WO2023081767A1 - Procédés d'immunothérapie - Google Patents

Procédés d'immunothérapie Download PDF

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WO2023081767A1
WO2023081767A1 PCT/US2022/079234 US2022079234W WO2023081767A1 WO 2023081767 A1 WO2023081767 A1 WO 2023081767A1 US 2022079234 W US2022079234 W US 2022079234W WO 2023081767 A1 WO2023081767 A1 WO 2023081767A1
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days
pharmaceutical composition
subject
administration
immune cells
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PCT/US2022/079234
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Michelle Brenda PIRES
Aaron Martin
Daniel T. MACLEOD
Alan F. List
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Precision Biosciences, Inc.
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
    • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
    • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001111—Immunoglobulin superfamily
    • A61K39/001112—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
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    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5156—Animal cells expressing foreign proteins
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)

Definitions

  • the invention relates to the field of oncology and immunotherapy.
  • the invention relates to allogeneic cellular immunotherapy and lymphodepletion regimens.
  • T cell adoptive immunotherapy is a promising approach for cancer treatment.
  • the immunotherapy treatment methods disclosed herein utilize isolated human T cells that have been genetically-modified to enhance their specificity for a specific tumor associated antigen. Genetic modification may involve the expression of a chimeric antigen receptor or an exogenous T cell receptor to graft antigen specificity onto the T cell. In contrast to exogenous T cell receptors, chimeric antigen receptors derive their specificity from the variable domains of a monoclonal antibody.
  • T cells expressing chimeric antigen receptors induce tumor immunoreactivity in a major histocompatibility complex non-restricted manner.
  • T cell adoptive immunotherapy has been utilized as a clinical therapy for a number of cancers, including B cell malignancies (e.g., acute lymphoblastic leukemia, B cell non-Hodgkin lymphoma, acute myeloid leukemia, and chronic lymphocytic leukemia), multiple myeloma, neuroblastoma, glioblastoma, advanced gliomas, ovarian cancer, mesothelioma, melanoma, prostate cancer, pancreatic cancer, and others.
  • B cell malignancies e.g., acute lymphoblastic leukemia, B cell non-Hodgkin lymphoma, acute myeloid leukemia, and chronic lymphocytic leukemia
  • multiple myeloma e.g., neuroblastoma, glioblastoma, advanced gliomas, ovarian cancer, mesothelioma, melanoma, prostate cancer, pancreatic cancer, and others.
  • CAR T cells expressing an endogenous T cell receptor may recognize major and minor histocompatibility antigens following administration to an allogeneic patient, which can lead to the development of graft-versus-host-disease (GVHD).
  • GVHD graft-versus-host-disease
  • clinical trials have largely focused on the use of autologous CAR T cells, wherein a patient’s T cells are isolated, genetically-modified to incorporate a chimeric antigen receptor, and then re-infused into the same patient.
  • An autologous approach provides immune tolerance to the administered CAR T cells; however, this approach is constrained by both the time and expense necessary to produce patient-specific CAR T cells after a patient’s cancer has been diagnosed.
  • CAR T cells prepared using T cells from a third party, healthy donor, that have reduced expression, or have no detectable cell surface expression of an endogenous T cell receptor (e.g., an alpha/beta T cell receptor) and do not initiate GvHD upon administration.
  • an endogenous T cell receptor e.g., an alpha/beta T cell receptor
  • Such products could be generated and validated in advance of diagnosis and could be made available to patients as soon as necessary. Therefore, a need exists for the development of allogeneic CAR T cells that lack an endogenous T cell receptor in order to prevent the occurrence of GvHD.
  • Clinical outcomes in CAR T therapy correlate with engraftment, expansion, and persistence of CAR T cells.
  • a lymphodepletion regimen consisting of cyclophosphamide and fludarabine precedes CAR T infusion. This creates niches for infused CAR T cells and stimulates beneficial homeostatic cytokine production. As these compounds are also toxic to CAR T cells, administering the proper doses of both the conditioning drugs and the cell therapies with appropriate timing can be a challenge.
  • the present disclosure describes methods and compositions for protecting CAR T cells from fludarabine toxicity by knocking down the gene deoxycytidine kinase (dCK), which converts fludarabine from the prodrug form to an active compound resulting in Fludarabine resistant allogeneic CAR T (FluR CAR T) useful for cellular immunotherapies.
  • dCK deoxycytidine kinase
  • the invention provides a method of reducing the number of target cells in a subject, the method comprising: (a) administering to the subject a lymphodepletion regimen comprising an effective dose of one or more chemotherapeutic lymphodepletion agents; and (b) administering to the subject an effective dose of a pharmaceutical composition comprising a population of human immune cells, wherein a plurality of the human immune cells are genetically-modified human immune cells; wherein the genetically- modified human immune cells comprise a cell surface engineered antigen receptor having specificity for an antigen on the target cells; wherein the genetically-modified human immune cells exhibit reduced expression of deoxycytidine kinase (dCK) protein compared to control cells; wherein the one or more chemotherapeutic lymphodepletion agents includes a nucleoside analog; and wherein the method reduces the number of the target cells in the subject.
  • the number of target cells in the subject is reduced relative to the same method wherein the genetically-modified human immune
  • the invention provides a method for reducing host rejection of genetically-modified human immune cells in a subject, the method comprising: (a) administering to the subject a lymphodepletion regimen comprising an effective dose of one or more chemotherapeutic lymphodepletion agents; and (b) administering to the subject an effective dose of a pharmaceutical composition comprising a population of human immune cells, wherein a plurality of the human immune cells are the genetically-modified human immune cells; wherein the genetically-modified human immune cells comprise a cell surface engineered antigen receptor having specificity for an antigen on target cells in the subject; wherein the genetically-modified human immune cells exhibit reduced expression of dCK protein compared to control cells; wherein the one or more chemotherapeutic lymphodepletion agents includes a nucleoside analog; and wherein rejection of the genetically-modified human immune cells by host immune cells is reduced (e.g., reduced relative to control genetically-modified human immune cells that are not modified to have reduced expression of d
  • the invention provides a method for reducing nucleoside analog- induced killing of genetically-modified human immune cells in a subject, the method comprising: (a) administering to the subject a lymphodepletion regimen comprising an effective dose of one or more chemotherapeutic lymphodepletion agents; and (b) administering to the subject an effective dose of a pharmaceutical composition comprising a population of human immune cells, wherein a plurality of the human immune cells are genetically-modified human immune cells; wherein the genetically-modified human immune cells comprise a cell surface engineered antigen receptor having specificity for an antigen on target cells in the subject; wherein the genetically-modified human immune cells exhibit reduced expression of dCK protein compared to control cells; wherein the one or more chemotherapeutic lymphodepletion agents includes the nucleoside analog; and wherein nucleoside analog-induced killing of the genetically-modified human immune cells is reduced (e.g., reduced relative to control genetically-modified human immune cells that are not
  • the genetically-modified human immune cells exhibit greater resistance (e.g., cell survival, cell expansion, target cell killing) to the nucleoside analog compared to control cells that do not exhibit reduced expression of dCK protein.
  • the human immune cells are human T cells. In some embodiments, the human immune cells are human natural killer (NK cells). In some embodiments, the human immune cells are human macrophages. In some embodiments, the human immune cells are human B cells.
  • the human immune cells are not derived from the subject.
  • the engineered antigen receptor is a chimeric antigen receptor (CAR). In some embodiments, the engineered antigen receptor is an exogenous T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR exogenous T cell receptor
  • the genetically-modified human immune cells comprise in their genome a polynucleotide comprising a nucleic acid sequence encoding the engineered antigen receptor.
  • the polynucleotide comprises an exogenous promoter that is operably linked to the nucleic acid sequence encoding the engineered antigen receptor.
  • the promoter is a Pol II promoter.
  • the Pol II promoter is a JET promoter or an EFl-alpha promoter.
  • the polynucleotide comprises a termination sequence.
  • the polynucleotide is positioned within a gene, and expression of the gene is disrupted by the polynucleotide.
  • the gene is a T cell receptor alpha gene.
  • the gene is a T cell receptor alpha constant region (TRAC) gene.
  • the gene is a T cell receptor beta gene.
  • the gene is a T cell receptor beta constant region (TRBC) gene.
  • the gene is a TRAC gene, and the polynucleotide is positioned within SEQ ID NO: 1.
  • the gene is a TRAC gene, and the polynucleotide is positioned between nucleotide 13 and 14 of SEQ ID NO: 1.
  • the genetically-modified human immune cells do not have detectable cell surface expression of an endogenous alpha/beta TCR.
  • the genetically- modified human immune cells do not have detectable cell surface expression of an endogenous CD3.
  • the genetically-modified human immune cells comprise an inhibitory molecule that is inhibitory against dCK.
  • the inhibitory molecule is an inhibitory nucleic acid molecule.
  • the inhibitory nucleic acid molecule is an RNA interference (RNAi) molecule.
  • the RNAi molecule is a short hairpin RNA (shRNA). In some embodiments, the RNAi molecule is a small interfering RNA (siRNA). In some embodiments, the RNAi molecule is a microRNA (miRNA).
  • the RNAi molecule is a microRNA- adapted shRNA (shRNAmiR).
  • shRNAmiR comprises, from 5' to 3': (a) a 5' miR scaffold domain; (b) a 5' miR basal stem domain; (c) a passenger strand; (d) a miR loop domain; (e) a guide strand; (f) a 3' miR basal stem domain; and (g) a 3' miR scaffold domain.
  • the miR loop domain is a miR-30a loop domain, a miR- 15 loop domain, a miR- 16 loop domain, a miR- 155 loop domain, a miR-22 loop domain, a miR- 103 loop domain, or a miR- 107 loop domain.
  • the miR loop domain is a miR-30a loop domain.
  • the miR-30a loop domain comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to SEQ ID NO: 19.
  • the miR-30a loop domain comprises a nucleic acid sequence of SEQ ID NO: 19.
  • the shRNAmiR comprises a microRNA-E (miR-E) scaffold, a miR-30 (e.g., miR-30a) scaffold, a miR-15 scaffold, a miR-16 scaffold, a miR-155 scaffold, a miR-22 scaffold, a miR- 103 scaffold, or a miR- 107 scaffold.
  • the shRNAmiR comprises a miR-E scaffold.
  • the shRNAmiR comprises a structure wherein: (a) the 5' miR scaffold domain comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to SEQ ID NO: 17; (b) the 5' miR basal stem domain comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to SEQ ID NO: 18; (c) the 3' miR basal stem domain comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least
  • the shRNAmiR comprises a structure wherein: (a) the 5' miR scaffold domain comprises a nucleic acid sequence of SEQ ID NO: 17; (b) the 5' miR basal stem domain comprises a nucleic acid sequence of SEQ ID NO: 18; (c) the 3' miR basal stem domain comprises a nucleic acid sequence of SEQ ID NO: 20; and (d) the 3' miR scaffold domain comprises a nucleic acid sequence of SEQ ID NO: 21.
  • the shRNAmiR has a structure wherein the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 7 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the shRNAmiR has a structure wherein the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 9 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the shRNAmiR has a structure wherein the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 11 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 12.
  • the shRNAmiR has a structure wherein the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 13 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the shRNAmiR has a structure wherein the passenger strand comprises a nucleic acid sequence of SEQ ID NO: 15 and the guide strand comprises a nucleic acid sequence of SEQ ID NO: 16.
  • the shRNAmiR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to a sequence set forth in SEQ ID NO: 2.
  • the shRNAmiR comprises a nucleic acid sequence set forth in SEQ ID NO: 2.
  • the shRNAmiR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to a sequence set forth in SEQ ID NO: 3.
  • the shRNAmiR comprises a nucleic acid sequence set forth in SEQ ID NO: 3.
  • the shRNAmiR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to a sequence set forth in SEQ ID NO: 4.
  • the shRNAmiR comprises a nucleic acid sequence set forth in SEQ ID NO: 4.
  • the shRNAmiR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to a sequence set forth in SEQ ID NO: 5.
  • the shRNAmiR comprises a nucleic acid sequence set forth in SEQ ID NO: 5.
  • the shRNAmiR comprises a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, sequence identity to a sequence set forth in SEQ ID NO: 6.
  • the shRNAmiR comprises a nucleic acid sequence set forth in SEQ ID NO: 6.
  • the genetically-modified human immune cells exhibit a reduction of dCK protein expression of between about 1% to about 99% compared to control cells. In some embodiments, the genetically-modified human immune cells exhibit a reduction of dCK protein expression of between about 5% to about 95% compared to control cells. In some embodiments, the genetically-modified human immune cells exhibit a reduction of dCK protein expression of between about 30% to about 90% compared to control cells. In some embodiments, the genetically-modified human immune cells exhibit a reduction of dCK protein expression of between about 50% to about 85% compared to control cells. In some embodiments, the genetically-modified human immune cells exhibit a reduction of dCK protein expression of between about 60% to about 80% compared to control cells.
  • the genetically-modified human immune cells exhibit a reduction of dCK protein expression of between about 65% to about 75% compared to control cells. In some embodiments, the genetically-modified human immune cells exhibit a reduction of dCK protein expression of about 70% compared to control cells.
  • the genetically-modified human immune cells comprise in their genome an inhibitor polynucleotide comprising a nucleic acid sequence encoding the inhibitory molecule.
  • the inhibitor polynucleotide comprises an exogenous promoter that is operably linked to the nucleic acid sequence encoding the inhibitory molecule.
  • the exogenous promoter is a Pol II or a Pol III promoter.
  • the Pol II promoter is a JET promoter or an EFl -alpha promoter.
  • the Pol III promoter is a U6 promoter.
  • the inhibitor polynucleotide comprises a termination sequence.
  • inhibitor polynucleotide is positioned within a gene, and expression of the gene is disrupted by the inhibitor polynucleotide.
  • the gene is a T cell receptor alpha gene.
  • the gene is a TRAC gene.
  • the gene is a T cell receptor beta gene.
  • the gene is a TRBC gene.
  • the gene is a TRAC gene, and the inhibitor polynucleotide is positioned within SEQ ID NO: 1.
  • the gene is a TRAC gene, and the inhibitor polynucleotide is positioned between nucleotide 13 and 14 of SEQ ID NO: 1.
  • the genetically-modified human immune cells comprise in their genomes a cassette comprising the nucleic acid sequence encoding the engineered antigen receptor and the nucleic acid sequence encoding the inhibitory molecule.
  • the cassette comprises a first exogenous promoter that is operably linked to the nucleic acid sequence encoding the engineered antigen receptor, and a second exogenous promoter that is operably linked to the nucleic acid sequence encoding the inhibitory molecule.
  • the first exogenous promoter is a Pol II promoter.
  • the second exogenous promoter is a Pol II promoter or a Pol III promoter.
  • the Pol II promoter is a JET promoter or an EFl -alpha promoter. In some embodiments, the Pol III promoter is a U6 promoter. In some such embodiments, the cassette comprises a first termination sequence 5' downstream of the nucleic acid sequence encoding the engineered antigen receptor, and a second termination sequence 5' downstream of the nucleic acid sequence encoding the inhibitory molecule. In some embodiments, the cassette comprises an exogenous promoter that is operably linked to the nucleic acid sequence encoding the engineered antigen receptor and the nucleic acid sequence encoding the inhibitory molecule. In some such embodiments, the exogenous promoter is a Pol II promoter.
  • the Pol II promoter is a JET promoter or an EFl -alpha promoter.
  • the cassette comprises a termination sequence downstream of the nucleic acid sequence encoding the engineered antigen receptor and the nucleic acid sequence encoding the inhibitory molecule.
  • the cassette is positioned within a gene, wherein expression of the gene is disrupted by the inhibitor polynucleotide.
  • the gene is a T cell receptor alpha gene.
  • the gene is a TRAC gene.
  • the gene is a T cell receptor beta gene.
  • the gene is a TRBC gene.
  • the gene is a TRAC gene, and the cassette is positioned within SEQ ID NO: 1.
  • the gene is a TRAC gene, and the cassette is positioned between nucleotide 13 and 14 of SEQ ID NO: 1.
  • the cassette comprises a first exogenous promoter (e.g., a Pol II promoter), a nucleic acid sequence encoding an engineered antigen receptor (e.g., a CAR or an exogenous TCR) described herein that is operably linked to the first exogenous promoter, a first termination sequence (e.g., a polyA sequence) that terminates expression of the engineered antigen receptor, a second exogenous promoter (e.g., a Pol II or Pol III promoter), a nucleic acid sequence encoding an inhibitory molecule (e.g., shRNAmiR) described herein that is operably linked to the second exogenous promoter, and a second termination sequence (e.g., a polyA sequence) that terminates expression of the inhibitory molecule.
  • a first exogenous promoter e.g., a Pol II promoter
  • a first termination sequence e.g., a polyA sequence
  • an inhibitory molecule e
  • the cassette comprises an exogenous promoter (e.g., a Pol II promoter), a nucleic acid sequence encoding an engineered antigen receptor (e.g., a CAR or an exogenous TCR) described herein, a nucleic acid sequence encoding an inhibitory molecule (e.g., shRNAmiR) described herein, and a termination sequence (e.g., a polyA sequence) that terminates expression of the engineered antigen receptor and the inhibitory molecule, wherein the exogenous promoter is operably linked to both the nucleic acid sequence encoding the engineered antigen receptor and the nucleic acid sequence encoding the inhibitory molecule.
  • a Pol II promoter e.g., a Pol II promoter
  • an engineered antigen receptor e.g., a CAR or an exogenous TCR
  • an inhibitory molecule e.g., shRNAmiR
  • a termination sequence e.g., a polyA sequence
  • the genetically-modified human immune cells comprise an inactivated dCK gene. In some such embodiments, the genetically-modified human immune cells exhibit a reduction of dCK protein expression of about 100% compared to control cells.
  • up to about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or 100% of the human immune cells in the population are genetically-modified human immune cells described herein.
