozogamicin dosing cycle 1; “C2” refers to gemtuzumab ozogamicin dosing cycle 2; and “C3” refers to gemtuzumab ozogamicin dosing cycle 3. “PB” refers to peripheral blood, and “BM” refers to bone marrow. FIG. 10D shows neutrophil and platelet counts after gemtuzumab ozogamicin dosing in Cohort 1 (0.5 mg/m2). The first gemtuzumab ozogamicin dose was given on days +68 (Patient 1), +74 (Patient 5), and +66 (Patient 6) post-hematopoietic cell transplant. FIG. 10E shows an increase in CD33 negative myeloid cells during gemtuzumab ozogamicin dosing. Results show that editing of CD33 persisted over time and treatment with gemtuzumab ozogamicin selected for CD33 negative cells. indicates that Patient l’s CD33 flow was contaminated by the presence of relapsed disease after the third gemtuzumab ozogamicin dose.
FIG. 11 is a table showing measurable residual disease status in the bone marrow and peripheral blood of Patient 1 following gemtuzumab ozogamicin (GO) administration at a single dose of 0.5 mg/m2 in each dosing cycle. “D28,” “D60,” and “D100” refer to days 28, 60, and 100, respectively, following HCT infusion with trem-cel. “D100*” indicates that the visit was 5 days post-GO infusion in Cl. “C1D1” refers to gemtuzumab ozogamicin dosing cycle 1, day 1. “C2D1” refers to gemtuzumab ozogamicin dosing cycle 2, day 1. “C3D1” refers to gemtuzumab ozogamicin dosing cycle 3, day 1. “C3D6” refers to gemtuzumab ozogamicin dosing cycle 3, day 6.
FIG. 12 is a graph showing the clinical course for Patient 2. Absolute neutrophil count (ANC) is shown on the left axis, and platelet count is shown on the right axis. The backup graft was infused on day 57 following the initial HCT infusion with trem-cel.
FIGs. 13A-13F show effects associated with administration of 0.5 mg/m2 x 1 gemtuzumab ozogamicin or 1.0 mg/m2 x 1 gemtuzumab ozogamicin following HCT with trem-cel. FIG. 13A shows a graph representing pharmacokinetic data associated with administration of 0.5 mg/m2 x 1 gemtuzumab ozogamicin in Cohort 1 (Patients 1, 5, and 6) and 1.0 mg/m2 x 1 gemtuzumab ozogamicin in Cohort 2 (Patients 7 and 8) following HCT with trem-cel. “hP67.6 Cone” refers to the concentration (in nanograms per mililiter (ng/mL)) of the antibody comprised in gemtuzumab ozogamicin. FIG. 13B shows a table representing pharmacokinetic data associated with administration of 0.5 mg/m2 x 1 gemtuzumab ozogamicin in Cohort 1 and 1.0 mg/m2 x 1 gemtuzumab ozogamicin in Cohort 2 following HCT with trem-cel. “N” refers to the number of subjects in each cohort. “SD” refers to standard deviation; “CV” refers to coefficient of variation; “T1/2” refers to the half-life;
“Cmax” refers to maximum concentration; “AUCiast” refers to the area under the curve from time = 0 to the last measurable time point; “AUCinf” refers to the area under the curve from time = 0 extrapolated to infinity; “Vz” refers to the volume of distribution; and “CL” refers to clearance. FIG. 13C is a table showing the pharmacokinetics associated with administration of 0.5 mg/m2 x 1 gemtuzumab ozogamicin or 1.0 mg/m2 x 1 gemtuzumab ozogamicin following HCT with trem-cel (VBP101) 1 as compared to administration of 0.5 mg/m2 x 1 gemtuzumab ozogamicin or 1.0 mg/m2 x 1 gemtuzumab ozogamicin in relapse refractory CD33+ AML patients in a reference clinical trial (0903A1-101-US; FDA Briefing Document Oncologic Drugs Advisory Committee Meeting July 11, 2017, BLA 761060 My lotarg (gemtuzumab ozogamicin)). “hP67.6” refers to the antibody comprised in gemtuzumab ozogamicin; “Cmax” refers to maximum concentration; “AUCinf” refers to the area under the curve from time = 0 extrapolated to infinity. FIG. 13D shows analyses of absolute neutrophil count (“ANC”) associated with administration of 0.5 mg/m2 x 1 gemtuzumab ozogamicin in Patients 1, 5, and 6 and 1.0 mg/m2 x 1 gemtuzumab ozogamicin in Patient 7 following HCT. FIG. 13E shows results obtained from analyses of CD33 editing in monocytes in peripheral blood (“PB”) samples from Cohort 1 (Patients 1, 5, and 6) after administration of multiple cycles of 0.5 mg/m2 gemtuzumab ozogamicin and from Cohort 2 (Patient 7) after administration of 1.0 mg/m2 x 1 gemtuzumab ozogamicin following HCT. FIG. 13F shows results obtained from analyses of CD33 editing in myeloid cells in peripheral blood (“PB”) samples from Cohort 1 (Patients 1, 5, and 6) after administration of multiple cycles of 0.5 mg/m2 gemtuzumab ozogamicin and from Cohort 2 (Patient 7) after administration of 1.0 mg/m2 x 1 gemtuzumab ozogamicin following HCT. DETAILED DESCRIPTION
The present disclosure provides targeted therapeutic approaches for use in treating hematopoietic malignancies that overcome limitations in existing therapies. For example, current CD33-targeted therapies for acute myeloid leukemia (AML) are limited by “on-target, off-leukemia” cytotoxicity directed toward normal healthy myeloid lineage cells expressing CD33. The loss of the noncancerous CD33+ cells can deplete the hematopoietic system of the patient. To address this depletion, the subject can be administered rescue cells (e.g., hematopoietic cells) comprising a modification in the CD33 gene, such as tremtelectogene empogeditemcel (trem-cel). These CD33-modified cells can be resistant to the anti-CD33 cancer therapy and can therefore maintain or repopulate the hematopoietic system during or after anti-CD33 therapy. In this way, the normal myeloid compartment is protected from the on-target effects of CD33-targeted agents, resulting in an improved therapeutic index for these agents and better patient outcomes.
Cells
Aspects of the present disclosure related to genetically engineered hematopoietic cells (also referred to herein as eHSCs or eHSPCs), or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen. In some embodiments, genetically engineered hematopoietic cells of the disclosure (e.g., a hematopoietic stem cells (HSC) or hematopoietic progenitor cells (HPC)) having a modification of the gene encoding CD33 are genetically engineered using any genetic editing methods known in the art.
In some embodiments, genetically engineered hematopoietic cells of the disclosure (e.g., a hematopoietic stem cells (HSC) or hematopoietic progenitor cells (HPC)) having a modification of the gene encoding CD33 are genetically engineered using a nuclease and/or a gRNA described herein. In some embodiments, a cell (e.g., HSC or HPC) having a modification of CD33 and a modification of a second lineage- specific cell surface antigen is made using a nuclease and/or a gRNA described herein. It is understood that the cell can be made by contacting the cell itself with the nuclease and/or a gRNA, or the cell can be the daughter cell of a cell that was contacted with the nuclease and/or gRNA. In some embodiments, a cell described herein (e.g., an HSC or HPC) is capable of reconstituting the hematopoietic system of a subject. In some embodiments, a cell described herein (e.g., an HSC or HPC) is capable of one or more of (e.g., all of): engrafting in a human subject, producing myeloid lineage cells, and producing and lymphoid lineage cells. In some embodiments, the cell comprises only one genetic modification. In some embodiments, the cell is only genetically modified at the CD33 locus, such as in a sequence of exon 3 of CD33. In some embodiments, the cell is genetically modified at a second locus. In some embodiments, the cell does not comprise a transgenic protein, e.g., does not comprise a chimeric antigen receptor (CAR).
The terms “CD33 antigen” and “CD33 protein” are used interchangeably herein and refer to the CD33 protein, or a portion or fragment thereof, such as a portion that is targeted by an anti-CD33 agent, such as a cytotoxic agent comprising an anti-CD33 antigen-binding domain, such as gemtuzumab ozogamicin.
In some embodiments, a genetically engineered hematopoietic cell described herein comprises substantially no CD33 protein (CD33 antigen). In some embodiments, a genetically engineered hematopoietic cell described herein comprises substantially no wildtype CD33 protein but comprises a mutant CD33 protein. In some embodiments, the mutant CD33 protein is not bound by an agent that targets CD33 for therapeutic purposes. As used herein, a genetically engineered hematopoietic cell that has been genetically engineered such that the cell has reduced or no expression of CD33 may be referred to as “CD33KO eHSCs” or “CD33KO eHSPCs.” In some embodiments, the population of genetically engineered CD33KO eHSPCs, or descendants thereof, are tremtelectogene empogeditemcel (trem-cel).
In some embodiments, the cell is a circulating blood cell, e.g., a reticulocyte, megakaryocyte erythroid progenitor (MEP) cell, myeloid progenitor cell (CMP/GMP), lymphoid progenitor (LP) cell, hematopoietic stem cell (HSC), or hematopoietic progenitor cell (HPC), which may be referred to as hematopoietic stem and progenitor cells (HSPCs), or endothelial cell (EC). In some embodiments, the cell is a bone marrow cell (e.g., a reticulocyte, an erythroid cell (e.g., erythroblast), megakaryocyte-erythroid progenitor cell (MEP cell), myeloid progenitor cell (CMP/GMP), lymphocyte predominant (LP) cell, erythroid progenitor (EP) cell, HSC, multipotent progenitor (MPP) cell, endothelial cell (EC), hemogenic endothelial (HE) cell, or mesenchymal stem cell). In some embodiments, the cell is a myeloid progenitor cell (e.g., a common myeloid progenitor (CMP) cell or granulocyte macrophage progenitor (GMP) cell). In some embodiments, the cell is a lymphoid progenitor cell, e.g., a common lymphoid progenitor (CLP) cell. In some embodiments, the cell is an erythroid progenitor cell (e.g., an MEP cell). In some embodiments, the cell is a hematopoietic stem/progenitor cell (e.g., a long-term HSC (LT-HSC), short term HSC (ST- HSC), MPP cell, or lineage restricted progenitor (LRP) cell). In some embodiments, the cell is a CD34+ cell, CD34+CD90+ cell, CD34+CD38+ cell, CD34+CD90+CD49+CD38+ CD45RA cell, CD105+ cell, CD31+, or CD133+ cell, or a CD34+CD90+ CD133+ cell. In some embodiments, the cell is an umbilical cord blood CD34+ HSPC, umbilical cord venous endothelial cell, umbilical cord arterial endothelial cell, amniotic fluid CD34+ cell, amniotic fluid endothelial cell, placental endothelial cell, or placental hematopoietic CD34+ cell. In some embodiments, the cell is a mobilized peripheral blood hematopoietic CD34+ cell (after the patient is treated with a mobilization agent, e.g., granulocyte colony-stimulating factor (G-CSF), etoposide, cyclophosphamide, and/or plerixafor). In some embodiments, the cell is a peripheral blood endothelial cell, or a population thereof.
In some embodiments, the cells are hematopoietic cells, e.g., hematopoietic stem cells. Hematopoietic stem cells (HSCs) are typically capable of giving rise to both myeloid and lymphoid progenitor cells that further give rise to myeloid cells (e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, erythrocytes, platelets, etc.) and lymphoid cells (e.g., T cells, B cells, NK cells), respectively. HSCs are characterized by the expression of the cell surface marker CD34 (e.g., CD34+), which can be used for the identification and/or isolation of HSCs, and absence of cell surface markers associated with commitment to a cell lineage.
In some embodiments, a population of genetically engineered hematopoietic cells described herein comprises a plurality of hematopoietic stem cells. In some embodiments, a population of genetically engineered hematopoietic cells described herein comprises a plurality of hematopoietic progenitor cells. In some embodiments, a population of genetically engineered hematopoietic cells described herein comprises a plurality of hematopoietic stem cells and a plurality of hematopoietic progenitor cells.
In some embodiments, a hematopoietic stem cell (HSC) refers to cells of a stem cell lineage that give rise to all the blood cell types including the erythroid (erythrocytes or red blood cells (RBCs)), myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, megakaryocytes/platelets, and dendritic cells), and lymphoid (T-cells, B-cells, NK-cells). In some embodiments, the cells used herein are selected from the group consisting of a circulating blood cell, a mobilized blood cell, a bone marrow cell, a myeloid progenitor cell, a lymphoid progenitor cell, a multipotent progenitor cell, a lineage restricted progenitor cell, an endothelial cell, or a mesenchymal stromal cell. In some embodiments, the HSC is from a non-cord blood source, an umbilical cord source, or a cord blood source. In one embodiment, the HSC is a CD34+ cell. In some embodiments, the HSC cell is capable of differentiating in vivo after transplantation into the subject. In some embodiments, the HSC cell is capable of differentiating into B cells, T cells, erythroid cells, and/or myeloid cells. In some embodiments, the HSC cell is capable of reconstituting hematopoiesis in the subject. In some embodiments, the hematopoietic stem cell has at least one of the cell surface marker characteristic of hematopoietic progenitor cells: CD34+, CD59+, Thyl/CD90+, CD381o/-, and C-kit/CDl 17+. In some embodiments, the hematopoietic progenitor are CD34+.
In some embodiments, a hematopoietic stem cell is a peripheral blood stem cell obtained from a subject after the subject has been treated with granulocyte colony stimulating factor (G-CSF) (optionally in combination with plerixafor, etoposide, and/or cyclophosphamide). In some embodiments, CD34+ cells are enriched using CliniMACS® Cell Selection System (Miltenyi Biotec). In some embodiments, CD34+ cells are stimulated in serum-free medium (e.g., CellGrow SCGM media, CellGenix) with cytokines (e.g., SCF, rhTPO, rhFLT3) before genome editing. In some embodiments, addition of SRI and dmPGE2 and/or other factors is contemplated to improve long-term engraftment.
In some embodiments, a population of genetically engineered hematopoietic cells for administration in accordance with the disclosure can be allogeneic hematopoietic progenitor cells obtained from one or more donors. As used herein, “allogeneic” refers to a hematopoietic progenitor cell or biological samples comprising hematopoietic progenitor cells obtained from one or more different donors of the same species, where the genes at one or more loci are not identical. For example, a hematopoietic cell population being administered to a subject can be derived from umbilical cord blood obtained from one more unrelated donor subjects or from one or more non-identical siblings. In some embodiments, syngeneic hematopoietic cell populations can be used, such as those obtained from genetically identical animals or from identical twins. In some embodiments, the hematopoietic cells are autologous cells; that is, the hematopoietic progenitor cells are obtained or isolated from a subject and administered to the same subject (z.e., the donor and recipient are the same).
In some embodiments, a population of genetically engineered hematopoietic cells described herein comprises a plurality of genetically engineered hematopoietic stem cells. In some embodiments, a population of genetically engineered hematopoietic cells described herein comprises a plurality of genetically engineered hematopoietic progenitor cells. In some embodiments, population of genetically engineered hematopoietic cells described herein comprises a plurality of genetically engineered hematopoietic stem cells and a plurality of genetically engineered hematopoietic progenitor cells. In some embodiments, the HSCs or HPCs are obtained from a subject, such as a human subject. Methods of obtaining HSCs are described, e.g., in PCT Application No. PCT/US2016/057339 (PCT Publication No. WO 2017/066760), which is herein incorporated by reference in its entirety. In some embodiments, the HSCs are peripheral blood HSCs. In some embodiments, the mammalian subject is a non-human primate, a rodent (e.g., mouse or rat), a bovine, a porcine, an equine, or a domestic animal. In some embodiments, the HSCs are obtained from a human subject, such as a human subject having a hematopoietic malignancy. In some embodiments, the HSCs or HPCs are obtained from a healthy donor. In some embodiments, the HSCs or HPCs are obtained from the subject to whom the gemtuzumab ozogamicin will be subsequently administered. HSCs or HPCs that are administered to the same subject from which the cells were obtained are referred to as autologous cells, whereas HSCs or HPCs that are obtained from a subject who is not the subject to whom the cells will be administered are referred to as allogeneic cells.
In some embodiments, a population of genetically engineered hematopoietic cells is a heterogeneous population of cells, e.g. heterogeneous population of genetically engineered hematopoietic cells containing different CD33 mutations. In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of copies of CD33 in the population of cells have a mutation effected by a genome editing approach described herein, e.g. by a CRISPR/Cas system using a gRNA described herein. By way of example, a population can comprise a plurality of different CD33 mutations and each mutation of the plurality contributes to the percent of copies of CD33 in the population of cells that have a mutation.
In some embodiments, the expression of CD33 on the genetically engineered hematopoietic cell is compared to the expression of CD33 on a naturally occurring hematopoietic cell (e.g., a wild-type counterpart). In some embodiments, the genetic engineering results in a reduction in the expression level of CD33 by at least 50%, at least 60%, at least 70%, at least 80%, 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%, or at least 99% as compared to the expression of CD33 on a naturally occurring hematopoietic cell (e.g., a wild-type counterpart). For example, in some embodiments, the genetically engineered hematopoietic cell expresses less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of CD33 as compared to a naturally occurring hematopoietic cell (e.g., a wild-type counterpart).
In some embodiments, the genetic engineering results in a reduction in the expression level of wild-type CD33 by at least 50%, at least 60%, at least 70%, at least 80%, 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%, or at least 99% as compared to the expression of the level of wild-type CD33 on a naturally occurring hematopoietic cell (e.g., a wild-type counterpart). For example, in some embodiments, the genetically engineered hematopoietic cell expresses less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of CD33 as compared to a naturally occurring hematopoietic cell (e.g., a wildtype counterpart).
In some embodiments, the genetic engineering results in a reduction in the expression level of wild-type lineage- specific cell surface antigen (e.g., CD33) by at least 50%, at least 60%, at least 70%, at least 80%, 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%, or at least 99% as compared to a suitable control (e.g., a cell or plurality of cells). In some embodiments, the suitable control comprises the level of the wild-type lineage- specific cell surface antigen measured or expected in a plurality of non-engineered cells from the same subject. In some embodiments, the suitable control comprises the level of the wild-type lineage- specific cell surface antigen measured or expected in a plurality of cells from a healthy subject. In some embodiments, the suitable control comprises the level of the wild-type lineage- specific cell surface antigen measured or expected in a population of cells from a pool of healthy individuals (e.g., 10, 20, 50, or 100 individuals). In some embodiments, the suitable control comprises the level of the wild-type lineage- specific cell surface antigen measured or expected in a subject in need of a treatment described herein, e.g., an anti-CD33 therapy, e.g., wherein the subject has a cancer, wherein cells of the cancer express CD33. In some embodiments, the suitable control comprises the level of the wild-type lineage- specific cell surface antigen measured in the cells prior to being subjected to genetic engineering to reduce or eliminate expression of CD33. In some embodiments, a population of genetically engineered hematopoietic cells is referred to as “tremtelectogene empogeditemcel” or “trem-cel.” Accordingly, trem-cel is a product comprising genome-edited hematopoietic stem and progenitor allogeneic donor cells wherein CD33 has been deleted using genome engineering. In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of copies of CD33 in of the cells in trem-cel have a mutation effected by a genome editing approach described herein, e.g. by a CRISPR/Cas system using a gRNA described herein. In some embodiments, trem-cel is manufactured with a CD33 editing efficacy of at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, trem-cel is manufactured with 88% CD33 editing efficacy. In some embodiments, the cells in trem-cel comprises reduction in the expression level of CD33 by at least 50%, at least 60%, at least 70%, at least 80%, 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%, or at least 99% as compared to the expression of CD33 on a naturally occurring hematopoietic cell (e.g., a wild-type counterpart). For example, in some embodiments, the genetically engineered hematopoietic cells of trem-cel express less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of CD33 as compared to a naturally occurring hematopoietic cell (e.g., a wild-type counterpart that has not be genetically engineered). In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cells in trem-cel are CD34+ cells. In some embodiments, 97% of the cells in trem-cel are CD34+.
In some embodiments, a method of making the genetically engineered hematopoietic cells described herein comprises a step of providing a wild-type cell, e.g., a wild-type hematopoietic stem or progenitor cell. In some embodiments, the wild-type cell is an unedited cell comprising (e.g., expressing) two functional copies of the lineage- specific cell surface antigen (e.g., CD33). In some embodiments, the cell comprises a CD33 gene sequence according to SEQ ID NO: 16. In some embodiments, the cell comprises a CD33 gene sequence encoding a CD33 protein that is encoded in SEQ ID NO: 16, e.g., the CD33 gene sequence may comprise one or more silent mutations relative to SEQ ID NO: 16. In some embodiments, the wild-type cell expresses the lineage- specific cell surface antigen (e.g., CD33), or gives rise to a more differentiated cell that expresses the lineage- specific cell surface antigen at a level comparable to (or within 90%-110%, 80%-120%, 70%-130%, 60- 140%, or 50%-150% of) HL60 or MOLM-13 cells. In some embodiments, the wild-type cell binds an antibody that binds the lineage- specific cell surface antigen (e.g., an anti-CD33 antibody, e.g., P67.6), or gives rise to a more differentiated cell that binds the antibody at a level comparable to (or within 90%-110%, 80%-120%, 70%-130%, 60-140%, or 50%-150% of) binding of the antibody to HL60 or MOLM-13 cells. Antibody binding may be measured, for example, by flow cytometry.
In some aspects, the genetically engineered hematopoietic stem or progenitor cell comprises a genetic mutation in the exon 3 of an endogenous CD33 gene, wherein the genetic mutation is at a site described herein (see, Table 1). One aspect of the present disclosure provides a genetically engineered hematopoietic stem and/or progenitor cell comprises a genetic mutation in exon 3 of an endogenous CD33 gene, wherein the genetic mutation is at a site targeted by a gRNA, such as any of the gRNAs presented in Table 1.
In some embodiments, an engineered cell described herein comprises two mutations, the first mutation being in CD33 and the second mutation being in a second lineage- specific cell surface antigen. Such a cell can, in some embodiments, be resistant to two agents: an anti-CD33 agent (e.g., gemtuzumab ozogamicin) and an agent targeting the second lineagespecific cell surface antigen. In some embodiments, such a cell can be produced using two or more gRNAs described herein, e.g., a gRNA of Table 3 and a second gRNA. In some embodiments, the cell can be produced using, e.g. , a ZFN or TALEN. The disclosure also provides populations comprising cells described herein.
In some embodiments, the second mutation is at a gene encoding a lineage- specific cell-surface antigen, such as any of the lineage- specific cell-surface antigens described herein.
Typically, a mutation effected by the methods and compositions provided herein, e.g., a mutation in a target gene, such as, for example, CD33 results in a loss of function of a gene product encoded by the target gene, e.g., in the case of a mutation in the CD33 gene, in a loss of function of a CD33 protein. In some embodiments, the loss of function is a reduction in the level of expression of the gene product, e.g., reduction to a lower level of expression, or a complete abolishment of expression of the gene product. In some embodiments, the mutation results in the expression of a non-functional variant of the gene product. For example, in the case of the mutation generating a premature stop codon in the encoding sequence, a truncated gene product, or, in the case of the mutation generating a nonsense or mis sense mutation, a gene product characterized by an altered amino acid sequence, which renders the gene product non-functional. In some embodiments, the function of a gene product is binding or recognition of a binding partner. In some embodiments, the reduction in expression of the gene product, e.g., of CD33, of the second lineage-specific cell-surface antigen, or both, is to less than or equal to 50%, less than or equal to 40%, less than or equal to 30%, less than or equal to 20%, less than or equal to 10%, less than or equal to 5%, less than or equal to 2%, or less than or equal to 1% of the level in a wild-type or non-engineered counterpart cell.
In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or more of copies of CD33 in the population of genetically engineered hematopoietic cells generated by the methods and/or using the compositions provided herein have a mutation. In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of copies of the second lineage- specific cell surface antigen in the population of genetically engineered hematopoietic cells have a mutation. In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of copies of CD33 and of the second lineagespecific cell surface antigen in the population of genetically engineered hematopoietic cells have a mutation. In some embodiments, the population comprises one or more wild-type cells. In some embodiments, the population comprises one or more cells that comprise one wild-type copy of CD33. In some embodiments, the population comprises one or more cells that comprise one wild-type copy of the second lineage- specific cell surface antigen.
Gemtuzumab ozogamicin (GO)
Aspects of the present disclosure related to administration of gemtuzumab ozogamicin to a subject in need thereof. Gemtuzumab ozogamicin is a recombinant, humanized anti- CD33 monoclonal antibody (IgG4 K antibody hP67.6) linked with (covalently attached to) the cytotoxic antitumor antibiotic calicheamicin (N-acetyl-y-calicheamicin) via a bifunctional linker (4-(4-acetylphenoxy) butanoic acid). Gemtuzumab ozogamicin is available commercially as Mylotarg® (Wyeth Pharmaceuticals, Philadelphia, Pa.). The antibody portion of gemtuzumab ozogamicin, referred to as hP67.6, binds specifically to the CD33 antigen.
Gemtuzumab ozogamicin contains amino acid sequences of which approximately 98.3% are of human origin. The constant region and framework regions contain human sequences while the complementarity-determining regions are derived from a murine antibody (P67.6) that binds CD33. This antibody is linked to N-acetyl-gamma calicheamicin via a bifunctional linker. Gemtuzumab ozogamicin has approximately 50% of the antibody loaded with 4-6 moles calicheamicin per mole of antibody. The remaining 50% of the antibody is not linked to the calicheamicin derivative. Gemtuzumab ozogamicin has a molecular weight of 151 to 153 kDa. Gemtuzumab ozogamicin and methods for making it are described in U.S. Pat. Nos. 5,733,001; 5,739,116; 5,767,285; 5,877,296; 5,606,040; 5,712,374; and 5,714,586, which are incorporated by reference herein in their entirety.
Amino acid sequence of the heavy chain sequence of gemtuzumab ozogamicin/Mylotarg® EVQLVQSGAEVKKPGS SVKVSCKASGYTI TDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNP TNTAYMELS SLRSEDTAFYYCVNGNPWLAYWGQGTLVTVS SASTKGP SVFPLAPCSRSTSESTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQS SGLYSLS SWTVP S S SLGTKTYTCNVDHKP SNTKVDKRVESKYGPPCPPC PAPEFLGGP SVFLFPPKPKDTLMI SRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWS VLTVLHQDWLNGKEYKCKVSNKGLP S S IEKTI SKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYP SDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK ( SEQ ID NO : 26 )
Amino acid sequence of the light chain sequence of gemtuzumab ozogamicin/Mylotarg® DIQLTQSP STLSASVGDRVTI TCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVP SRFSGSGSGTEF TLTI S SLQPDDFATYYCQQTKEVPWSFGQGTKVEVKRTVAAP SVFIFPP SDEQLKSGTASWCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC ( SEQ ID NO : 27 )
Gemtuzumab ozogamicin is an antibody-drug conjugate (ADC) comprising an anti- CD33 antibody conjugated to a toxin or drug molecule. Binding of the antibody or fragment thereof to the corresponding antigen allows for delivery of the toxin or drug molecule to a cell that presents CD33 on its cell surface (e.g., target cell), thereby resulting in death of the target cell.
Administration of gemtuzumab ozogamicin interacts with and induces cytotoxicity of cells expressing CD33, however as described herein, administration of gemtuzumab ozogamicin may induce cytotoxicity of not only cancer cells expressing CD33 but also normal, healthy cells that also express CD33, e.g. i “on-target, off-leukemia” effects.
Methods of Treatment and Administration
Aspects of the present disclosure provide methods involving administering to a subject an effective amount of a population of genetically engineered hematopoietic cells (also referred to herein as eHSCs or eHSPCs), as described herein, and gemtuzumab ozogamicin in a dosing regimen comprising at least one dosing cycle, wherein the dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin. In some embodiments, the subject is diagnosed with a hematopoietic malignancy and is directed to undergo a combination treatment involving administration of a population of genetically engineered hematopoietic cells, as described herein, and gemtuzumab ozogamicin in a dosing regimen comprising at least one dosing cycle, e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more dosing cycles, wherein each dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin. As will be understood by one of ordinary skill in the art, a combination treatment involves more than one aspect of the treatment that may be performed together (e.g., administered at the same time or in a single composition) but also encompasses more than one treatment within a treatment regimen aimed to treat the malignancy, or any symptom or manifestation thereof. For example, some methods described herein involve the combination treatment to treat a hematopoietic malignancy (e.g., acute myeloid leukemia) involving administering an effective amount of a population of genetically engineered hematopoietic cells, as described herein, and gemtuzumab ozogamicin in a dosing regimen comprising at least one dosing cycle (e.g., one, two, three, four dosing cycles), wherein each dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin. In some embodiments, the methods described herein involve the combination treatment to treat a premalignant stage of a hematopoietic malignancy (e.g., myelodysplastic syndrome (MDS)) involving administering an effective amount of a population of genetically engineered hematopoietic cells, as described herein, and gemtuzumab ozogamicin in a dosing regimen comprising at least one dosing cycle (e.g., one, two, three, four dosing cycles), wherein each dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin. Some combination treatment methods provided herein comprise sequential administration of a population of genetically engineered hematopoietic cells (e.g., CD33KO eHSPCs) and gemtuzumab ozogamicin/Mylotarg®, including, for example, administration of the dosing regimen of gemtuzumab ozogamicin/Mylotarg® after administration of the genetically engineered hematopoietic cells.
In some embodiments, an effective number of genetically engineered hematopoietic stem cells, e.g., C 33-modil'icd hematopoietic stem cells described herein, is administered in combination with a dosing regimen of gemtuzumab ozogamicin. In some embodiments, an effective number of cells comprising a modified CD33 and a modified second lineagespecific cell surface antigen are administered in combination with a dosing regimen of gemtuzumab ozogamicin.
In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, e.g., C 33-modil'icd hematopoietic stem cells described herein, comprises about 102 cells/kilogram to about IO10 cells/kilogram body weight of a subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, e.g., C 33-modil'icd hematopoietic stem cells described herein, comprises about 104 cells/kilogram to about 108 cells/kilogram body weight of a subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, e.g., C -modil'icd hematopoietic stem cells described herein, comprises about 106 cells/kilogram to about 108 cells/kilogram body weight of a subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, e.g., C 55-modil'icd hematopoietic stem cells described herein, comprises about 105 cells/kilogram to about 107 cells/kilogram body weight of a subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, e.g., C 55-modil'icd hematopoietic stem cells described herein, comprises about 106 cells/kilogram to about 107 cells/kilogram body weight of a subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, e.g., C 55-modil'icd hematopoietic stem cells described herein, comprises about 105 cells/kilogram, about 106 cells/kilogram, about 107 cells/kilogram, or about 108 cells/kilogram body weight of a subject. In some embodiments, an effective amount a population of genetically engineered hematopoietic stem cells, e.g., C 55-modil'icd hematopoietic stem cells described herein, comprises at least 102 cells, at least 103 cells, at least 104 cells, at least 105 cells, at least 2 x 105 cells, at least 3 x 105 cells, at least 4 x 105 cells, at least 5 x 105 cells, at least 6 x 105 cells, at least 7 x 105 cells, at least 8 x 105 cells, at least 9 x 105 cells, at least 106 cells, at least 2 x 106 cells, at least 3 x 106 cells, at least 4 x 106 cells, at least 5 x 106 cells, at least 6 x 106 cells, at least 7 x 106 cells, at least 8 x 106 cells, at least 9 x 106 cells, at least 1 x 107 cells, or multiples thereof.
In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, e.g., CD33-modified hematopoietic stem cells described herein, comprises about 1.0 x 105, about 2.0 x 105, about 3.0 x 105, about 4.0 x 105, about 5.0 x 105, about 6.0 x 105, about 7.0 x 105, about 8.0 x 105, about 9.0 x 105, about 1.0 x 106, about 2.0 x 106, about 3.0 x 106, about 4.0 x 106, about 5.0 x 106, about 6.0 x 106, about 7.0 x 106, about 8.0 x 106, about 9.0 x 106, about 1.0 x 107, about 2.0 x 107, about 3.0 x 107, about 4.0 x 107, 5.0 x 107, about 6.0 x 107, about 7.0 x 107, about 8.0 x 107, about 9.0 x 107, or about 1.0 x 108 cells/kilogram body weight of a subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, e.g., CD33-modified hematopoietic stem cells described herein, comprises about 3.0 x 106 cells/kilogram body weight of a subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, e.g., CD33-modified hematopoietic stem cells described herein, comprises between about 6 x 106 - 8 x 106 cells/kilogram body weight of a subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic stem cells, e.g., CD33-modified hematopoietic stem cells described herein, comprises about 7.6 x 106 cells/kilogram body weight of a subject. In some embodiments, an effective amount of trem-cel comprises about 3 x 106 cells/kilogram body weight of a subject. In some embodiments, an effective amount of trem-cel comprises about 106 cells/kilogram body weight of a subject. In some embodiments, an effective amount of trem-cel comprises about 5 x 105 cells/kilogram body weight of a subject. In some embodiments, an effective amount of trem-cel comprises about 105 cells/kilogram body weight of a subject.
In some embodiments, an effective amount of trem-cel comprises about 1.0 x 105, about 2.0 x 105, about 3.0 x 105, about 4.0 x 105, about 5.0 x 105, about 6.0 x 105, about 7.0 x 105, about 8.0 x 105, about 9.0 x 105, about 1.0 x 106, about 2.0 x 106, about 3.0 x 106, about 4.0 x 106, about 5.0 x 106, about 6.0 x 106, about 7.0 x 106, about 8.0 x 106, about 9.0 x 106, about 1.0 x 107, about 2.0 x 107, about 3.0 x 107, about 4.0 x 107, 5.0 x 107, about 6.0 x 107, about 7.0 x 107, about 8.0 x 107, about 9.0 x 107, or about 1.0 x 108 cells/kilogram body weight of a subject. In some embodiments, an effective amount of trem-cel comprises between about 6 x 106 - 8 x 106 cells/kilogram body weight of a subject. In some embodiments, an effective amount of trem-cel comprises about 7.6 x 106 cells/kilogram body weight of a subject. In some embodiments, an effective amount of trem-cel comprises about 3 x 106 cells/kilogram body weight of a subject. In some embodiments, an effective amount of trem-cel comprises less than about 3 x 106 cells/kilogram body weight of a subject.
Hematopoietic stem cells, e.g., CD34+ hematopoietic stem cells, that can, at least in some embodiments, serve as the starting material for generating the genetically engineered hematopoietic stem cells, e.g., CD33-modified hematopoietic stem cells described herein, can be derived from one or more donors or can be obtained from an autologous source. In some embodiments, the genetically engineered hematopoietic stem cells, e.g., CD33-modified hematopoietic stem cells described herein, are expanded in culture prior to administration to a subject in need thereof.
A typical number of cells, e.g., immune cells or hematopoietic cells, administered to a mammal (e.g., a human) can be, for example, in the range of one million to 100 billion cells; however, amounts below or above this exemplary range are also within the scope of the present disclosure.
In some embodiments, gemtuzumab ozogamicin is administered in a dosing regimen comprising multiple dosing cycles (e.g., 2, 3, 4 dosing cycles) each comprising administration of an effective amount of gemtuzumab ozogamicin, e.g., in combination with the population of genetically engineered hematopoietic stem cells, e.g., CDAi-modil'icd hematopoietic stem cells described herein. In some embodiments, an effective amount of a gemtuzumab ozogamicin is about 0.01 mg/m2 to about 6.0 mg/m2body surface area of a subject. In some embodiments, an effective amount of a gemtuzumab ozogamicin is about 0.01 mg/m2 to about 9.0 mg/m2 body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin (e.g. , in each dosing cycle) is about 0.05 mg/m2 to about 2.5 mg/m2, about 0.1 mg/m2 to about 2.0 mg/m2, about 0.1 mg/m2 to about 1.0 mg/m2, about 1.0 mg/m2 to about 2.0 mg/m2, or about 1.5 mg/m2 to about 2.5 mg/m2, 0.05 mg/m2 to about 6.0 mg/m2, about 0.1 mg/m2 to about 6.0 mg/m2, about 0.1 mg/m2 to about 3.0 mg/m2, about 1.0 mg/m2 to about 6.0 mg/m2, about 1.5 mg/m2 to about 2.5 mg/m2, about 3.0 mg/m2 to about 6.0 mg/m2 body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 0.05 mg/m2, about 0.1 mg/m2, about 0.25 mg/m2, about 0.5 mg/m2, about 0.6 mg/m2, about 0.7 mg/m2, about 0.8 mg/m2, about 0.9 mg/m2, about 1.0 mg/m2, about 1.1 mg/m2, about 1.2 mg/m2, about 1.3 mg/m2, about 1.4 mg/m2, about 1.5 mg/m2, about 1.6 mg/m2, about 1.7 mg/m2, about 1.8 mg/m2, about 1.9 mg/m2, about 2.0 mg/m2, about 2.1 mg/m2, about 2.2 mg/m2, about 2.3 mg/m2, about
2.4 mg/m2, 2.5 mg/m2, about 2.6 mg/m2, about 2.7 mg/m2, about 2.8 mg/m2, about
2.9 mg/m2, about 3.0 mg/m2, about 3.1 mg/m2, about 3.2 mg/m2, about 3.3 mg/m2, about
3.4 mg/m2, 3.5 mg/m2, about 3.6 mg/m2, about 3.7 mg/m2, about 3.8 mg/m2, about
3.9 mg/m2, about 4.0 mg/m2, about 4.1 mg/m2, about 4.2 mg/m2, about 4.3 mg/m2, about
4.4 mg/m2, 4.5 mg/m2, about 4.6 mg/m2, about 4.7 mg/m2, about 4.8 mg/m2, about
4.9 mg/m2, about 5.0 mg/m2, about 5.1 mg/m2, about 5.2 mg/m2, about 5.3 mg/m2, about
5.4 mg/m2, 5.5 mg/m2, about 5.6 mg/m2, about 5.7 mg/m2, about 5.8 mg/m2, about
5.9 mg/m2, about 6.0 mg/m2, about 6.1 mg/m2, about 6.2 mg/m2, about 6.3 mg/m2, about
6.4 mg/m2, about 6.5 mg/m2, about 6.6 mg/m2, about 6.7 mg/m2, about 6.8 mg/m2, about
6.9 mg/m2, about 7.0 mg/m2, about 7.1 mg/m2, about 7.2 mg/m2, about 7.3 mg/m2, about
7.4 mg/m2, about 7.5 mg/m2, about 7.6 mg/m2, about 7.7 mg/m2, about 7.8 mg/m2, about
7.9 mg/m2, about 8.0 mg/m2, about 8.1 mg/m2, about 8.2 mg/m2, about 8.3 mg/m2, about
8.4 mg/m2, about 8.5 mg/m2, about 8.6 mg/m2, about 8.7 mg/m2, about 8.8 mg/m2, about
8.9 mg/m2, or about 9.0 mg/m2 body surface area of a subject.
In some embodiments, an effective amount of gemtuzumab ozogamicin is about 0.5 mg/m2 body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 1.0 mg/m2 body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 2.0 mg/m2 body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 3.0 mg/m2 body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 4.0 mg/m2body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 5.0 mg/m2body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 6.0 mg/m2body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 7.0 mg/m2body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 8.0 mg/m2body surface area of a subject. In some embodiments, an effective amount of gemtuzumab ozogamicin is about 9.0 mg/m2body surface area of a subject.
In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g., C 55-modil'icd hematopoietic stem cells described herein, is about 104 cells/kilogram to about 108 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 0.1 mg/m2 to about 3.0 mg/m2 body surface area of the subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33-modified hematopoietic stem cells described herein, is about 106 cells/kilogram to about 107 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 0.1 mg/m2 to about 6.0 mg/m2 body surface area of the subject.
In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33-modified hematopoietic stem cells described herein, is about 3.0 x 106 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 0.1 mg/m2, about 0.25 mg/m2, about 0.5 mg/m2, about 1.0 mg/m2, about 2.0 mg/m2, about 3.0 mg/m2, about 4.0 mg/m2, about 5.0 mg/m2, or about 6.0 mg/m2 body surface area of the subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein, is about 3.0 x 106 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 0.5 mg/m2 body surface area of the subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein, is about 3.0 x 106 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 1.0 mg/m2 body surface area of the subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein, is about 3.0 x 106 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 2.0 mg/m2body surface area of the subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein, is about 3.0 x 106 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 3.0 mg/m2body surface area of the subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein, is about 3.0 x 106 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 4.0 mg/m2body surface area of the subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein, is about 3.0 x 106 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 5.0 mg/m2body surface area of the subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein, is about 3.0 x 106 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 6.0 mg/m2body surface area of the subject.
In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g., C 33-modil'icd hematopoietic stem cells described herein, is about 7.6 x 106 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 0.1 mg/m2, about 0.25 mg/m2, about 0.5 mg/m2, about 1.0 mg/m2, about 2.0 mg/m2, about 3.0 mg/m2, about 4.0 mg/m2, about 5.0 mg/m2, or about 6.0 mg/m2 body surface area of the subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein, is about 7.6 x 106 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 0.5 mg/m2 body surface area of the subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein, is about 7.6 x 106 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 1.0 mg/m2 body surface area of the subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein, is about 7.6 x 106 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 2.0 mg/m2body surface area of the subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein, is about 7.6 x 106 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 3.0 mg/m2body surface area of the subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein, is about 7.6 x 106 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 4.0 mg/m2body surface area of the subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein, is about 7.6 x 106 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 5.0 mg/m2body surface area of the subject. In some embodiments, an effective amount of a population of genetically engineered hematopoietic cells, e.g. CD33- modified hematopoietic stem cells described herein, is about 7.6 x 106 cells/kilogram body weight of a subject, and an effective amount of gemtuzumab ozogamicin administered in each dosing cycle is about 6.0 mg/m2body surface area of the subject.
In some embodiments, a population of genetically engineered hematopoietic cells and the dosing regimen of gemtuzumab ozogamicin are administered in temporal proximity. As used herein, in some embodiments, temporal proximity refers to the timing of the administration of the population of genetically engineered hematopoietic cells relative to the administration of gemtuzumab ozogamicin. It should be appreciated that no particular ordering is implied in the use of this terminology unless an ordering is expressly stated. For example, administration of the population of genetically engineered hematopoietic cells and a gemtuzumab ozogamicin in temporal proximity can include administration of the hematopoietic cells prior to, following, or at approximately the same time as the administration of the dosing regimen of gemtuzumab ozogamicin. Furthermore, the treatments may be admixed or in separate volumes. For example, in some embodiments, administration in combination includes administration in the same course of treatment, e.g., in the course of treating a cancer with an anti-CD33 therapy, the subject may be administered an effective number of C -modil'icd cells concurrently or sequentially, e.g., before, during, or after the treatment, with gemtuzumab ozogamicin.
In some embodiments, administering in temporal proximity comprises administering a population of genetically engineered hematopoietic stem cells and gemtuzumab ozogamicin within a single treatment regimen. In some embodiments, administering in temporal proximity comprises administering a population of genetically engineered hematopoietic stem cells and a gemtuzumab ozogamicin dosing regimen simultaneously or concurrently. In some embodiments, administering in temporal proximity comprises administering a population of genetically engineered hematopoietic stem cells and a gemtuzumab ozogamicin dosing regimen sequentially (e.g., administering either treatment before the other treatment). In some embodiments, a population of genetically engineered hematopoietic stem cells is administered prior to a gemtuzumab ozogamicin dosing regimen. In some embodiments, administering in temporal proximity comprises administering a population of genetically engineered hematopoietic stem cells within 120 days (e.g., within 90 days, within 60 days, within 30 days, within 20 days, within 10 days, within 7 days, or within 1 day) of administering a dosing regimen of gemtuzumab ozogamicin. In some embodiments, administering in temporal proximity comprises administering a population of genetically engineered hematopoietic stem cells to a subject within 120 days (e.g., within 90 days, within 60 days, within 30 days, within 20 days, within 10 days, within 7 days, or within 1 day) prior to administering a dosing regimen of gemtuzumab ozogamicin to the subject. In some embodiments, administering in temporal proximity comprises administering a population of genetically engineered hematopoietic stem cells within 365 days (e.g., within 300 days, within 250 days, within 200 days, within 150 days, or within 100 days) of administering a dosing regimen of gemtuzumab ozogamicin.