  • between about 20% to about 99% of the human immune cells in the population are genetically-modified human immune cells described herein. In some embodiments, between about 30% to about 99% of the human immune cells in the population are genetically-modified human immune cells described herein. In some embodiments, between about 40% to about 99% of the human immune cells in the population are genetically-modified human immune cells described herein. In some embodiments, between about 50% to about 99% of the human immune cells in the population are genetically- modified human immune cells described herein. In some embodiments, between about 60% to about 99% of the human immune cells in the population are genetically-modified human immune cells described herein.
  • between about 70% to about 99% of the human immune cells in the population are genetically-modified human immune cells described herein. In some embodiments, between about 80% to about 99% of the human immune cells in the population are genetically-modified human immune cells described herein. In some embodiments, between about 90% to about 99% of the human immune cells in the population are genetically-modified human immune cells described herein. In some embodiments, between about 50% to about 80% of the human immune cells in the population are genetically-modified human immune cells described herein. In some embodiments, between about 60% to about 70% of the human immune cells in the population are genetically-modified human immune cells described herein.
  • the nucleoside analog is fludarabine.
  • the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) to the subject at a dose between about 10 to about 40 mg/m 2 /day. In some embodiments, the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) to the subject at a dose between about 20 to about 40 mg/m 2 /day. In some embodiments, the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) to the subject at a dose of about 30 mg/m 2 /day.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) daily for at least one day, or for multiple days, within 7 days prior to administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 5 days and ending 3 days prior to administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 4 days and ending 2 days prior to administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 3 days and ending 1 day prior to administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 2 days prior and ending on the same day as administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 1 day prior and ending 1 day after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 5 days and ending 4 days prior to administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 4 days and ending 3 days prior to administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 3 days and ending 2 days prior to administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 2 days and ending 1 day prior to administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 1 day prior and ending on the same day as administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) to the subject daily starting on the same day as and ending 1 day after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 2 days and ending 4 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 3 days and ending 5 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 4 days and ending 6 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 5 days and ending 7 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 6 days and ending 8 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 7 days and ending 9 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 8 days and ending 10 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 9 days and ending 11 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 10 days and ending 12 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 11 days and ending 13 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 12 days and ending 14 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 13 days and ending 15 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 14 days and ending 16 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 2 days and ending 3 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 3 days and ending 4 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 4 days and ending 5 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 5 days and ending 6 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 6 days and ending 7 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 7 days and ending 8 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 8 days and ending 9 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 9 days and ending 10 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 10 days and ending 11 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 11 days and ending 12 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 12 days and ending 13 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 13 days and ending 14 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject daily starting 14 days and ending 15 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject once 2 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject once 3 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject once 4 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject once 5 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject once 6 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises readministering the nucleoside analog (e.g., fludarabine) to the subject once 7 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject once 8 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises readministering the nucleoside analog (e.g., fludarabine) to the subject once 9 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject once 10 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises readministering the nucleoside analog (e.g., fludarabine) to the subject once 11 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject once 12 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises readministering the nucleoside analog (e.g., fludarabine) to the subject once 13 days after administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject once 14 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 1 day, starting 2 days after the last dose of the nucleoside analog. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 2 days, starting 2 days after the last dose of the nucleoside analog. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 3 days, starting 2 days after the last dose of the nucleoside analog.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 1 day, starting 3 days after the last dose of the nucleoside analog. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 2 days, starting 3 days after the last dose of the nucleoside analog. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 3 days, starting 3 days after the last dose of the nucleoside analog.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 1 day, starting 4 days after the last dose of the nucleoside analog. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 2 days, starting 4 days after the last dose of the nucleoside analog. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 3 days, starting 4 days after the last dose of the nucleoside analog.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 1 day, starting 5 days after the last dose of the nucleoside analog. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 2 days, starting 5 days after the last dose of the nucleoside analog. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 3 days, starting 5 days after the last dose of the nucleoside analog.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 1 day, starting 6 days after the last dose of the nucleoside analog. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 2 days, starting 6 days after the last dose of the nucleoside analog. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 3 days, starting 6 days after the last dose of the nucleoside analog.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 1 day, starting 7 days after the last dose of the nucleoside analog. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 2 days, starting 7 days after the last dose of the nucleoside analog. In some embodiments, the lymphodepletion regimen comprises re-administering the nucleoside analog (e.g., fludarabine) to the subject a second time daily for 3 days, starting 7 days after the last dose of the nucleoside analog.
  • the nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) to the subject daily at a dose of about 30 mg/m 2 /day, starting 3 days and ending 1 day prior to administration of the pharmaceutical composition.
  • the nucleoside analog is re-administered to the subject daily at a dose of about 30 mg/m 2 /day, starting 7 days and ending 9 days after administration of the pharmaceutical composition.
  • the nucleoside analog is readministered to the subject daily at a dose of about 30 mg/m 2 /day, starting 8 days and ending 10 days after administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering the nucleoside analog (e.g., fludarabine) to the subject daily at a dose of about 30 mg/m 2 /day, starting 2 days prior and ending the same day as administration of the pharmaceutical composition.
  • the nucleoside analog is re-administered to the subject daily at a dose of about 30 mg/m 2 /day, starting 7 days and ending 9 days after administration of the pharmaceutical composition.
  • the nucleoside analog is re-administered to the subject daily at a dose of about 30 mg/m 2 /day, starting 8 days and ending 10 days after administration of the pharmaceutical composition.
  • the one or more chemotherapeutic agents includes cyclopho sphamide .
  • the lymphodepletion regimen comprises administering cyclophosphamide to the subject at a dose between about 400 to about 1500 mg/m 2 /day. In some embodiments, the lymphodepletion regimen comprises administering cyclophosphamide to the subject at a dose between about 500 to about 1000 mg/m 2 /day. In some embodiments, the lymphodepletion regimen comprises administering cyclophosphamide to the subject at a dose of about 500 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering cyclophosphamide to the subject daily for at least one day, or for multiple days, within 7 days prior to administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering cyclophosphamide to the subject daily beginning 6 days and ending 4 days prior to administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises administering cyclophosphamide to the subject daily beginning 5 days and ending 3 days prior to administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen comprises administering cyclophosphamide to the subject daily beginning 4 days and ending 2 days prior to administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering cyclophosphamide to the subject daily at a dose of about 500 mg/m 2 /day, starting 5 days and ending 3 days prior to administration of the pharmaceutical composition, and administering the nucleoside analog (e.g., fludarabine) to the subject daily at a dose of about 30 mg/m 2 /day, starting 3 days and ending 1 day prior to administration of the pharmaceutical composition.
  • the nucleoside analog is re-administered to the subject daily at a dose of about 30 mg/m 2 /day, starting 7 days and ending 9 days after administration of the pharmaceutical composition.
  • the nucleoside analog is readministered to the subject daily at a dose of about 30 mg/m 2 /day, starting 8 days and ending 10 days after administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering cyclophosphamide to the subject daily at a dose of about 500 mg/m 2 /day, starting 5 days and ending 3 days prior to administration of the pharmaceutical composition, and administering the nucleoside analog (e.g., fludarabine) to the subject daily at a dose of about 30 mg/m 2 /day, starting 2 days prior and ending the same day as administration of the pharmaceutical composition.
  • the nucleoside analog is re-administered to the subject daily at a dose of about 30 mg/m 2 /day, starting 7 days and ending 9 days after administration of the pharmaceutical composition.
  • the nucleoside analog is re-administered to the subject daily at a dose of about 30 mg/m 2 /day, starting 8 days and ending 10 days after administration of the pharmaceutical composition.
  • the pharmaceutical composition is administered to the subject at a dose between about 0.3xl0 6 to about 6.0xl0 6 genetically-modified human immune cells/kg. In some embodiments, the pharmaceutical composition is administered to the subject at a dose of about 0.5xl0 6 to about 3.0xl0 6 genetically-modified human immune cells/kg. In some embodiments, the pharmaceutical composition is administered to the subject at a dose of about 0.5xl0 6 genetically-modified human immune cells/kg. In some embodiments, the pharmaceutical composition is administered to the subject at a dose of about l.OxlO 6 genetically-modified human immune cells/kg.
  • the pharmaceutical composition is administered to the subject at a dose of about 1.5xl0 6 genetically-modified human immune cells/kg. In some embodiments, the pharmaceutical composition is administered to the subject at a dose of about 2.0xl0 6 genetically-modified human immune cells/kg. In some embodiments, the pharmaceutical composition is administered to the subject at a dose of about 2.5xl0 6 genetically-modified human immune cells/kg. In some embodiments, the pharmaceutical composition is administered to the subject at a dose of about 3.0xl0 6 genetically-modified human immune cells/kg. In some embodiments, the pharmaceutical composition is administered to the subject at a dose of about 270xl0 6 genetically-modified human immune cells. In some embodiments, the pharmaceutical composition is administered to the subject at a dose of about 500xl0 6 genetically-modified human immune cells.
  • the lymphodepletion regimen comprises administering to the subject an effective amount of a biological lymphodepletion agent.
  • the biological lymphodepletion agent is an antibody.
  • the antibody has specificity for a cell surface antigen present on endogenous T cells.
  • the cell surface antigen is CD3.
  • the cell surface antigen is CD52.
  • the lymphodepletion regimen does not comprise administering to the subject a biological lymphodepletion agent.
  • the target cells are cancer cells.
  • the method reduces the size of the cancer in the subject.
  • the method eradicates the cancer in the subject.
  • the method is a method of immunotherapy .
  • Figure 1A illustrates an AAV vector comprising a donor template for CAR T cell production.
  • Figure IB illustrates an experimental workflow to characterize CAR T cell proliferative capacity and resistance properties to fludarabine.
  • Figure 2A shows the reduction of dCK mRNA abundance in CAR T cells comprising the dCK shRNAmiR.
  • Figure 2B shows the number of viable CAR T cells over time in vitro in the presence or absence of fludarabine.
  • Figure 2C provides a table summarizing enrichment of CD3-negative/CAR-positive cells observed following treatment of fludarabine- resistant (FluR) CAR T cells with fludarabine.
  • Figure 3 provides a table summarizing experimental groups for Example 2.
  • Figure 4 shows cell killing by CAR T cells in a real-time cell analysis (RTCA) assay in the presence or absence of fludarabine.
  • RTCA real-time cell analysis
  • Figure 5 summarized the cytotoxicity observed in Figure 4.
  • Figure 6 illustrates the outline for an in vivo mouse study to evaluate FluR CAR T cells in the presence and absence of fludarabine.
  • Figure 7 shows the ventral average total flux observed in the in vivo study conducted in Example 3 of the present disclosure.
  • Figure 8 shows ventral flux images of mice evaluated in Example 3.
  • SEQ ID NO: 1 sets forth the nucleic acid sequence of the TRC 1-2 recognition sequence within the TRAC gene.
  • SEQ ID NO: 2 sets forth the nucleic acid sequence of a 72136 dCK-specific shRNAmiR.
  • SEQ ID NO: 3 sets forth the nucleic acid sequence of a 72137 dCK-specific shRNAmiR.
  • SEQ ID NO: 4 sets forth the nucleic acid sequence of a 72138 dCK-specific shRNAmiR.
  • SEQ ID NO: 5 sets forth the nucleic acid sequence of a 72139 dCK-specific shRNAmiR.
  • SEQ ID NO: 6 sets forth the nucleic acid sequence of a 72140 dCK-specific shRNAmiR.
  • SEQ ID NO: 7 sets forth the nucleic acid sequence of the passenger strand of the
  • SEQ ID NO: 8 sets forth the nucleic acid sequence of the guide strand of the 72136 dCK shRNAmiR.
  • SEQ ID NO: 9 sets forth the nucleic acid sequence of the passenger strand of the
  • SEQ ID NO: 10 sets forth the nucleic acid sequence of the guide strand of the 72137 dCK shRNAmiR.
  • SEQ ID NO: 11 sets forth the nucleic acid sequence of the passenger strand of the
  • SEQ ID NO: 12 sets forth the nucleic acid sequence of the guide strand of the 72138 dCK shRNAmiR.
  • SEQ ID NO: 13 sets forth the nucleic acid sequence of the passenger strand of the
  • SEQ ID NO: 14 sets forth the nucleic acid sequence of the guide strand of the 72139 dCK shRNAmiR.
  • SEQ ID NO: 15 sets forth the nucleic acid sequence of the passenger strand of the
  • SEQ ID NO: 16 sets forth the nucleic acid sequence of the guide strand of the 72140 dCK shRNAmiR.
  • SEQ ID NO: 17 sets forth the nucleic acid sequence of a 5' miR-E scaffold domain.
  • SEQ ID NO: 18 sets forth the nucleic acid sequence of a 5' miR-E basal stem domain.
  • SEQ ID NO: 19 sets forth the nucleic acid sequence of a miR-30a loop domain.
  • SEQ ID NO: 20 sets forth the nucleic acid sequence of a 3' miR-E basal stem domain.
  • SEQ ID NO: 21 sets forth the nucleic acid sequence of a 3' miR-E scaffold domain.
  • a can mean one or more than one.
  • a cell can mean a single cell or a multiplicity of cells.
  • deoxycytidine kinase refers to the protein encoded by the human deoxycytidine kinase gene set forth in NCBI Gene ID No. 1633 (i.e., the Homo sapiens DCK gene), and naturally-occurring variants of the gene which still encode a wild-type dCK protein.
  • the dCK protein phosphorylates several deoxyribonucleosides and their nucleoside analogs, and in the present disclosure, metabolizes nucleoside analogs used for lymphodepletion regimens (e.g., fludarabine) from their prodrug form to an active form.
  • lymphodepletion or “lymphodepletion regimen” refers to the administration to a subject of one or more agents (e.g., chemotherapeutic lymphodepletion agents or biological lymphodepletion agents) capable of reducing endogenous lymphocytes in the subject for immunotherapy; e.g., a reduction of one or more lymphocytes (e.g., B cells, T cells, and/or NK cells) by at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or up to 100% relative to a control (e.g., relative to a starting amount in the subject undergoing treatment, relative to a pre-determined threshold, or relative to an untreated subject).
  • agents e.g., chemotherapeutic lymphodepletion agents or biological lymphodepletion agents
  • lymphocytes e.g
  • biological lymphodepletion agent refers to a biological material, such an antibody, antibody fragment, antibody conjugate, or the like, that can be administered as part of a lymphodepletion regimen to reduce endogenous lymphocytes in the subject for immunotherapy.
  • biological lymphodepletion agents can have specificity for antigens present on lymphocytes; e.g., CD52 or CD3.
  • chemotherapeutic lymphodepletion agents refers to non- biological materials, such as small molecules, that can be administered as part of a lymphodepletion regimen to reduce endogenous lymphocytes in the subject for immunotherapy.
  • the chemotherapeutic lymphodepleting agent can be lymphodepleting but non-myeloablative.
  • nucleoside analogs refers to a certain class of compounds useful in chemotherapy and lymphodepletion, particularly those that are metabolized by deoxycytidine kinase such that they are converted from a prodrug form to an active form.
  • Nucleoside analogs useful in the invention can include, for example, fludarabine, cytarabine, gemcitabine, and decitabine.
  • an effective dose of a lymphodepletion agent refers to an amount sufficient to effect beneficial or desirable biological and/or clinical results.
  • an effective dose of a lymphodepletion agent is sufficient to reduce endogenous lymphocytes in the subject ; e.g., a reduction of one or more lymphocytes (e.g., B cells, T cells, and/or NK cells) by at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or up to 100% relative to a control (e.g., relative to a starting amount in the subject undergoing treatment of a disease, condition or disorder, relative to a pre-determined threshold, or relative to an untreated subject).
  • a control e.g., relative to a starting amount in the subject undergoing treatment of a disease, condition or disorder, relative to a pre-determined threshold, or
  • the effective dose is equivalent to the suggested, recommended or allowed dose (for adults or children) provided in the drug product labeling for a lymphodepletion agent.
  • an effective dose of an immunosuppressant agent is sufficient to reduce an immune response in the subject; e.g., a reduction in number of one or more immune cell types, activation of one or more lymphocyte type, or levels of one or more cytokines by at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or up to 100% relative to a control (e.g., relative to a starting amount in the subject, relative to a pre-determined threshold, or relative to an untreated subject).
  • a control e.g., relative to a starting amount in the subject, relative to a pre-determined threshold, or relative to an untreated subject.
  • the effective dose is equivalent to the suggested, recommended or allowed dose (for adults or children) provided in the drug product labeling for an immunosuppressant agent.
  • an effective dose of a pharmaceutical composition comprising a population of human immune cells, wherein the population comprises a plurality of genetically-modified human immune cells, and wherein the genetically-modified human immune cells express an engineered antigen receptor having specificity for an antigen on target cells, when administered in concert with a lymphodepletion regimen, is sufficient to reduce the target cells by at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or up to 100% relative to a control (e.g., relative to a starting amount in the subject, relative to a pre-determined threshold, or relative to an untreated subject).
  • the effective dose is equivalent to the suggested, recommended or
  • treatment refers to the administration of a pharmaceutical composition disclosed herein, comprising a population of human immune cells to a subject having a disease, disorder or condition.
  • the subject can have a disease such as cancer, and treatment can represent immunotherapy for the treatment of the disease.
  • Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, a partial or complete reduction in the number of cancer cells present in the subject, and remission or improved prognosis.
  • treatment includes the administration of a lymphodepletion regimen to reduce endogenous lymphocytes in the subject for immunotherapy.
  • target cells refers to cells that are desired to be reduced in number using the presently disclosed methods.
  • the target cells express an antigen that can be targeted with genetically-modified human immune cells comprising an engineered antigen receptor, wherein the engineered antigen receptor comprises an extracellular ligand-binding domain having specificity for the antigen.
  • the antigen that is targeted with genetically-modified immune cells according to the presently disclosed methods is on the surface of the target cells.
  • the target cells can be viral, bacterial, fungal, or human cells.
  • the target cells can be disease-causing cells or cells associated with a particular disease state (e.g., autoimmune disease, cancer) or infection, such as cells infected with a virus, bacteria, fungus, or parasite.