In some embodiments, administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen at least 60 days after administration of the population of genetically engineered hematopoietic cells. In some embodiments, administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen between 20-60 days after administration of the population of genetically engineered hematopoietic cells, if the subject experiences early relapse. In some embodiments, administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen between 30-60 days after administration of the population of genetically engineered hematopoietic cells, if the subject experiences early relapse. In some embodiments, administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen between 40-60 days after administration of the population of genetically engineered hematopoietic cells, if the subject experiences early relapse. In some embodiments, administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen between 50-60 days after administration of the population of genetically engineered hematopoietic cells, if the subject experiences early relapse.
In some embodiments, the subject is evaluated based on one or more parameters, such as level of engraftment, following administration of the population of genetically engineered hematopoietic cells, and described herein, prior to administration of gemtuzumab ozogamicin. In some embodiments, the subject has a CD33-negative absolute neutrophil count (ANC) above a threshold value (e.g., at least 500 cells/pL, at least 1000 cells/pL) prior to administration of the gemtuzumab ozogamicin.
In some embodiments, the dosing regimen of gemtuzumab ozogamicin comprises dosing cycles, each of which involves administering an effective amount of gemtuzumab ozogamicin. In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in multiple doses, for example at a regular interval (e.g., every day, every two days, every three days, every four days, every five days, every six days, every week (e.g., every 7 days), every two weeks (e.g., every 14 days), every three weeks (e.g., every 21 days), every four weeks (e.g., every 28 days), every month, every two months, every three months, every four months, every five months, or every six months). In some embodiments, each dosing cycle of gemtuzumab ozogamicin is administered in multiple doses of the effective amount every four weeks. For example, in some embodiments, an effective amount of gemtuzumab ozogamicin is administered in a first dosing cycle, which may be followed by one or more subsequent dosing cycles of the effective amount, where each dosing cycle is separated by approximately four weeks (e.g., 28 days). In some embodiments, each dosing cycle is separated by about two weeks to about six weeks (e.g., about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about three weeks to about five weeks, or about four weeks to about six weeks). In some embodiments, an effective amount of gemtuzumab ozogamicin is administered to a subject in at least one dose, at least two doses, at least three doses, at least four doses, between one and six doses, between one and four doses, or between one and three doses. In some embodiments, the doses of gemtuzumab ozogamicin in a dosing cycle are administered to a subject daily for the duration of the dosing cycle. In some embodiments, the doses of gemtuzumab ozogamicin in a dosing cycle are administered to a subject weekly for the duration of the dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is about 0.5 mg/m2. In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in multiple doses of about 0.5 mg/m2 every four weeks. In some embodiments, the effective amount of gemtuzumab ozogamicin is 0.5 mg/m2 and is administered to the subject in a single dose per dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 0.5 mg/m2 and is administered to the subject in a multiple, fractionated dose per dosing cycle, such that the total gemtuzumab ozogamicin administered to the subject per dosing cycle is 0.5 mg/m2.
In some embodiments, the effective amount of gemtuzumab ozogamicin is about 1.0 mg/m2. In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in multiple doses of about 1.0 mg/m2 every four weeks. In some embodiments, the effective amount of gemtuzumab ozogamicin is 1.0 mg/m2 and is administered to the subject in a single dose per dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 1.0 mg/m2 and is administered to the subject in a multiple, fractionated dose per dosing cycle, such that the total gemtuzumab ozogamicin administered to the subject per dosing cycle is 1.0 mg/m2.
In some embodiments, the effective amount of gemtuzumab ozogamicin is about 2.0 mg/m2. In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in multiple doses of about 2.0 mg/m2 every four weeks. In some embodiments, the effective amount of gemtuzumab ozogamicin is 2.0 mg/m2 and is administered to the subject in a single dose per dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 2.0 mg/m2 and is administered to the subject in a multiple, fractionated dose per dosing cycle, such that the total gemtuzumab ozogamicin administered to the subject per dosing cycle is 2.0 mg/m2.
In some embodiments, the effective amount of gemtuzumab ozogamicin is about 3.0 mg/m2. In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in multiple doses of about 3.0 mg/m2 every four weeks. In some embodiments, the effective amount of gemtuzumab ozogamicin is 3.0 mg/m2 and is administered to the subject in a single dose per dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 3.0 mg/m2 and is administered to the subject in a multiple, fractionated dose per dosing cycle, such that the total gemtuzumab ozogamicin administered to the subject per dosing cycle is 3.0 mg/m2.
In some embodiments, the effective amount of gemtuzumab ozogamicin is about 4.0 mg/m2. In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in multiple doses of about 4.0 mg/m2 every four weeks. In some embodiments, the effective amount of gemtuzumab ozogamicin is 4.0 mg/m2 and is administered to the subject in a single dose per dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 4.0 mg/m2 and is administered to the subject in a multiple, fractionated dose per dosing cycle, such that the total gemtuzumab ozogamicin administered to the subject per dosing cycle is 4.0 mg/m2.
In some embodiments, the effective amount of gemtuzumab ozogamicin is about 5.0 mg/m2. In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in multiple doses of about 5.0 mg/m2 every four weeks. In some embodiments, the effective amount of gemtuzumab ozogamicin is 5.0 mg/m2 and is administered to the subject in a single dose per dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 5.0 mg/m2 and is administered to the subject in a multiple, fractionated dose per dosing cycle, such that the total gemtuzumab ozogamicin administered to the subject per dosing cycle is 5.0 mg/m2.
In some embodiments, the effective amount of gemtuzumab ozogamicin is about 6.0 mg/m2. In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in multiple doses of about 6.0 mg/m2 every four weeks. In some embodiments, the effective amount of gemtuzumab ozogamicin is 6.0 mg/m2 and is administered to the subject in a single dose per dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 6.0 mg/m2 and is administered to the subject in a multiple, fractionated dose per dosing cycle, such that the total gemtuzumab ozogamicin administered to the subject per dosing cycle is 6.0 mg/m2.
In some embodiments, the effective amount of gemtuzumab ozogamicin is about 9.0 mg/m2. In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in three doses of about 3.0 mg/m2. In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in two doses, e.g., one dose of about 6.0 mg/m2 and one dose of about 3.0 mg/m2. In some embodiments, the effective amount of gemtuzumab ozogamicin is 9.0 mg/m2 and is administered to the subject in a multiple, fractionated dose per dosing cycle, such that the total gemtuzumab ozogamicin administered to the subject per dosing cycle is 9.0 mg/m2.
In some embodiments, a subject in need of treatment in accordance with the present disclosure has been identified as having newly-diagnosed, de novo CD33-positive AML. When given as part of a combination regimen for the treatment of newly-diagnosed, de novo CD33-positive AML, the recommended treatment course including gemtuzumab ozogamicin consists of 1 induction cycle and 2 consolidation cycles. For the induction cycle, the recommended dose of gemtuzumab ozogamicin is 3 mg/m2 (up to one 4.5 mg vial) on days 1, 4, and 7 in combination with daunorubicin and cytarabine. For subjects requiring a second induction cycle, gemtuzumab ozogamicin is not administered during the second induction cycle. For the consolidation cycles, the recommended dose of gemtuzumab ozogamicin is 3 mg/m2 on day 1 (up to one 4.5 mg vial) in combination with daunorubicin and cytarabine.
In some embodiments, a subject in need of treatment in accordance with the present disclosure has been identified as having newly-diagnosed CD33-positive AML. In some embodiments, the dosing regimen of gemtuzumab ozogamicin comprises 2, 3, or 4 dosing cycles, each comprising administration of an effective amount of gemtuzumab ozogamicin, wherein the effective amount is 0.1 mg/m2 - 3.0 mg/m2 body surface area of the subject. In some embodiments, the effective amount is administered in a single dose, e.g.t that is administered on day 1 of each dosing cycle. In some embodiments, the effective amount is administered in multiple doses, e.g.t that are administered on prescribed days of each dosing cycle.
In some embodiments, the effective amount is administered in multiple doses, e.g.t at least 2, 3, 4, 5, or more doses, which may be referred to as fractionated dosing.
In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in two doses in a dosing cycle. In some embodiments, a first dose is administered to the subject on day 1 of the dosing cycle and a second dose is administered on any of days 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 of the dosing cycle.
In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in three doses in a dosing cycle. In some embodiments, the effective amount is administered in multiple doses, and a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered on any of days 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 of the dosing cycle, and a third dose is administered on any one of days 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28, such that administration of the third dose is after administration of the second dose. In some embodiments, the effective amount is administered in multiple doses, and a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered in day 4 of the dosing cycle, and a third dose is administered on day 7 of the dosing cycle. In some embodiments, the effective amount is administered in multiple doses, and a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered in day 7 of the dosing cycle, and a third dose is administered on day 14 of the dosing cycle.
In some embodiments, the effective amount of gemtuzumab ozogamicin is administered in four doses in a dosing cycle. In some embodiments, the effective amount is administered in multiple doses, and a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered on any of days 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 of the dosing cycle, a third dose is administered on any one of days 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28, such that administration of the third dose is after administration of the second dose, and a fourth dose is administered on any one of days 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28, such that administration of the fourth dose is after administration of the third dose. In some embodiments, the effective amount is administered in multiple doses, and a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered in day 7 of the dosing cycle, a third dose is administered on day 14 of the dosing cycle, and a fourth dose is administered on day 21 of the dosing cycle.
As will be evident to one of ordinary skill in the art, each of the doses of the multiple doses of a dosing cycle may be of the same or different amounts of gemtuzumab ozogamicin. In some embodiments, the effective amount of gemtuzumab ozogamicin is 9 mg/m2 and is administered in multiple doses of 3 mg/m2, wherein a first dose of 3 mg/m2 is administered to the subject on day 1, a second dose of 3 mg/m2 is administered to the subject day 4 of the dosing cycle, and a third dose of 3 mg/m2 is administered to the subject day 7 of the dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 9 mg/m2 and is administered in multiple doses, wherein a first dose of 6 mg/m2 is administered to the subject and a second dose of 3 mg/m2 is administered on a subsequent day of the dosing cycle. In some embodiments, the effective amount of gemtuzumab ozogamicin is 9 mg/m2 and is administered in multiple doses, wherein a first dose of 6 mg/m2 is administered to the subject on day 1 of the dosing cycle, a second dose of 3 mg/m2 is administered on day 8 of the dosing cycle.
In some embodiments, the effective amount is administered in multiple doses, and a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered to the subject once the plasma concentration of gemtuzumab ozogamicin in the subject is less than a threshold value, and a third dose is administered to the subject once the plasma concentration of gemtuzumab ozogamicin in the subject is less than a threshold value following administration of the second dose. In some embodiments, the methods further comprise monitoring the plasma concentration of gemtuzumab ozogamicin. In some embodiments, if the plasma concentration of gemtuzumab ozogamicin is below a threshold value, one or more additional doses of gemtuzumab ozogamicin are administered.
In some embodiments, a threshold value of gemtuzumab ozogamicin in the plasma of a subject comprises an AUC of 60,000 nanograms (ng) of gemtuzumab ozogamicin x hours (h) since the last measurable time point that gemtuzumab ozogamicin was administered to the subject per mililiter (mL) of plasma sample (ng x h/mL) or less.
In some embodiments, the subject is monitored following administration of one or more doses of gemtuzumab ozogamicin in a dosing cycle and may be administered one or more additional doses, based on factors such as the presence of minimal residual disease (MRD), pharmacokinetic measures, toxicity, and/or other functional tests.
In some embodiments, a subject is pretreated with one or more of a corticosteroid, antihistamine, and acetaminophen prior to administration of gemtuzumab ozogamicin. In some embodiments, the subject is pretreated approximately 1 hour (e.g., about 30 minutes to 1.5 hours, about 45 minutes to 1.5 hours, about 1 to 2 hours, or about 45 minutes to 1 hour) prior to administration of gemtuzumab ozogamicin. In some embodiments, a subject is pretreated with approximately 650 mg acetaminophen (e.g., orally) and approximately 50 mg diphenhydramine (e.g., orally or intravenously) 1 hour prior to administration of gemtuzumab ozogamicin. In some embodiments, a subject is pretreated with approximately 1 mg/kg methylprednisolone or an equivalent dose of an alternative corticosteroid within 30 minutes prior to administration of gemtuzumab ozogamicin. Pediatric subjects may be pretreated with acetaminophen 15 mg/kg (maximum of 650 mg), diphenhydramine 1 mg/kg (maximum of 50 mg), and 1 mg/kg methylprednisolone orally or intravenously; additional doses of acetaminophen and diphenhydramine may be administered every 4 hours after the initial pretreatment dose. Pretreatment may be repeated with the same dose of methylprednisolone or an equivalent corticosteroid for any sign of an infusion reaction, such as fever, chills, hypotension, or dyspnea during the infusion or within 4 hours afterwards.
In some embodiments, the subject does not have a homozygous dominant genotype for CD33 single nucleotide polymorphism (SNP) rs 12459419. In some embodiments, the subject does not have acute promyelocytic leukemia or chronic myeloid leukemia. In some embodiments, the subject does not have a genetic translocation associated with acute promyelocytic leukemia or chronic myeloid leukemia, optionally wherein the genetic translocation is t(15; 17)(q22; q21) or t(9; 22)(q34; ql l). In some embodiments, the subject has not previously received autologous or allogeneic stem cell transplantation. In some embodiments, the subject has not previously received gemtuzumab ozogamicin.
In some embodiments, the method further comprises determining a percent donor chimerism and/or a level of CD33-negative myeloid hematopoiesis in a peripheral blood sample from the subject.
In some embodiments, gemtuzumab ozogamicin is reconstituted from a lyophilized form prior to administration. In some embodiments, the lyophilized form comprises approximately 4.5 mg of a lyophilized cake or powder. In some embodiments, the lyophilized form comprises a lyophilized cake or powder in a single-dose vial for reconstitution and/or dilution.
In some embodiments, a subject has been preconditioned prior to administration of gemtuzumab ozogamicin and/or a population of genetically engineered hematopoietic stem cells. In some embodiments, preconditioning of a subject comprises administering one or more chemotherapeutic agents to the subject. Examples of chemotherapeutic agents include, without limitation, busulfan, melphalan, fludarabine, cyclophosphamide, and thiotepa. In some embodiments, preconditioning comprises total body irradiation of a subject. In some embodiments, preconditioning comprises administering antibodies that bind human T cells (e.g., rabbit anti-thymocyte globulins (rATG), equine anti-thymocyte globulins (eATG)). In some embodiments, preconditioning occurs within two weeks (e.g., within 14 days, within 12 days, within 10 days, within 9 days, within 7 days) prior to administration of gemtuzumab ozogamicin and/or hematopoietic cells. In some embodiments, preconditioning occurs over a period of about one day to about ten days. In some embodiments, preconditioning occurs over a period of about nine days.
Further aspects of the disclosure relate to methods of treating a subject (e.g. , a human subject, such as one having or suspected of having a hematopoietic malignancy) which comprise administering gemtuzumab ozogamicin in an amount which is based on measurements of CD33 antigen density on hematopoietic cells in a biological sample from a subject. CD33 antigen density refers to the number of CD33 molecules on a cell, e.g., the surface of hematopoietic cell. Generally, cells or tissues thereof having higher a higher density of CD33 antigens relative to cells with reduced or eliminated expression of wild-type CD33 (e.g., hematopoietic cancer cells comprising a mutated gene encoding a mutant CD33 or hematopoietic cells that are genetically engineered in a gene encoding CD33) are capable of binding to higher amounts of gemtuzumab ozogamicin and, therefore, may exhibit higher levels of gemtuzumab ozogamicin-dependent cytotoxic effects. The CD33 antigen density on hematopoietic cells in a subject from subject who has received hematopoietic cell transplant with a population of genetically engineered hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen, would be expected to be lower than the CD33 antigen density on hematopoietic cells that express wild-type CD33 and/or have not been genetically engineered.
In some embodiments, the effective amount of gemtuzumab ozogamicin administered to the subject is 0.1 mg/m2 - 6.0 mg/m2 body surface area of the subject. In some embodiments, a method comprises administering an effective amount of gemtuzumab ozogamicin administering and an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen. In some embodiments, the effective amount of the population of genetically modified hematopoietic cells, or descendants thereof is 5 x 107 cells/kilogram body weight of the subject. Administration of gemtuzumab ozogamicin and/or the population of genetically modified hematopoietic cells, or descendants thereof can be performed using any of the methods provided by the disclosure.
Non-limiting examples of biological samples (e.g., the first biological sample and/or the second biological sample) include whole blood samples, plasma samples, blood samples processed to enrich for white blood cells, bone marrow samples, or peripheral blood samples. The first biological sample can be obtained from the subject at a first time point and the second biological sample can be obtained from the subject at a second time point occurring after the first time point. Alternatively, the second biological sample is obtained from a counterpart subject (e.g., a sample obtained directly from the second subject or obtained from a banked sample collected from the second subject).
Methods for detecting CD33 antigen density can involve, without limitation, quantitative protein expression analyses (e.g., flow cytometry, enzyme-linked immunosorbent assay (ELISA), electrochemiluminescence assays, such as meso-scale detection (MSD) assays), western blot), quantitative RNA analyses (e.g., real-time quantitative polymerase chain reaction (RT-qPCR)), or any combination thereof. In some embodiments, detecting CD33 antigen density comprises flow cytometry. In some embodiments, flow cytometry is performed using an antibody or other labeling agent comprising an antigen-binding fragment that binds to the same regions of CD33 that is targeted by gemtuzumab ozogamicin. In some embodiments, the antibody is hP67.6 or the labeling agent comprising antigen-binding fragment thereof.
In some embodiments, a first biological sample obtained from a subject is identified as having hematopoietic cells comprising a lower density of CD33 (e.g., wild-type CD33) relative to a second biological sample. In some embodiments, the lower density of CD33 (e.g., wild-type CD33) in the first biological sample relative to the second biological sample is characterized by an expression level of CD33 (e.g., wild-type CD33) in the second biological sample which is 95% or less than an expression level of CD33 (e.g., wild-type CD33) in the first biological sample. In some embodiments, the lower density of CD33 in the first biological sample relative to the second biological sample is characterized by an expression level of CD33 in the first biological sample that is 90-95%, 85-90%, 80-85%, 75- 80%, 70-75%, 60-70%, 50-60%, 40-50%, 30-40%, 20-30%, or less than 20% than the expression level of CD33 in the second biological sample. In some embodiments, the lower density of CD33 (e.g., wild-type CD33) in the first biological sample relative to the second biological sample is characterized by an expression level of CD33 in the first biological sample that is 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or less than 10% than the expression level of CD33 in the second biological sample. In some embodiments, a lower density of CD33 (e.g., wild-type CD33) is measured in a first biological sample which was obtained from a subject experiencing remission from a hematopoietic malignancy and a second biological sample is obtained from the subject after the subject has experienced or is suspected of experiencing a relapse of the hematopoietic malignancy.
In some embodiments, the effective amount of gemtuzumab ozogamicin administered to the subject is 2.0 mg/m2 or less (e.g., 0.1-0.25 mg/m2, 0.25-0.5 mg/m2, 0.5-1.0 mg/m2, or 1.0-2.0 mg/m2) when the expression level of CD33 in the first biological sample is 75% or less than the expression level of CD33 in the second biological sample. In some embodiments, the effective amount of gemtuzumab ozogamicin administered to the subject is about 0.1 mg/m2, 0.25 mg/m2, about 0.5 mg/m2, about 1.0 mg/m2, or about 2.0 mg/m2 when the expression level of CD33 in the first biological sample is 50% or less than the expression level of CD33 in the second biological sample. In some embodiments, the effective amount of gemtuzumab ozogamicin administered to the subject is more than 2.0 mg/m2 (e.g., 2.0- 3.0 mg/m2, 3.0-4.0 mg/m2, 4.0-5.0 mg/m2, or 5.0-6.0 mg/m2) when the expression level of CD33 in the first biological sample is more than 75% of the expression level of CD33 in the second biological sample. In some embodiments, a composition of the disclosure (e.g., a population of hematopoietic cells, gemtuzumab ozogamicin dosing regimen) may be administered via a route such as, but not limited to, enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra- amniotic, intraarticular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intraci sternal (within the cistema magna cerebellomedularis), intracorneal (within the cornea), dental intracornal, intracoronary (within the coronary arteries), intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidermal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), intralymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intramyocardial (within the myocardium), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intrapro static (within the prostate gland), intrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumor (within a tumor), intratympanic (within the aurus media), intravascular (within a vessel or vessels), intraventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique (topical route administration which is then covered by a dressing which occludes the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis and spinal. As will be appreciated by one of ordinary skill in the art, administration of the population of genetically engineered hematopoietic cells and the gemtuzumab ozogamicin may be performed by the same administration route (e.g., intravenous infusion) or by different administration routes.
Modes of administration include injection, infusion, instillation, and/or ingestion. "Injection" includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracap sular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrastemal injection and infusion. In some examples, the route is intravenous. For the delivery of cells, administration by injection or infusion can be made. In some embodiments, a population of genetically engineered hematopoietic stem cells can be administered systemically. The phrases “systemic administration,” “administered systemically,” “peripheral administration,” and “administered peripherally” refer to the administration of a population of progenitor cells other than directly into a target site, tissue, or organ, such that it enters, instead, the subject's circulatory system and, thus, is subject to metabolism and other like processes.
The efficacy of a treatment having a composition for the treatment of a hematopoietic malignancy (e.g., AML) can be determined by the skilled clinician. However, a treatment is considered “effective treatment,” if any one or all of the signs or symptoms of the hematopoietic malignancy are altered in a beneficial manner, or other clinically accepted symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of an individual to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein. Treatment includes any treatment of a disease in an individual or an animal (some nonlimiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.