  • the target cells are cancer cells.
  • the target cells can be reduced using the presently disclosed methods.
  • the methods result in a reduction in the number of the target cells within the subject when compared to a control (e.g., relative to a starting amount in the subject prior to treatment according to the presently disclosed methods, relative to a predetermined threshold, relative to the same method wherein the genetically-modified human immune cells do not exhibit reduced expression of dCK protein compared to control cells, or relative to an untreated subject).
  • the number of target cells in the subject may be reduced by a percentage using the methods described herein. Such a reduction may be up to 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or up to 100%.
  • immune cell refers to any cell that is part of the immune system (innate and/or adaptive) and is of hematopoietic origin.
  • Non-limiting examples of immune cells include lymphocytes, B cells, T cells, monocytes, macrophages, dendritic cells, granulocytes, megakaryocytes, monocytes, macrophages, natural killer cells, myeloid-derived suppressor cells, innate lymphoid cells, platelets, red blood cells, thymocytes, leukocytes, neutrophils, mast cells, eosinophils, basophils, and granulocytes.
  • T cell and “T lymphocyte” are used interchangeably herein and refer to a white blood cell of the lymphocyte subtype that expresses T cell receptors on the cell membrane.
  • T cells develop in the thymus gland and include both CD8+ T cells and CD4+ T cells, as well as natural killer T cells, memory T cells, gamma delta T cells, and any other lymphocytic cell that expresses a T cell receptor.
  • human natural killer cell or “human NK cell” or “natural killer cell” or “NK cell” refers to a type of cytotoxic lymphocyte critical to the innate immune system.
  • the role NK cells play is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response.
  • NK cells provide rapid responses to virally infected cells and respond to tumor formation, acting at around 3 days after infection.
  • Human NK cells, and cells derived therefrom, include isolated NK cells that have not been passaged in culture, NK cells that have been passaged and maintained under cell culture conditions without immortalization, and NK cells that have been immortalized and can be maintained under cell culture conditions indefinitely.
  • the human NK cell is a differentiated induced pluripotent stem cell (iPSC); e.g., an iPSC derived from a human somatic cell.
  • iPSC differentiated induced pluripotent stem cell
  • T cell receptor alpha gene or “TCR alpha gene” refer to the locus in a T cell which encodes the T cell receptor alpha subunit.
  • the T cell receptor alpha gene can refer to NCBI Gene ID number 6955, before or after rearrangement. Following rearrangement, the T cell receptor alpha gene comprises an endogenous promoter, rearranged V and J segments, the endogenous splice donor site, an intron, the endogenous splice acceptor site, and the T cell receptor alpha constant region locus, which comprises the subunit coding exons.
  • T cell receptor alpha constant region or “TCR alpha constant region” or “TRAC” refers to a coding sequence of the T cell receptor alpha gene.
  • the TCR alpha constant region includes the wild-type sequence, and functional variants thereof, identified by NCBI Gene ID NO. 28755.
  • T cell receptor beta gene or “TCR beta gene” refers to the locus in a T cell which encodes the T cell receptor beta subunit.
  • the T cell receptor beta gene can refer to NCBI Gene ID number 6957.
  • the term “detectable cell surface expression of an endogenous alpha/beta TCR” refers to the ability to detect one or more components of the TCR complex (e.g., an alpha/beta TCR complex) on the cell surface of a T cell (e.g., a CAR T cell), or a population of T cells (e.g., CAR T cells) described herein, using standard experimental methods. Such methods can include, for example, immuno staining and/or flow cytometry specific for components of the TCR itself, such as a TCR alpha or TCR beta chain, or for components of the assembled cell surface TCR complex, such as CD3. Methods for detecting cell surface expression of an endogenous TCR (e.g., an alpha/beta TCR) on an immune cell include those described in MacLeod et al. (2017) Molecular Therapy 25(4): 949- 961.
  • the term “no detectable cell surface expression of CD3” refers to lack of detection of CD3 on the surface of a T cell (e.g., a CAR T cell) described herein, or population of T cells (e.g., CAR T cells) described herein, as detected using standard experimental methods in the art. Methods for detecting cell surface expression of CD3 on an immune cell include those described in MacLeod et al. (2017).
  • exogenous T cell receptor refers to a TCR whose sequence is introduced into the genome of an immune effector cell (e.g., a human T cell) that may or may not endogenously express the TCR.
  • an exogenous TCR on an immune effector cell can confer specificity for a specific epitope or antigen (e.g., an epitope or antigen preferentially present on the surface of a cancer cell or other diseasecausing cell or particle).
  • exogenous T cell receptors can comprise alpha and beta chains or, alternatively, may comprise gamma and delta chains.
  • Exogenous TCRs useful in the invention may have specificity to any antigen or epitope of interest.
  • exogenous TCRs can include an extracellular ligand-binding domain comprising an antibody, or antibody fragment, having specificity for a target antigen.
  • an antibody fragment can be, for example, a single-chain variable fragment (scFv).
  • An “exogenous T cell receptor” or “exogenous TCR” can also refer to a cell surface TCR complex that incorporates one or more genetically-modified and/or exogenous TCR components (e.g., a TRuC; see for example, WO2016187349, WO2018026953, WO2018067993, WO2018098365, WO2018119298, and WO202 1035170).
  • a nucleic acid sequence encodes an “exogenous T cell receptor” or “exogenous TCR”
  • this can refer to a sequence encoding one or more genetically-modified and/or exogenous TCR complex components that, when expressed, associate with endogenous TCR components to form a functional modified TCR complex on the cell surface.
  • antibody refers to a protein, or polypeptide sequence derived from an immunoglobulin molecule which specifically binds with an antigen.
  • Antibodies can be polyclonal or monoclonal, multiple or single chain, or intact immunoglobulins, and may be derived from natural sources or from recombinant sources.
  • Antibodies can be tetramers of immunoglobulin molecules.
  • the terms “antigen” or “Ag” refers to a molecule that is capable of being bound specifically by an antibody, or otherwise provokes an immune response. This immune response may involve either antibody production, or the activation of specific immunologically-competent cells, or both.
  • the terms “tumor associated antigen” or “tumor antigen” or “hyperproliferative disorder antigen” or “antigen associated with a hyperproliferative disorder” refers to antigens that are common to specific hyperproliferative disorders.
  • the hyperproliferative disorder antigens of the present disclosure are derived from, cancers including but not limited to primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, NHL, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.
  • the term “monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present disclosure may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phagedisplay methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • an anti-CD52 antibody refers to an antibody, or antibody fragment or conjugate, having specificity for a CD52 protein expressed on the cell surface of human T cells.
  • an anti-CD52 antibody can be a monoclonal antibody.
  • an anti-CD52 antibody can be alemtuzumab (i.e., CAMPATH).
  • an anti-CD52 antibody can be ALLO-647 (Allogene Therapeutics, San Francisco, CA).
  • an anti-CD3 antibody refers to an antibody, or antibody fragment or conjugate, having specificity for a CD3 protein expressed on the cell surface of human T cells.
  • an anti-CD3 antibody can be a monoclonal antibody.
  • an anti-CD3 antibody can be muromonab-CD3 (Orthoclone OKT3TM), otelixizumab, teplizumab, foralumab, visilizumab, or derivatives thereof which have specificity for CD3.
  • chimeric antigen receptor refers to an engineered receptor that confers or grafts specificity for an antigen onto an immune effector cell (e.g., a human T cell).
  • a chimeric antigen receptor comprises at least an extracellular ligand-binding domain or moiety, a transmembrane domain, and an intracellular domain, wherein the intracellular domain comprises one or more signaling domains and/or costimulatory domains.
  • the extracellular ligand-binding domain or moiety is an antibody, or antibody fragment.
  • antibody fragment can refer to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VE or VH), camelid VHH domains, multi- specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody.
  • An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005).
  • Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).
  • Fn3 fibronectin type III
  • the extracellular ligand-binding domain or moiety is in the form of a single-chain variable fragment (scFv) derived from a monoclonal antibody, which provides specificity for a particular epitope or antigen (e.g., an epitope or antigen preferentially present on the surface of a cell, such as a cancer cell or other disease-causing cell or particle).
  • scFv single-chain variable fragment
  • the scFv is attached via a linker sequence.
  • the scFv is murine, humanized, or fully human.
  • the extracellular ligand-binding domain of a chimeric antigen receptor can also comprise an autoantigen (see, Payne et al. (2016), Science 353 (6295): 179-184), that can be recognized by autoantigen- specific B cell receptors on B lymphocytes, thus directing T cells to specifically target and kill autoreactive B lymphocytes in antibody-mediated autoimmune diseases.
  • CARs can be referred to as chimeric autoantibody receptors (CAARs), and their use is encompassed by the invention.
  • the extracellular ligand-binding domain of a chimeric antigen receptor can also comprise a naturally-occurring ligand for an antigen of interest, or a fragment of a naturally-occurring ligand which retains the ability to bind the antigen of interest.
  • the intracellular stimulatory domain includes one or more cytoplasmic signaling domains that transmit an activation signal to the T cell following antigen binding.
  • cytoplasmic signaling domains can include, without limitation, a CD3 zeta signaling domain.
  • the intracellular stimulatory domain can also include one or more intracellular costimulatory domains that transmit a proliferative and/or cell- survival signal after ligand binding.
  • the co- stimulatory domain can comprise one or more TRAF-binding domains.
  • TRAF binding-domains may include, for example, those set forth in SEQ ID NOs: 9-11.
  • Such intracellular co- stimulatory domains can be any of those known in the art and can include, without limitation, those co-stimulatory domains disclosed in WO 2018/067697 including, for example, Novel 6 (“N6”).
  • co-stimulatory domains can include 4-1BB (CD137), CD27, CD28, CD8, 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, or any combination thereof.
  • a chimeric antigen receptor further includes additional structural elements, including a transmembrane domain that is attached to the extracellular ligand-binding domain via a hinge or spacer sequence.
  • the transmembrane domain can be derived from any membranebound or transmembrane protein.
  • the transmembrane polypeptide can be a subunit of the T-cell receptor (e.g., an a, p, y or polypeptide constituting CD3 complex), IL2 receptor p55 (a chain), p75 (P chain) or y chain, subunit chain of Fc receptors (e.g., Fey receptor III) or CD proteins such as the CD8 alpha chain.
  • the transmembrane domain is a CD8 alpha domain.
  • the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine.
  • the hinge region refers to any oligo or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain.
  • a hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • Hinge regions may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region.
  • the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence or may be an entirely synthetic hinge sequence.
  • a hinge domain can comprise a part of a human CD8 alpha chain, FcyRllla receptor or IgGl.
  • the hinge region can be a CD8 alpha domain.
  • chimeric antigen receptor T cell or “CAR T cell” refers to a human T cell modified to comprise a transgene encoding a CAR, wherein the CAR is expressed on the cell surface of the T cell.
  • the term “proliferate in vivo” refers to an expansion in the number of genetically-modified human immune cells described herein in a subject following administration during immunotherapy. Such proliferation or expansion can be determined by methods known in the art and those shown in the examples herein, which include, for example, utilizing PCR analysis to determine the number of copies of a transgene (e.g., a CAR or exogenous TCR transgene) per mg of DNA isolated from peripheral blood mononuclear cells over a time course following administration of the pharmaceutical composition comprising the genetically-modified human immune cells.
  • a transgene e.g., a CAR or exogenous TCR transgene
  • cancer should be understood to encompass any neoplastic disease (whether invasive or metastatic) which is characterized by abnormal and uncontrolled cell division causing malignant growth or tumor.
  • the term “response,” “complete response,” “complete response with incomplete blood count recovery,” “refractory disease,” “partial response,” “disease progression” or “progressive disease,” “refractory disease,” “relapse” or “relapsed disease” each refer to assessments of disease state and response in subjects following treatment according to the methods disclosed herein.
  • transgene refers to a nucleic acid molecule that encodes a polypeptide or RNA that is heterologous to the vector sequences flanking the coding sequence or is intended for transfer or has been transferred to a non-native cell or genomic locus.
  • the terms “recombinant” or “engineered,” with respect to a protein means having an altered amino acid sequence as a result of the application of genetic engineering techniques to nucleic acids that encode the protein and cells or organisms that express the protein.
  • the term “recombinant” or “engineered” means having an altered nucleic acid sequence as a result of the application of genetic engineering techniques. Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation, and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion.
  • Genetic engineering techniques include, but are not limited to, PCR and DNA cloning technologies; transfection, transformation, and other gene transfer technologies; homologous recombination; site-directed mutagenesis; and gene fusion.
  • a protein having an amino acid sequence identical to a naturally-occurring protein, but produced by cloning and expression in a heterologous host is not considered recombinant or engineered.
  • exogenous or heterologous in reference to a nucleotide sequence or amino acid sequence are intended to mean a sequence that is purely synthetic, that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • endogenous in reference to a nucleotide sequence or protein is intended to mean a sequence or protein that is naturally comprised within or expressed by a cell.
  • wild-type refers to the most common naturally occurring allele (i.e., polynucleotide sequence) in the allele population of the same type of gene, wherein a polypeptide encoded by the wild-type allele has its original functions.
  • wild-type also refers to a polypeptide encoded by a wild-type allele. Wild-type alleles (i.e., polynucleotides) and polypeptides are distinguishable from mutant or variant alleles and polypeptides, which comprise one or more mutations and/or substitutions relative to the wildtype sequence(s).
  • Wild-type nucleases are distinguishable from recombinant or non- naturally-occurring nucleases.
  • the term “wild-type” can also refer to a cell, an organism, and/or a subject which possesses a wild-type allele of a particular gene, or a cell, an organism, and/or a subject used for comparative purposes.
  • the term “genetically-modified” refers to a cell or organism in which, or in an ancestor of which, a genomic DNA sequence has been deliberately modified by recombinant technology. As used herein, the term “genetically-modified” encompasses the term “transgenic.”
  • modification means any insertion, deletion, or substitution of an amino acid residue in the recombinant sequence relative to a reference sequence (e.g., a wild-type or a native sequence).
  • the term “inactivation” or “inactivated” or “disrupted” or “disrupts” or “disrupts expression” or “disrupting a target sequence” refers to the introduction of a mutation (e.g., frameshift mutation) that interferes with the gene function and prevents expression and/or function of the polypeptide/expression product encoded thereby.
  • a mutation e.g., frameshift mutation
  • nuclease-mediated inactivation or disruption of a gene can result in the expression of a truncated protein and/or expression of a protein that does not retain its wild-type function.
  • introduction of a donor template into a gene can result in no expression of an encoded protein, expression of a truncated protein, and/or expression of a protein that does not retain its wild-type function.
  • the term with respect to both amino acid sequences and nucleic acid sequences refers to a measure of the degree of similarity of two sequences based upon an alignment of the sequences that maximizes similarity between aligned amino acid residues or nucleotides, and which is a function of the number of identical or similar residues or nucleotides, the number of total residues or nucleotides, and the presence and length of gaps in the sequence alignment.
  • a variety of algorithms and computer programs are available for determining sequence similarity using standard parameters.
  • sequence similarity is measured using the BLASTp program for amino acid sequences and the BLASTn program for nucleic acid sequences, both of which are available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/), and are described in, for example, Altschul et al. (1990), J. Mol. Biol. 215:403-410; Gish and States (1993), Nature Genet. 3:266-272; Madden et al. (1996), Meth. Enzymol.266:131-141; Altschul et al. (1997), Nucleic Acids Res. 25:33 89-3402); Zhang et al. (2000), J. Comput. Biol.
  • recombinant DNA construct As used herein, the term “recombinant DNA construct,” “recombinant construct,” “expression cassette,” “expression construct,” “chimeric construct,” “construct,” and “recombinant DNA fragment” are used interchangeably herein and are single or doublestranded polynucleotides.
  • a recombinant construct comprises an artificial combination of nucleic acid fragments, including, without limitation, regulatory and coding sequences that are not found together in nature.
  • a recombinant DNA construct may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source and arranged in a manner different than that found in nature. Such a construct may be used by itself or may be used in conjunction with a vector.
  • control refers to a cell that provides a reference point for measuring changes in genotype or phenotype of a genetically-modified cell.
  • a control cell may comprise, for example: (a) a wild-type cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the genetically- modified cell; (b) a cell of the same genotype as the genetically-modified cell but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest); or, (c) a cell genetically identical to the genetically-modified cell but which is not exposed to conditions or stimuli or further genetic modifications that would induce expression of altered genotype or phenotype.
  • vector or “recombinant DNA vector” may be a construct that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell. If a vector is used, then the choice of vector is dependent upon the method that will be used to transform host cells as is well known to those skilled in the art.
  • Vectors can include, without limitation, plasmid vectors and recombinant AAV vectors, or any other vector known in the art suitable for delivering a gene to a target cell. The skilled artisan is well aware of the genetic elements that must be present on the vector in order to successfully transform, select and propagate host cells comprising any of the isolated nucleotides or nucleic acid sequences of the invention.
  • a “vector” also refers to a virus (i.e., a viral vector).
  • Viruses can include, without limitation retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAVs).
  • inhibitory molecule refers to any molecule (e.g., chemical compound, DNA, RNA) that reduces the expression of a target gene in a cell and levels of the encoded gene product as compared to a control cell (e.g., one which does not comprise or has not been introduced to the inhibitory molecule).
  • a control cell e.g., one which does not comprise or has not been introduced to the inhibitory molecule
  • inhibitory nucleic acid molecule refers to a nucleic acid molecule that can function as an inhibitory molecule by reducing the expression of a target gene or that encodes such an inhibitory molecule.
  • a non-limiting example of an inhibitory nucleic acid molecule is an RNA interference (RNAi) molecule that reduces the expression of a target gene via RNA interference.
  • RNA interference or “RNAi” refers to a phenomenon in which the introduction of double- stranded RNA (dsRNA) into a diverse range of organisms and cell types causes degradation of the complementary mRNA.
  • siRNAs small interfering RNAs
  • Dicer small interfering RNAs
  • the siRNAs subsequently assemble with protein components into an RNA- induced silencing complex (RISC), unwinding in the process.
  • RISC RNA- induced silencing complex
  • Activated RISC then binds to complementary transcript by base pairing interactions between the siRNA antisense strand and the mRNA.
  • the bound mRNA is cleaved and sequence specific degradation of mRNA results in gene silencing. See, for example, U.S. Pat. No. 6,506,559.
  • siRNA refers to small interfering RNA, also known as short interfering RNA or silencing RNA.
  • siRNAs can be, for example, 18 to 30, 20 to 25, 21 to 23 or 21 nucleotide-long double-stranded RNA molecules.
  • An “shRNA” as used herein is a short hairpin RNA, which is a sequence of RNA that makes a tight hairpin turn that can also be used to silence gene expression via RNA interference.
  • shRNA can by operably linked to the U6 promoter for expression. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA.
  • shRNA disclosed herein can comprise a sequence complementary to at least 13 nucleotides, at least 14 nucleotides, at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, at least 22 nucleotides, or 23 nucleotides of the mRNA of a target protein.
  • miRNA or “microRNA” or “miR” refers to mature microRNAs (miRNAs) that are endogenously encoded ⁇ 22 nt long RNAs that post- transcriptionally reduce the expression of target genes. miRNAs are found in plants, animals, and some viruses and are generally expressed in a highly tissue- or developmental- stagespecific fashion.
  • a “stem- loop structure” refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion), In some cases, the loop may also be very short and thereby not be recognized by Dicer, leading to Dicer- independent shRNAs (comparable to the endogenous miR0431).
  • the term “hairpin” is also used herein to refer to stem-loop structures. The actual primary sequence of nucleotides within the stem-loop structure is not critical to the practice of the description as long as the secondary structure is present. As is known in the art, the secondary structure does not require exact base-pairing. Thus, the stem may include one or more base mismatches. Alternatively, the base-pairing may be exact (i.e., not include any mismatches).
  • shRNAmiR and “microRN A- adapted shRNA” refer to shRNA sequences embedded within a microRNA scaffold.
  • a shRNAmiR molecule mimics naturally-occurring pri-miRNA molecules in that they comprise a hairpin flanked by sequences necessary for efficient processing and can be processed by the Drosha enzyme into pre-miRNAs, exported into the cytoplasm, and cleaved by Dicer, after which the mature miRNA can enter the RISC.
  • the microRNA scaffold can be derived from naturally- occurring microRNA, pre-miRNAs, or pri-miRNAs or variants thereof.
  • the shRNA sequences which the shRNAmiR is based upon is of a different length from miRNAs (which are 22 nucleotides long) and the miRNA scaffold must therefore be modified in order to accommodate the longer or shorter shRNA sequence length.
  • microRNA flanking sequences refers to nucleotide sequences comprising microRNA processing elements.
  • MicroRNA processing elements are the minimal nucleic acid sequences which contribute to the production of mature microRNA from primary microRNA or precursor microRNA. Often these elements are located within a 40 nucleotide sequence that flanks a microRNA stem-loop structure. In some instances, the microRNA processing elements are found within a stretch of nucleotide sequences of between 5 and 4,000 nucleotides in length that flank a microRNA stem-loop structure.
  • MicroRNA flanking sequences used in the shRNAmiR molecules can be naturally-occurring sequences flanking naturally-occurring microRNA or can be variants thereof.
  • MicroRNA flanking sequences include miR scaffold domains and miR basal stem domains.
  • shRNAmiR molecules used in the presently disclosed compositions and methods can comprise in the 5' to 3' direction: (a) a 5' miR scaffold domain; (b) a 5' miR basal stem domain; (c) a passenger strand; (d) a miR loop domain; (e) a guide strand; (f) a 3' miR basal stem domain; and (g) a 3' miR scaffold domain.
  • miR scaffold domain as it relates to a shRNAmiR refers to a nucleotide sequence that can flank either the 5' or 3' end of a microRNA or shRNA in a shRNAmiR molecule and can be derived from a naturally-occurring microRNA flanking sequence or a variant thereof.
  • the miR basal stem domain sequence separates the shRNA sequence (passenger and guide strand, and miR loop domain) and the scaffold domains.
  • the 5' miR scaffold domain can comprise a restriction enzyme (e.g., type IIS restriction enzyme) recognition sequence at or near its 3' end and the 3' miR scaffold domain can comprise a restriction enzyme recognition sequence at or near its 5' end, thus facilitating the insertion of a shRNA sequence.
  • the secondary structure of the miR scaffold domain is more important than the actual sequence thereof.
  • miR basal stem domain refers to sequences immediately flanking the passenger and guide strand sequences that comprise the base of the hairpin stem below the passenger: guide duplex.
  • the 5' and 3' miR basal stem domains are complementary (fully or partially) in sequence to one another.
  • the 5' and 3' miR basal stem domains comprise sequences that when hybridized together, form two mismatch bubbles, each comprising one or two mismatched base pairs.
  • the term “passenger strand” as it relates to a shRNAmiR refers to the sequence of the shRNAmiR, which is complementary (fully or partially) to the guide sequence.
  • guide strand refers to the sequence of the shRNAmiR that has complementarity (full or partial) with the target mRNA sequence for which a reduction in expression is desired.
  • a “miR loop domain” as it relates to a shRNAmiR refers to the singlestranded loop sequence at one end of the passengerguide duplex of the shRNAmiR.
  • the miR loop domain can be derived from a naturally-occurring pre-microRNA loop sequence or a variant thereof.
  • variable As used herein, the recitation of a numerical range for a variable is intended to convey that the present disclosure may be practiced with the variable equal to any of the values within that range. Thus, for a variable which is inherently discrete, the variable can be equal to any integer value within the numerical range, including the end-points of the range. Similarly, for a variable which is inherently continuous, the variable can be equal to any real value within the numerical range, including the end-points of the range.
  • the present disclosure provides methods and compositions for protecting genetically- modified immune cells from the toxic effects of nucleoside analogs (e.g., fludarabine) by knocking down the gene deoxycytidine kinase (dCK), which metabolizes nucleoside analogs used for lymphodepletion regimens (e.g., fludarabine) from their prodrug form to an active form.
  • nucleoside analogs e.g., fludarabine
  • dCK gene deoxycytidine kinase
  • genetically-modified immune cells having reduced expression of dCK can be enriched by incubation of a cell population with a nucleoside analog such as fludarabine, thus, generating a population of nucleoside analog (e.g., fludarabine) resistant genetically-modified immune cells (e.g., CAR T cells).
  • Genetically-modified immune cells having reduced expression of dCK may have greater persistence in vivo during immunotherapy when a nucleoside analog, such as fludarabine, is administered during lymphodepletion and in some embodiments, after administration of the genetically-modified immune cells.
  • a nucleoside analog such as fludarabine
  • the genetically-modified human immune cells exhibit reduced expression of deoxy cytidine kinase (dCK).
  • dCK deoxy cytidine kinase
  • the genetically-modified human immune cells having reduced expression of dCK exhibit greater resistance to nucleoside analogs compared to control cells that do not exhibit reduced expression of dCK.
  • the genetically-modified human immune cells comprise an inhibitory molecule that is inhibitory against dCK, resulting in reduced expression of dCK.
  • the inhibitory molecule comprises an inhibitory nucleic acid molecule.
  • the inhibitory nucleic acid molecule comprises or encodes an RNA interference (RNAi) molecule.
  • RNAi RNA interference
  • the RNAi molecule is a short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), or a microRNA-adapted shRNA (shRNAmiR).
  • RNAi molecule may target any region of a dCK mRNA.
  • Representative dCK mRNA and protein sequences are known in the art.
  • a non-limiting example of a dCK mRNA sequence is NCBI Acc. No. NM_000788.3 and a dCK protein sequence is NCBI Acc. No. NP_000779.1.
  • the expression of dCK is reduced by between 5% and about 95%, between 30% and 90%, between 50% and 85%, between 60% and 80%, between 65% and 75%, including but not limited to at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or up to about 99% compared to a control cell (e.g., a cell not comprising an inhibitory molecule).
  • a control cell e.g., a cell not comprising an inhibitory molecule
  • Reduced expression of dCK can be measured by any method known in the art, including measuring the levels of dCK mRNA or protein or measuring the amount of dCK enzymatic activity (i.e., metabolization of nucleoside analogs from their prodrug form to an active form) or a downstream effect of reduced dCK expression, such as the effects of a nucleoside analog on the proliferation and survival of cells having reduced dCK expression as compared to control cells.
  • dCK enzymatic activity i.e., metabolization of nucleoside analogs from their prodrug form to an active form
  • a downstream effect of reduced dCK expression such as the effects of a nucleoside analog on the proliferation and survival of cells having reduced dCK expression as compared to control cells.
  • the shRNAmiR molecule used in the presently disclosed methods can comprise a microRNA scaffold in that the structure of the shRNAmiR molecule can mimic that of a naturally-occurring microRNA (or pri-miRNA or pre-miRNA) or a variant thereof. Sequences of microRNAs (and pri-miRNAs and pre-miRNAs) are known in the art.
  • suitable miR scaffolds for the presently disclosed shRNAmiRs include miR-E, miR-30 (e.g., miR-30a), miR-15, miR-16, miR-155, miR-22, miR-103, and miR-107.
  • the shRNAmiR used in the presently disclosed compositions and methods comprises a mir-E scaffold.
  • the mir-E scaffold is a synthetically-derived variant of miR-30a and its genesis is described in International Publication No. WO 2014/117050, which is incorporated by reference in its entirety.
  • the shRNAmiR molecules useful in the presently disclosed methods can comprise the following domains in the 5' to 3' direction: (a) a 5' miR scaffold domain; (b) a 5' miR basal stem domain; (c) a passenger strand; (d) a miR loop domain; (e) a guide strand; (f) a 3' miR basal stem domain; and (g) a 3' miR scaffold domain.
  • the miR scaffold domains and basal stem domains flank the miRNA stem-loop and are referred to herein as microRNA flanking sequences that comprise the microRNA processing elements (the minimal nucleic acid sequences which contribute to the production of mature microRNA from primary microRNA or precursor microRNA).
  • microRNA processing elements are located within a 40 nucleotide sequence that flanks a microRNA stem-loop structure.
  • the microRNA processing elements are found within a stretch of nucleotide sequences of between 5 and 4,000 nucleotides in length that flank a microRNA stem-loop structure.
  • the miRNA flanking sequences are about 3 to about 4,000 nt in length and can be present on either or both the 5' and 3' ends of the shRNAmiR molecule.
  • the minimal length of the microRNA flanking sequence of the shRNAmiR molecule is about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, about 140, about 150, about 200, and any integer therein between.
  • the maximal length of the microRNA flanking sequence of the shRNAmiR molecule is about 2,000, about 2,100, about 2,200, about 2,300, about 2,400, about 2,500, about 2,600, about 2,700, about 2,800, about 2,900, about 3,000, about 3,100, about 3,200, about 3,300, about 3,400, about 3,500, about 3,600, about 3,700, about 3,800, about 3,900, about 4,000, and any integer therein between.
  • the microRNA flanking sequences may be native microRNA flanking sequences or artificial microRNA flanking sequences.
  • a native microRNA flanking sequence is a nucleotide sequence that is ordinarily comprised within naturally existing systems with microRNA sequences (i.e., these sequences are found within the genomic sequences surrounding the minimal microRNA hairpin in vivo).
  • Artificial microRNA flanking sequences are nucleotides sequences that are not found to be flanking microRNA sequences in naturally existing systems.
  • the artificial microRNA flanking sequences may be flanking sequences found naturally in the context of other microRNA sequences. Alternatively, they may be composed of minimal microRNA processing elements which are found within naturally occurring flanking sequences and inserted into other random nucleic acid sequences that do not naturally occur as flanking sequences or only partially occur as natural flanking sequences.
  • the 5' miR scaffold domain is about 10 to about 150 nucleotides in length, including but not limited to about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, and about 150 nucleotides long. In some of these embodiments, the 5' miR scaffold domain is about 111 nucleotides in length.
  • the 5' miR scaffold domain may comprise a 3' sequence that is a recognition sequence for a type IIS restriction enzyme. In some of these embodiments, the 5' miR scaffold domain comprises a Xhol recognition sequence on its 3' end.
  • the 5' miR scaffold domain has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the sequence set forth as SEQ ID NO: 17.
  • the 5' miR scaffold domain has the sequence set forth as SEQ ID NO: 17.
  • the 5' miR basal stem domain of the shRNAmiR can be about 5 to about 30 nucleotides in length in some embodiments, including but not limited to about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, and about 30 nucleotides long. In some of these embodiments, the 5' miR basal stem domain is about 20 nucleotides in length.
  • the 5' miR basal stem domain has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the sequence set forth as SEQ ID NO: 18.
  • the 5' miR basal stem domain has the sequence set forth as SEQ ID NO: 18.
  • the shRNAmiR molecules useful in the presently disclosed methods comprise a stem-loop structure, wherein the stem is comprised of the hybridized passenger and guide strands and the loop is single-stranded.
  • the miR loop domain can be derived from a naturally-occurring pre-microRNA or pri-microRNA loop sequence or a variant thereof.
  • the miR loop domain has the sequence of a loop domain from any one of miR-30 (e.g., miR-30a), miR-15, miR-16, miR-155, miR-22, miR-103, and miR-107.
  • the shRNAmiR comprises a miR-30a loop domain, the sequence of which is set forth as SEQ ID NO: 19.
  • the miR loop domain is about 5 to about 30 nucleotides in length, including but not limited to about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, and about 30 nucleotides long. In some of these embodiments, the miR loop domain is about 15 nucleotides in length.
  • the miR loop domain has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the sequence set forth as SEQ ID NO: 19.
  • the miR loop domain has the sequence set forth as SEQ ID NO: 19.
  • the 3' miR basal stem domain of the shRNAmiR can be about 5 to about 30 nucleotides in length in some embodiments, including but not limited to about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, and about 30 nucleotides long. In some of these embodiments, the 3' miR basal stem domain is about 18 nucleotides in length.
  • the 3' miR basal stem domain has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the sequence set forth as SEQ ID NO: 20.
  • the 3' miR basal stem domain has the sequence set forth as SEQ ID NO: 20.
  • the 3' miR scaffold domain is about 50 to about 150 nucleotides in length, including but not limited to about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, or about 150 nucleotides long. In some of these embodiments, the 3' miR scaffold domain is about 116 nucleotides in length.
  • the 3' miR scaffold domain has at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or more sequence identity to the sequence set forth as SEQ ID NO: 21.
  • the 3' miR scaffold domain has the sequence set forth as SEQ ID NO: 21.
  • the guide strand of the shRNAmiR is the sequence that targets the mRNA, leading to reduction in abundance of the protein encoded by the mRNA. After the guide strand binds to its target mRNA, RISC either degrades the target transcript and/or prevents the target transcript from being loaded into the ribsome for translation.
  • the guide strand is of sufficient complementarity with the target mRNA in order to lead to reduced expression of the target mRNA. In some embodiments, the guide strand is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99% or 100% complementary to the target mRNA sequence.
  • the guide strand hybridizes with the target mRNA within a coding sequence.
  • the guide strand can comprise 1, 2, 3, 4, 5, or more mismatching nucleotides with the target mRNA sequence.
  • the guide strand hybridizes with the target mRNA in a non-coding region, such as a 5' or 3' untranslated region (UTR).
  • UTR 5' or 3' untranslated region
  • the guide strand is about 15 to about 25 nucleotides in length, including but not limited to about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, and about 25 nucleotides long. In some of these embodiments, the guide strand is about 22 nucleotides in length.
  • the shRNA sequence from which the shRNAmiR is derived is less than 22 nucleotides in length, which is the length of most naturally-occurring microRNAs
  • an additional nucleotide is added to the shRNA sequence and in certain embodiments, this additional nucleotide is one that is complementary with the corresponding position within the target mRNA.
  • the passenger strand of the shRNAmiR is the sequence that is fully or partially complementary with the guide strand sequence.
  • the passenger strand is about 15 to about 25 nucleotides in length, including but not limited to about 15 to about 25 nucleotides in length, including but not limited to about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, and about 25 nucleotides long.
  • the passenger strand is about 22 nucleotides in length.
  • the passenger strand can be at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99% or 100% complementary to the guide strand sequence.
  • the passenger strand can comprise 1, 2, 3, 4, 5, or more mismatching nucleotides with the guide strand. In certain embodiments, however, the guide:passenger strand duplex does not comprise any mismatching nucleotides. In general, guide/passenger strand sequences should be selected that do not form any secondary structures within themselves. Further, the use of guide/passenger strand sequences that target sites within an mRNA that comprise singlenucleotide polymorphisms should be avoided. Guide/passenger strand sequences that are specific for the target mRNA are preferred to avoid any off-target effects (i.e., reduction in expression of non-target mRNAs).
  • any program known in the art that models the predicted secondary structure of a RNA molecule can be used, including but not limited to Mfold, RNAfold, and UNAFold.
  • Any program known in the art that can predict the efficiency of a shRNA or miRNA guide/passenger sequence to target a particular mRNA can be used to select suitable guide/passenger strand sequences, including but not limited to those disclosed in Agarwal et al. (2015) eLife 4:e05005; and Knott et al. (2014) Mol Cell 56(6):796-807, each of which is incorporated herein in its entirety.
  • shRNAmiR molecules that target dCK may comprise any passenger and corresponding guide sequence that is complementary (fully or partially) to a sequence within the dCK gene.
  • the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 7 and 8, respectively (e.g., dCK 72136 shRNAmiR).
  • the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 9 and 10, respectively (e.g., dCK 72137 shRNAmiR).
  • the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 11 and 12, respectively (e.g., dCK 72138 shRNAmiR).
  • the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 13 and 14, respectively (e.g., dCK 72139 shRNAmiR). In other embodiments, the passenger and guide sequence of the shRNAmiR comprise the sequences set forth as SEQ ID NO: 15 and 16, respectively (e.g., dCK 72140 shRNAmiR).