Also provided herein are clinical preparations comprising populations of cells comprising any of the genetically modified hematopoietic cells, or descendants thereof, described herein. In some embodiments, the composition comprises a population of at least 1 x 106 cells per milliliter (mL) in a medium, wherein the population of cells comprise genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen. In some embodiments, the population comprises at least 2 x 106 cells per mL, at least 3 x 106 cells per mL, at least 4 x 106 cells per mL, at least 5 x 106 cells per mL, at least 6 x 106 cells per mL, at least at least 7 x 106 cells per mL, at least 8 x 106 cells per mL, or at least 9 x 106 cells per mL.
In some embodiments, the medium has a volume between about 5-150 mL. In some embodiments, the medium has a volume between about 10-100 mL. In some embodiments, the medium has a volume between about 25-75 mL. In some embodiments, the medium has a volume between about 30-70 mL. In some embodiments, the medium has a volume between about 40-60 mL. In some embodiments, the medium has a volume of about 45 mL. In some embodiments, the medium has a volume of about 30 mL. In some embodiments, the medium has a volume of about 35 mL. In some embodiments, the medium has a volume of about 40 mL. In some embodiments, the medium has a volume of about 50 mL. In some embodiments, the medium has a volume of about 55 mL. In some embodiments, the medium has a volume of about 60 mL. In some embodiments, the medium has a volume of about 70 mL. In some embodiments, the medium has a volume between about 40-50 mL. In some embodiments, the medium has a volume of about 40 mL, 41 mL, 42 mL, 43 mL, 44 mL, 45 mL, 46 mL, 47 mL, 48 mL, 49 mL, or about 50 mL. In some embodiments, the medium has a volume of about 45 mL. In some embodiments, the composition comprises a population of between about 1 x 106 - 1 x 108 cells total in the medium. In some embodiments, the composition comprises a population of about 1 x 107, 2 x 107, 3 x 107, 4 x 107, 5 x 107, 6 x 107, 7 x 107, 8 x 107, 9 x 107, or 1 x 108 cells total in the medium. In some embodiments, the population comprises at least 0.5 x 106 cells per mL, at least 1 x 106 cells per mL, at least 2 x 106 cells per mL, at least 3 x 106 cells per mL, at least 4 x 106 cells per mL, at least 5 x 106 cells per mL, at least 6 x 106 cells per mL, at least at least 7 x 106 cells per mL, at least 8 x 106 cells per mL, or at least 9 x 106 cells per mL. In some embodiments, the population comprises at least 0.5 x 106 cells per mL, at least 1 x 106 cells per mL, at least 2 x 106 cells per mL, at least 3 x 106 cells per mL, at least 4 x 106 cells per mL, at least 5 x 106 cells per mL, at least 6 x 106 cells per mL, at least at least 7 x 106 cells per mL, at least 8 x 106 cells per mL, or at least 9 x 106 cells per mL. In some embodiments, the cell population comprises at least 1 x 109 viable cells, at least 2 x 109 viable cells, at least 3 x 109 viable cells, at least 4 x 109 viable cells, at least 5 x 109 viable cells, at least 6 x 109 viable cells, at least 7 x 109 viable cells, at least 8 x 109 viable cells, at least 9 x 109 viable cells, at least 1 x IO10 viable cells, at least 2 x IO10 viable cells, at least 3 x IO10 viable cells, at least 4 x IO10 viable cells, at least 5 x IO10 viable cells, at least 6 x IO10 viable cells, at least 7 x IO10 viable cells, at least 8 x IO10 viable cells, at least 9 x IO10 viable cells, or at least 1 x 1011 viable cells, wherein, in some embodiments, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of the cells of the population are genetically modified hematopoietic cells, or descendants thereof, having reduced or eliminated expression of a CD33 antigen.
In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells of population are genetically modified hematopoietic cells, or descendants thereof, having reduced or eliminated expression of a CD33 antigen.
In some embodiments, the medium is a cryopreservation medium comprising a cryoprotectant. Non-limiting examples of cryoprotects include cetamide, agarose, alginate, 1-analine, albumin, ammonium acetate, butanediol, chondroitin sulfate, chloroform, choline, diethylene glycol, dimethyl acetamide, dimethyl formamide, dimethylsulfoxide (DMSO), erythritol, ethanol, ethylene glycol, formamide, glucose, glycerol, a-glycerol phosphate, glycerol monoacetate, glycine, hydroxyethyl starch, inositol, lactose, magnesium chloride, magnesium sulfate, maltose, mannitol, mannose, methanol, methyl acetamide, methylformamide, methyl urea, phenol, pluronic polyol, polyethylene glycol, polyvinylpyrrolidone, proline, propylene glycol, pyridine N-oxide, ribose, serine, sodium bromide, sodium chloride, sodium iodide, sodium nitrate, sodium sulfate, sorbitol, sucrose, trehalose, triethylene glycol, trimethylamine acetate, urea, valine, and xylose In some embodiments, the cryoprotectant comprises dimethylsulfoxide (DMSO). In some embodiments, the cryoprotectant comprises DMSO in an amount of about 10% (v/v).
In some embodiments, the composition is in a frozen state. In some embodiments, the composition has been subjected to a cryopreservation process. As will be evident one of ordinary skill in the art, cryopreservation processes are methods aimed, for example, to preserve (viable) cells by cooling and storing a sample comprising the cells at a low temperature (e.g., at or below -80 °C). In some embodiments, the cryopreservation process is controlled-rate freezing.
Genomic Editing
Aspects of the present disclosure relate to populations of genetically engineered hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen. The gene encoding CD33 may be engineered by any means known in the art such that the cell has reduced or eliminated expression of a CD33 antigen.
The term “binds”, as used herein with reference to a gRNA interaction with a target domain, refers to the gRNA molecule and the target domain forming a complex. The complex may comprise two strands forming a duplex structure, or three or more strands forming a multi-stranded complex. The binding may constitute a step in a more extensive process, such as the cleavage of the target domain by a Cas endonuclease. In some embodiments, the gRNA binds to the target domain with perfect complementarity, and in other embodiments, the gRNA binds to the target domain with partial complementarity, e.g., with one or more mismatches. In some embodiments, when a gRNA binds to a target domain, the full targeting domain of the gRNA base pairs with the targeting domain. In other embodiments, only a portion of the target domain and/or only a portion of the targeting domain base pairs with the other. In an embodiment, the interaction is sufficient to mediate a target domain-mediated cleavage event.
A “Cas9 molecule” as that term is used herein, refers to a molecule or polypeptide that can interact with a gRNA and, in concert with the gRNA, home or localize to a site which comprises a target domain. Cas9 molecules include naturally occurring Cas9 molecules and engineered, altered, or modified Cas9 molecules that differ, e.g., by at least one amino acid residue, from a naturally occurring Cas9 molecule. The terms “gRNA” and “guide RNA” are used interchangeably throughout and refer to a nucleic acid that promotes the specific targeting or homing of a gRNA/Cas9 molecule complex to a target nucleic acid. A gRNA can be unimolecular (having a single RNA molecule), sometimes referred to herein as sgRNAs, or modular (comprising more than one, and typically two, separate RNA molecules). A gRNA may bind to a target domain in the genome of a host cell. The gRNA (e.g., the targeting domain thereof) may be partially or completely complementary to the target domain. The gRNA may also comprise a “scaffold sequence,” (e.g., a tracrRNA sequence), that recruits a Cas9 molecule to a target domain bound to a gRNA sequence (e.g., by the targeting domain of the gRNA sequence). The scaffold sequence may comprise at least one stem loop structure and recruits an endonuclease. Exemplary scaffold sequences can be found, for example, in Jinek, et al. Science (2012) 337(6096):816-821, Ran, et al. Nature Protocols (2013) 8:2281-2308, PCT Publication No. WO2014/093694, and PCT Publication No. WO2013/176772.
The term “mutation” is used herein to refer to a genetic change (e.g., insertion, deletion, or substitution) in a nucleic acid compared to a reference sequence, e.g., the corresponding wild-type nucleic acid. In some embodiments, a mutation to a gene detargetizes the protein produced by the gene. As used herein, the term “detargetizes” refers to mutating a gene such that the protein produced by the gene is no longer recognized (“targeted”), or is recognized to a lesser extent, by an agent that binds to the corresponding wildtype protein that has not been mutated. In some embodiments, the gene is mutated such that is produces a protein that lacks an epitope that is bound/recognized by an antigen that targets the protein. In some embodiments, a detargetized CD33 protein is not bound by, or is bound at a lower level by, an agent that targets CD33.
The “targeting domain” of the gRNA is complementary to the “target domain” on the target nucleic acid. The strand of the target nucleic acid comprising the nucleotide sequence complementary to the core domain of the gRNA is referred to herein as the “complementary strand” of the target nucleic acid. Guidance on the selection of targeting domains can be found, e.g., in Fu Y et al, Nat Biotechnol (2014) 32: 279-284 (doi: 10.1038/nbt.2808) and Sternberg SH et al., Nature (2014) 507(7490): 62-7 (doi: 10.1038/naturel3011).
Nucleases
In some embodiments, a cell (e.g., HSC or HPC) described herein is made using a nuclease described herein. Exemplary nucleases include Cas molecules (e.g., Cas9 or Casl2a), TALENs, ZFNs, and meganucleases. In some embodiments, a nuclease is used in combination with a CD33 gRNA described herein (e.g.. according to Table 3).
Cas9 molecules
In some embodiments, a CD33 gRNA described herein is complexed with a Cas9 molecule. Various Cas9 molecules can be used. In some embodiments, a Cas9 molecule is selected that has the desired PAM specificity to target the gRNA/Cas9 molecule complex to the target domain in CD33. In some embodiments, genetically engineering a cell also comprises introducing one or more (e.g., 1, 2, 3 or more) Cas9 molecules into the cell.
Cas9 molecules of a variety of species can be used in the methods and compositions described herein. In embodiments, the Cas9 molecule is of, or derived from, .S'. pyogenes (SpCas9), .S', aureus (SaCas9) or .S', thermophilus. Additional suitable Cas9 molecules include those of, or derived from, Staphylococcus aureus, Neisseria meningitidis (NmCas9), Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., Cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Brady rhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni (CjCas9), Campylobacter lari, Candidatus punic eispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tistrella mobilis, Treponema sp., or Verminephrobacter eiseniae.
In some embodiments, the Cas9 molecule is a naturally occurring Cas9 molecule. In some embodiments, the Cas9 molecule is an engineered, altered, or modified Cas9 molecule that differs, e.g., by at least one amino acid residue, from a reference sequence, e.g., the most similar naturally occurring Cas9 molecule or a sequence of Table 50 of PCT Publication No. W02015/157070, which is herein incorporated by reference in its entirety.
A naturally occurring Cas9 molecule typically comprises two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe; each of which further comprises domains described, e.g., in PCT Publication No. WO 2015/157070, e.g., in Figs. 9A-9B therein, which is incorporated herein by reference in its entirety.
The REC lobe comprises the arginine-rich bridge helix (BH), the RECI domain, and the REC2 domain. The REC lobe appears to be a Cas9-specific functional domain. The BH domain is a long alpha helix and arginine rich region and comprises amino acids 60-93 of the sequence of .S'. pyogenes Cas9. The RECI domain is involved in recognition of the repeat: anti-repeat duplex, e.g., of a gRNA or a tracrRNA. The RECI domain comprises two RECI motifs at amino acids 94 to 179 and 308 to 717 of the sequence of .S', pyogenes Cas9. These two RECI domains, though separated by the REC2 domain in the linear primary structure, assemble in the tertiary structure to form the RECI domain. The REC2 domain, or parts thereof, may also play a role in the recognition of the repeat: anti-repeat duplex. The REC2 domain comprises amino acids 180-307 of the sequence of .S', pyogenes Cas9.
The NUC lobe comprises the RuvC domain (also referred to herein as RuvC-like domain), the HNH domain (also referred to herein as HNH-like domain), and the PAM- interacting (PI) domain. The RuvC domain shares structural similarity to retroviral integrase superfamily members and cleaves a single strand, e.g., the non-complementary strand of the target nucleic acid molecule. The RuvC domain is assembled from the three split RuvC motifs (RuvC I, RuvCII, and RuvCIII, which are often commonly referred to in the art as RuvCI domain, or N-terminal RuvC domain, RuvCII domain, and RuvCIII domain) at amino acids 1-59, 718-769, and 909-1098, respectively, of the sequence of .S'. pyogenes Cas9. Similar to the RECI domain, the three RuvC motifs are linearly separated by other domains in the primary structure, however in the tertiary structure, the three RuvC motifs assemble and form the RuvC domain. The HNH domain shares structural similarity with HNH endonucleases, and cleaves a single strand, e.g., the complementary strand of the target nucleic acid molecule. The HNH domain lies between the RuvC II-III motifs and comprises amino acids 775-908 of the sequence of .S', pyogenes Cas9. The PI domain interacts with the PAM of the target nucleic acid molecule and comprises amino acids 1099-1368 of the sequence of .S'. pyogenes Cas9.
Crystal structures have been determined for naturally occurring bacterial Cas9 molecules (Jinek et al., Science (2014) 343(6176): 1247997) and for .S'. pyogenes Cas9 with a guide RNA (e.g., a synthetic fusion of crRNA and tracrRNA) (Nishimasu et al., Cell (2014) 156:935-949; and Anders et al., Nature (2014) doi: 10.1038/naturel3579).
In some embodiments, a Cas9 molecule described herein has nuclease activity, e.g., double strand break activity. In some embodiments, the Cas9 molecule has been modified to inactivate one of the catalytic residues of the endonuclease. In some embodiments, the Cas9 molecule is a nickase and produces a single stranded break. See, e.g., Dabrowska et al. Frontiers in Neuroscience (2018) 12(75). It has been shown that one or more mutations in the RuvC and HNH catalytic domains of the enzyme may improve Cas9 efficiency. See, e.g., Sarai et al. Currently Pharma. Biotechnol. (2017) 18(13). In some embodiments, the Cas9 molecule is fused to a second domain, e.g., a domain that modifies DNA or chromatin, e.g., a deaminase or demethylase domain. In some such embodiments, the Cas9 molecule is modified to eliminate its endonuclease activity.
In some embodiments, a Cas9 molecule described herein is administered together with a template for homology directed repair (HDR). In some embodiments, a Cas9 molecule described herein is administered without an HDR template.
In some embodiments, the Cas9 molecule is modified to enhance specificity of the enzyme (e.g., reduce off-target effects, maintain robust on-target cleavage). In some embodiments, the Cas9 molecule is an enhanced specificity Cas9 variant (e.g., eSPCas9). See, e.g., Slaymaker et al. Science (2016) 351 (6268): 84-88. In some embodiments, the Cas9 molecule is a high fidelity Cas9 variant (e.g., SpCas9-HFl). See, e.g., Kleinstiver et al. Nature (2016) 529: 490-495.
Various Cas9 molecules are known in the art and may be obtained from various sources and/or engineered/modified to modulate one or more activities or specificities of the enzymes. In some embodiments, the Cas9 molecule has been engineered/modified to recognize one or more PAM sequence. In some embodiments, the Cas9 molecule has been engineered/modified to recognize one or more PAM sequence that is different than the PAM sequence the Cas9 molecule recognizes without engineering/modification. In some embodiments, the Cas9 molecule has been engineered/modified to reduce off-target activity of the enzyme.
In some embodiments, the nucleotide sequence encoding the Cas9 molecule is modified further to alter the specificity of the endonuclease activity (e.g., reduce off-target cleavage, decrease the endonuclease activity or lifetime in cells, increase homology-directed recombination and reduce non-homologous end joining). See, e.g., Komor et al. Cell (2017) 168: 20-36. In some embodiments, the nucleotide sequence encoding the Cas9 molecule is modified to alter the PAM recognition of the endonuclease. For example, the Cas9 molecule SpCas9 recognizes PAM sequence NGG, whereas relaxed variants of the SpCas9 comprising one or more modifications of the endonuclease (e.g., VQR SpCas9, EQR SpCas9, VRER SpCas9) may recognize the PAM sequences NGA, NGAG, NGCG. PAM recognition of a modified Cas9 molecule is considered “relaxed” if the Cas9 molecule recognizes more potential PAM sequences as compared to the Cas9 molecule that has not been modified. For example, the Cas9 molecule SaCas9 recognizes PAM sequence NNGRRT, whereas a relaxed variant of the SaCas9 comprising one or more modifications (e.g., KKH SaCas9) may recognize the PAM sequence NNNRRT. In one example, the Cas9 molecule FnCas9 recognizes PAM sequence NNG, whereas a relaxed variant of the FnCas9 comprising one or more modifications of the endonuclease (e.g., RHA FnCas9) may recognize the PAM sequence YG. In one example, the Cas9 molecule is a Cpfl endonuclease comprising substitution mutations S542R and K607R and recognize the PAM sequence TYCV. In one example, the Cas9 molecule is a Cpfl endonuclease comprising substitution mutations S542R, K607R, and N552R and recognize the PAM sequence TATV. See, e.g., Gao et al. Nat. Biotechnol. (2017) 35(8): 789-792.
In some embodiments, more than one (e.g., 2, 3, or more) Cas molecules, e.g., Cas9 molecules, are used. In some embodiments, at least one of the Cas9 molecule is a Cas9 enzyme. In some embodiments, at least one of the Cas molecules is a Cpfl enzyme. In some embodiments, at least one of the Cas9 molecules is derived from Streptococcus pyogenes. In some embodiments, at least one of the Cas9 molecules is derived from Streptococcus pyogenes and at least one Cas9 molecules is derived from an organism that is not Streptococcus pyogenes.
In some embodiments, the Cas9 molecule is a base editor. Base editor endonuclease generally comprises a catalytically inactive Cas9 molecule fused to a function domain. See, e.g., Eid et al. Biochem. J. (2018) 475(11): 1955-1964; Rees et al. Nature Reviews Genetics (2018) 19:770-788. In some embodiments, the catalytically inactive Cas9 molecule is dCas9. In some embodiments, the catalytically inactive Cas9 molecule (dCas9) is fused to one or more uracil glycosylase inhibitor (UGI) domains. In some embodiments, the endonuclease comprises a dCas9 fused to an adenine base editor (ABE), for example an ABE evolved from the RNA adenine deaminase TadA. In some embodiments, the endonuclease comprises a dCas9 fused to cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDAl, activation- induced cytidine deaminase (AID)). In some embodiments, the catalytically inactive Cas9 molecule has reduced activity and is nCas9. In some embodiments, the Cas9 molecule comprises a nCas9 fused to one or more uracil glycosylase inhibitor (UGI) domains. In some embodiments, the Cas9 molecule comprises a nCas9 fused to an adenine base editor (ABE), for example an ABE evolved from the RNA adenine deaminase TadA. In some embodiments, the Cas9 molecule comprises a nCas9 fused to cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDAl, activation-induced cytidine deaminase (AID)).
Examples of base editors include, without limitation, BE1, BE2, BE3, HF-BE3, BE4, BE4max, BE4-Gam, YE1-BE3, EE-BE3, YE2-BE3, YEE-CE3, VQR-BE3, VRER-BE3, SaBE3, SaBE4, SaBE4-Gam, Sa(KKH)-BE3, Target-AID, Target-AID-NG, xBE3, eA3A- BE3, BE-PLUS, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR- ABE, VRER-ABE, Sa(KKH)-ABE, and CRISPR-SKIP. Additional examples of base editors can be found, for example, in U.S. Publication No. 2018/0312825A1, U.S Publication No. 2018/0312828A1, and PCT Publication No. WO 2018/165629A1, which are incorporated by reference herein in their entireties.
In some embodiments, the base editor has been further modified to inhibit base excision repair at the target site and induce cellular mismatch repair. Any of the Cas9 molecules described herein may be fused to a Gam domain (bacteriophage Mu protein) to protect the Cas9 molecule from degradation and exonuclease activity. See, e.g., Eid et al. Biochem. J. (2018) 475(11): 1955-1964.
In some embodiments, the Cas9 molecule belongs to class 2 type V of Cas endonuclease. Class 2 type V Cas endonucleases can be further categorized as type V-A, type V-B, type V-C, and type V-U. See, e.g., Stella et al. Nature Structural & Molecular Biology (2017). In some embodiments, the Cas molecule is a type V-A Cas endonuclease, such as a Cpfl nuclease. In some embodiments, the Cas9 molecule is a type V-B Cas endonuclease, such as a C2cl endonuclease. See, e.g., Shmakov et al. Mol Cell (2015) 60: 385-397. In some embodiments, the Cas molecule is Mad7™ (from Inscripta). Alternatively or in addition, the Cas9 molecule is a Cpfl nuclease or a variant thereof. As will be appreciated by one of skill in the art, the Cpfl nuclease may also be referred to as Cas 12a. See, e.g., Strohkendl et al. Mol. Cell (2018) 71: 1-9. In some embodiments, a composition or method described herein involves, or a host cell expresses, a Cpfl nuclease derived from Provetella spp. or Francis ella spp., Acidaminococcus sp. (AsCpfl), Lachnospiraceae bacterium (LpCpfl), or Eubacterium rectale. In some embodiments, the nucleotide sequence encoding the Cpfl nuclease may be codon optimized for expression in a host cell. In some embodiments, the nucleotide sequence encoding the Cpfl endonuclease is further modified to alter the activity of the protein. In some embodiments, catalytically inactive variants of Cas molecules (e.g., of Cas9 or Cas 12a) are used according to the methods described herein. A catalytically inactive variant of Cpfl (Cas 12a) may be referred to dCasl2a. As described herein, catalytically inactive variants of Cpfl maybe fused to a function domain to form a base editor. See, e.g., Rees et al. Nature Reviews Genetics (2018) 19:770-788. In some embodiments, the catalytically inactive Cas9 molecule is dCas9. In some embodiments, the endonuclease comprises a dCasl2a fused to one or more uracil glycosylase inhibitor (UGI) domains. In some embodiments, the Cas9 molecule comprises a dCasl2a fused to an adenine base editor (ABE), for example an ABE evolved from the RNA adenine deaminase TadA. In some embodiments, the Cas molecule comprises a dCasl2a fused to cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDAl, activation-induced cytidine deaminase (AID)).