  • the dCK-targeted shRNAmiR may comprise a sequence having 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NOs: 2-6.
  • the shRNAmiR comprises the sequence set forth in SEQ ID NO: 2.
  • the genetically-modified immune cell comprises an inhibitory molecule that reduces the expression of dCK
  • the genetically-modified immune cell is less susceptible (i.e., resistant) to the effects of a nucleoside analog (e.g., fludarabine) on cell proliferation and survival.
  • a nucleoside analog e.g., fludarabine
  • genetically-modified immune cells having reduced expression of dCK can be enriched by incubation of a cell population with a purine nucleoside analog such as fludarabine.
  • genetically-modified immune cells having reduced expression of dCK may have greater persistence in vivo during immunotherapy when a purine nucleoside analog such as fludarabine is administered during the course of therapy.
  • the genetically-modified immune cell comprising an inhibitory molecule that reduces the expression of dCK exhibits resistance to a nucleoside analog (e.g., fludarabine), including but not limited to at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% greater cell survival, cell expansion, and target cell killing in the presence of a nucleoside analog compared to a control cell (e.g., a cell not comprising the inhibitory molecule that reduces the expression of dCK).
  • a nucleoside analog e.g., fludarabine
  • the methods comprise a lymphodepletion regimen wherein one or more effective doses of one or more lymphodepletion agents are administered to the subject in order to reduce the number of endogenous lymphocytes prior to administration of the pharmaceutical composition comprising a population of human immune cells, wherein a plurality of the human immune cells are genetically-modified human immune cells.
  • the lymphodepletion regimen comprises a nucleoside analog (e.g., fludarabine), which is a chemotherapeutic lymphodepletion agent.
  • the lymphodepletion regimen used in the presently disclosed methods can comprise one or more additional lymphodepletion agents such as biological lymphodepletion agents, chemotherapeutic lymphodepletion agents, or a combination thereof.
  • a biological lymphodepletion agent can be, for example, any biological material, such an antibody, antibody fragment, antibody conjugate, or the like, that can be administered as part of a lymphodepletion regimen to reduce endogenous lymphocytes in the subject for immunotherapy.
  • Such biological lymphodepletion agents can include, for example, a monoclonal antibody, or a fragment thereof.
  • the biological lymphodepletion agent has specificity for a T cell antigen; i.e., an antigen expressed on the cell surface of T cells. Examples of such antigens include, without limitation, CD52 and CD3.
  • the biological lymphodepletion agent is an antibody, such as a monoclonal antibody, having specificity for CD52.
  • Such antibodies can include, for example, alemtuzumab (i.e., CAMPATH), ALLO-647 (Allogene Therapeutics, San Francisco, CA), derivatives thereof, which bind CD52, or any other CD52 antibody.
  • the biological lymphodepletion agent is an antibody, such as a monoclonal antibody, having specificity for CD3.
  • an anti-CD3 antibody can be muromonab-CD3 (Orthoclone OKT3TM), otelixizumab, teplizumab, foralumab, visilizumab, or derivatives thereof, which have specificity for CD3.
  • lymphodepletion regimens of the invention include the administration of one or more chemotherapeutic lymphodepletion agents.
  • Pre-treatment or pre-conditioning patients prior to cell therapies with one or more chemotherapeutic lymphodepletion agents improves the efficacy of the cellular therapy by reducing the number of endogenous host lymphocytes in the subject, thereby providing a more optimal environment for administered cells to proliferate once administered to the subject.
  • An effective dose of one or more chemotherapeutic lymphodepletion agents can result in the reduction of one or more endogenous lymphocytes (e.g., B cells, T cells, and/or NK cells) in the subject by at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or up to 100% relative to a control; e.g., relative to a starting amount in the subject undergoing treatment, relative to a pre-determined threshold, or relative to an untreated subject.
  • endogenous lymphocytes e.g., B cells, T cells, and/or NK cells
  • 1, 2, 3, 4, or more chemotherapeutic lymphodepletion agents may be included in the lymphodepletion regimen.
  • Chemotherapeutic lymphodepletion agents can refer to non-biological materials, such as small molecules, that can be administered as part of a lymphodepletion regimen to reduce endogenous lymphocytes in the subject for immunotherapy.
  • the chemotherapeutic lymphodepleting agent can be lymphodepleting but non-myeloablative.
  • Chemotherapeutic lymphodepletion agents can include those known in the art including, without limitation, nucleoside analog (e.g., fludarabinejs (such as fludarabine, pentostatin, azathioprine, mercaptopurine such as 6-mercaptopurine, clofarabine, cladribine, and thiopurines such as thioguanine), and compounds capable of inducing interstrand crosslinks within DNA (such as cisplatin, mitomycin C, carmustine, psoralen or nitrogen mustard- derived alkylating agents like cyclophosphamide, ifosfamide, chlorambucil, uramustine, melphalan, and bendamustine).
  • nucleoside analog e.g., fludarabinejs (such as fludarabine, pentostatin, azathioprine, mercaptopurine such as 6-mercaptopurine, clofarabine, cladribine
  • chemotherapeutic lymphodepletion agents useful in the presently disclosed methods include daunorubicin, L- asparaginase, methotrexate, prednisone, dexamethasone, and nelarabine.
  • the lymphodepletion regimen comprises one or more chemotherapeutic lymphodepletion agents, wherein the one or more chemotherapeutic lymphodepletion agents comprises fludarabine.
  • the one or more chemotherapeutic lymphodepletion agents further comprises cyclophosphamide.
  • the lymphodepletion regimen administered during the method of the invention can be administered in an amount effective (i.e., an effective dose) to deplete or reduce the quantity of endogenous lymphocytes in the subject, for example, by 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, relative to a control, e.g., relative to a starting amount in the subject undergoing treatment, relative to a pre-determined threshold, or relative to an untreated subject, prior to administration of the pharmaceutical composition.
  • an effective dose i.e., an effective dose
  • the reduction in lymphocyte count can be monitored using conventional techniques known in the art, such as by flow cytometry analysis of cells expressing characteristic lymphocyte cell surface antigens in a blood sample withdrawn from the subject at varying intervals during treatment with the antibody.
  • the physician may conclude the lymphodepletion therapy and may begin preparing the subject for administration of the pharmaceutical composition.
  • the one or more chemotherapeutic lymphodepletion agents can be administered one day to one month (e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, or 30 days) prior to administration of the pharmaceutical compositions described herein.
  • one or more chemotherapeutic lymphodepletion agents are administered to the subject two or more days prior to administration of the pharmaceutical composition.
  • one or more chemotherapeutic lymphodepletion agents are administered to the subject within seven days prior to administration of the pharmaceutical composition. In certain embodiments, administration of one or more chemotherapeutic lymphodepletion agents ends at least one day, at least two days, or at least three days prior to administration of the pharmaceutical composition.
  • a chemotherapeutic lymphodepletion agent is administered as a single dose per day on each of eight consecutive days, as a single dose per day on each of seven consecutive days, as a single dose per day on each of six consecutive days, as a single dose per day on each of five consecutive days, as a single dose per day on each of four consecutive days, as a single dose per day on each of three consecutive days, as a single dose per day on each of two consecutive days, or as a single dose on one day, prior to administration of the pharmaceutical composition comprising a population of human immune cells, wherein a plurality of the human immune cells are genetically-modified human immune cells.
  • a chemotherapeutic lymphodepletion agent is administered as a single dose per day for at least one day, or for multiple days, within seven days prior to administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) at a dose between about 1 mg/m 2 /day and about 60 mg/m 2 /day. In some of these embodiments, the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) at a dose between about 10 mg/m 2 /day to about 40 mg/m 2 /day. In certain embodiments, the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) at a dose between about 20 mg/m 2 /day and 40 mg/m 2 /day.
  • a nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) at a dose of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 45, about 50, about 55, or about 60 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) at a dose of about 30 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) once daily starting 5 days and ending 3 days prior to administration of the pharmaceutical composition, starting 4 days and ending 2 day prior to administration of the pharmaceutical composition, starting 3 days and ending 1 day prior to administration of the pharmaceutical composition, starting 2 days before and ending the day of administration of the pharmaceutical composition, starting 1 day before and ending 1 day after administration of the pharmaceutical composition, starting 5 days and ending 4 days prior to administration of the pharmaceutical composition, starting 4 days and ending 3 days prior to administration of the pharmaceutical composition, starting 3 days and ending 2 days prior to administration of the pharmaceutical composition, starting 2 days and ending 1 day prior to administration of the pharmaceutical composition, starting 1 day before and ending the day of administration of the pharmaceutical composition, starting the day of administration of the pharmaceutical composition and ending the day after administration of the pharmaceutical composition, starting 2 days and ending 4 days after administration of the pharmaceutical composition, starting 3 days and ending 5 days after administration of the pharmaceutical composition, starting 4 days and ending 6 days after administration of the pharmaceutical composition, or starting 5 days and ending 7 days
  • the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) once daily starting 5 days prior to administration of the pharmaceutical composition and ending 4 days, 3 days, 2 days, or 1 day prior to administration of the pharmaceutical composition, ending the day of administration of the pharmaceutical composition, or ending 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the pharmaceutical composition.
  • a nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) once daily starting 4 days prior to administration of the pharmaceutical composition and ending 3 days, 2 days, or 1 day prior to administration of the pharmaceutical composition, ending the day of administration of the pharmaceutical composition, or ending 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the pharmaceutical composition.
  • a nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) once daily starting 3 days prior to administration of the pharmaceutical composition and ending 2 days or 1 day prior to administration of the pharmaceutical composition, ending the day of administration of the pharmaceutical composition, or ending 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the pharmaceutical composition.
  • a nucleoside analog e.g., fludarabine
  • the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) once daily starting 2 days prior to administration of the pharmaceutical composition and ending 1 day prior to administration of the pharmaceutical composition, ending the day of administration of the pharmaceutical composition, or ending 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) once daily starting 1 day prior to administration of the pharmaceutical composition and ending the day of administration of the pharmaceutical composition, or ending 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) once daily starting the day of administration of the pharmaceutical composition and ending 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) once daily starting 1 day after administration of the pharmaceutical composition and ending 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) once daily starting 2 days after administration of the pharmaceutical composition and ending 3 days, 4 days, 5 days, 6 days, or 7 days after administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) once daily starting 3 days after administration of the pharmaceutical composition and ending 4 days, 5 days, 6 days, or 7 days after administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) once daily starting 4 days after administration of the pharmaceutical composition and ending 5 days, 6 days, or 7 days after administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) once daily starting 5 days after administration of the pharmaceutical composition and ending 6 days or 7 days after administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering a nucleoside analog (e.g., fludarabine) once daily starting 6 days after administration of the pharmaceutical composition and ending 7 days after administration of the pharmaceutical composition.
  • the nucleoside analog (e.g., fludarabine) is re-administered once daily to the subject. In some of these embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily to the subject after administration of the pharmaceutical composition. In certain embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily to the subject after administration of the pharmaceutical composition for a total of 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days.
  • the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 2 days and ending 4 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 3 days and ending 5 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 4 days and ending 6 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 5 days and ending 7 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 6 days and ending 8 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 7 days and ending 9 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 8 days and ending 10 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 9 days and ending 11 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 10 days and ending 12 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 11 days and ending 13 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 12 days and ending 14 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 13 days and ending 15 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 14 days and ending 16 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 2 days and ending 3 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 3 days and ending 4 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 4 days and ending 5 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 5 days and ending 6 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 6 days and ending 7 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 7 days and ending 8 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 8 days and ending 9 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 9 days and ending 10 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 10 days and ending 11 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 11 days and ending 12 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 12 days and ending 13 days after administration of the pharmaceutical composition. In some embodiments, the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 13 days and ending 14 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the nucleoside analog (e.g., fludarabine) is re-administered once daily starting 14 days and ending 15 days after administration of the pharmaceutical composition.
  • the nucleoside analog (e.g., fludarabine) is readministered to the subject once daily starting 2 days and ending 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or 16 days after administration of the pharmaceutical composition comprising a population of human immune cells, wherein a plurality of the human immune cells are genetically-modified human immune cells.
  • the nucleoside analog (e.g., fludarabine) is readministered to the subject once daily starting 3 days and ending 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or 16 days after administration of the pharmaceutical composition.
  • the nucleoside analog (e.g., fludarabine) is re-administered to the subject once daily starting 4 days and ending 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or 16 days after administration of the pharmaceutical composition.
  • the nucleoside analog (e.g., fludarabine) is re-administered to the subject once daily starting 5 days and ending 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or 16 days after administration of the pharmaceutical composition.
  • the nucleoside analog (e.g., fludarabine) is re-administered to the subject once daily starting 6 days and ending 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or 16 days after administration of the pharmaceutical composition.
  • the nucleoside analog (e.g., fludarabine) is re-administered to the subject once daily starting 7 days and ending 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or 16 days after administration of the pharmaceutical composition.
  • the nucleoside analog (e.g., fludarabine) is re-administered to the subject once daily starting 8 days and ending 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or 16 days after administration of the pharmaceutical composition.
  • the nucleoside analog (e.g., fludarabine) is re-administered to the subject once daily starting 9 days and ending 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, or 16 days after administration of the pharmaceutical composition.
  • the nucleoside analog (e.g., fludarabine) is re-administered to the subject once daily starting 10 days and ending 11 days, 12 days, 13 days, 14 days, 15 days, or 16 days after administration of the pharmaceutical composition.
  • the nucleoside analog (e.g., fludarabine) is re-administered to the subject once daily starting 11 days and ending 12 days, 13 days, 14 days, 15 days, or 16 days after administration of the pharmaceutical composition.
  • the nucleoside analog (e.g., fludarabine) is re-administered to the subject once daily starting 12 days and ending 13 days, 14 days, 15 days, or 16 days after administration of the pharmaceutical composition.
  • the nucleoside analog (e.g., fludarabine) is re-administered to the subject once daily starting 13 days and ending 14 days, 15 days, or 16 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the nucleoside analog is re-administered to the subject once daily starting 14 days and ending 15 days or 16 days after administration of the pharmaceutical composition.
  • the nucleoside analog e.g., fludarabine
  • the nucleoside analog is re-administered to the subject once daily starting 15 days and ending 16 days after administration of the pharmaceutical composition.
  • the nucleoside analog (e.g., fludarabine) is re-administered once to the subject 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days after administration of the pharmaceutical composition comprising a population of human immune cells, wherein a plurality of the human immune cells are genetically-modified human immune cells.
  • the nucleoside analog (e.g., fludarabine) is re-administered to the subject at a dose between about 1 mg/m 2 /day and about 60 mg/m 2 /day. In some of these embodiments, the nucleoside analog (e.g., fludarabine) is re-administered at a dose between about 10 mg/m 2 /day to about 40 mg/m 2 /day. In certain embodiments, the nucleoside analog (e.g., fludarabine) is re-administered at a dose between about 20 mg/m 2 /day and 40 mg/m 2 /day.
  • the nucleoside analog (e.g., fludarabine) is readministered at a dose of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 45, about 50, about 55, or about 60 mg/m 2 /day.
  • the nucleoside analog (e.g., fludarabine) is readministered at a dose of about 30 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering one or more effective doses of a nucleoside analog (e.g., fludarabine) (e.g., fludarabine) and a compound capable of inducing interstrand cross-links within DNA (e.g., cyclophosphamide).
  • a nucleoside analog e.g., fludarabine
  • a compound capable of inducing interstrand cross-links within DNA e.g., cyclophosphamide.
  • the lymphodepletion regimen further comprises administering cyclophosphamide.
  • cyclophosphamide is administered to the subject at a dose of about 100 to about 2000 mg/m 2 /day, about 200 to about 1800 mg/m 2 /day, about 300 to about 1700 mg/m 2 /day, about 400 to about 1500 mg/m 2 /day, or about 500 to about 1000 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1100, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, or about 2000 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering cyclophosphamide at a dose of about 500 mg/m 2 /day.
  • the lymphodepletion regimen comprises administering cyclophosphamide once daily starting 6 days and ending 4 days prior to administration of the pharmaceutical composition, starting 5 days and ending 3 days prior to administration of the pharmaceutical composition, or starting 4 days and ending 2 days prior to administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering cyclophosphamide once daily starting 8 days prior to administration of the pharmaceutical composition and ending 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day prior to administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering cyclophosphamide once daily starting 7 days prior to administration of the pharmaceutical composition and ending 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day prior to administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering cyclophosphamide once daily starting 6 days prior to administration of the pharmaceutical composition and ending 5 days, 4 days, 3 days, 2 days, or 1 day prior to administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering cyclophosphamide once daily starting 5 days prior to administration of the pharmaceutical composition and ending 4 days, 3 days, 2 days, or 1 day prior to administration of the pharmaceutical composition.
  • the lymphodepletion regimen comprises administering cyclophosphamide once daily starting 4 days prior to administration of the pharmaceutical composition and ending 3 days, 2 days, or 1 day prior to administration of the pharmaceutical composition. In still other embodiments, the lymphodepletion regimen comprises administering cyclophosphamide once daily starting 3 days prior to administration of the pharmaceutical composition and ending 2 days or 1 day prior to administration of the pharmaceutical composition. In yet other embodiments, the lymphodepletion regimen comprises administering cyclophosphamide once daily starting 2 days prior to administration of the pharmaceutical composition and ending 1 day prior to administration of the pharmaceutical composition.
  • the one or more chemotherapeutic lymphodepletion agents can be administered to the subject using any acceptable route of administration.
  • the nucleoside analog is administered to the subject intravenously.
  • the alkylating agent e.g., cyclophosphamide
  • the lymphodepletion regimen does not comprise administering an effective dose of a biological lymphodepletion agent. In some embodiments, the lymphodepletion regimen does not comprise administering a biological lymphodepletion agent.
  • a biological lymphodepletion agent include monoclonal antibodies or fragments thereof. Such monoclonal antibodies or fragments thereof can have specificity for a T cell antigen.