Alternatively or in addition, the Cas9 molecule may be a Cas 14 endonuclease or variant thereof. Cas 14 endonucleases are derived from archaea and tend to be smaller in size (e.g., 400-700 amino acids). Additionally, Casl4 endonucleases do not require a PAM sequence. See, e.g., Harrington et al. Science (2018) 362 (6416).
Any of the Cas9 molecules described herein may be modulated to regulate levels of expression and/or activity of the Cas9 molecule at a desired time. For example, it may be advantageous to increase levels of expression and/or activity of the Cas9 molecule during particular phase(s) of the cell cycle. It has been demonstrated that levels of homology- directed repair are reduced during the Gi phase of the cell cycle, therefore increasing levels of expression and/or activity of the Cas9 molecule during the S phase, G2 phase, and/or M phase may increase homology-directed repair following the Cas endonuclease editing. In some embodiments, levels of expression and/or activity of the Cas9 molecule are increased during the S phase, G2 phase, and/or M phase of the cell cycle. In one example, the Cas9 molecule fused to the N-terminal region of human Geminin. See, e.g., Gutschner et al. Cell Rep. (2016) 14(6): 1555-1566. In some embodiments, levels of expression and/or activity of the Cas9 molecule are reduced during the Gi phase. In one example, the Cas9 molecule is modified such that it has reduced activity during the Gi phase. See, e.g., Lomova et al. Stem Cells (2018) 37(2): 284-294.
Alternatively or in addition, any of the Cas9 molecules described herein may be fused to an epigenetic modifier (e.g., a chromatin-modifying enzyme, e.g., DNA methylase, histone deacetylase). See, e.g., Kungulovski et al. Trends Genet. (2016) 32(2): 101- 113. Cas9 molecule fused to an epigenetic modifier may be referred to as “epieffectors” and may allow for temporal and/or transient endonuclease activity. In some embodiments, the Cas9 molecule is a dCas9 fused to a chromatin-modifying enzyme.
Zinc Finger Nucleases
In some embodiments, a cell or cell population described herein is produced using zinc finger (ZFN) technology. In some embodiments, the ZFN recognizes a target domain described herein, e.g., in Table 1. In general, zinc finger mediated genomic editing involves use of a zinc finger nuclease, which typically comprises a zinc finger DNA binding domain and a nuclease domain. The zinc finger binding domain may be engineered to recognize and bind to any target domain of interest, e.g., may be designed to recognize a DNA sequence ranging from about 3 nucleotides to about 21 nucleotides in length, or from about 8 to about 19 nucleotides in length. Zinc finger binding domains typically comprise at least three zinc finger recognition regions e.g., zinc fingers).
Restriction endonucleases (restriction enzymes) capable of sequence- specific binding to DNA (at a recognition site) and cleaving DNA at or near the site of binding are known in the art and may be used to form ZFN for use in genomic editing. For example, Type IIS restriction endonucleases cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. In one example, the DNA cleavage domain may be derived from the FokI endonuclease.
TALENs
In some embodiments, a cell or cell population described herein is produced using TALEN technology. In some embodiments, the TALEN recognizes a target domain described herein, e.g., in Table 1. In general, TALENs are engineered restriction enzymes that can specifically bind and cleave a desired target DNA molecule. A TALEN typically contains a Transcriptional Activator-Like Effector (TALE) DNA-binding domain fused to a DNA cleavage domain. The DNA binding domain may contain a highly conserved 33-34 amino acid sequence with a divergent 2 amino acid RVD (repeat variable dipeptide motif) at positions 12 and 13. The RVD motif determines binding specificity to a nucleic acid sequence and can be engineered to specifically bind a desired DNA sequence. In one example, the DNA cleavage domain may be derived from the FokI endonuclease. In some embodiments, the FokI domain functions as a dimer, using two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. A TALEN specific to a target gene of interest can be used inside a cell to produce a double-stranded break (DSB). A mutation can be introduced at the break site if the repair mechanisms improperly repair the break via non-homologous end joining. For example, improper repair may introduce a frame shift mutation. Alternatively, a foreign DNA molecule having a desired sequence can be introduced into the cell along with the TALEN. Depending on the sequence of the foreign DNA and chromosomal sequence, this process can be used to correct a defect or introduce a DNA fragment into a target gene of interest, or introduce such a defect into the endogenous gene, thus decreasing expression of the target gene.
Some exemplary, non-limiting embodiments of endonucleases and nuclease variants suitable for use in connection with the guide RNAs and genetic engineering methods provided herein have been described above. Additional suitable nucleases and nuclease variants will be apparent to those of skill in the art based on the present disclosure and the knowledge in the art. The disclosure is not limited in this respect. gRNA Sequences and Configurations
A gRNA can comprise a number of domains. In an embodiment, a unimolecular, sgRNA, or chimeric, gRNA comprises, e.g., from 5' to 3': a targeting domain (which is complementary to a target nucleic acid in the CD33 gene; a first complementarity domain; a linking domain; a second complementarity domain (which is complementary to the first complementarity domain); a proximal domain; and optionally, a tail domain. Each of these domains is now described in more detail.
The targeting domain may comprise a nucleotide sequence that is complementary, e.g., at least 80, 85, 90, or 95% complementary, e.g., fully complementary, to the target sequence on the target nucleic acid. The targeting domain is part of an RNA molecule and will therefore comprise the base uracil (U), while any DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, in an embodiment, it is believed that the complementarity of the targeting domain with the target sequence contributes to specificity of the interaction of the gRNA/Cas9 molecule complex with a target nucleic acid. It is understood that in a targeting domain and target sequence pair, the uracil bases in the targeting domain will pair with the adenine bases in the target sequence. In an embodiment, the target domain itself comprises in the 5' to 3' direction, an optional secondary domain, and a core domain. In an embodiment, the core domain is fully complementary with the target sequence. In an embodiment, the targeting domain is 5 to 50 nucleotides in length. The targeting domain may be between 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length. In some embodiments, the targeting domain is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the targeting domain is between 10-30, or between 15-25, nucleotides in length.
In some embodiments, a targeting domain comprises a core domain and a secondary targeting domain, e.g., as described in PCT Publication No. WO 2015/157070, which is incorporated by reference in its entirety. In an embodiment, the core domain comprises about 8 to about 13 nucleotides from the 3' end of the targeting domain (e.g., the most 3' 8 to 13 nucleotides of the targeting domain). In an embodiment, the secondary domain is positioned 5' to the core domain. In many embodiments, the core domain has exact complementarity with the corresponding region of the target sequence. In other embodiments, the core domain can have 1 or more nucleotides that are not complementary with the corresponding nucleotide of the target sequence.
The first complementarity domain is complementary with the second complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In an embodiment, the first complementarity domain is 5 to 30 nucleotides in length. In an embodiment, the first complementarity domain comprises 3 subdomains, which, in the 5' to 3' direction are: a 5' subdomain, a central subdomain, and a 3' subdomain. In an embodiment, the 5' subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9 nucleotides in length. In an embodiment, the central subdomain is 1, 2, or 3, e.g., 1, nucleotide in length. In an embodiment, the 3' subdomain is 3 to 25, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. The first complementarity domain can share homology with, or be derived from, a naturally occurring first complementarity domain. In an embodiment, it has at least 50% homology with a .S'. pyogenes, S. aureus or .S', thermophilus, first complementarity domain.
The sequence and placement of the above-mentioned domains are described in more detail in PCT Publication No. WO 2015/157070, which is herein incorporated by reference in its entirety, including p. 88-112 therein.
A linking domain serves to link the first complementarity domain with the second complementarity domain of a unimolecular gRNA. The linking domain can link the first and second complementarity domains covalently or non-covalently. In an embodiment, the linkage is covalent. In an embodiment, the linking domain is, or comprises, a covalent bond interposed between the first complementarity domain and the second complementarity domain. In some embodiments, the linking domain comprises one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the linking domain comprises at least one non-nucleotide bond, e.g., as disclosed in PCT Publication No. WO 2018/126176, the entire contents of which are incorporated herein by reference.
The second complementarity domain is complementary, at least in part, with the first complementarity domain, and in an embodiment, has sufficient complementarity to the second complementarity domain to form a duplexed region under at least some physiological conditions. In some embodiments, the second complementarity domain can include a sequence that lacks complementarity with the first complementarity domain, e.g., a sequence that loops out from the duplexed region. In some embodiments, the second complementarity domain is 5 to 27 nucleotides in length. In some embodiments, the second complementarity domain is longer than the first complementarity region. In an embodiment, the complementary domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the second complementarity domain comprises 3 subdomains, which, in the 5' to 3' direction are: a 5' subdomain, a central subdomain, and a 3' subdomain. In some embodiments, the 5' subdomain is 3 to 25, e.g., 4 to
22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, or 25, nucleotides in length. In some embodiments, the central subdomain is 1, 2, 3, 4, or 5 nucleotides in length. In some embodiments, the 3' subdomain is 4 to 9, e.g., 4, 5, 6, 7, 8 or 9, nucleotides in length. In some embodiments, the 5' subdomain and the 3' subdomain of the first complementarity domain, are respectively, complementary, e.g., fully complementary, with the 3' subdomain and the 5' subdomain of the second complementarity domain.
In some embodiments, the proximal domain is 5 to 20 nucleotides in length. In some embodiments, the proximal domain can share homology with or be derived from a naturally occurring proximal domain. In some embodiments, it has at least 50% homology with a proximal domain from 5. pyogenes, S. aureus, or 5. thermophilus.
A broad spectrum of tail domains is suitable for use in gRNAs. In an embodiment, the tail domain is 0 (absent), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the tail domain nucleotides are from or share homology with a sequence from the 5' end of a naturally occurring tail domain. In some embodiments, the tail domain includes sequences that are complementary to each other and which, under at least some physiological conditions, form a duplexed region. In some embodiments, the tail domain is absent or is 1 to 50 nucleotides in length. In some embodiments, the tail domain can share homology with or be derived from a naturally occurring proximal tail domain. In some embodiments, it has at least 50% homology with an .S'. pyogenes, S. aureus, or .S'. thermophilus, tail domain. In some embodiments, the tail domain includes nucleotides at the 3' end that are related to the method of in vitro or in vivo transcription.
In some embodiments, a modular gRNA comprises: a first strand comprising, e.g., from 5' to 3': a targeting domain (which is complementary to a target nucleic acid in the CD33 gene), and a first complementarity domain; and a second strand, comprising, preferably from 5' to 3': optionally, a 5' extension domain, a second complementarity domain, a proximal domain, and optionally, a tail domain.
In some embodiments, the gRNA is chemically modified. For instance, the gRNA may comprise one or more modification chosen from phosphorothioate backbone modification, 2'-O-Me-modified sugars (e.g., at one or both of the 3’ and 5’ termini), 2’F- modified sugar, replacement of the ribose sugar with the bicyclic nucleotide-cEt, 3 'thioPACE (MSP), or any combination thereof. Suitable gRNA modifications are described, e.g., in Rahdar et al. PNAS (2015) 112 (51) E7110-E7117 and Hendel et al., Nat Biotechnol. (2015) Sep; 33(9): 985-989, each of which is incorporated herein by reference in its entirety. In some embodiments, a gRNA described herein comprises one or more 2'-O-methyl-3'- phosphorothioate nucleotides, e.g., at least 2, 3, 4, 5, or 6 2'-O-methyl-3 '-phosphorothioate nucleotides. In some embodiments, a gRNA described herein comprises modified nucleotides (e.g., 2'-O-methyl-3'-phosphorothioate nucleotides) at the three terminal positions and the 5’ end and/or at the three terminal positions and the 3’ end. In some embodiments, the gRNA may comprise one or more modified nucleotides, e.g., as described in PCT Publication Nos. WO/2017/214460, WO/2016/089433, and WO/2016/164356, which are incorporated by reference their entirety.
In some embodiments, a gRNA described herein is chemically modified. For example, the gRNA may comprise one or more 2’-0 modified nucleotide, e.g., 2’-O-methyl nucleotide. In some embodiments, the gRNA comprises a 2’-0 modified nucleotide, e.g., 2’- O-methyl nucleotide at the 5’ end of the gRNA. In some embodiments, the gRNA comprises a 2’-0 modified nucleotide, e.g., 2’-O-methyl nucleotide at the 3’ end of the gRNA. In some embodiments, the gRNA comprises a 2’-O-modified nucleotide, e.g., 2’-O-methyl nucleotide at both the 5’ and 3’ ends of the gRNA. In some embodiments, the gRNA is 2’-O-modified, e.g. 2’-O-methyl-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, and the third nucleotide from the 5’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified, e.g. 2’-O-methyl-modified at the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified, e.g. 2’-O-methyl-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified, e.g. 2’-O-methyl-modified at the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and at the fourth nucleotide from the 3’ end of the gRNA. In some embodiments, the nucleotide at the 3’ end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3’ end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2’-O- modified, e.g. 2’-O-methyl-modified, at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA. In some embodiments, the 2’-O-methyl nucleotide comprises a phosphate linkage to an adjacent nucleotide. In some embodiments, the 2’-O-methyl nucleotide comprises a phosphorothioate linkage to an adjacent nucleotide. In some embodiments, the 2’-O-methyl nucleotide comprises a thioPACE linkage to an adjacent nucleotide.
In some embodiments, the gRNA may comprise one or more 2’-O-modified and 3’phosphorous-modified nucleotide, e.g., a 2’-O-methyl 3’phosphorothioate nucleotide. In some embodiments, the gRNA comprises a 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’phosphorothioate nucleotide at the 5’ end of the gRNA. In some embodiments, the gRNA comprises a 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O- methyl 3’phosphorothioate nucleotide at the 3’ end of the gRNA. In some embodiments, the gRNA comprises a 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’phosphorothioate nucleotide at the 5’ and 3’ ends of the gRNA. In some embodiments, the gRNA comprises a backbone in which one or more non-bridging oxygen atoms has been replaced with a sulfur atom. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g. 2’-O-methyl 3’phosphorothioate-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, and the third nucleotide from the 5’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g. 2’-O-methyl 3’phosphorothioate-modified at the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g. 2’-O-methyl 3’phosphorothioate-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g. 2’-O-methyl 3’phosphorothioate-modified at the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA. In some embodiments, the nucleotide at the 3’ end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3’ end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g. 2’-O-methyl 3’phosphorothioate-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA.
In some embodiments, the gRNA may comprise one or more 2’-O-modified and 3’- phosphorous-modified, e.g., 2’-O-methyl 3’thioPACE nucleotide. In some embodiments, the gRNA comprises a 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’thioPACE nucleotide at the 5’ end of the gRNA. In some embodiments, the gRNA comprises a 2’-O-modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’thioPACE nucleotide at the 3’ end of the gRNA. In some embodiments, the gRNA comprises a 2’-O- modified and 3’phosphorous-modified, e.g., 2’-O-methyl 3’thioPACE nucleotide at the 5’ and 3’ ends of the gRNA. In some embodiments, the gRNA comprises a backbone in which one or more non-bridging oxygen atoms have been replaced with a sulfur atom and one or more non-bridging oxygen atoms have been replaced with an acetate group. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g. 2’-O-methyl 3’ thioPACE-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, and the third nucleotide from the 5’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g. 2’-O-methyl 3 ’thioPACE-modified at the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g. 2’-O-methyl 3 ’thioPACE-modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g. 2’-O-methyl 3 ’thioPACE- modified at the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA. In some embodiments, the nucleotide at the 3’ end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3’ end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2’-O-modified and 3’phosphorous-modified, e.g. 2’-O-methyl 3 ’thioPACE- modified at the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA.
In some embodiments, the gRNA comprises a chemically modified backbone. In some embodiments, the gRNA comprises a phosphorothioate linkage. In some embodiments, one or more non-bridging oxygen atoms have been replaced with a sulfur atom. In some embodiments, the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, and the third nucleotide from the 5’ end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and at the fourth nucleotide from the 3’ end of the gRNA each comprise a phosphorothioate linkage. In some embodiments , the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA each comprise a phosphorothioate linkage.
In some embodiments, the gRNA comprises a thioPACE linkage. In some embodiments, the gRNA comprises a backbone in which one or more non-bridging oxygen atoms have been replaced with a sulfur atom and one or more non-bridging oxygen atoms have been replaced with an acetate group. In some embodiments, the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, and the third nucleotide from the 5’ end of the gRNA each comprise a thioPACE linkage. In some embodiments, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA each comprise a thioPACE linkage. In some embodiments, the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end of the gRNA, the nucleotide at the 3’ end of the gRNA, the second nucleotide from the 3’ end of the gRNA, and the third nucleotide from the 3’ end of the gRNA each comprise a thioPACE linkage. In some embodiments, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and at the fourth nucleotide from the 3’ end of the gRNA each comprise a thioPACE linkage. In some embodiments, the nucleotide at the 5’ end of the gRNA, the second nucleotide from the 5’ end of the gRNA, the third nucleotide from the 5’ end, the second nucleotide from the 3’ end of the gRNA, the third nucleotide from the 3’ end of the gRNA, and the fourth nucleotide from the 3’ end of the gRNA each comprise a thioPACE linkage.
Some exemplary, non-limiting embodiments of modifications, e.g., chemical modifications, suitable for use in connection with the guide RNAs and genetic engineering methods provided herein have been described above. Additional suitable modifications, e.g., chemical modifications, will be apparent to those of skill in the art based on the present disclosure and the knowledge in the art, including, but not limited to those described in Hendel, A. et al., Nature Biotech. (2015) Vol 33, No. 9; in PCT Publication Nos. WO/2017/214460; in WO/2016/089433; and/or in WO/2016/164356; each one of which is herein incorporated by reference in its entirety. gRNAs targeting CD33
The present disclosure provides a number of useful gRNAs that can target an endonuclease to human CD33. In some embodiments, the gRNA used in the methods described herein target a sequence in exon 3 of CD33. Table 1 below illustrates target domains in human endogenous CD33 that can be bound by gRNAs described herein. Table 1. Exemplary Cas9 target site sequences of human CD33 are provided, as are exemplary targeting domain sequences useful for targeting such sites.
Figure imgf000066_0001
*For each target site, the first sequence represents the DNA target domain sequence, the second sequence represents the reverse complement thereof, and the third sequence represents an exemplary targeting domain sequence of a gRNA that can be used to target the respective target site.
The CD33 (CCDS33084.1) cDNA sequence is provided below as SEQ ID NO: 16.
Exon 3 is underlined.
ATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCCCTGGCTATGGATCCAAATTT
CTGGCTGCAAGTGCAGGAGTCAGTGACGGTACAGGAGGGTTTGTGCGTCCTCGTGCCCTGCA
CTTTCTTCCATCCCATACCCTACTACGACAAGAACTCCCCAGTTCATGGTTACTGGTTCCGG
GAAGGAGCCATTATATCCAGGGACTCTCCAGTGGCCACAAACAAGCTAGATCAAGAAGTACA
GGAGGAGACTCAGGGCAGATTCCGCCTCCTTGGGGATCCCAGTAGGAACAACTGCTCCCTGA
GCATCGTAGACGCCAGGAGGAGGGATAATGGTTCATACTTCTTTCGGATGGAGAGAGGAAGT
ACCAAATACAGTTACAAATCTCCCCAGCTCTCTGTGCATGTGACAGACTTGACCCACAGGCC
CAAAATCCTCATCCCTGGCACTCTAGAACCCGGCCACTCCAAAAACCTGACCTGCTCTGTGT
CCTGGGCCTGTGAGCAGGGAACACCCCCGATCTTCTCCTGGTTGTCAGCTGCCCCCACCTCC
CTGGGCCCCAGGACTACTCACTCCTCGGTGCTCATAATCACCCCACGGCCCCAGGACCACGG
CACCAACCTGACCTGTCAGGTGAAGTTCGCTGGAGCTGGTGTGACTACGGAGAGAACCATCC
AGCTCAACGTCACCTATGTTCCACAGAACCCAACAACTGGTATCTTTCCAGGAGATGGCTCA
GGGAAACAAGAGACCAGAGCAGGAGTGGTTCATGGGGCCATTGGAGGAGCTGGTGTTACAGC
CCTGCTCGCTCTTTGTCTCTGCCTCATCTTCTTCATAGTGAAGACCCACAGGAGGAAAGCAG CCAGGACAGCAGTGGGCAGGAATGACACCCACCCTACCACAGGGTCAGCCTCCCCGAAACAC CAGAAGAAGTCCAAGTTACATGGCCCCACTGAAACCTCAAGCTGTTCAGGTGCCGCCCCTAC TGTGGAGATGGATGAGGAGCTGCATTATGCTTCCCTCAACTTTCATGGGATGAATCCTTCCA AGGACACCTCCACCGAATACTCAGAGGTCAGGACCCAGTGA (SEQ ID NO: 16)
Exon 3 of CD33 is provided separately below as SEQ ID NO: 17. Underlining indicates the regions complementary to gRNA A, gRNA B, gRNA C, gRNA D (or the reverse complement thereof). Note that the target regions for gRNA A, gRNA B, and gRNA D partially overlap. ACTTGACCCACAGGCCCAAAATCCTCATCCCTGGCACTCTAGAACCCGGCCACTCCAAAAAC CTGACCTGCTCTGTGTCCTGGGCCTGTGAGCAGGGAACACCCCCGATCTTCTCCTGGTTGTC AGCTGCCCCCACC T C C C T G G GCCCCAGGACTACTCACTCCTCGGTGCTCATAATCACCCCAC GGCCCCAGGACCACGGCACCAACCTGACCTGTCAGGTGAAGTTCGCTGGAGCTGGTGTGACT ACGGAGAGAACCATCCAGCTCAACGTCACCT (SEQ ID NO: 17)
Dual gRNA compositions and uses thereof
In some embodiments, a gRNA described herein (e.g., a gRNA having a target domain sequence of Table 1) can be used in combination with a second gRNA, e.g., for directing nucleases to two sites in a genome. For instance, in some embodiments, it is desired to produce a hematopoietic cell that is deficient for CD33 and a second lineagespecific cell surface antigen, e.g., so that the cell can be resistant to two agents: an anti-CD33 agent and an agent targeting the second lineage- specific cell surface antigen. In some embodiments, it is desirable to contact a cell with two different gRNAs that target different regions of CD33, in order to make two cuts and create a deletion between the two cut sites. Accordingly, the disclosure provides various combinations of gRNAs.
In some embodiments, two or more (e.g., 2, 3, 4, or more) gRNAs described herein are admixed. In some embodiments, each gRNA is in a separate container. In some embodiments, a kit described herein (e.g., a kit comprising one or more gRNAs according to Table 1) also comprises a Cas9 molecule, or a nucleic acid encoding the Cas9 molecule.
In some embodiments, the first and second gRNAs are gRNAs according to Table 1 or variants thereof.
In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence of Table 1 or a variant thereof) and the second gRNA targets a gene encoding a lineage- specific cell-surface antigen chosen from: CD5, CD6, CD7, BCMA, CD19, CD20, CD30, R0R1, B7H6, B7H3, CD23, CD38, C-type lectin like molecule-1, CS1, IL-5, Ll-CAM, PSCA, PSMA, CD138, CD133, CD70, CD7, CD13, NKG2D, NKG2D ligand, CLEC12A, CD11, CD123, CD56, CD34, CD14, CD66b, CD41, CD61, CD62, CD235a, CD 146, CD326, LMP2, CD22, CD52, CD 10, CD3/TCR, CD79/BCR, and CD26.
In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding a lineage- specific cell-surface antigen associated with a specific type of cancer, such as, without limitation, CD20, CD22 (Non-Hodgkin's lymphoma, B-cell lymphoma, chronic lymphocytic leukemia (CLL)), CD52 (B-cell CLL), CD33 (acute myeloid leukemia (AML)), CD 10 (gplOO) (Common (pre-B) acute lymphocytic leukemia and malignant melanoma), CD3/T-cell receptor (TCR) (T-cell lymphoma and leukemia), CD79/B-cell receptor (BCR) (B-cell lymphoma and leukemia), CD26 (epithelial and lymphoid malignancies), human leukocyte antigen (HLA)-DR, HLA-DP, and HLA-DQ (lymphoid malignancies), RCAS1 (gynecological carcinomas, biliary adenocarcinomas and ductal adenocarcinomas of the pancreas) as well as prostate specific membrane antigen.
In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding a lineage- specific cell-surface antigen chosen from: CD5, CD6, CD7, CD13, CD19, CD22, CD20, CD25, CD30, CD32, CD38, CD44, CD45, CD47, CD56, 96, CD117, CD123, CD135, CD174, CLL-1, BCMA, folate receptor 0, IL1RAP, MUC1, NKG2D/NKG2DL, TIM-3, or WT1.
In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding a lineage- specific cell-surface antigen chosen from: CD la, CD lb, CDlc, CD Id, CDle, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CDl la, CDl lb, CDl lc, CDl ld, CDwl2, CD13, CD14, CD15, CD16, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32a, CD32b, CD32c, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD61, CD62E, CD62L, CD62P, CD63, CD64a, CD65, CD65s, CD66a, CD66b, CD66c, CD66F, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75S, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85A, CD85C, CD85D, CD85E, CD85F, CD85G, CD85H, CD85I, CD85J, CD85K, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD121a, CD121b, CD122, CD123, CD124, CD125, CD126, CD127, CD129, CD130, CD131, CD132, CD133, CD134, CD135, CD136, CD137, CD138, CD139, CD140a, CD140b, CD141, CD142, CD143, CD14, CDwl45, CD146, CD147, CD148, CD150, CD152, CD152, CD153, CD154, CD155, CD156a, CD156b, CD156c, CD157, CD158bl, CD158b2, CD158d, CD158el/e2, CD158f, CD158g, CD158h, CD158i, CD158j, CD158k, CD159a, CD159c, CD160, CD161, CD163, CD164, CD165, CD166, CD167a, CD168, CD169, CD170, CD171, CD172a, CD172b, CD172g, CD173, CD174, CD175, CD175s, CD176, CD177, CD178, CD179a, CD179b, CD180, CD181, CD182, CD183, CD184, CD185, CD186, CD191, CD192, CD193, CD194, CD195, CD196, CD197, CDwl98, CDwl99, CD200, CD201, CD202b, CD203c, CD204, CD205, CD206, CD207, CD208, CD209, CD210a, CDw210b, CD212, CD213al, CD213a2, CD215, CD217, CD218a, CD218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232, CD233, CD234, CD235a, CD235b, CD236, CD236R, CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD252, CD253, CD254, CD256, CD257, CD258, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD272, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD286, CD288, CD289, CD290, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD3OOa, CD3OOc, CD3OOe, CD301, CD302, CD303, CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD359, CD360, CD361, CD362 or CD363.
In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence of according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding a lineage- specific cell-surface antigen chosen from: CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLECL1); epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (CD2); ganglioside GD3 (aNeu5Ac(2- 8)aNeu5Ac(2-3)bDGalp(l-4)bDGlep(l-l)Cer); TNF receptor family member B cell maturation (BCMA), Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (R0R1); Fms- Like tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPC AM); B7H3 (CD276); KIT (CD117); Interleukin- 13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL-l lRa); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR- beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor I receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), Proteasome (Prosome, Macropain) Subunit, Beta Type 9 (LMP2); glycoprotein 100 (gplOO); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr- abl); tyrosinase; ephrin type-A receptor 2 (EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer); transglutaminase 5 (TGS5); high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta- specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex; locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE-la); Melanoma- associated antigen 1 (MAGE-A1), ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen- 1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumor antigen- 1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-1AP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl- transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin Bl; v- myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY- TES1); lymphocyte- specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7); intestinal carboxy esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte- associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like modulecontaining mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).
In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding a lineage- specific cell-surface antigen chosen from: CDl la, CD18, CD19, CD20, CD31, CD34, CD44, CD45, CD47, CD51, CD58, CD59, CD63, CD97, CD99, CD100, CD102, CD123, CD127, CD133, CD135, CD157, CD172b, CD217, CD300a, CD305, CD317, CD321, and CLL1.
In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding a lineage- specific cell-surface antigen chosen from: CD 123, CLL1, CD38, CD135 (FLT3), CD56 (NCAM1), CD117 (c-KIT), FRp (FOLR2), CD47, CD82, TNFRSF1B (CD120B), CD191, CD96, PTPRJ (CD148), CD70, LILRB2 (CD85D), CD25 (IL2Ralpha), CD44, CD96, NKG2D Ligand, CD45, CD7, CD15, CD19, CD20, CD22, CD37, CD82, CD312 (EMR2).
In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding a lineage- specific cell-surface antigen chosen from: CD7, CDl la, CD15, CD18, CD19, CD20, CD22, CD25, CD31, CD34, CD37, CD38, CD44, CD45, CD47, CD51, CD56, CD58, CD59, CD63, CD70, CD82, CD85D, CD96, CD97, CD99, CD100, CD102, CD117, CD120B, CD123, CD127, CD133, CD135, CD148, CD157, CD172b, CD191, CD217, CD300a, CD305, CD317, CD321, CLL1, FRp (FOLR2), or NKG2D Ligand.
In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA having a targeting sequence according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding CLL-1. In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA according to Table 1 or a variant thereof) and the second gRNA targets a gene encoding CD 123.
Table 2. Exemplary gRNA targeting (spacer) sequences.
Figure imgf000072_0001
Figure imgf000073_0001
Some of the embodiments, advantages, features, and uses of the technology disclosed herein will be more fully understood from the Examples below. The Examples are intended to illustrate some of the benefits of the present disclosure and to describe particular embodiments but are not intended to exemplify the full scope of the disclosure and, accordingly, do not limit the scope of the disclosure.
EXAMPLES
Example 1. Generation of genetically engineered hematopoietic cells comprising a modified gene encoding CD33
The Cas9 sgRNAs indicated in Table 1 were designed based on the SpCas9 PAM (5'- NGG-3') with close proximity to the target region and evaluated for predicted specificity by minimizing potential off-target sites in the human genome with an online search algorithm (e.g., the Benchling algorithm, Doench et al 2016, Hsu et al 2013).
Cas9 sgRNAs are synthesized using the gRNA targeting domains provided below and the Cas9 sgRNA scaffold sequence 5'-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC
UUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3' (SEQ ID NO: 18). For example, the nucleotide sequence of sgRNA A is
5'-CCCCAGGACGACGCACGCCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUA AGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3' (SEQ ID NO: 19, targeting domain sequence in bold).
For example, the nucleotide sequence of sgRNA B is
5'-ACCGAGGAGGGAGGAGGCCGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUA AGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3' (SEQ ID NO: 20, targeting domain sequence in bold).
For example, the nucleotide sequence of sgRNA C is
5'-GGGGGGGGCAGCGGACAACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUA AGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3' (SEQ ID NO: 21, targeting domain sequence in bold).
For example, the nucleotide sequence of sgRNA D is
5'-CGGGGCGCAGAAGCACCCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUA AGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3' (SEQ ID NO: 22, targeting domain sequence in bold).
For example, the nucleotide sequence of sgRNA E is
5'-CCGCACGAGACGGGACCCACGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUA AGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU-3' (SEQ ID NO: 23, targeting domain sequence in bold).
All designed synthetic sgRNAs are produced with chemically modified nucleotides at the three terminal positions at both the 5' and 3' ends. The modified nucleotides contained 2'- O-methyl-3'-phosphorothioate (abbreviated as “ms”) and the ms-sgRNAs are HPLC -purified. Cas9 protein is purchased from Synthego.
For example, the nucleotide sequence of sgRNA A, showing the modified nucleotides, is 5'-CmsCmsCmsCAGGACGACGCACGCCGGUUUUAGAGCUAGAAAUAGCAAGUUAA AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmsU msUmsU-3' (SEQ ID NO: 24, targeting domain sequence in bold).
For example, the nucleotide sequence of sgRNA E, showing the modified nucleotides, is
5'-CmsCmsGmsCACGAGACGGGACCCACGUUUUAGAGCUAGAAAUAGCAAGUUAA AAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUmsU msUmsU-3' (SEQ ID NO: 25, targeting domain sequence in bold). Peripheral blood mononuclear cells are collected from healthy donor subject by apheresis following hematopoietic stem cell mobilization. The donor CD34+ cells are electroporated with Cas9 protein and any of the indicated CD33 -targeting Cas9 gRNAs disclosed herein, e.g., having the targeting domain sequences provided in Tables 1 and 3, e.g., gRNA A, gRNA, B, gRNA C, gRNA D, or gRNA E.
Table 3. Exemplary CD33 gRNA targeting domain sequences.
Figure imgf000075_0001
The edited cells are cultured for less than 48 hours. Upon harvest, the cells are washed, resuspended in the final formulation, and cryopreserved.
A representative sample of the edited HSCs is evaluated for viability and expression of CD33, or absence thereof, by staining for CD33 using an anti-CD33 antibody e.g., P67.7) and analyzed by flow cytometry. Edited CD33KO eHSPC populations exhibiting at least 70% cell viability and at least 45% CD33 editing efficiency (i.e., absence of CD33 expression in at least 45% of the cells in the cell population) at 48 hours after electroporation are used for HCT.
Example 2: Treatment of a subject having AML with CD33KO eHSPC generated using gRNA A, and the CD33-targeted ADC gemtuzumab ozogamicin/Mylotarg®
A subject having CD33-positive AML is treated with an allogeneic HCT comprising CD33KO eHSPCs and with the ADC gemtuzumab ozogamicin/Mylotarg®.
For the HCT, a population of cells comprising CD34+ hematopoietic stem cells is obtained from a healthy donor who is HLA matched at 8/8 loci (HLA-A, -B, -C, DRB1) to the subject.
After G-CSF/plerixafor mobilization, up to two apheresis procedures are performed in order to obtain a target of 10 x 106 viable cells/kg (where kg refers to recipient subject weight) from the donor for processing and subsequent administration to the recipient subject. From this apheresis product, at least 3.0 x 106 viable cells/kg (recipient weight) undergo minimal manipulation and are cryopreserved to serve as a back-up stem cell source, e.g., for use as a rescue dose. The remainder of the apheresis product is used for processing and preparation of the CD33KO eHSPC population for HCT. The CD33KO eHSPC population for HCT is prepared by enriching the apheresis product for CD34+ cells, followed by electroporation and editing with a CD33 gRNA/Cas9 complex using any of the CD33 gRNAs as described in Example 1.
The edited cells are subsequently placed in culture for <48 hours. Upon harvest, after the culture duration is finished, cells are washed, resuspended in the final formulation, and cryopreserved. Cell viability and editing efficiency are confirmed using a representative sample as described in Example 1, and CD33KO eHSPC populations meeting the criteria set forth in Example 1 (at least 70% viability and at least 45% CD33 editing efficiency) are used for HCT. A population for administration to a subject comprises a CD33KO eHSPC population satisfying these viability and editing efficiency criteria of at least 3 x 106 cells/kg body weight of the recipient subject, and preferably comprises at least 4 x 106 cells/kg, 5 x 106 cells/kg, 6 x 106 cells/kg, or 7 x 106 cells/kg of the recipient subject.
After completion of the conditioning regimen, the subject receives an HCT comprising the thawed CD33KO eHSPCs via an intravenous (IV) infusion. The day of the HCT is day 0 of the treatment regimen.
The subject is assessed for CD33KO eHSPC engraftment at day 28 by measuring the absolute peripheral neutrophil count (ANC) for CD33KO (CD33-) neutrophils in the subject. The subject is deemed to exhibit neutrophil recovery (also referred to as successful CD33KO neutrophil engraftment) if the subject exhibits an absolute peripheral CD33KO neutrophil count of >500 cells/pL CD33- ANC at 28 days after CD33KO eHSPC HCT.
A bone marrow biopsy is obtained from the subject on day 60 in order to assess disease status and hematopoietic recovery. In addition, percent donor chimerism and CD33- negative (CD33-) myeloid hematopoiesis are determined from the peripheral blood at this time. If the subject exhibits successful CD33- HSC engraftment and CD33- hematopoiesis at day 60, the subject is subsequently administered gemtuzumab ozogamicin/My lotarg®. The CD33- ANC is monitored in the subject prior to administration of gemtuzumab ozogamicin/My lotarg®, and the subject should preferably have >1000/dL CD33- ANC prior to receiving gemtuzumab ozogamicin/My lotarg®.
Administration of gemtuzumab ozogamicin/Mylotarg® is preferably initiated within 30 days of the bone marrow biopsy at day 60, i.e., is preferably initiated by day 90. However, initiation of gemtuzumab ozogamicin/Mylotarg® may be delayed up to day 120 or later if a subject’s clinical status, e.g., in view of comorbidities, including, for example, HCT- related comorbidities, necessitate such a delay, or in order to allow attainment of >1000/dL CD33- ANC in a subject. If gemtuzumab ozogamicin/Mylotarg® is initiated more than 30 days after the Day 60 bone marrow biopsy, a repeat bone marrow biopsy is completed prior to starting gemtuzumab ozogamicin/Mylotarg®.
Gemtuzumab ozogamicin/Mylotarg® is administered to the subject at a dose within the range of 0.1mg/m2 to 6 mg/m2, e.g., at a dose of 0.1mg/m2, 0.25 mg/m2, 0.5 mg/m2, lmg/m2, 2 mg/m2, 3 mg/m2 , 4 mg/m2, 5 mg/m2, or 6 mg/m2 .
Gemtuzumab ozogamicin/Mylotarg® is administered to the subject in a regimen of 4- week (28d) treatment cycles, wherein the subject receives the entire amount of a respective treatment cycle, e.g., at 0.5mg/m2 of gemtuzumab ozogamicin/Mylotarg®, on day 1 of the respective 4-week (28d) treatment cycle. Alternatively, the subject may receive the amount of gemtuzumab ozogamicin/Mylotarg® fractionated between multiple doses administered on separate prescribed days, e.g., on days 1, 4, and 7 of the respective 4-week (28d) dosing cycle, or one days 1, 8, and 16 of the 4-week dosing cycle. As yet a further alternative, the subject may receive the amount of gemtuzumab ozogamicin/Mylotarg® fractionated between multiple doses each administered once the plasma concentration of gemtuzumab ozogamicin falls below a threshold level.
At completion of the last gemtuzumab ozogamicin/Mylotarg® treatment cycle, the subject is monitored for disease status and hematopoietic chimerism and is monitored for these parameters at six months, 1 year, 2 years, and then annually for up to 15 years after completion of the final treatment cycle.
Example 3: Clinical scale manufacturing of human CD33KO eHSPC
Two clinical-scale batches of allogeneic CRISPR/Cas9 genome edited hematopoietic stem/progenitor cells (HSPCs) lacking the CD33 protein were manufactured for the treatment of human leukocyte antigen (HLA)-matched patients with high-risk CD33+ acute myeloid leukemia (AML). The resulting HSPC populations are suitable for infusion into HLA- matched human patients with AML undergoing hematopoietic cell transplant, e.g., patients who are known to be at high-risk for leukemia relapse and mortality post-transplantation.
The final HSPC populations were formulated at a volume of 45mL in cryopreservation media ready for cryopreservation, storage in the vapor phase of liquid nitrogen, subsequent thawing and administration via intravenous (IV) infusion to a recipient patient.
Each batch was manufactured from leukapheresis starting material obtained from a single donor to generate a one-donor-to-one -recipient HLA matched product, allowing for the manufacture of the product for a specifically matched patient.
For each batch, a healthy donor was subjected to a leukapheresis procedure. Leukapheresis starting material was collected and stored at 2-8 °C before initiation of cell manipulation. Cell number and viability of the leukapheresis starting material was tested by flow cytometry. Cell viability was confirmed to be >80%. A sample was removed for cell analysis and other assessments.
A leukapheresis rescue dose was removed from the leukapheresis material to obtain a volume comprising 3xl06 CD34+ cells/kg of patient weight. The rescue dose material was processed and cryopreserved and then stored in the vapor phase of liquid nitrogen at < -140 °C.
After rescue dose removal, the leukapheresis starting material was processed to remove red blood cells, platelets, and plasma. The processed material was then enriched for CD34-positive cells and then transferred into 250 mL conical tubes.
A Cas9/gRNA ribonucleoprotein (RNP) complex was prepared prior to electroporation by mixing Cas9 protein and gRNA E under sterile conditions. Cells in the 250 mL conical tube were spun down at 200 xg, resuspended in electroporation buffer, mixed with the prepared RNP complex, and electroporated in a single-use sterile electroporation cassette.
Post-electroporation, the cells were removed from the cassette, transferred to culture media, and incubated in suspension culture at 37 °C and 5% CO2. Cell cultures were monitored for cell count and viability. Once cells recovered from electroporation (determined by cell viability being > 80%), the cells were washed to reduce cellular debris and other residuals, and resuspended in serum-free, animal component-free, and defined cryopreservation medium containing 10% DMSO. The cells were formulated at a volume of 45 mL and cryopreserved in a controlled rate freezer (CRF). Samples were taken to determine cell counts, viability, percentage of cells expressing particular markers (e.g., CD34, CD3, CD19, CD14, CD56). editing efficiency, and residual Cas9 as shown in Table 4 below. Table 4. Analysis of exemplary cell preparations
Figure imgf000079_0001
BLLQ= below lower limit of quantification.
Example 4: CD33-Deleted Hematopoietic Stem and Progenitor Cells Display Engraftment after Hematopoietic Cell Transplant (HCT) and Tolerate Post-HCT Gemtuzumab Ozogamicin (GO) Treatment Without Cytopenias.
This example describes an evaluation of the safety of infusion of engineered CD33- deficient hematopoietic cells (referred to as trem-cel; formerly known as VOR33) and gemtuzumab ozogamicin (GO; tradename My lotarg®) in human acute myeloid leukemia (AML) patients who are at a high risk of relapse post-hematopoietic cell transplant (HCT).
As shown in FIG. 1, open-label first-in-human trial referred to as VBP101 evaluates CD33-positive AML patients who are at high risk of relapse undergo myeloablative HCT with trem-cel followed by treatment with low-dose gemtuzumab ozogamicin. Part 1 of the study will enroll 9-18 patients in 3 cohorts and who were treated with escalating doses of gemtuzumab ozogamicin (0.5-2.0 mg/m2) in 28-day dosing cycles for up to 4 cycles. The safety of trem-cel will be assessed and the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D) of gemtuzumab ozogamicin will be determined. In Part 2 of the study, an additional 12 patients will be enrolled to further evaluate the safety of trem- cel and the preliminary efficacy of trem-cel and gemtuzumab ozogamicin at RP2D.
As shown in FIG. 1, eligible subjects are between 18-70 years of age with CD33+ AML and be candidates for myeloablative conditioning (MAC) HCT. Subjects are also considered at a high risk of relapse but have had previously been subjected to autologous or allogeneic HCT and have not previously been treated with GO (GO-naive).
Subjects are subjected to a 9 day conditioning regimen consisting of busulfan/melphalan/fludarabine/rATG or total body irradiation/cyclophosphamide/ thiotepa/rATG; Following infusion with an CD33-deleted allograft (e.g., trem-cel), cells are allowed to recover and engraft for 60 days and then are subjected to anti-CD33 maintenance therapy involving a dosing regimen of gemtuzumab ozogamicin comprising up to 4 dosing cycles (each 28 days) of gemtuzumab ozogamicin in escalating doses (an effective amount of 0.5-2.0 mg/m2).
The primary endpoint of the study is successful neutrophil engraftment at 28 days post-HCT. Key secondary endpoints include, platelet engraftment, persistence of CD33- negative engraftment, primary and secondary graft failure, graft- versus-host disease, transplant-related mortality, and GO pharmacokinetic assessment.