  • the monoclonal antibody or fragment thereof is an anti-CD52 monoclonal antibody or fragment thereof, or an anti-CD3 antibody or fragment thereof. In certain embodiments, the monoclonal antibody is alemtuzumab or ALLO-647.
  • the lymphodepletion regimen includes administration of a biological lymphodepletion agent in an amount no greater than 1.0 mg/kg during the 7 day period preceding administration of the pharmaceutical composition. In some embodiments, the lymphodepletion regimen includes administration of a biological lymphodepletion agent in an amount no greater than 0.75 mg/kg, 0.5 mg/kg, 0.25 mg/kg, or 0.1 mg/kg during the 7 day period preceding administration of the pharmaceutical composition. In certain embodiments, the lymphodepletion regimen includes administration of a biological lymphodepletion agent in an amount no greater than 0.1 mg/kg during the 7 day period preceding administration of the pharmaceutical composition. In some examples, the lymphodepletion regimen includes no more than a minimal effective dose of a biological lymphodepletion agent.
  • the invention provides methods that utilize genetically-modified human immune cells and populations thereof and provides methods for producing the same.
  • the genetically-modified human immune cells used in the presently disclosed methods are human immune cells.
  • the immune cells are T cells, or cells derived therefrom.
  • the immune cells are natural killer (NK) cells, or cells derived therefrom.
  • the immune cells are B cells, or cells derived therefrom.
  • the immune cells are monocyte or macrophage cells or cells derived therefrom.
  • Immune cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • any number of T cell lines, NK cell lines, B cell lines, monocyte cells lines, or macrophage cell lines available in the art may be used.
  • immune cells are obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan.
  • cells from the circulating blood of an individual are obtained by apheresis.
  • the human immune cells utilized in the presently disclosed methods are not derived from the subject which is administered the pharmaceutical compositions disclosed herein.
  • immune cells useful for the methods can be derived from induced pluripotent stem cells (iPSCs) that have been differentiated into immune cells.
  • iPSCs induced pluripotent stem cells
  • the genetically-modified human immune cells used in the presently disclosed methods comprise a cell surface engineered antigen receptor.
  • engineered antigen receptors include but are not limited to chimeric antigen receptors (CAR)s and exogenous T cell receptors (TCR)s.
  • CAR chimeric antigen receptors
  • TCR exogenous T cell receptors
  • a CAR utilized in the presently disclosed methods will comprise at least an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain comprises a target- specific binding element otherwise referred to as an extracellular ligand-binding domain or moiety.
  • the intracellular domain, or cytoplasmic domain comprises at least one costimulatory domain and one or more signaling domains.
  • a CAR or exogenous TCR useful in the invention comprises an extracellular ligand-binding domain.
  • the choice of ligand-binding domain depends upon the type and number of ligands that define the surface of a target cell.
  • the ligand-binding domain may be chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.
  • cell surface markers that may act as ligands for the ligand-binding domain in a CAR or exogenous TCR can include those associated with viruses, bacterial and parasitic infections, autoimmune disease, and cancer cells.
  • a CAR or exogenous TCR is engineered to target a cancer-specific antigen of interest by way of engineering a desired ligand-binding moiety that specifically binds to an antigen on a cancer (i.e., tumor) cell.
  • cancer antigen tumor antigen
  • cancer- specific antigen tumor-specific antigen
  • tumor-specific antigen refer to antigens that are common to specific hyperproliferative disorders such as cancer.
  • the extracellular ligand-binding domain of the CAR or exogenous TCR is specific for any antigen or epitope of interest, particularly any cancer antigen or epitope of interest.
  • the antigen of the target is a tumor-associated surface antigen, such as ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), EGFR variant III (EGFRvIII), CD19, CD20, CD22, CD30, CD40, CD79B, IL1RAP, glypican 3 (GPC3), CLL-1, disialoganglioside GD2, ductal- epithelial mucine, gp36, TAG-72, glycosphingolipids, glioma-associated antigen, B-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE- 1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hs
  • HER2/neu tumor-associated
  • the extracellular ligand-binding domain or moiety is an antibody, or antibody fragment.
  • An antibody fragment can, for example, be at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multi- specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody.
  • An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005).
  • Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies).
  • Fn3 fibronectin type III
  • the extracellular ligand-binding domain or moiety is in the form of a single-chain variable fragment (scFv) derived from a monoclonal antibody, which provides specificity for a particular epitope or antigen (e.g., an epitope or antigen preferentially present on the surface of a cell, such as a cancer cell or other disease-causing cell or particle).
  • scFv single-chain variable fragment
  • the scFv is attached via a linker sequence.
  • the scFv is murine, humanized, or fully human.
  • the extracellular ligand-binding domain of a chimeric antigen receptor or exogenous TCR can also comprise an autoantigen (see, Payne et al. (2016), Science 353 (6295): 179- 184), that can be recognized by autoantigen-specific B cell receptors on B lymphocytes, thus directing genetically-modified human immune cells to specifically target and kill autoreactive B lymphocytes in antibody-mediated autoimmune diseases.
  • CARs can be referred to as chimeric autoantibody receptors (CAARs), and their use is encompassed by the invention.
  • the extracellular ligand-binding domain of a chimeric antigen receptor or exogenous TCR can also comprise a naturally-occurring ligand for an antigen of interest, or a fragment of a naturally-occurring ligand which retains the ability to bind the antigen of interest.
  • the ligand-binding domain of the CAR or exogenous TCR is an scFv.
  • the scFv comprises a heavy chain variable (VH) domain and a light chain variable (VL) domain from a monoclonal antibody having specificity for a cancer cell antigen.
  • the scFv comprises a VH domain and a VL domain obtained from a CD19-specific antibody.
  • the scFv comprises a VH domain and a VL domain obtained from a CD20-specific antibody.
  • the scFv comprises a VH domain and a VL domain obtained from a BCMA-specific antibody.
  • a CAR can comprise a transmembrane domain which links the extracellular ligandbinding domain with the intracellular signaling and co- stimulatory domains via a hinge region or spacer sequence.
  • the transmembrane domain can be derived from any membranebound or transmembrane protein.
  • the transmembrane polypeptide can be a subunit of the T-cell receptor (e.g., an a, p, y or polypeptide constituting CD3 complex), IL2 receptor p55 (a chain), p75 (P chain) or y chain, subunit chain of Fc receptors (e.g., Fey receptor III) or CD proteins such as the CD8 alpha chain.
  • the transmembrane domain can be synthetic and can comprise predominantly hydrophobic residues such as leucine and valine.
  • the hinge region refers to any oligo or polypeptide that functions to link the transmembrane domain to the extracellular ligand-binding domain.
  • a hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • Hinge regions may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of an antibody constant region.
  • the hinge region may be a synthetic sequence that corresponds to a naturally occurring hinge sequence or may be an entirely synthetic hinge sequence.
  • a hinge domain can comprise a part of a human CD8 alpha chain, FcyRllla receptor or IgGl.
  • Intracellular signaling domains of a CAR are responsible for activation of at least one of the normal effector functions of the cell in which the CAR has been placed and/or activation of proliferative and cell survival pathways.
  • effector function refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines.
  • the intracellular signaling domain can include one or more cytoplasmic signaling domains that transmit an activation signal to the T cell following antigen binding.
  • the intracellular stimulatory domain can also include one or more intracellular costimulatory domains that transmit a proliferative and/or cell- survival signal after ligand binding.
  • the co- stimulatory domain can comprise one or more TRAF-binding domains.
  • Such intracellular co- stimulatory domains can be any of those known in the art and can include, without limitation, those co-stimulatory domains disclosed in WO 2018/067697.
  • co-stimulatory domains can include 4-1BB (CD137), CD27, CD28, CD8, 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, or any combination thereof.
  • the co-stimulatory domain is an N6 domain.
  • the co-stimulatory domain is a 4- IBB co- stimulatory domain.
  • the genetically-modified human immune cell comprises a nucleic acid sequence encoding an exogenous TCR.
  • exogenous TCRs can comprise alpha and beta chains or, alternatively, may comprise gamma and delta chains.
  • Exogenous TCRs useful in the invention may have specificity to any antigen or epitope of interest.
  • the extracellular ligand-binding domain of an exogenous TCR can comprise an antibody or antibody fragment, such as an scFv, fused to one of the TCR complex subunits.
  • the CARs or exogenous TCRs described herein can have, for example, specificity for cancer cell antigens.
  • cancers can include, without limitation, carcinoma, lymphoma, sarcoma, blastomas, leukemia, cancers of B cell origin, breast cancer, gastric cancer, neuroblastoma, osteosarcoma, lung cancer, melanoma, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, rhabdomyosarcoma, leukemia, and Hodgkin lymphoma.
  • cancers and disorders include but are not limited to pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B cell lymphoma, salvage post allogenic bone marrow transplantation, and the like. These cancers can be treated using a combination of CARs that target, for example, CD 19, CD20, CD22, and/or ROR1.
  • a genetically-modified human immune cell or population thereof of the present disclosure targets carcinomas, lymphomas, sarcomas, melanomas, blastomas, leukemias, and germ cell tumors, including but not limited to cancers of B-cell origin, neuroblastoma, osteosarcoma, prostate cancer, renal cell carcinoma, liver cancer, gastric cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, renal cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, non-Hodgkin lymphoma, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine
  • cancers of B-cell origin include, without limitation, B -lineage acute lymphoblastic leukemia, B-cell chronic lymphocytic leukemia, B-cell lymphoma, diffuse large B cell lymphoma, pre-B ALL (pediatric indication), mantle cell lymphoma, follicular lymphoma, marginal zone lymphoma, Burkitt’s lymphoma, multiple myeloma, and B-cell non-Hodgkin lymphoma.
  • cancers can include, without limitation, cancers of B cell origin or multiple myeloma.
  • the cancer of B cell origin is acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), or nonHodgkin lymphoma (NHL).
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • NHL nonHodgkin lymphoma
  • MCL mantle cell lymphoma
  • DLBCL diffuse large B cell lymphoma
  • genetically-modified human immune cells useful in the presently disclosed methods comprise an inactivated TCR alpha gene and/or an inactivated TCR beta gene.
  • Inactivation of the TCR alpha gene and/or TCR beta gene to generate the genetically-modified human immune cells used in the present disclosure occurs in at least one or both alleles where the TCR alpha gene and/or TCR beta gene is being expressed. Accordingly, inactivation of one or both genes prevents expression of the endogenous TCR alpha chain or the endogenous TCR beta chain protein. Expression of these proteins is required for assembly of the endogenous alpha/beta TCR on the cell surface.
  • inactivation of the TCR alpha gene and/or the TCR beta gene results in genetically-modified human immune cells that have no detectable cell surface expression of the endogenous alpha/beta TCR.
  • the endogenous alpha/beta TCR incorporates CD3. Therefore, cells with an inactivated TCR alpha gene and/or TCR beta chain can have no detectable cell surface expression of CD3.
  • the inactivated gene is a TCR alpha constant region (TRAC) gene.
  • the TCR alpha gene, the TRAC gene, the TCR beta gene, or the TRBC gene is inactivated by insertion of a transgene encoding the CAR or exogenous TCR and/or an inhibitory nucleic acid sequence encoding an inhibitory molecule. Insertion of the CAR or exogenous TCR transgene and/or inhibitory nucleic acid sequence disrupts expression of the endogenous TCR alpha chain or TCR beta chain and, therefore, prevents assembly of an endogenous alpha/beta TCR on the T cell surface.
  • the CAR or exogenous TCR transgene and/or inhibitory nucleic acid sequence is inserted into the TRAC gene.
  • a CAR or exogenous TCR transgene and/or inhibitory nucleic acid sequence is inserted into the TRAC gene at an engineered meganuclease recognition sequence comprising SEQ ID NO: 1.
  • the CAR or exogenous TCR transgene and/or inhibitory nucleic acid sequence is inserted into SEQ ID NO: 1 between nucleotide positions 13 and 14.
  • Human immune cells used in the present disclosure may require activation prior to introduction of a nuclease and/or an exogenous sequence of interest to generate CAR T cells.
  • human immune cells can be contacted with anti-CD3 and anti-CD28 antibodies that are soluble or conjugated to a support (e.g., beads) for a period of time sufficient to activate the cells.
  • Immune cells used in the invention can be further modified to express one or more inducible suicide genes, the induction of which provokes cell death and allows for selective destruction of the cells in vitro or in vivo.
  • a suicide gene can encode a cytotoxic polypeptide, a polypeptide that has the ability to convert a non-toxic pro-drug into a cytotoxic drug, and/or a polypeptide that activates a cytotoxic gene pathway within the cell. That is, a suicide gene is a nucleic acid that encodes a product that causes cell death by itself or in the presence of other compounds.
  • a representative example of such a suicide gene is one that encodes thymidine kinase of herpes simplex virus.
  • genes that encode thymidine kinase of varicella zoster virus and the bacterial gene cytosine deaminase that can convert 5-fluorocytosine to the highly toxic compound 5- fluorouracil are also include as non-limiting examples genes that encode caspase- 9, caspase-8, or cytosine deaminase. In some examples, caspase-9 can be activated using a specific chemical inducer of dimerization (CID).
  • a suicide gene can also encode a polypeptide that is expressed at the surface of the cell that makes the cells sensitive to therapeutic and/or cytotoxic monoclonal antibodies.
  • a suicide gene can encode recombinant antigenic polypeptide comprising an antigenic motif recognized by the anti-CD20 mAb Rituximab and an epitope that allows for selection of cells expressing the suicide gene.
  • a suicide gene can encode recombinant antigenic polypeptide comprising an antigenic motif recognized by the anti-CD20 mAb Rituximab and an epitope that allows for selection of cells expressing the suicide gene.
  • the RQR8 polypeptide described in WO2013153391 which comprises two Rituximab-binding epitopes and a QBEndlO-binding epitope.
  • Rituximab can be administered to a subject to induce cell depletion when needed.
  • a suicide gene may include a QBEndlO-binding epitope expressed in combination with a truncated EGFR polypeptide.
  • the invention utilizes a population of human immune cells that includes a plurality of genetically-modified human immune cells expressing a cell surface CAR or exogenous TCR.
  • a population of human immune cells that includes a plurality of genetically-modified human immune cells expressing a cell surface CAR or exogenous TCR.
  • cells in the population are a genetically-modified human immune cell as described herein.
  • at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 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%, at least 96%, at least 97%, at least 98%, at least 99%, or up to 100%, of cells in the population are genetically-modified human immune cells that express a CAR or exogenous TCR and have an inactivated TCR alpha and/or beta gene.
  • between about 20% to about 99% of the human immune cells in the population are genetically-modified human immune cells. In particular embodiments, between about 30% to about 99% of the human immune cells in the population are genetically-modified human immune cells. In particular embodiments, between about 40% to about 99% of the human immune cells in the population are genetically-modified human immune cells. In particular embodiments, between about 50% to about 99% of the human immune cells in the population are genetically-modified human immune cells. In particular embodiments, between about 60% to about 99% of the human immune cells in the population are genetically-modified human immune cells. In particular embodiments, between about 70% to about 99% of the human immune cells in the population are genetically-modified human immune cells.
  • between about 80% to about 99% of the human immune cells in the population are genetically-modified human immune cells. In particular embodiments, between about 90% to about 99% of the human immune cells in the population are genetically-modified human immune cells. In particular embodiments, between about 50% to about 80% of the human immune cells in the population are genetically-modified human immune cells. In particular embodiments, between about 60% to about 70% of the human immune cells in the population are genetically-modified human immune cells.
  • the present disclosure uses human immune cells, or populations of human immune cells comprising a plurality of genetically-modified human immune cells that have been modified to express a CAR or an exogenous TCR and to reduce the expression of dCK.
  • Such human immune cells can be modified in a number of ways in order to introduce a transgene encoding a CAR or exogenous TCR and/or an inhibitory nucleic acid sequence encoding an inhibitory molecule into the genome of the cell, such that the CAR or exogenous TCR, and/or inhibitory nucleic acid sequence is expressed by the cell.
  • a transgene encoding a CAR or exogenous TCR and/or an inhibitory nucleic acid sequence can be introduced into the genome of an immune cell by random integration.
  • the transgene and/or inhibitory nucleic acid sequence can be randomly integrated by transducing the cell with a lentivirus comprising the transgene and/or inhibitory nucleic acid sequence.
  • a transgene encoding a CAR or exogenous TCR and/or inhibitory nucleic acid sequence can be introduced by targeted insertion at a specified location in the genome.
  • targeted integration can be achieved by use of a site- specific, engineered nuclease that generates a cleavage site at a particular location in the genome (e.g., within a target gene), and insertion of a donor template comprising the transgene and/or inhibitory nucleic acid sequence into the cleavage site.
  • the genetically-modified human immune cells comprise an inactivated TCR alpha gene and/or an inactivated TCR beta gene.
  • the inactivated gene can be a TCR alpha constant region (TRAC) gene or a T cell receptor beta constant region (TRBC) gene.
  • TCR alpha constant region TRAC
  • TRBC T cell receptor beta constant region
  • inactivation of one or more of these genes results in genetically-modified human immune cells that do not have detectable cell surface expression of an endogenous alpha/beta TCR and, in some embodiments, do not have detectable cell surface expression of CD3 which is part of the TCR complex.
  • inactivation of the TCR alpha gene, TCR beta gene, the TRAC gene, and/or the TRBC gene can result from the insertion of a transgene and/or an inhibitory nucleic acid sequence into one of these endogenous genes. Insertion of the transgene and/or inhibitory nucleic acid sequence disrupts expression of the polypeptide encoded by the gene; e.g., the endogenous TCR alpha chain or the endogenous TCR beta chain.
  • the transgene encodes the CAR or exogenous TCR, which is expressed by the cell and localized to the cell surface.
  • the inhibitory polynucleotide comprises a nucleic acid sequence encoding an inhibitory molecule that inhibits the expression of the dCK protein.
  • Insertion of one or more donor templates comprising the CAR or exogenous TCR transgene and/or the inhibitory nucleic acid sequence can be achieved by use of an engineered nuclease to generate a cleavage site within a recognition sequence in the genome, such as within the TCR alpha gene, the TRAC gene, the TCR beta gene, or the TRBC gene.
  • nucleases for disrupting expression of an endogenous TCR gene has been disclosed, including the use of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), megaTALs, and CRISPR systems (e.g., Osborn et al. (2016), Molecular Therapy 24(3): 570-581; Eyquem et al. (2017), Nature 543: 113-117; U.S. Patent No. 8,956,828; U.S. Publication No. US2014/0301990; U.S. Publication No.
  • TRC 1-2 meganucleases which have specificity for the TRC 1-2 recognition sequence (SEQ ID NO: 1) in exon 1 of the TRAC gene.
  • SEQ ID NO: 1 The ‘439 and ‘451 publications also disclosed methods for targeted insertion of a CAR coding sequence or an exogenous TCR coding sequence into a cleavage site in the TCR alpha constant region gene.
  • Any engineered nuclease can be used for targeted insertion of the donor template, including an engineered meganuclease, a zinc finger nuclease, a TALEN, a compact TALEN, a CRISPR system nuclease, or a megaTAL.
  • ZFNs zinc-finger nucleases
  • ZFNs can be engineered to recognize and cut pre-determined sites in a genome.
  • ZFNs are chimeric proteins comprising a zinc finger DNA-binding domain fused to a nuclease domain from an endonuclease or exonuclease (e.g., Type Ils restriction endonuclease, such as the FokI restriction enzyme).
  • the zinc finger domain can be a native sequence or can be redesigned through rational or experimental means to produce a protein which binds to a pre-determined DNA sequence ⁇ 18 basepairs in length. By fusing this engineered protein domain to the nuclease domain, it is possible to target DNA breaks with genome-level specificity.
  • ZFNs have been used extensively to target gene addition, removal, and substitution in a wide range of eukaryotic organisms (reviewed in S. Durai et al., Nucleic Acids Res 33, 5978 (2005)).
  • TAL-effector nucleases can be generated to cleave specific sites in genomic DNA.
  • a TALEN comprises an engineered, site-specific DNA- binding domain fused to an endonuclease or exonuclease (e.g., Type Ils restriction endonuclease, such as the FokI restriction enzyme) (reviewed in Mak, et al. (2013) Curr Opin Struct Biol. 23:93-9).
  • the DNA binding domain comprises a tandem array of TAL-effector domains, each of which specifically recognizes a single DNA basepair.
  • Compact TALENs are an alternative endonuclease architecture that avoids the need for dimerization (Beurdeley, et al. (2013) Nat Commun. 4:1762).
  • a Compact TALEN comprises an engineered, site-specific TAL-effector DNA-binding domain fused to the nuclease domain from the I-TevI homing endonuclease or any of the endonucleases listed in Table 2 in U.S. Application No. 20130117869.
  • Compact TALENs do not require dimerization for DNA processing activity, so a Compact TALEN is functional as a monomer.
  • a CRISPR system comprises two components: (1) a CRISPR nuclease; and (2) a short “guide RNA” comprising a ⁇ 20 nucleotide targeting sequence that directs the nuclease to a location of interest in the genome.
  • the CRISPR system may also comprise a tracrRNA.
  • a meganuclease can be an endonuclease that is derived from LCrel and can refer to an engineered variant of LCrel that has been modified relative to natural LCrel with respect to, for example, DNA-binding specificity, DNA cleavage activity, DNA-binding affinity, or dimerization properties.
  • Methods for producing such modified variants of LCrel are known in the art (e.g. WO 2007/047859, incorporated by reference in its entirety).
  • a meganuclease as used herein binds to double-stranded DNA as a heterodimer.
  • a meganuclease may also be a “single-chain meganuclease” in which a pair of DNA-binding domains is joined into a single polypeptide using a peptide linker.
  • Nucleases referred to as megaTALs are single-chain endonucleases comprising a transcription activator-like effector (TALE) DNA binding domain with an engineered, sequence-specific homing endonuclease.
  • TALE transcription activator-like effector
  • the CAR or exogenous TCR transgene and/or inhibitory nucleic acid sequence can be inserted at any position within the TCR alpha gene, the TCR beta gene, the TRAC gene, or the TRBC gene, such that insertion of the transgene and/or inhibitory nucleic acid sequence results in disrupted expression of the endogenous polypeptide; i.e., the endogenous TCR alpha chain or the endogenous TCR beta chain.
  • the CAR or exogenous TCR transgene and/or the inhibitory nucleic acid sequence can be inserted in the TRAC gene at a meganuclease recognition sequence comprising SEQ ID NO: 1.
  • the transgene and/or the inhibitory nucleic acid sequence is inserted between positions 13 and 14 of SEQ ID NO: 1.
  • the nucleases used to practice the invention are singlechain meganucleases.
  • a single-chain meganuclease comprises an N-terminal subunit and a C-terminal subunit joined by a linker peptide.
  • Each of the two domains recognizes half of the recognition sequence (i.e., a recognition half-site) and the site of DNA cleavage is at the middle of the recognition sequence near the interface of the two subunits.
  • DNA strand breaks are offset by four base pairs such that DNA cleavage by a meganuclease generates a pair of four base pair, 3' single-strand overhangs.
  • nuclease-mediated insertion using engineered single-chain meganucleases has been disclosed in International Publication Nos. WO 2017/062439 and WO 2017/062451.
  • Nuclease-mediated insertion of the donor template can also be accomplished using an engineered single-chain meganuclease comprising SEQ ID NO: 17.
  • RNA encoding the engineered nuclease is delivered to the cell because this reduces the likelihood that the gene encoding the engineered nuclease will integrate into the genome of the cell.
  • an RNA interference (RNAi) molecule e.g., shRNA, siRNA, miRNA, or shRNAmiR
  • an mRNA encoding the same is delivered to the cell.
  • the mRNA encoding an engineered nuclease or RNAi molecule can be produced using methods known in the art such as in vitro transcription.
  • the mRNA comprises a modified 5' cap.
  • modified 5' caps are known in the art and can include, without limitation, an anti-reverse cap analogs (ARCA) (US7074596), 7-methyl- guanosine, CleanCap® analogs, such as Cap 1 analogs (Trilink; San Diego, CA), or enzymatically capped using, for example, a vaccinia capping enzyme or the like.
  • the mRNA may be polyadenylated.
  • the mRNA may contain various 5' and 3' untranslated sequence elements to enhance expression of the encoded engineered nuclease or RNAi molecule and/or stability of the mRNA itself.
  • Such elements can include, for example, posttranslational regulatory elements such as a woodchuck hepatitis virus posttranslational regulatory element.
  • the mRNA may contain modifications of naturally-occurring nucleosides to nucleoside analogs. Any nucleoside analogs known in the art are envisioned for use in the present methods. Such nucleoside analogs can include, for example, those described in US 8,278,036.
  • nucleoside modifications can include a modification of uridine to pseudouridine, and/or a modification of uridine to N1 -methyl pseudouridine.
  • Purified nuclease proteins can be delivered into cells to cleave genomic DNA, which allows for homologous recombination or non-homologous end-joining at the cleavage site with an exogenous nucleic acid molecule encoding a polypeptide of interest as described herein, by a variety of different mechanisms known in the art, including those further detailed herein.
  • RNAi molecules can be delivered to cells using any of the methods known in the art, including those further detailed herein.
  • a nucleic acid encoding an engineered nuclease or an RNAi molecule can be introduced into the cell using a single-stranded DNA template.
  • the single-stranded DNA can further comprise a 5' and/or a 3' AAV inverted terminal repeat (ITR) upstream and/or downstream of the sequence encoding the engineered nuclease or RNAi molecule.
  • the single- stranded DNA can further comprise a 5' and/or a 3' homology arm upstream and/or downstream of the sequence encoding the engineered nuclease or RNAi molecule.
  • genes encoding a nuclease or RNAi molecule are introduced into a cell using a linearized DNA template.
  • linearized DNA templates can be produced by methods known in the art.
  • a plasmid DNA encoding a nuclease or RNAi molecule can be digested by one or more restriction enzymes such that the circular plasmid DNA is linearized prior to being introduced into a cell.
  • the nuclease proteins, DNA/mRNA encoding the nuclease, RNAi molecules, or DNA/mRNA encoding the RNAi molecule are coupled to a cell penetrating peptide or targeting ligand to facilitate cellular uptake.
  • cell penetrating peptides known in the art include poly-arginine (Jearawiriyapaisarn, et al. (2008) Mol Ther. 16:1624-9), TAT peptide from the HIV virus (Hudecz et al. (2005), Med. Res. Rev. 25: 679-736), MPG (Simeoni, et al. (2003) Nucleic Acids Res.
  • engineered nucleases, DNA/mRNA encoding nucleases, RNAi molecules, or DNA/mRNA encoding the RNAi molecule are coupled covalently or non-covalently to an antibody that recognizes a specific cell-surface receptor expressed on target cells such that the nuclease protein/DNA/mRNA binds to and is internalized by the target cells.
  • engineered nuclease protein/DNA/mRNA or RNAi molecule/DNA/mRNA can be coupled covalently or non- covalently to the natural ligand (or a portion of the natural ligand) for such a cell-surface receptor.
  • nuclease proteins, DNA/mRNA encoding nucleases, RNAi molecules, or DNA/mRNA encoding RNAi molecules are encapsulated within biodegradable hydrogels for injection or implantation within the desired region of the liver (e.g., in proximity to hepatic sinusoidal endothelial cells or hematopoietic endothelial cells, or progenitor cells which differentiate into the same).
  • Hydrogels can provide sustained and tunable release of the therapeutic pay load to the desired region of the target tissue without the need for frequent injections, and stimuli-responsive materials (e.g., temperature- and pH- responsive hydrogels) can be designed to release the payload in response to environmental or externally applied cues (Kang Derwent et al. (2008) Trans Am Ophthalmol Soc. 106:206- 214).
  • stimuli-responsive materials e.g., temperature- and pH- responsive hydrogels
  • nuclease proteins, DNA/mRNA encoding nucleases, RNAi molecules, or DNA/mRNA encoding RNAi molecules are coupled covalently or, preferably, non-covalently to a nanoparticle or encapsulated within such a nanoparticle using methods known in the art (Sharma, et al. (2014) Biomed Res Int. 2014).
  • a nanoparticle is a nanoscale delivery system whose length scale is ⁇ 1 pm, preferably ⁇ 100 nm.
  • Such nanoparticles may be designed using a core composed of metal, lipid, polymer, or biological macromolecule, and multiple copies of the nuclease proteins, RNAi molecules, mRNA, or DNA can be attached to or encapsulated within the nanoparticle core. This increases the copy number of the protein/mRNA/DNA that is delivered to each cell and, so, increases the intracellular expression of each nuclease to maximize the likelihood that the target recognition sequences will be cut.
  • Nanoparticles may be further modified with polymers or lipids (e.g., chitosan, cationic polymers, or cationic lipids) to form a core-shell nanoparticle whose surface confers additional functionalities to enhance cellular delivery and uptake of the payload (Jian et al. (2012) Biomaterials. 33(30): 7621-30).
  • Nanoparticles may additionally be advantageously coupled to targeting molecules to direct the nanoparticle to the appropriate cell type and/or increase the likelihood of cellular uptake. Examples of such targeting molecules include antibodies specific for cell- surface receptors and the natural ligands (or portions of the natural ligands) for cell surface receptors.
  • the nuclease proteins, DNA/mRNA encoding the nucleases, the RNAi molecules, or DNA/mRNA encoding the RNAi molecules are encapsulated within liposomes or complexed using cationic lipids (see, e.g., LIPOFECTAMINETM, Life Technologies Corp., Carlsbad, CA; Zuris et al. (2015) Nat Biotechnol. 33: 73-80; Mishra et al. (2011) J Drug Deliv. 2011:863734).
  • the liposome and lipoplex formulations can protect the payload from degradation, enhance accumulation and retention at the target site, and facilitate cellular uptake and delivery efficiency through fusion with and/or disruption of the cellular membranes of the target cells.
  • nuclease proteins, DNA/mRNA encoding nucleases, RNAi molecules, or DNA/mRNA encoding RNAi molecules are encapsulated within polymeric scaffolds (e.g., PLGA) or complexed using cationic polymers (e.g., PEI, PLL) (Tamboli et al. (2011) Ther Deliv. 2(4): 523-536).
  • Polymeric scaffolds e.g., PLGA
  • cationic polymers e.g., PEI, PLL
  • Polymeric carriers can be designed to provide tunable drug release rates through control of polymer erosion and drug diffusion, and high drug encapsulation efficiencies can offer protection of the therapeutic payload until intracellular delivery to the desired target cell population.
  • nuclease proteins, DNA/mRNA encoding nucleases, RNAi molecules, or DNA/mRNA encoding RNAi molecules are combined with amphiphilic molecules that self-assemble into micelles (Tong et al. (2007) J Gene Med. 9(11): 956-66).
  • Polymeric micelles may include a micellar shell formed with a hydrophilic polymer (e.g., polyethyleneglycol) that can prevent aggregation, mask charge interactions, and reduce nonspecific interactions.
  • nuclease proteins, DNA/mRNA encoding nucleases, RNAi molecules, or DNA/mRNA encoding RNAi molecules are formulated into an emulsion or a nanoemulsion (z.e., having an average particle diameter of ⁇ Inm) for administration and/or delivery to the target cell.
  • emulsion refers to, without limitation, any oil-in-water, water-in-oil, water-in-oil-in-water, or oil-in-water-in-oil dispersions or droplets, including lipid structures that can form as a result of hydrophobic forces that drive apolar residues (e.g., long hydrocarbon chains) away from water and polar head groups toward water, when a water immiscible phase is mixed with an aqueous phase.
  • lipid structures include, but are not limited to, unilamellar, paucilamellar, and multilamellar lipid vesicles, micelles, and lamellar phases.
  • Emulsions are composed of an aqueous phase and a lipophilic phase (typically containing an oil and an organic solvent). Emulsions also frequently contain one or more surfactants. Nanoemulsion formulations are well known, e.g., as described in US Pat. Nos. 6,015,832, 6,506,803, 6,635,676, 6,559,189, and 7,767,216, each of which is incorporated herein by reference in its entirety.
  • nuclease proteins, DNA/mRNA encoding nucleases, RNAi molecules, or DNA/mRNA encoding RNAi molecules are covalently attached to, or non- covalently associated with, multifunctional polymer conjugates, DNA dendrimers, and polymeric dendrimers (Mastorakos et al. (2015) Nanoscale. 7(9): 3845-56; Cheng et al. (2008) J Pharm Sci. 97(1): 123-43).
  • the dendrimer generation can control the payload capacity and size, and can provide a high pay load capacity.
  • display of multiple surface groups can be leveraged to improve stability, reduce nonspecific interactions, and enhance cell-specific targeting and drug release.
  • the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art.
  • the expression vector can be transferred into a host cell by physical, chemical, or biological means.
  • Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like.
  • Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al.
  • a preferred method for the introduction of a polynucleotide into a host cell is calcium phosphate transfection.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors, and include viral vectors.
  • genes encoding a nuclease or RNAi molecules are delivered using a virus.
  • viruses are known in the art and include retroviruses, lentiviruses, adenoviruses, and adeno-associated viruses (AAVs) (reviewed in Vannucci, et al. (2013 New Microbiol. 36:1-22).
  • AAVs useful in the invention can have any serotype that allows for transduction of the virus into a target cell type and expression of the nuclease gene or RNAi molecule in the target cell.
  • AAVs have a serotype of AAV2 or AAV6.
  • AAVs can be single-stranded AAVs or alternatively, can be self-complementary such that they do not require second-strand DNA synthesis in the host cell (McCarty, et al. (2001) Gene Ther. 8:1248-54).
  • nuclease genes are delivered in DNA form (e.g. plasmid) and/or via a virus (e.g. AAV) they must be operably linked to a promoter.
  • a promoter such as endogenous promoters from the virus (e.g. the LTR of a lentiviral vector) or the well-known cytomegalovirus- or SV40 virus-early promoters.
  • nuclease genes are operably linked to a promoter that drives gene expression preferentially in the target cell.
  • nuclease genes are operably linked to a synthetic promoter, such as a JeT promoter (US 6555674).
  • One or more donor templates comprising the CAR or exogenous TCR transgene and/or the inhibitory nucleic acid sequence is inserted into a cleavage site in the targeted genes.
  • the donor template comprises a 5' homology arm and a 3' homology arm flanking the transgene and/or inhibitory nucleic acid sequence and elements of the insert.
  • Such homology arms have sequence homology to corresponding sequences 5' upstream and 3' downstream of the nuclease recognition sequence where a cleavage site is produced.
  • homology arms can have a length of at least 50 base pairs, preferably at least 100 base pairs, and up to 2000 base pairs or more, and can have at least 90%, preferably at least 95%, or more, sequence homology to their corresponding sequences in the genome.
  • the cassettes or template nucleic acids of the invention may not require an exogenous promoter in order for the encoded sequences to be expressed. Further, in such cases, the cassettes or template nucleic acids may comprise elements (e.g., splice acceptor sequences, 2A or IRES sequences, and the like) necessary for the nucleic acids to be operably linked to the endogenous promoter. In other embodiments, the cassettes or template nucleic acids of the invention comprise one or more exogenous promoters that are operably linked to the nucleic acid sequences and drive expression of the CAR or exogenous TCR and/or inhibitory nucleic acid sequence.
  • elements e.g., splice acceptor sequences, 2A or IRES sequences, and the like
  • the donor template comprises at least two cassettes, wherein the CAR or exogenous TCR transgene is operably linked to a first promoter and the inhibitory nucleic acid sequence is operably linked to a second promoter.
  • the first and second promoter are identical. In other embodiments, the first and second promoter are different from one another.
  • the promoter operably linked to the CAR or exogenous TCR transgene is a Pol II promoter.
  • the promoter operably linked to the inhibitory nucleic acid sequence is a Pol II or Pol III promoter.