The first patient (Patient 1) was a 64-year-old female patient (weight 69.9 kg) diagnosed with AML including the following high-risk features: highly complex (adverse) cytogenetics, myelodysplastic syndrome (MDS)-related changes (MRC), and TP53 mutation. The patient required 2 courses of cytarabine/daunorubicin to achieve complete response (CR). However, she relapsed after 3 cycles of high dose cytarabine (HiDAC). After 2 cycles of venetoclax and decitabine, she achieved CR2. However, her bone marrow (BM) contained 1.8% measurable residual disease (MRD). A 10/10 HLA matched unrelated donor (MUD) was identified and consented. Donor cells were mobilized using granulocyte colony stimulating factor (G-CSF) and plerixafor. In order to reduce AML relapse post-HCT, a CD33 CRISPR/Cas9 gene-edited donor allograft was developed to enable post-HCT CD33- directed therapies while protecting healthy donor cells from on-target myelosuppression. Hematopoietic cells deficient in CD33 e.g., gemtuzumab ozogamicin) were manufactured with 97% CD34+ cells and 88% gene editing efficacy. The patient received myeloablative conditioning (MAC) with busulfan/melphalan/fludarabine/rabbit anti-thymocyte globulin (ATG) prior to trem-cel infusion of 7.6 x 106 cells/kg.
The second patient (Patient 2) was a 32-year-old male (120.7 kg) diagnosed with AML after myeloid sarcoma partially resected from the small bowel and omentum. Initial cytogenetics were Inv 16 and +22. Subsequent adverse risk t(3;3) was identified. Initial treatment consisted of 7+3 cytarabine/daunorubicin with complete response (CR) Minimal residual disease was less than 0.1%. The patient then received 3 cycles of HiDAC, remaining in CR with persistence of abdominal disease by positron emission tomography (PET). A 10/10 HLA matched unrelated donor (MUD) was identified and consented. Donor cells were mobilized with G-CSF and plerixafor. Trem-cel was manufactured with 87% CD33 gene editing efficacy; the patient’s trem-cel dose was 3.2 x 106 CD34+ cells/kg. The patient received MAC with busulfan/melphalan/fludarabine/rabbit ATG prior to trem-cel infusion. The third patient (Patient 3) was a 55-year-old female (114.1 kg) with AML with myelodysplastic syndrome (MDS)-related changes (MRC). Cytogenetics were normal. DNMT3A, IDH2, and SMC1A mutations were detected. Minimal residual disease was less than 0.1%. A 10/10 HLA matched unrelated donor (MUD) was identified and consented. Donor cells were mobilized using granulocyte colony stimulating factor (G-CSF) and plerixafor. Trem-cel was manufactured with 80% CD33 gene editing efficacy; the patient’s trem-cel dose was 2.6 x 106 CD34+ cells/kg. The patient received MAC with busulfan/melphalan/fludarabine/equine ATG prior to trem-cel infusion.
The fourth patient (Patient 4) was a 68-year-old male (72.4 kg) with AML with myelodysplastic syndrome (MDS)-related changes (MRC). Patient 4 exhibited complex cytogenetics and active disease. NRAS, ZRSR2, and TET2 mutations were detected. Minimal residual disease was 16%. A 10/10 HLA matched unrelated donor (MUD) was identified and consented. Donor cells were mobilized using granulocyte colony stimulating factor (G-CSF) and plerixafor. Trem-cel was manufactured with 89% CD33 gene editing efficacy; the patient’s trem-cel dose was 5.8 x 106 CD34+ cells/kg. The patient received MAC with busulfan/melphalan/fludarabine/rabbit ATG prior to trem-cel infusion.
The fifth patient (Patient 5) was a 66-year-old male (102.1 kg) with secondary AML. The patient exhibited normal cytogenetics. KIT D816V, CBL, SRSF2, RUNX1/2, and BCORL1 mutations were detected. Minimal residual disease was 0.1% MRD. A 10/10 HLA matched unrelated donor (MUD) was identified and consented. Donor cells were mobilized using granulocyte colony stimulating factor (G-CSF) and plerixafor. Trem-cel was manufactured with 85% CD33 gene editing efficacy; the patient’s trem-cel dose was 4.6 x 106 CD34+ cells/kg. The patient received MAC with busulfan/melphalan/fludarabine/rabbit ATG prior to trem-cel infusion.
Following trem-cel infusion, neutrophil engraftment was observed between days 10- 11 post HCT (z.e., at 10 days post HCT in Patients 1 and 3-5 and at day 11 in Patient 2). Platelet recovery was observed at 22 days post HCT in Patient 1, 15 days post HCT in Patient 4, and 17 days post HCT in Patient 2 (FIGs. 2A-4D).
Donor chimerism analysis in Patient 1 indicated whole blood and myeloid cells were 100% of donor origin, which was maintained through day 100 (D100) of the assessment. Donor chimerism of 97% was observed in T cells at the D100 assessment. Indel analysis of whole blood showed 95.2%, 95.9%, and 99.4% gene editing efficiency at D28, D60, and D100, respectively, in bulk cells (FIG. 5A). CD33-deficient cells administered to Patient 1 (e.g., trem-cel) had 92.4% CD33 editing. CD33 editing was largely consistent across bulk cells and cells of myeloid and lymphoid lineages (FIG. 5A). An increase in CD33 editing (-3%) in bulk cells, monocytes, and B cells at D100 reflects the elimination of CD33+ cells and enrichment of CD33-negative cells after 1 dosing cycle of 0.5 mg/m2 and 1.0 mg/m2 of gemtuzumab ozogamicin. Enrichment of B cells (95.6% at D60 to 98.5% at D100) was unexpected as they are not high- CD33 expressing cells. CD33 editing correlates well with CD33 expression in monocytes (FIG. 5A).
At 28 days post HCT (D+28), donor chimerism analysis of Patients 3-4 indicated whole blood, monocytes, NK cells, and B cells were 100% of donor origin which was maintained, in Patient 3, through day 60 (D+60). Similar analyses in Patient 2 indicated whole blood cells were 94% of donor origin at D+28, and myeloid cells were 99-100% of donor origin. Donor chimerism of 4%, 99%, and 52% were observed in the T cell population at D+28 in Patients 2-4, respectively. Indel analysis of whole blood showed -86-96% gene editing efficiency at D+28 post HCT in Patients 2-4. By D+60 post HCT, 87.9% gene editing efficiency was detected in Patient 3 (FIG. 5B). All patients showed successful neutrophil engraftment within 28 days of HCT with trem-cel. The median time to neutrophil engraftment was 10 days. Five out of six patients had successful platelet recovery within 28 days of HCT with trem-cel. The median time to platelet recovery was 16 days. Complete donor chimerism was observed in monocytes (FIG. 5C) suggesting engraftment and reconstitution of these cells from trem-cel. Trem-cel exhibited greater than 85% CD33 gene editing at the time of infusion and the editing frequency was maintained over time in monocytes (FIG. 5D). Greater than 80% of the monocytes and myeloid cells are CD33- negative up to 180 days post-transplant with trem-cel, indicating in-vivo long-term persistence of CD33-editing (FIG. 5E).
At the D28 assessment, flow cytometry analyses of peripheral blood (PB) and bone marrow (BM) samples from patients were analyzed for CD33 expression. For PB samples from Patient 1, approximately 94% of monocytes and approximately 95% of myeloid cells were found to be CD33-negative (FIG. 7A). For PB samples from Patient 2, approximately 93% of monocytes and 99% of myeloid cells were found to be CD33-negative. For PB samples from Patient 3, approximately 82% of monocytes and approximately 86% of myeloid cells were found to be CD33-negative. For PB samples of Patient 4, approximately 90% of monocytes and approximately 95% of myeloid cells were found to be CD33-negative. For BM from Patient 1, approximately 95% of maturing myeloid, approximately 92% of maturing monocyte, and approximately 94% of CD34+ myeloblasts were found to be CD33- negative with development patterns comparable to non-edited cells at day 28 post-HCT. For BM from Patient 2, approximately 98% of maturing myeloid cells and approximately 91% of monocytes that were found to be CD33-negative. For BM of Patient 3, approximately 86% of myeloid cells and approximately 80% of monocytes were found to be CD33-negative. For BM of Patient 4, approximately 94% of maturing myeloid cells and 90% of monocytes were found to be CD33-negative (FIGs. 6A-6B and 7A-7B).
Patient 1 received gemtuzumab ozogamicin at 0.5 mg/m2 at 68 days post-HCT during the first dosing cycle of the anti-CD33 maintenance therapy period. Plasma samples were collected pre-GO dosing and post-GO dosing on day 1 of the first gemtuzumab ozogamicin dosing cycle (C1D1) (1, 2, 3, 4, and 6 hours) and on day 8 of the first gemtuzumab ozogamicin dosing cycle (C1D8) and analyzed for hP67.6 (the anti-CD33 portion of gemtuzumab ozogamicin) by ELISA. Pharmacokinetic parameters were calculated using a non-compartmental analysis method in Phoenix WinNonlin Version 9.3 using the concentration data measured after drug infusion and the actual dosing and pharmacokinetic (PK) sampling information. Surprisingly, the Cmax and AUC values observed following administration of a dosing cycle involving a single dose of 0.5 mg/m2 gemtuzumab ozogamicin corresponded to gemtuzumab ozogamicin doses of 1-2 mg/m2 and 4-5mg/m2, respectively, based on pop PK analysis (ODAC, FDA, 2017) (FIGs. 8A-8D). The half-life of gemtuzumab ozogamicin was found to be 64.56 hours, which closely matched a previously published report, where the half-life of gemtuzumab ozogamicin was reported to be 62 hours in patients dosed with 9 mg/m2 of gemtuzumab ozogamicin/Mylotarg® (see, Hibma J, et al. Clinical Pharmacokinet. 2019; 58(3):335-347. 2. Mylotarg® ODAC Brief 2017). Collectively, this data indicated that the therapeutic threshold of gemtuzumab ozogamicin (AUC 51,400 ng h/mL) was achieved at a lower dose compared to the CD33+ setting. As such, these results suggested that the effective amount of gemtuzumab ozogamicin per dosing cycle in a CD33-negative setting may be lower than that previously used in CD33+ individuals.
Following the dosing regimen comprising three dosing cycles with gemtuzumab ozogamicin administered in a single dose of 0.5 mg/m2, no decreases in neutrophil or platelet counts were observed in Patient 1 through day 147 (FIGs. 9, 10A, and 10D). No elevation in liver function tests (LFTs) were observed. Additionally, CD33-negative cells were increased after GO treatment as demonstrated by flow cytometry (FIGs. 10C and 10E). This data indicated that patient cell counts were protected and the CD33-negative cell fraction in the blood were stable even with repeated dosing of gemtuzumab ozogamicin. Measurable residual disease (MRD) was assessed throughout the course of the study in the bone marrow and peripheral blood tissue of the patient (FIGs. 10B and 11).
Serious adverse events (AEs) after trem-cel dosing in Patient 1 included Grade 3 renal colic attributable to nephrolithiasis and Grade 3 deep venous thrombosis. Infectious AEs included Grade 1 and Grade 2 skin infection, Grade 2 CMV reactivation, and Grade 2 BK virus (urine) which resolved or, in other instances, showed signs of resolving. Hepatic AEs included Grade 1 and Grade 2 aspartate aminotransferase (AST)/alanine transaminase (ALT) elevations and both were attributed to anti-fungal therapy and ultimately resolved. No trem- cel- or GO-related AEs were reported for up to 3 cycles. Prior to HCT, the patient had 1.8% MRD in the BM and none was observed post-HCT at day 28 and day 60. MRD in the BM was detected at D101 (0.3%) and D129 (2.2%) (FIG. 10B).
After primary neutrophil engraftment at D+l 1, Patient 2 developed cytopenias after D+28 in the setting of coronavirus hKUl infection, trimethoprim-sulfamethoxazole (TMP- SMP) exposure, and subsequent eosinophilia. Early sepsis had also developed following initial infusion of the graft. The backup graft was administered for secondary graft failure on D+57, and neutrophil engraftment and platelet recovery were observed after 26 and 30 days, respectively, following back-up infusion (FIG. 12).
Patient 3 had neutrophil engraftment at D+10 did not have platelet recovery as of D+l 17. The patient received courses of steroids, IVIg, eltrombopag, and romiplostim for treatment of presumed immune thrombocytopenia. The D+60 BM biopsy was hypocellular and showed decreased megakaryocytes. A positive platelet reactive antibody was identified on D+53 and characterized as an anti-HLA Class I Ab. D+100 BM biopsy was normocellular with normal megakaryocytes. As of D+l 17 the platelet count was 15,000/pL without transfusion in prior 11 days.
In sum, trem-cel was found to be well-tolerated. No related AEs were reported in Patients 4 and 5, and no unexpected AEs were reported. All 5 patients transplanted with trem-cel demonstrated primary neutrophil engraftment (days 10-11), which was similar to patients who received non-edited CD34 selected grafts. All patients achieved high levels of myeloid donor chimerism by day +28. A high level of CD33-negative hematopoiesis was achieved (>80%) in the 4 patients evaluable to day +28.
In Patient 1, a high level of CD33-negative hematopoiesis was achieved (>90%) and increased over the course of gemtuzumab ozogamicin treatment. The pattern of myeloid development derived from the CD33-deleted graft was similar to a reference patient with a non-edited graft. Additionally, CD33-negative cells were enriched after gemtuzumab ozogamicin treatment across lineages. The CD33 deletion was observed at the genetic level across all lineages of Patient 1, suggesting editing of a progenitor hematopoietic cell. In the context of a CD33-deleted graft, dosing cycles of a single dose of 0.5 mg/m2 gemtuzumab ozogamicin demonstrated a Cmax and AUC comparable to doses of 1-2 mg/m2 and 4-5 mg/m2, respectively, in an AML patient. At a gemtuzumab ozogamicin dose of 0.5 mg/m2, neutrophil and platelet counts remained stable through multiple cycles of GO, including in the presence of MRD. This suggests protection from gemtuzumab ozogamicin hemato toxicity.
Further analyses were performed to compare the effects associated with administration of 0.5 mg/m2 x 1 gemtuzumab ozogamicin or 1.0 mg/m2 x 1 gemtuzumab ozogamicin following HCT with trem-cel to previously reported effects observed in subjects that received the same doses of gemtuzumab ozogamicin but did not receive HCT with CD33-edited cells. When 0.5 mg/m2 gemtuzumab ozogamicin was administered following HCT with CD33-edited cells, an 8-fold increase in Cmax and a 24-fold increase in AUCinf in AUC relative to effects reported in the 0901A1-101-US study (FIGs. 13A-13C). When 1.0 mg/m2 gemtuzumab ozogamicin was administered following HCT with CD33-edited cells, a 6-fold increase in Cmax and 24-fold increase in AUCinf increase in AUC relative to effects reported in the 0901A1-101-US study (FIGs. 13A-13C). Analyses which were performed at select time points between day 63 and day 105 showed no decrease in absolute neutrophil counts in Patient 7 following administration of 1.0 mg/m2 gemtuzumab ozogamicin compared to Patients 1, 5, and 6 following administration of 0.5 mg/m2 gemtuzumab ozogamicin (FIG. 13D). An enrichment of CD33-negative monocytes and myeloid cells was observed postexposure to gemtuzumab ozogamicin at dose levels of 0.5 mg/m2 and 1.0 mg/m2 (FIGs. 13E- 13F) was consistent with protection of CD33-negative cells but the eradication of the CD33- positive cells by gemtuzumab ozogamicin.
Example 5: CD33 Antigen Density and Gemtuzumab Ozogamicin Dosing
CD33 antigen density refers to the number of CD33 molecules present on a cell. This Example specifically relates to the density of CD33 antigens, e.g., CD33 antigens capable of binding gemtuzumab ozogamicin, as well as diagnostic and therapeutic methods related to the same.
Generally, cells having higher a higher density of CD33 antigens relative to cells with reduced or eliminated expression of wild-type CD33 e.g., hematopoietic cancer cells comprising a mutated gene encoding a mutant CD33 or hematopoietic cells that are genetically engineered in a gene encoding CD33) will be capable of binding to higher amounts of gemtuzumab ozogamicin. CD33 antigen density can be used to refer to the number of CD33 molecules expressed at the cell surface of an individual cell (e.g., a hematopoietic cell). However, CD33 antigen density can also be used to refer to the number of cells in a tissue, such as a peripheral blood or bone marrow, expressing CD33 antigens capable of binding to gemtuzumab ozogamicin.
CD33 antigen density may be used to inform gemtuzumab ozogamicin dosing regimens in subjects (e.g., human subjects). Methods for determining CD33 antigen density will involve quantitative analyses of CD33 protein levels at the cell surface. For example, cells (e.g., hematopoietic cells expressing wild-type CD33) are contacted with an anti-CD33 antibody, such as hP67.6 (the antibody component of gemtuzumab ozogamicin), which is fluorescently labeled or contacted with an anti-CD33 antibody which is not fluorescently labeled prior to being contacted with a fluorescently labeled secondary antibody. Flow cytometry analyses are performed to measure the amount of fluorescent signal corresponding to antibody-bound cells. Control samples are used to normalize fluorescent signals. Genetically engineered cells comprising reduced or eliminated expression of CD33 can be further assessed by DNA sequencing analyses to correlate DNA editing efficiency to the fluorescent signals corresponding to antibody -bound cells measured by flow cytometry. Estimations of CD33 antigen density can be further compared with pharmacokinetic and/or pharmacodynamic data via methods described herein to determine correlations between CD33 antigen density and response to gemtuzumab ozogamicin treatment.
The overall antigen density of CD33 may be influential in the expected PK and PD of a given dose of gemtuzumab ozogamicin, consistent with the principles of target mediated drug disposition. For instance, the large reservoir of CD33 expression can be assumed from both malignant and normal myeloid cells in relapsed and refractor AML patients, requiring a sufficient dose of gemtuzumab ozogamicin (z.e., > 2 mg/m2) for efficacy. Total CD33+ receptor expression per cell, as well as the total CD33+ cell counts can both drive the relative antigen density within a given population. Importantly, due to reduction in both the overall disease burden and the proportion of nonmalignant CD33+ cells in the body following hematopoietic cell transplant with trem-cel, the dose of gemtuzumab ozogamicin required to reach an effective outcome (z.e., targeted cell killing of CD33+ blast cells) may be lower than that used in R/R AML patients with higher disease burden and a CD33+ normal myeloid compartment. Interim pharmacokinetic data following a first dose of gemtuzumab ozogamicin administered at the first dose level (cohort 1, 0.5 mg/m2) showed that the observed exposures after a single dose of 0.5 mg/m2 was found to be within the predicted therapeutic range of 3 mg/m2 gemtuzumab ozogamicin for all patients. Moreover, the observed Cmax values for the patients suggested a low risk of veno-occlusive disease (VOD). Consistent with the principles of target mediated drug disposition, populations with substantially lower CD33+ antigen density may require a lower dose to achieve similar pharmacokinetic exposures and pharmacodynamic responses (Peletier, 2012). Thus, estimation of antigen density may be used to determine a therapeutic dose of gemtuzumab ozogamicin in the post-trem-cel HCT setting.
EQUIVALENTS AND SCOPE
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the exemplary embodiments described herein. The scope of the present disclosure is not intended to be limited to the above description.
Articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between two or more members of a group are considered satisfied if one, more than one, or all of the group members are present, unless indicated to the contrary or otherwise evident from the context. The disclosure of a group that includes “or” between two or more group members provides embodiments in which exactly one member of the group is present, embodiments in which more than one members of the group are present, and embodiments in which all of the group members are present. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.
It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitation, element, clause, or descriptive term, from one or more of the claims or from one or more relevant portion of the description, is introduced into another claim. For example, a claim that is dependent on another claim can be modified to include one or more of the limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of making or using the composition according to any of the methods of making or using disclosed herein or according to methods known in the art, if any, are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.
Where elements are presented as lists, it is to be understood that every possible individual element or subgroup of the elements is also disclosed, and that any element or subgroup of elements can be removed from the group. It is also noted that the term “comprising” is intended to be open and permits the inclusion of additional elements, features, or steps. It should be understood that, in general, where an embodiment, is referred to as comprising particular elements, features, or steps, embodiments, that consist, or consist essentially of, such elements, features, or steps, are provided as well. For purposes of brevity those embodiments have not been individually spelled out herein, but it will be understood that each of these embodiments is provided herein and may be specifically claimed or disclaimed.
Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in some embodiments, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. For purposes of brevity, the values in each range have not been individually spelled out herein, but it will be understood that each of these values is provided herein and may be specifically claimed or disclaimed. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.
In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein. The disclosure contemplates all combinations of any one or more of the foregoing embodiments, as well as combinations with any one or more of the embodiments set forth in the detailed description and examples.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. All publications, patent applications, patents, and other references (e.g., sequence database reference numbers) mentioned herein are incorporated by reference in their entirety. For example, all GenBank, Unigene, and Entrez sequences referred to herein, e.g., in any Table herein, are incorporated by reference. Unless otherwise specified, the sequence accession numbers specified herein, including in any Table herein, refer to the database entries current as of May 23, 2019. When one gene or protein references a plurality of sequence accession numbers, all of the sequence variants are encompassed.

Claims

CLAIMS What is claimed is:
1. A method, comprising administering to a subject an effective amount of a population of genetically engineered hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen; and administering to the subject gemtuzumab ozogamicin in a dosing regimen comprising a dosing cycle, wherein the dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin; wherein the effective amount of gemtuzumab ozogamicin is 0.1 mg/m2 - 6.0 mg/m2 body surface area of the subject.
2. The method of claim 1, wherein the dosing regimen comprises at least two, at least three, or at least four dosing cycles, wherein each dosing cycle of the dosing regimen comprises administration of the effective amount of gemtuzumab ozogamicin.
3. The method of claim 1 or 2, wherein the dosing cycle or each dosing cycle is about 4 weeks or less.
4. The method of any one of claims 1-3, wherein the effective amount of gemtuzumab ozogamicin is administered to the subject in a single dose.
5. The method of claim 4, wherein the single dose of gemtuzumab ozogamicin is about 0.1 mg/m2, about 0.25 mg/m2, about 0.5 mg/m2, about 1.0 mg/m2, about 2.0 mg/m2, about 3.0 mg/m2, about 4.0 mg/m2, about 5.0 mg/m2, or about 6.0 mg/m2 body surface area of the subject.
6. The method of claim 5, wherein the single dose of gemtuzumab ozogamicin is about 0.5 mg/m2 body surface area of the subject.
7. The method of claim 6, wherein the single dose of gemtuzumab ozogamicin is about 0.5 mg/m2 body surface area of the subject and the dosing cycle is about 4 weeks.
8. The method of any one of claims 1-3, wherein the effective amount of gemtuzumab ozogamicin is administered to the subject in multiple doses in the dosing cycle or each dosing cycle.
9. The method of claim 8, wherein each of the multiple doses of gemtuzumab ozogamicin is about 0.1 mg/m2, about 0.25 mg/m2, about 0.5 mg/m2, about 1.0 mg/m2, about 2.0 mg/m2, about 3.0 mg/m2, about 4.0 mg/m2, about 5.0 mg/m2, or about 6.0 mg/m2 body surface area of the subject.
10. The method of claim 9, wherein each of the multiple doses of gemtuzumab ozogamicin is about 0.5 mg/m2 body surface area of the subject.
11. The method of any one of claims 8-10, wherein the multiple doses of gemtuzumab ozogamicin comprises two doses of gemtuzumab ozogamicin.
12. The method of claim 11, wherein a first dose is administered to the subject on day 1 of the dosing cycle and a second dose is administered on day 7 of the dosing cycle.
13. The method of claim 11, wherein a first dose is administered to the subject on day 1 of the dosing cycle and a second dose is administered on day 14 of the dosing cycle.
14. The method of any one of claims 8-10, wherein the multiple doses of gemtuzumab ozogamicin comprises three doses of gemtuzumab ozogamicin.
15. The method of claim 14, wherein a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered on day 4 of the dosing cycle, and a third dose is administered on day 7 of the dosing cycle.
16. The method of claim 14, wherein a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered on day 7 of the dosing cycle, and a third dose is administered on day 14 of the dosing cycle.
17. The method of claim 14, wherein a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered to the subject once the plasma concentration of gemtuzumab ozogamicin in the subject is less than a threshold value, and a third dose is administered to the subject once the plasma concentration of gemtuzumab ozogamicin in the subject is less than a threshold value following administration of the second dose.
18. The method of any one of claims 8-10, wherein the multiple doses of gemtuzumab ozogamicin comprises four doses of gemtuzumab ozogamicin.
19. The method of claim 18, wherein the multiple doses of gemtuzumab ozogamicin are administered to the subject weekly in the dosing cycle.
20. The method of claim 18 or 19, wherein a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered on day 7 of the dosing cycle, a third dose is administered on day 14 of the dosing cycle, and a fourth dose is administered on day 21 of the dosing cycle.
21. The method of any one of claims 1-20, wherein the population of genetically engineered hematopoietic cells and the dosing cycle are administered in temporal proximity.
22. The method of claim 21, wherein administering in temporal proximity comprises administering the dosing cycle of the dosing regimen at least 60 days after administration of the population of genetically engineered hematopoietic cells.
23. The method of claim 21, wherein the administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen between 40-60 days after administration of the population of genetically engineered hematopoietic cell, if the subject experiences early relapse.
24. The method of any one of claims 1-23, wherein the population of genetically engineered hematopoietic cells are administered prior to the gemtuzumab ozogamicin.
25. The method of any one of claims 1-24, wherein the population of genetically engineered hematopoietic cells are administered in a single treatment regimen.
26. The method of any one of claims 1-25, wherein the population of genetically engineered hematopoietic cells and/or the gemtuzumab ozogamicin are administered intravenously.
27. The method of any one of claims 1-26, wherein the effective amount of the population of genetically engineered hematopoietic cells is about 106 cells/kilogram body weight of the subject to about 5 x 107 cells/kilogram body weight of the subject.
28. The method of claim 27, wherein the effective amount of the population of genetically engineered hematopoietic cells is about 7.5 x 106 cells/kilogram body weight of the subject.
29. The method of claim 27, wherein the effective amount of the population of genetically engineered hematopoietic cells is about 3 x 106 cells/kilogram body weight of the subject.
30. The method of claim 27, wherein the effective amount of the population of genetically engineered hematopoietic cells is about 106 cells/kilogram body weight of the subject.
31. The method of any one of claims 1-30, wherein the population of genetically engineered hematopoietic cells are thawed from a cryopreserved form prior to administration.
32. The method of any one of claims 1-31, wherein the gemtuzumab ozogamicin is reconstituted from a lyophilized form prior to administration.
33. The method of any one of claims 1-32, wherein the subject has been preconditioned prior to administering the population of hematopoietic cells and gemtuzumab ozogamicin.
34. The method of any one of claims 1-33, further comprising preconditioning the subject prior to administering the population of hematopoietic cells and gemtuzumab ozogamicin.
35. The method of claim 33 or 34, wherein the preconditioning comprises administering one or more chemotherapeutic agents to the subject.
36. The method of any one of claims 33-35, wherein the preconditioning comprises total body irradiation of the subject.
37. The method of claim 35 or 36, wherein the one or more chemotherapeutic agents are selected from the group consisting of busulfan, melphalan, fludarabine, cyclophosphamide, and thiotepa.
38. The method of any one of claims 33-37, wherein the preconditioning comprises administering antibodies that bind human T cells, optionally wherein the antibodies comprise rabbit anti-thymocyte globulins (rATG).
39. The method of any one of claims 1-38, wherein the subject has, or has been diagnosed with, a hematopoietic malignancy or a hematopoietic pre-malignant disease, and wherein the hematopoietic malignancy is characterized by the presence of CD33-positive malignant cells, or wherein the hematopoietic pre-malignant disease is characterized by the presence of CD33-positive pre-malignant cells.
40. The method of any one of claims 1-39, wherein the subject has, or has been diagnosed with, CD33-positive acute myeloid leukemia.
41. The method of any one of claims 1-40, wherein the subject has, or has been diagnosed with, CD33-positive myelodysplastic syndrome.
42. The method of any one of claims 1-41, wherein the subject has, or has been diagnosed with, CD33-positive myelodysplastic syndrome and wherein the subject is at high risk of developing acute myeloid leukemia or refractory cytopenias.
43. The method of any one of claims 1-32 or 39-42, wherein the subject is naive to chemotherapy and/or radiation therapy, optionally wherein the subject is naive to any treatment aimed to address a hematopoietic malignancy or hematopoietic pre-malignant disease.
44. The method of any one of claims 1-43, wherein the subject has previously received chemotherapy.
45. The method of any one of claims 1-44, wherein the subject has previously received induction therapy.
46. The method of any one of claims 1-45, wherein the subject has previously entered a complete hematological remission, optionally wherein the complete hematological remission is characterized by an incomplete recovery of peripheral counts.
47. The method of any one of claims 1-46, wherein the subject has one or more risk factors associated with early leukemia relapse.
48. The method of claim 47, wherein the one or more risk factors associated with early leukemia relapse are selected from the group consisting of: bone marrow in morphological complete remission with presence of intermediate or high-risk disease-related genetics; presence of minimal residual disease (MRD) post cyto-reductive therapy; bone marrow with persistent leukemia blasts post cyto-reductive therapy; and bone marrow blast count of about 10% or less.
49. The method of any one of claims 1-48, wherein the subject does not have acute promyelocytic leukemia or chronic myeloid leukemia.
50. The method of any one of claims 1-49, wherein the subject has not previously received a stem cell transplantation.
51. The method of any one of claims 1-50, wherein the subject has not previously received gemtuzumab ozogamicin.
52. The method of any one of claims 1-51, further comprising determining a percent donor chimerism and/or a level of CD33-negative myeloid hematopoiesis in a peripheral blood sample from the subject.
53. The method of any one of claims 1-52, wherein the subject has a CD33-negative absolute neutrophil count (ANC) of at least 500 cells/pL prior to receiving the dosing regimen.
54. The method of any one of claims 1-52, wherein the subject has a CD33-negative absolute neutrophil count (ANC) of at least 1,000 cells/pL prior to receiving the dosing regimen.
55. The method of any one of claims 1-54, wherein the hematopoietic cells are hematopoietic stem and progenitor cells.
56. The method of claim 55, wherein the hematopoietic stem cells are from bone marrow cells, cord blood cells, or peripheral blood mononuclear cells (PBMCs).
57. The method of claim 55 or 56, wherein the hematopoietic stem cells are CD34+/CD33’.
58. The method of any one of claims 1-57, wherein the hematopoietic cells are autologous.
59. The method of claim 58, wherein the method further comprises obtaining the autologous hematopoietic stem cells from the subject, optionally wherein the method further comprises genetically engineering the autologous stem cells to have reduced or eliminated expression of the CD33 antigen, and returning the genetically engineered hematopoietic stem cells to the subject.
60. The method of any one of claims 1-57, wherein the hematopoietic cells are allogeneic.
61. The method of claim 60, wherein the hematopoietic cells are allogeneic hematopoietic stem cells obtained from a donor having an HLA haplotype that matches with the HLA haplotype of the subject.
62. The method of any one of claims 1-57, 60, or 61, further comprising obtaining hematopoietic cells from a donor having an HLA haplotype that matches with the HLA haplotype of the subject.
63. The method of any one of claims 1-62, further comprising preparing the hematopoietic cells by modifying an endogenous gene of the hematopoietic cells encoding the CD33 antigen.
64. The method of claim 63, wherein the whole or a portion of the endogenous gene encoding the CD33 cell-surface antigen is deleted.
65. The method of claim 63 or 64, wherein the whole or the portion of the endogenous gene is deleted using genome editing.
66. The method of claim 65, wherein the genome editing involves a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a CRISPR-Cas system.
67. The method of any one of claims 1-66, wherein the population of genetically engineered hematopoietic cells, or descendants thereof, are tremtelectogene empogeditemcel (trem-cel).
68. A method, comprising administering to a subject: gemtuzumab ozogamicin in a dosing regimen comprising a dosing cycle, wherein the dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin; wherein the effective amount of gemtuzumab ozogamicin is 0.1 mg/m2 - 6.0 mg/m2 body surface area of the subject; and wherein the subject is receiving or has received an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen.
69. The method of claim 68, wherein the dosing cycle comprises at least two, at least three, or at least four dosing cycles, wherein each dosing cycle comprises administration of an effective amount of gemtuzumab ozogamicin.
70. The method of claim 68 or 69, wherein the dosing cycle or each dosing cycle is about 4 weeks or less.
71. The method of any one of claims 68-70, wherein the effective amount of gemtuzumab ozogamicin is administered to the subject in a single dose.
72. The method of claim 71, wherein the single dose of gemtuzumab ozogamicin is about 0.1 mg/m2, about 0.25 mg/m2, about 0.5 mg/m2, about 1.0 mg/m2, about 2.0 mg/m2, about 3.0 mg/m2, about 4.0 mg/m2, about 5.0 mg/m2, or about 6.0 mg/m2 body surface area of the subject.
73. The method of claim 72, wherein the single dose of gemtuzumab ozogamicin is about 0.5 mg/m2 body surface area of the subject.
74. The method of claim 73, wherein the single dose of gemtuzumab ozogamicin is about 0.5 mg/m2 body surface area of the subject and the dosing cycle is about 4 weeks.
75. The method of any one of claims 68-70, wherein the effective amount of gemtuzumab ozogamicin is administered to the subject in multiple doses.
76. The method of claim 75, wherein each of the multiple doses of gemtuzumab ozogamicin is about 0.1 mg/m2, about 0.25 mg/m2, about 0.5 mg/m2, about 1.0 mg/m2, about 2.0 mg/m2, about 3.0 mg/m2, about 4.0 mg/m2, about 5.0 mg/m2, or about 6.0 mg/m2 body surface area of the subject.
77. The method of claim 76, wherein each of the multiple doses of gemtuzumab ozogamicin is about 0.5 mg/m2 body surface area of the subject.
78. The method of any one of claims 75-77, wherein the multiple doses of gemtuzumab ozogamicin comprises two doses of gemtuzumab ozogamicin.
79. The method of claim 78, wherein a first dose is administered to the subject on day 1 of the dosing cycle and a second dose is administered on day 7 of the dosing cycle.
80. The method of claim 78, wherein a first dose is administered to the subject on day 1 of the dosing cycle and a second dose is administered on day 14 of the dosing cycle.
81. The method of any one of claims 75-77, wherein the multiple doses of gemtuzumab ozogamicin comprises three doses of gemtuzumab ozogamicin.
82. The method of claim 81, wherein a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered on day 4 of the dosing cycle, and a third dose is administered on day 7 of the dosing cycle.
83. The method of claim 81, wherein a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered on day 8 of the dosing cycle, and a third dose is administered on day 16 of the dosing cycle.
84. The method of claim 81, wherein a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered to the subject once the plasma concentration of gemtuzumab ozogamicin in the subject is less than a threshold value, and a third dose is administered to the subject once the plasma concentration of gemtuzumab ozogamicin in the subject is less than a threshold value following administration of the second dose.
85. The method of any one of claims 75-77, wherein the multiple doses of gemtuzumab ozogamicin comprise four doses of gemtuzumab ozogamicin.
86. The method of claim 85, wherein the four doses of gemtuzumab ozogamicin are administered to the subject weekly in the dosing cycle.
87. The method of claim 85 or 86, wherein a first dose is administered to the subject on day 1 of the dosing cycle, a second dose is administered on day 8 of the dosing cycle, a third dose is administered on day 15 of the at least one dosing cycle, and a fourth dose is administered on day 21 of the dosing cycle.
88. The method of any one of claims 68-87, wherein the population of genetically engineered hematopoietic cells and a first dosing cycle of gemtuzumab ozogamicin are administered in temporal proximity.
89. The method of claim 88, wherein administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen at least 60 days after administration of the population of genetically engineered hematopoietic cells.
90. The method of claim 88, wherein administering in temporal proximity comprises administering the first dosing cycle of the dosing regimen between 40-60 days after administration of the population of genetically engineered hematopoietic cell, if the subject experiences early relapse.
91. The method of any one of claims 68-89, wherein the subject received the population of genetically engineered hematopoietic cells prior to administration of gemtuzumab ozogamicin.
92. The method of any one of claims 68-91, wherein the subject received the population of genetically engineered hematopoietic cells in a single treatment regimen.
93. The method of any one of claims 68-92, wherein the administration of gemtuzumab ozogamicin is intravenous.
94. The method of any one of claims 68-93, wherein the effective amount of the population of genetically engineered hematopoietic cells is about 106 cells/kilogram body weight of the subject to about 5 x 107 cells/kilogram body weight of the subject.
95. The method of claim 94, wherein the effective amount of the population of genetically engineered hematopoietic cells is about 7.5 x 106 cells/kilogram body weight of the subject.
96. The method of claim 94, wherein the effective amount of the population of genetically engineered hematopoietic cells is about 3 x 106 cells/kilogram body weight of the subject.
97. The method of any one of claims 68-96, wherein the gemtuzumab ozogamicin is reconstituted from a lyophilized form prior to administration.
98. The method of any one of claims 68-97, wherein the subject has been preconditioned prior to receiving the population of hematopoietic cells and gemtuzumab ozogamicin.
99. The method of any one of claims 68-97, further comprising preconditioning the subject prior to administering the gemtuzumab ozogamicin.
100. The method of claim 98 or claim 99, wherein the preconditioning comprises administering one or more chemotherapeutic agents to the subject.
101. The method of any one of claims 98-100, wherein the preconditioning comprises total body irradiation of the subject.
102. The method of claim 100 or 101, wherein the chemotherapeutic agent is selected from the group consisting of busulfan, melphalan, fludarabine, cyclophosphamide, and thiotepa.
103. The method of any one of claims 98-102, wherein the preconditioning comprises administering antibodies that bind human T cells, optionally wherein the antibodies comprise rabbit anti-thymocyte globulins (rATG).
104. The method of any one of claims 68-103, wherein the subject has, or has been diagnosed with, a hematopoietic malignancy or a hematopoietic pre-malignant disease, and wherein the hematopoietic malignancy is characterized by the presence of CD33-positive malignant cells, or wherein the hematopoietic pre-malignant disease is characterized by the presence of CD33-positive pre-malignant cells.
105. The method of any one of claims 68-104, wherein the subject has, or has been diagnosed with, CD33-positive acute myeloid leukemia.
106. The method of any one of claims 68-105, wherein the subject has, or has been diagnosed with, CD33-positive myelodysplastic syndrome.
107. The method of any one of claims 68-106, wherein the subject has, or has been diagnosed with, CD33-positive myelodysplastic syndrome and wherein the subject is at high risk of developing acute myeloid leukemia or refractory cytopenias.
108. The method of any one of claims 68-107, wherein the subject is naive to chemotherapy and/or radiation therapy, optionally wherein the subject is naive to any treatment aimed to address a hematopoietic malignancy or hematopoietic pre-malignant disease.
109. The method of any one of claims 68-108, wherein the subject has previously received chemotherapy.
110. The method of any one of claims 68-109, wherein the subject has previously received induction therapy.
111. The method of any one of claims 68- 110, wherein the subject has previously entered a complete hematological remission, optionally wherein the complete hematological remission is characterized by an incomplete recovery of peripheral counts.
112. The method of any one of claims 68-111, wherein the subject has one or more risk factors associated with early leukemia relapse.
113. The method of claim 112, wherein the one or more risk factors associated with early leukemia relapse are selected from the group consisting of: bone marrow in morphological complete remission with presence of intermediate or high-risk disease-related genetics; presence of minimal residual disease (MRD) post cyto-reductive therapy; bone marrow with persistent leukemia blasts post cyto-reductive therapy; and bone marrow blast count of about 10% or less.
114. The method of any one of claims 68-113, wherein the subject does not have acute promyelocytic leukemia or chronic myeloid leukemia.
115. The method of any one of claims 68-114, wherein the subject has not previously received a stem cell transplantation.
116. The method of any one of claims 68-115, wherein the subject has not previously received gemtuzumab ozogamicin.
117. The method of any one of claims 68-116, further comprising determining a percent donor chimerism and/or a level of CD33-negative myeloid hematopoiesis in a peripheral blood sample from the subject.
118. The method of any one of claims 68-117, wherein the subject has a CD33-negative absolute neutrophil count (ANC) of at least 1,000 cells/pL prior to receiving the dosing regimen.
119. The method of any one of claims 68-117, wherein the subject has a CD33-negative absolute neutrophil count (ANC) of at least 500 cells/pL prior to receiving the dosing regimen.
120. The method of any one of claims 68-119, wherein the hematopoietic cells are hematopoietic stem and progenitor cells.
121. The method of claim 120, wherein the hematopoietic stem cells are from bone marrow cells, cord blood cells, or peripheral blood mononuclear cells (PBMCs).
122. The method of claim 120 or 121, wherein the hematopoietic stem cells are CD34+/CD33’.
123. The method of any one of claims 68-122, wherein the hematopoietic cells are autologous.
124. The method of any one of claims 68-122, wherein the hematopoietic cells are allogeneic.
125. The method of claim 124, wherein the hematopoietic cells are allogeneic hematopoietic stem cells obtained from a donor having an HLA haplotype that matches with the HLA haplotype of the subject.
126. The method of any one of claims 68-122 or 125, further comprising obtaining hematopoietic cells from a donor having an HLA haplotype that matches with the HLA haplotype of the subject.
127. The method of any one of claims 68-126, further comprising preparing the population of hematopoietic cells by modifying an endogenous gene of the hematopoietic cells encoding the CD33 antigen.
128. The method of claim 127, wherein the whole or a portion of the endogenous gene encoding the CD33 antigen is deleted.
129. The method of claim 127 or 128, wherein the whole or the portion of the endogenous gene is deleted using genome editing.
130. The method of claim 129, wherein the genome editing involves a zinc finger nuclease (ZFN), a transcription activator-like effector-based nuclease (TALEN), or a CRISPR-Cas system.
131. The method of any one of claims 68-130, wherein the population of genetically engineered hematopoietic cells, or descendants thereof, are tremtelectogene empogeditemcel (trem-cel).
132. A method comprising administering an effective amount of gemtuzumab ozogamicin to a subject, wherein the subject is identified as having hematopoietic cells comprising a lower density of wild-type CD33 in a first biological sample relative to a second biological sample.
133. The method of claim 132, wherein the first biological sample is obtained from the subject at a first time point and the second biological sample is obtained from the subject at a second time point; or wherein the second biological sample is obtained from a counterpart subject.
134. The method of claim 132 or 133, wherein the method further comprises: obtaining the first biological sample; and measuring the density of wild-type CD33 in the first biological sample.
135. The method of any one of claims 132-134, wherein the method further comprises obtaining the second biological sample; and measuring the density of wild-type CD33 in the second biological sample.
136. The method of any one of claims 132-135, wherein the lower density of wild-type CD33 in the first biological sample is 95% or less than the density of wild-type CD33 in the second biological sample.
137. The method of any one of claims 132-136, wherein the effective amount of gemtuzumab ozogamicin is 0.1 mg/m2 - 6.0 mg/m2 body surface area of the subject.
138. The method of any one of claims 132-136, wherein the effective amount of gemtuzumab ozogamicin is administered to the subject in a single dose or multiple doses of the effective amount.
139. The method of any one of claims 132-138, wherein: the method further comprises administering an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen; or the subject is receiving or has received an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen.
140. A method comprising
(a) measuring density of wild-type CD33 on a population of hematopoietic cells in a first biological sample of a subject;
(b) comparing the density of wild-type CD33 on the population of hematopoietic cells in the first biological sample to the density of wild-type CD33 on a population of hematopoietic cells in a second biological sample; and
(c) determining an effective amount of gemtuzumab ozogamicin for administration to the subject based on (b).
141. The method of claim 140, further comprising administering the effective amount of gemtuzumab ozogamicin for administration to the subject.
142. The method of claim 140 or 142, wherein the density of wild-type CD33 on the population of hematopoietic cells in the first biological sample is lower than the density of wild-type CD33 on a population of hematopoietic cells in a second biological sample.
143. The method of any one of claims 140-142, wherein the effective amount of gemtuzumab ozogamicin is 0.1 mg/m2 - 6.0 mg/m2 body surface area of the subject.
144. The method of any one of claims 140-143, wherein the method comprises administering to the first subject an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen.
145. The method of any one of claims 140-143, wherein the first subject has received an effective amount of a population of genetically modified hematopoietic cells, or descendants thereof, comprising a modified gene encoding CD33 that is engineered to have reduced or eliminated expression of a CD33 antigen.
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