  • the CAR or exogenous TCR transgene comprises a first transcriptional termination sequence and the inhibitory nucleic acid sequence comprises a second transcriptional termination sequence.
  • the first and second transcriptional termination sequence are identical. In other embodiments, the first and second transcriptional termination sequence are different from one another.
  • the donor template comprises a single cassette comprising a CAR or exogenous TCR transgene and an inhibitory nucleic acid sequence, wherein the cassette comprises a single exogenous promoter operably linked to both the CAR or exogenous TCR transgene and the inhibitory nucleic acid sequence.
  • the single exogenous promoter is a Pol II promoter.
  • the single cassette further comprises a single transcriptional termination sequence downstream of the transgene and inhibitory nucleic acid sequence.
  • the first and second cassettes can be in the same orientation. This orientation can be either 5' to 3' relative to the homology arms or, alternatively, 3' to 5'.
  • the first cassette may be 5' to the second cassette, or the second cassette may be 5' to the first cassette.
  • the first and second cassettes can be in different orientations in the donor template.
  • the first cassette may be oriented 5' to 3', whereas the second cassette may be oriented 3' to 5'.
  • the first cassette may be oriented 3' to 5' and the second cassette may be oriented 5' to 3'.
  • the cassettes are in opposite orientations, they may be oriented in a “tail-to-tail” configuration, such that the first cassette is oriented 3' to 5' and is positioned 5' to the second cassette, which is oriented 5' to 3'.
  • the second cassette is oriented 3' to 5' and is positioned 5' to the first cassette, which is oriented 5' to 3'.
  • the cassettes are in opposite orientations, they may be oriented in a “head-to-head” configuration, such that the first cassette is oriented 5' to 3' and is positioned 5' to the second cassette, which is oriented 3' to 5'.
  • the second cassette is oriented 5' to 3' and is positioned 5' to the first cassette, which is oriented 3' to 5'.
  • each of the coding sequences can be present in the genome in the same orientation or in different orientations from each other.
  • one coding sequence can be on the plus strand of the double- stranded DNA and another coding sequence on the minus strand.
  • the inhibitory nucleic acid sequence is 3' downstream of the transgene encoding the CAR or exogenous TCR. In alternative embodiments, the inhibitory nucleic acid sequence is 5' upstream of the CAR/TCR-encoding transgene.
  • the CAR or exogenous TCR transgene and/or the inhibitory nucleic acid sequence is operably linked to a Pol II promoter.
  • a suitable Pol II promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto.
  • CMV immediate early cytomegalovirus
  • EF-la Elongation Growth Factor-la
  • constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the present disclosure should not be limited to the use of constitutive promoters.
  • inducible promoters are also contemplated as part of the present disclosure.
  • the use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired.
  • inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.
  • the promoter driving expression of the engineered antigen receptor is a JeT promoter (see, WO/2002/012514).
  • the promoters are selected based on the desired outcome. It is recognized that different applications can be enhanced by the use of different promoters in the cassettes to modulate the timing, location and/or level of expression of the polynucleotides disclosed herein.
  • Such expression constructs may also contain, if desired, a promoter regulatory region (e.g., one conferring inducible, constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific/selective expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
  • Promoters particularly useful for driving expression of an RNA interference molecule are well known in the art and can include, without limitation, pol III promoters, such as U6 or Hl.
  • the transgene encoding the CAR or exogenous TCR and/or the inhibitory nucleic acid sequence can further comprise additional control sequences.
  • the sequence can include homologous recombination enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences, selectable marker sequences (e.g., antibiotic resistance genes), origins of replication, and the like.
  • Sequences encoding engineered nucleases can also include at least one nuclear localization signal. Examples of nuclear localization signals are known in the art (see, e.g., Eange et al., J. Biol. Chem., 2007, 282:5101-5105).
  • a single donor template comprising the CAR or exogenous TCR transgene and the inhibitory nucleic acid sequence is inserted into the cleavage site of a target gene.
  • a first donor template comprising a CAR or exogenous TCR transgene is inserted into a first cleavage site of a first target gene
  • a second donor template comprising an inhibitory nucleic acid sequence is inserted into a second cleavage site of a second target gene.
  • the first and second cleavage site are within the same target gene.
  • the first and second target gene are different from each other.
  • the first donor template is introduced into a cell and subsequently into the genome before the second donor template is introduced. In other embodiments, the first donor template is introduced into a cell and subsequently into the genome after the second donor template is introduced. In yet other embodiments, the first and second donor template are introduced into a cell simultaneously.
  • a donor template comprising the CAR or exogenous TCR transgene and/or inhibitory nucleic acid sequence can be introduced into the cell by any of the means previously discussed.
  • the donor template is introduced by way of a virus, such as a recombinant AAV.
  • AAVs useful for introducing an exogenous nucleic acid can have any serotype that allows for transduction of the virus into the cell and insertion of the exogenous nucleic acid sequence into the cell genome.
  • the AAVs have a serotype of AAV2 or AAV6.
  • AAVs can be single-stranded AAVs or, alternatively, can be self-complementary such that they do not require second-strand DNA synthesis in the host cell.
  • the transgene for the CAR or the exogenous TCR and/or the inhibitory nucleic acid sequence is operably-linked to a promoter such as, for example, a JeT promoter.
  • the nucleic acid molecule of the invention can optionally comprise an epitope which can be used to detect the presence of the encoded cell surface protein.
  • a CAR coding sequence may include a QBendlO epitope which allows for detection using an anti-CD34 antibody (see, WO2013/153391).
  • a cassette can also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors.
  • the selectable marker may be carried on a separate piece of DNA and used in a cotransfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic -resistance genes and fluorescent marker genes.
  • Expression may also be assessed by determining protein expression of the polypeptide targeted by the inhibitory nucleic acid sequence using any method known in the art.
  • the donor template comprising the CAR or exogenous TCR transgene and/or inhibitor polynucleotide can be introduced into the cell using a single-stranded DNA template.
  • the single- stranded DNA can comprise the exogenous sequence of interest and, in preferred embodiments, can comprise 5' and 3' homology arms to promote insertion of the nucleic acid sequence into the cleavage site by homologous recombination.
  • the single- stranded DNA can further comprise a 5' AAV inverted terminal repeat (ITR) sequence 5' upstream of the 5' homology arm, and a 3' AAV ITR sequence 3' downstream of the 3' homology arm.
  • ITR inverted terminal repeat
  • the donor template comprising the CAR or exogenous TCR transgene and/or inhibitor polynucleotide can be introduced into the cell by transfection with a linearized DNA template.
  • a plasmid DNA can be digested by one or more restriction enzymes such that the circular plasmid DNA is linearized prior to transfection into the cell.
  • the method of the invention comprises administering a pharmaceutical composition comprising a population of human immune cells, including a plurality of genetically- modified human immune cells.
  • a pharmaceutical composition comprising a population of human immune cells, including a plurality of genetically- modified human immune cells.
  • Such pharmaceutical compositions can be prepared in accordance with known techniques. See, e.g., Remington, The Science and Practice of Pharmacy (21st ed. 2005).
  • cells are typically admixed with a pharmaceutically acceptable carrier and the resulting composition is administered to a subject.
  • the carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject.
  • pharmaceutical compositions used in the invention can further comprise one or more additional agents useful in the treatment of a disease in the subject.
  • compositions of the invention can further include biological molecules, such as cytokines (e.g., IL-2, IL-7, IL- 15, and/or IL-21), which may promote in vivo cell proliferation and engraftment of genetically- modified human immune cells.
  • cytokines e.g., IL-2, IL-7, IL- 15, and/or IL-21
  • Pharmaceutical compositions comprising genetically- modified human immune cells used in the invention can be administered in the same composition as an additional agent or biological molecule or, alternatively, can be coadministered in separate compositions.
  • the present disclosure also provides genetically-modified human immune cells, or populations thereof, described herein for use as a medicament.
  • the present disclosure further provides the use of genetically-modified human immune cells or populations thereof described herein in the manufacture of a medicament for treating a disease in a subject in need thereof.
  • the medicament is useful for cancer immunotherapy in subjects in need thereof.
  • the method of the invention comprises administering to a subject a pharmaceutical composition comprising a population of human immune cells, wherein the population comprises a plurality of genetically-modified human immune cells.
  • the pharmaceutical composition administered to the subject can comprise an effective dose of genetically-modified human immune cells (e.g., CAR T cells or CAR NK cells) for treatment of a cancer or other disease and administration of the genetically-modified human immune cells of the invention represent an immunotherapy.
  • the administered genetically-modified human immune cells are able to reduce the proliferation, reduce the number, or kill target cells in the recipient.
  • genetically-modified human cells of the present disclosure are able to replicate and expand in vivo, resulting in long-term persistence that can lead to sustained control of a disease.
  • a pharmaceutical composition comprising the genetically-modified human immune cells or populations thereof described herein is administered at a dosage of 0.1 x 10 6 (i.e., 1 x 10 5 ) to 1.0 x 10 9 cells/kg body weight, including all integer values within those ranges.
  • the dosage is 0.3 x 10 6 to 6.0 x 10 6 cells/kg body weight, including all integer values within those ranges.
  • the dosage is 0.3 x 10 6 to 6.0 x 10 6 cells/kg body weight, including all integer values within those ranges. In other embodiments, the dosage is 0.5 x 10 6 to 3.0 x 10 6 cells/kg body weight, including all integer values within those ranges.
  • Dosages of genetically-modified human immune cells can include any of the dosages described herein. In some embodiments, cell compositions are administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.
  • the pharmaceutical composition is administered at a dose of between about 1 xlO 5 and about 1 xlO 9 , about 0.3 x 10 6 and about 6 x 10 6 , or about 0.5 x 10 6 and about 3 x 10 6 genetically-modified human immune cells/kg.
  • the pharmaceutical composition is administered at a dose of about 1 x 10 5 , about 2 x 10 5 , about 3 x 10 5 , about 4 x 10 5 , about 5 x 10 5 , about 6 x 10 5 , about 7 x 10 5 , about 8 x 10 5 , about 9 x 10 5 , about 1 x 10 6 , about 2 x 10 6 , about 3 x 10 6 , about 4 x 10 6 , about 5 x 10 6 , about 6 x 10 6 , about 7 x 10 6 , about 8 x 10 6 , about 9 x 10 6 , about 1 x 10 7 , about 2 x 10 7 , about 3 x 10 7 , about 4 x 10 7 , about 5 x 10 7 , about 6 x 10 7 , about 7 x 10 7 , about 8 x 10 7 , about 9 x 10 7 , about 1 x 10 8 , about 2 x 10 8 , about 3 x 10 8 , about 3 x
  • the pharmaceutical composition is administered at a dose of about 0.5 x 10 6 genetically- modified human immune cells/kg. In some of these embodiments, the pharmaceutical composition is administered at a dose of about 1 x 10 6 genetically-modified human immune cells/kg. In some of these embodiments, the pharmaceutical composition is administered at a dose of about 1.5 x 10 6 genetically-modified human immune cells/kg. In some of these embodiments, the pharmaceutical composition is administered at a dose of about 2 x 10 6 genetically-modified human immune cells/kg. In some of these embodiments, the pharmaceutical composition is administered at a dose of about 2.5 x 10 6 genetically-modified human immune cells/kg.
  • the pharmaceutical composition is administered at a dose of about 3 x 10 6 genetically-modified human immune cells/kg. In some of these embodiments, the pharmaceutical composition is administered at a dose of about 270 x 10 6 genetically-modified human immune cells/kg. In some of these embodiments, the pharmaceutical composition is administered at a dose of about 500 x 10 6 genetically-modified human immune cells/kg.
  • compositions comprising genetically-modified human immune cells include parenteral, (e.g., intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC), or infusion) administration.
  • parenteral e.g., intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC), or infusion
  • lymphodepletion regimens described herein include parenteral (e.g., intravenous (IV), intramuscular (IM), intradermal, subcutaneous (SC), or infusion) administration or oral administration.
  • the administration may be by continuous infusion or by single or multiple boluses.
  • the genetically-modified human immune cells or the one or more chemotherapeutic lymphodepletion agent is infused over a period of less than about 12 hours, less than about 10 hours, less than about 8 hours, less than about 6 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, or less than about 1 hour.
  • the infusion occurs slowly at first and then is increased over time.
  • compositions of the invention can be useful for treating any disease state such as, for example, diseases that can be targeted by adoptive immunotherapy.
  • the presently disclosed methods are useful in the treatment of cancer.
  • the presently disclosed methods comprise administering a pharmaceutical composition comprising genetically-modified human immune cells targeting a cancer cell antigen (i.e., an antigen expressed on the surface of a cancer cell) for the purpose of treating cancer.
  • cancers can include, without limitation, any of the cancers described herein.
  • the presently disclosed methods reduce at least one symptom of a cancer. Symptoms of cancers are well known in the art and can be determined by known techniques. Further, the presently disclosed methods can reduce the number of cancer cells or the size of a cancer (e.g., a tumor) in a subject. Methods for determining the number of cancer cells or the size of a cancer (e.g., a tumor) in a subject vary based on the cancer being treated. Such methods are well known in the art and reductions in cancer cell numbers and tumor number and/or size can be determined by known techniques. In some embodiments, the presently disclosed methods eradicate cancer (i.e., no detectable tumor or cancer cells) in the subject.
  • cancer i.e., no detectable tumor or cancer cells
  • the subject can be further administered an additional therapeutic agent or treatment, including, but not limited to gene therapy, radiation, surgery, or a chemotherapeutic agent(s) (i.e., chemotherapy).
  • an additional therapeutic agent or treatment including, but not limited to gene therapy, radiation, surgery, or a chemotherapeutic agent(s) (i.e., chemotherapy).
  • variants are intended to mean substantially similar sequences.
  • a “variant” polypeptide is intended to mean a polypeptide derived from the “native” polypeptide by deletion or addition of one or more amino acids at one or more internal sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native polypeptide.
  • a “native” polynucleotide or polypeptide comprises a parental sequence from which variants are derived.
  • Variant polypeptides encompassed by the embodiments are biologically active. That is, they continue to possess the desired biological activity of the native protein.
  • Such variants may result, for example, from human manipulation.
  • Biologically active variants of polypeptides described herein will have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%, sequence identity to the amino acid sequence of the native polypeptide, as determined by sequence alignment programs and parameters described elsewhere herein.
  • a biologically active variant of a polypeptide may differ from that polypeptide or subunit by as few as about 1-40 amino acid residues, as few as about 1-20, as few as about 1-10, as few as about 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • polypeptides may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al.
  • a “variant” comprises a deletion and/or addition of one or more nucleotides at one or more sites within the native polynucleotide.
  • variants of the nucleic acids of the embodiments will be constructed such that the open reading frame is maintained.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the polypeptides of the embodiments.
  • Variant polynucleotides include synthetically derived polynucleotides, such as those generated, for example, by using site- directed mutagenesis but which still encode a polypeptide or RNA.
  • variants of a particular polynucleotide of the embodiments will have at least about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.
  • Variants of a particular polynucleotide can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide.
  • deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the polypeptide. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by screening the polypeptide for its biological activity.
  • RNAi sequence featuring a dCK- specific shRNA sequence embedded into a micro-RNA backbone i.e, a shRNAmiR.
  • the resulting RNAi sequence demonstrated the potency of shRNA and the stability of a microRNA.
  • Precision BioSciences’ ARCUS gene editing technology and AAV- mediated targeted transgene insertion strategy, we disrupted the endogenous T cell receptor and inserted a transgene encoding a CD19-specific CAR and a dCK-specific RNAi sequence into the T cell receptor alpha constant (TRAC) locus.
  • TRC 1-2L.1592 meganuclease that generates a cleavage site in the TRAC gene at SEQ ID NO: 1 (i.e., the TRC 1-2 recognition sequence).
  • the donor human T cells were also transduced with an AAV comprising the construct illustrated in Figure 1, which comprises 5' and 3' homology arms (having homology to sequences upstream and downstream of the TRC 1-2 recognition sequence), flanking a JET promoter, a coding sequence for a CD19-specific CAR, a polyA sequence, a U6 promoter, a dCK-specific shRNAmiR (72136, set forth in SEQ ID NO: 2), and a cPPT termination sequence.
  • AAV comprising the construct illustrated in Figure 1, which comprises 5' and 3' homology arms (having homology to sequences upstream and downstream of the TRC 1-2 recognition sequence), flanking a JET promoter, a coding sequence for a CD19-specific CAR, a polyA sequence, a U6 promoter, a dCK-specific shRNAmiR (72136, set forth in SEQ ID NO: 2), and a cPPT termination sequence.
  • the homology arms promoted insertion of the donor template into the cleavage site generated by the TRC 1-2L.1592 meganuclease, allowing for expression of the CAR and shRNAmiR, and knockout of the TRAC gene (and subsequent knockout of the endogenous alpha/beta TCR on the cell surface).
  • Cells produced in this manner referred to as FluR CAR T cells, were exposed to CD 19+ target cells in vitro and in immune-deficient mice and CAR T proliferation and target killing were monitored in the presence and absence of fludarabine.
  • CAR T cells expressing a dCK shRNAmiR had reduced dCK mRNA abundance (Figure 2A), conferring resistance and the ability to proliferate in the presence fludarabine (Figure 2B), as well as the ability to work as a selection system helping in CAR enrichment (Figure 2C).
  • Anti-CD19 CAR+ cells with dcK knockdown (FluR CAR T’s) in the presence of fludarabine display efficient antitumor response to CD 19 expressing tumor cells in vitro
  • RTCA real-time cell analysis

Abstract

La présente invention concerne des procédés de réduction du nombre de cellules cibles chez un sujet, de réduction du rejet d'hôte de cellules immunitaires génétiquement modifiées, et/ou de réduction de l'élimination de cellules immunitaires génétiquement modifiées par des analogues de nucléosides. Plus particulièrement, les procédés de l'invention utilisent des cellules immunitaires génétiquement modifiées comprenant un récepteur antigénique modifié possédant une expression réduite de la protéine désoxycytidine kinase (dCK).
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